WO2004087195A2 - Therapeutic use of modulators of notch and/or kruppel-like factors - Google Patents

Therapeutic use of modulators of notch and/or kruppel-like factors Download PDF

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Publication number
WO2004087195A2
WO2004087195A2 PCT/GB2004/001379 GB2004001379W WO2004087195A2 WO 2004087195 A2 WO2004087195 A2 WO 2004087195A2 GB 2004001379 W GB2004001379 W GB 2004001379W WO 2004087195 A2 WO2004087195 A2 WO 2004087195A2
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klf
activity
polynucleotide
polypeptide
protein
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PCT/GB2004/001379
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French (fr)
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WO2004087195A3 (en
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Brian Robert Champion
Maria Lioumi
Grahame James Mckenzie
Lesley Lynn Young
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Lorantis Limited
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Publication of WO2004087195A3 publication Critical patent/WO2004087195A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A method is described for detecting, measuring or monitoring Notch signalling by determining the amount of a KLF protein, polypeptide or polynucleotide or determining the amount of a polynucleotide coding for such a protein or polypeptide. Methods of modulating the immune system by modulation of KLF activity and methods of modulating immune cell quiescence and proliferation are also described.

Description

Assays and Medical Treatments
Field of the invention
The present invention relates inter alia to monitoring, detecting or measuring Notch signalling and to modulation of the Notch signalling pathway in therapy.
Background of the invention
International Patent Publication No WO 98/20142 describes how manipulation of the Notch signalling pathway can be used in immunotherapy and in the prevention and/or treatment of T-cell mediated diseases. In particular, allergy, autom_mmιity, graft rejection, tumour induced aberrations to the T-cell system and infectious diseases caused, for example, by Plasmodium species, Microfilariae, Helminths, Mycobacteria, HIV, Cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, Haemophilus influenza type B, measles, Hepatitis C or Toxicara, may be targeted.
It has also been shown that it is possible to generate a class of regulatory T cells which are able to transmit tolerance to other T cells, a process termed infectious tolerance (WO98/20142). The functional activity of these cells can be mimicked by over- expression of a Notch ligand protein on then cell surfaces or on the surface of antigen presenting cells. In particular, regulatory T cells can be generated by over-expression of a member of the Delta or Senate family of Notch ligand proteins.
A description of the Notch signalling pathway and conditions affected by it may be found, for example, in our published PCT Applications as follows: PCT/GB97/03058 (filed on 6 November 1997 and published as WO 98/20142; claiming priority from GB 9623236.8 filed on 7 November 1996, GB 9715674.9 filed on 24 July 1997 and GB 9719350.2 filed on 11 September 1997); PCT/GB99/04233 (filed on 15 December 1999 and published as WO 00/36089; claiming priority from GB 9827604.1 filed on 15 December 1999);
PCT/GB00/04391 (filed on 17 November 2000 and published as WO 0135990; claiming priority from GB 9927328.6 filed on 18 November 1999); PCT/GB01/03503 (filed on 3 August 2001 and published as WO 02/12890; claiming priority from GB 0019242.7 filed on 4 August 2000);
PCT/GB02/02438 (filed on 24 May 2002 and published as WO 02/096952; claiming priority from GB 0112818.0 filed on 25 May 2001);
PCT/GB02/03381 (filed on 25 July 2002 and published as WO 03/012111 ; claiming priority from GB 0118155.1 filed on 25 July 2001);
PCT/GB02/03397 (filed on 25 July 2002 and published as WO 03/012441 ; claiming priority from GB0118153.6 filed on 25 July 2001, GB0207930.9 filed on 5 April 2002,
GB 0212282.8 filed on 28 May 2002 and GB 0212283.6 filed on 28 May 2002);
PCT/GB02/03426 (filed on 25 July 2002 and published as WO 03/011317; claiming priority from GB0118153.6 filed on 25 July 2001, GB0207930.9 filed on 5 April 2002,
GB 0212282.8 filed on 28 May 2002 and GB 0212283.6 filed on 28 May 2002);
PCT/GB02/04390 (filed on 27 September 2002 and published as WO 03/029293; claiming priority from GB 0123379.0 filed on 28 September 2001);
PCT/GB02/05137 (filed on 13 November 2002 and published as WO 03/041735; claiming priority from GB 0127267.3 filed on 14 November 2001 , PCT/GB02/03426 filed on 25 July 2002, GB 0220849.4 filed on 7 September 2002, GB 0220913.8 filed on
10 September 2002 and PCT/GB02/004390 filed on 27 September 2002);
PCT/GB02/05133 (filed on 13 November 2002 and published as WO 03/042246; claiming priority from GB 0127271.5 filed on 14 November 2001 and GB 0220913.8 filed on 10 September 2002). All of the foregoing are hereby incorporated herein by reference.
Reference is made also to Hoyne G.F. et al (1999) Int Arch Allergy Immunol 118:122-124; Hoyne et al. (2000) Immunology 100:281-288; Hoyne G.F. et al (2000) Intl Immunol 12:177-185; Hoyne, G. et al. (2001) Immunological Reviews 182:215-227), which are also incorporated herein by reference.
As disclosed in Dang et al (Int J Biochem Cell Biol 2000 Nov-Dec;32(ll-12):l 103-21) recent advances in molecular cloning have led to the identification of a large number of mammalian zinc finger-containing transcription factors that exhibit homology to the Drosophilamelanogaster protein, Kruppel. The amino acid sequences in the zinc finger domains of these K ppel-like factors (KLFs) are closely related to one another.
As described for example by Seddon (http://www.nj_i_r.mrc.ac.ulc/immcellbiol/seddon) all stages of development, differentiation and function of T lymphocytes are regulated by cellular interactions with the environment. The clonotypic T cell receptor (TCR) plays a vital role in the interpretation of these enviromnental cues at all stages of T cell fife. In the thymus, successful rearrangement of TCR β chains is signalled through the pre-TCR complex during the process of β selection, signals through the TCR regulate positive selection and also help decide which mature subset the T cell will differentiate into. In the periphery, recognition of cognate antigen instigates a program of activation and differentiation that allow the T cell to perform its immunological functions, and the qualitative nature of the TCR signal during this process can influence how the activated T cell differentiates and also affect formation of long term memory.
Only recently, however, has it been recognised that the state of quiescence that naϊve T cells enter after development and before activation is not a passive one of awaiting antigenic stimulation but rather an active process requiring specific environmental cues and program of transcriptional activity. Naϊve B cells are maintained in a similar state of quiescence that requires survival signals through the B cell surface Ig receptor that are mediated through activation of the nuclear factor (NF)-κB pathway. Members of the kruppel zinc finger family of transcription factors, such as basic Kmppel-like factor, lung Kmppel-like factor (LKLF) and gut-Kruppel-like factor are thought to be important for maintaining this programmed state. Less is known about what maintains T cell quiescence but LKLF, expressed in mature thymocytes and peripheral T cells, is known to be important.
It has now been found that KLF expression is modulated by Notch signalling, and that the Notch signalling pathway plays a role in cellular quiescence.
Statements of Invention
According to a first aspect of the invention there is provided a method for detecting, measuring or monitoring Notch signalling comprising determining the amount of a KLF protein or polypeptide or determining the amount of a polynucleotide coding for such a protein or polypeptide.
Suitably, the amount of the KLF protein, polypeptide or polynucleotide in a biological sample taken from a subject is determined. Such a sample, may, for example comprise blood, serum, urine, lymphatic fluid, or tissue.
In one embodiment the sample may comprise an immune cell. Suitably the sample comprises peripheral T-cells.
Alternatively the sample may comprise a cancer or tumour cell.
Alternatively the sample may comprise a stem cell.
According to a further aspect of the invention there is provided a method for detecting, measuring or monitoring Notch signalling comprising the steps of: i) obtaining a biological sample from a subject; and ii) contacting the biological sample with a binding agent that binds to a KLF protein, polypeptide or polynucleotide. Suitably the method comprises the steps of: . i) obtaining a biological sample from a subject; ii) contacting the biological sample with a binding agent that binds to a KLF protein, polypeptide or polynucleotide; and iii) detecting in the sample an amount of KLF protein, polypeptide or polynucleotide that binds to the binding agent.
Suitably the method comprises the steps of: i) obtaining a biological sample from the patient; ii) contacting the biological sample with a binding agent that binds to a KLF protein, polypeptide or polynucleotide; iii) detecting in the sample an amount of KLF protein, polypeptide or polynucleotide that binds to the binding agent; and iv) comparing the amount of KLF protein, polypeptide or polynucleotide to a reference value and therefrom determining the degree of Notch signalling.
Suitably the binding agent may be a protein or polypeptide. For example, the binding agent may be an antibody or antibody fragment which binds to a human KLF, preferably human KLF-2.
Alternatively the binding agent may be a polynucleotide, for example a polynucleotide "probe". Preferably such a polynucleotide may be a sequence of at least 10, preferably at least 20 nucleotide residues which hybridises to a nucleotide sequence of a human KLF. Preferably such a polynucleotide hybridises to a nucleotide sequence of a human KLF under stringent hybridisation conditions as described herein. Suitably such a probe may be labelled as described infra.
Suitably, the method may comprise the further step of amphfying a KLF polynucleotide in a sample and detecting the amplified polynucleotide. Suitably the amplification may be by PCR, for example by real-time PCR. According to a further aspect of the invention there is provided a method of detecting, measuring or monitoring Notch signalling in an immune cell by determining the amount of a KLF protein or polypeptide or a polynucleotide coding for such a protein or polypeptide in the cell. Suitably the immune cell may be a T-cell, a B-cell or an antigen presenting cell (APC).
According to a further aspect of the invention there is provided a method for detecting, measuring or monitoring immunological tolerance or activity comprising the step of determining the amount of a KLF protein or polypeptide or a polynucleotide coding for such a protein or polypeptide.
According to a further aspect of the invention there is provided a method for detecting, measuring or monitoring T-cell activity comprising the step of determining the amount of a KLF protein or polypeptide or a polynucleotide coding for such a protein or polypeptide.
According to a further aspect of the invention there is provided a method for detecting, measuring or monitoring the reactivity of a T-cell to an antigen comprising the step of detemiining the amount of a KLF protein or polypeptide or a polynucleotide coding for such a protein or polypeptide.
Suitably the method further comprises a step of comparing the amount of a KLF protein or polypeptide or a polynucleotide coding for such a protein or polypeptide with a reference amount.
According to a further aspect of the invention there is provided a diagnostic kit for monitoring or detecting Notch signalling comprising a binding agent that binds to a KLF protein, polypeptide or polynucleotide for detecting, measuring or monitoring Notch signalling. According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of an immune response in peripheral immune cells.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of an immune response to an antigen or antigenic determinant in peripheral immune cells.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of peripheral lymphocyte activity.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of peripheral T-cell activity.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of effector T-cell activity.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of helper (Th) T-cell activity.
According to a further aspect of the invention there is provided the use of an inhibitor of KLF activity for the manufacture of a medicament for increasing helper (Th) T-cell activity. Preferably when the inhibitor is a receptor or a nucleic acid sequence encoding a receptor, the receptor is activated. Thus, for example, when the agent is a nucleic acid sequence, the receptor is preferably constitutively active when expressed.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity for the manufacture of a medicament for decreasing helper (Th) T-cell activity.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of cytotoxic (Tc) T-cell activity.
According to a further aspect of the invention there is provided the use of an inhibitor of KLF activity for the manufacture of a medicament for increasing cytotoxic (Tc) T-cell activity.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity for the manufacture of a medicament for decreasing cytotoxic (Tc) T-cell activity.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of regulatory (T reg) T-cell activity.
According to a further aspect of the invention there is provided the use of an inhibitor of KLF activity for the manufacture of a medicament for inhibition of regulatory (T reg) T- cell activity. According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity for the manufacture of a medicament for enhancement of regulatory (T reg) T-cell activity.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of Tri regulatory T-cell activity.
According to a further aspect of the invention there is provided the use of an inhibitor of KLF activity for the manufacture of a medicament for inhibition of Tri regulatory T-cell activity.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity for the manufacture of a medicament for enhancing Tri regulatory T-cell activity.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of Th3 regulatory T-cell activity.
According to a further aspect of the invention there is provided the use of an inhibitor of KLF activity for the manufacture of a medicament for inhibition of Th3 regulatory T-cell activity.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity for the manufacture of a medicament for enhancing Th3 regulatory T-cell activity. According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity in the manufacture of a medicament for treatment of inflammation or an inflammatory condition.
According to a further aspect of the invention there is provided the use of a combination of: i) a KLF protein, polypeptide or polynucleotide or an enhancer or inhibitor of KLF activity; and ii) an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant; in the manufacture of a medicament for modulation of the immune system.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or an enhancer or inhibitor of KLF activity in the manufacture of a medicament for modulation of the immune system in simultaneous, contemporaneous, separate or sequential combination with an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant.
Suitably the KLF protein, polypeptide or polynucleotide or modulator of KLF activity is administered to a patient in vivo.
Alternatively the KLF protein, polypeptide or polynucleotide or modulator of KLF activity may be administered to a cell from a patient ex-vivo.
Suitably the KLF protein, polypeptide or polynucleotide or modulator of KLF activity may be used for treatment of allergy, graft rejection, graft-versus-host disease, cancer or infectious disease.
Suitably the modulator of KLF activity may be selected from polypeptides and fragments thereof, linear peptides, cyclic peptides, and nucleic acids which encode therefor, synthetic and natural compounds including low molecular weight organic or inorganic compounds and antibodies.
According to a further aspect of the invention there is provided a method of modulating the peripheral immune system comprising administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided a method of modulating the immune system comprising adniinistering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity in combination with a modulator of the Notch signalling pathway.
According to a further aspect of the invention there is provided an agonist of KLF, in combination with an agent capable of activating Notch signalling.
According to a further aspect of the invention there is provided an antagonist of KLF, in combination with an agent capable of inhibiting Notch signalling.
According to a further aspect of the invention there is provided a method for modulating an immune response in the peripheral immune system by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided a method for modulating an immune response to a selected antigen or antigenic determinant by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided a method for modulating peripheral lymphocyte activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity. According to a further aspect of the invention there is provided a method for modulating peripheral T-cell activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided a method for modulating effector T-cell activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided a method for modulating helper (Th) T-cell activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided a method for increasing helper (Th) T-cell activity by administering an inhibitor of KLF activity.
According to a further aspect of the invention there is provided a method for decreasing helper (Th) T-cell activity by administering a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity.
According to a further aspect of the invention there is provided a method for modulating cytotoxic (Tc) T-cell activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided a method for increasing cytotoxic (Tc) T-cell activity by administering an inhibitor of KLF activity.
According to a further aspect of the invention there is provided a method for decreasing cytotoxic (Tc) T-cell activity by administering an a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity. According to a further aspect of the invention there is provided a method for modulating regulatory (T reg) T-cell activity by adrninistering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided a method for decreasing regulatory (T reg) T-cell activity by administering an inhibitor of KLF activity.
According to a further aspect of the invention there is provided a method for increasing regulatory (T reg) T-cell activity by administering a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity.
According to a further aspect of the invention there is provided a method for modulating Tri regulatory T-cell activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided a method for inhibiting Tri regulatory T-cell activity by administering an inhibitor of KLF activity.
According to a further aspect of the invention there is provided a method for increasing Tri regulatory T-cell activity by administering a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity.
According to a further aspect of the invention there is provided a method for modulating Th3 regulatory T-cell activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided a method for inhibiting Th3 regulatory T-cell activity by administering an inhibitor of KLF activity. According to a further aspect of the invention there is provided a method for increasing Th3 regulatory T-cell activity by administering a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity.
According to a further aspect of the invention there is provided a method for modulating the immune system by simultaneously, contemporaneously, separately or sequentially administering a combination of: i) a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; and ii) an antigen or antigenic detenninant or a polynucleotide coding for an antigen or antigenic determinant.
Suitably such methods may be used for the modification of peripheral T-cell activity.
Suitably such methods may b e used for the treatment of autoimmune disease.
Suitably such methods may be used for the treatment of allergy, graft rejection or GvHD.
Suitably such methods may be used for the treatment of cancer.
Suitably such methods may be used for enhancing the immune response to cancer.
Suitably such methods may be used for enhancing the immune response to a pathogen.
According to a further aspect of the invention there is provided a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for use in affecting linked suppression. According to a further aspect of the invention there is provided a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for use in affecting infectious tolerance.
According to a further aspect of the invention there is provided a product comprising a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity in combination with a modulator of the Notch signalling pathway.
According to a further aspect of the invention there is provided a product comprising an inhibitor of KLF activity in combination with an inhibitor of the Notch signalling pathway.
According to a further aspect of the invention there is provided a product comprising a KLF protein, polypeptide or polynucleotide or an activator of KLF activity in combination with an activator of the Notch signalling pathway.
According to a further aspect of the invention there is provided a method for producing a lymphocyte or antigen presenting cell (APC) for promoting tolerance to an allergen or antigen which method comprises incubating a lymphocyte or APC obtained from a human or animal patient with (i) a KLF protein, polypeptide or polynucleotide or an agonist of KLF and (ii) the allergen or antigen.
Such methods may be used, for example, for producing an APC capable of inducing T cell tolerance.
Suitably such methods comprise incubating a T cell obtained from a human or animal patient with an antigen presenting cell (APC) in the presence of (i) a KLF protein, polypeptide or polynucleotide or an agonist of KLF activity and (ii) the allergen or antigen for producing ex vivo a T cell having tolerance to an allergen or antigen. According to a further aspect of the invention there is provided a method for producing a lymphocyte or APC for promoting tolerance to an allergen or antigen which method comprises incubating a lymphocyte or APC obtained from a human or animal patient with a lymphocyte or APC produced by a method as described above.
According to a further aspect of the invention there is provided the use of a lymphocyte or APC produced by a method as described above in suppressing an immune response in a mammal to the allergen or antigen.
According to a further aspect of the invention there is provided a method for treating a patient suffering from a disease characterised by inappropriate lymphocyte activity which method comprises administering to the patient a lymphocyte produced by a method as described above.
According to a further aspect of the invention there is provided a method for enhancing the reactivity of a T cell toward a tumour cell which method comprises: i) isolating a T cell from a patient having said tumour cell present in their body; ii) exposing the T cell to a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; and iii) re-introducing the T cell into the patient; wherein the T cell comprises a T cell receptor specific for a tumour antigen expressed by the tumour cell.
According to a further aspect of the invention there is provided a method for enhancing the reactivity of a T cell toward a tumour cell which method comprises: i) isolating an antigen presenting cell (APC) from a tumour present in the body of a patient; ii) exposing the APC to a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; and iii) re-introducing the APC into the patient. According to a further aspect of the invention there is provided a method for enhancing the reactivity of a T cell toward a tumour cell which method comprises: i) isolating a tumour cell from a tarnour present in the body of a patient; ii) exposing the tumour cell to a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; and iii) re-introducing the tumour cell into the patient.
Suitably in such methods the T cell may be a tumour infiltrating lymphocyte (TIL).
According to a further aspect of the invention there is provided a method of vaccinating a patient against a tumour which method comprises: i) administering a mmour antigen expressed by the tumour to a patient; and ii) exposing APCs present in the patient to a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided an assay method for modulators of KLF activity comprising contacting a KLF protein, polypeptide or polynucleotide, or a modulator of KLF activity, in the presence of Notch and a modulator of the Notch signalling pathway, with a candidate compound and determining if the compound affects the Notch signalling pathway.
According to a further aspect of the invention there is provided an assay method for identifying substances that modulate the activity of an KLF protein comprising: providing a preparation containing: a KLF protein, polypeptide or polynucleotide and a candidate substance; and detecting whether said candidate substance affects activity of the KLF protein, polypeptide or polynucleotide. Suitably the KLF-interacting protein may be Notch or a member of the Notch signalling pathway. Suitably such an assay may be conducted using an immune cell.
According to a further aspect of the invention there is provided the use of a KLF protein, polypeptide or polynucleotide or a KLF modulator identifiable using an assay as described above in the use or method as described above.
According to a further aspect of the invention there is provided a kit comprising in one or more containers (a) a modulator of the Notch signalling pathway and (b) a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
According to a further aspect of the invention there is provided a product comprising: i) a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; and ii) an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
According to a further aspect of the invention there is provided a pharmaceutical composition comprising: i) a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; and ii) an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
According to a further aspect of the invention there is provided a pharmaceutical composition comprising: i) a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; ii) an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant; and iii) a pharmaceutically acceptable carrier.
Suitably such a composition may be used for increasing effector T cell activity.
According to a further aspect of the invention there is provided a pharmaceutical kit comprising a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity and an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant.
According to a further aspect of the invention there is provided the use of an inhibitor of KLF activity in the manufacture of a medicament for use as an mununostimulant.
According to a further aspect of the invention there is provided the use of an inhibitor of KLF activity in the manufacture of a medicament for use in vaccination against a pathogen.
According to a further aspect of the invention, there is provided the use of an inhibitor of KLF activity in the manufacture of a medicament for use in vaccination against a tumour or pathogen.
According to a further aspect of the invention there is provided the use of an inhibitor of KLF activity in the manufacture of a medicament for increasing the immune response against a rumour or pathogen antigen or antigenic determinant.
According to a further aspect of the invention there is provided a method for stimulating the peripheral immune system by administering an inhibitor of KLF activity According to a further aspect of the invention there is provided a method for vaccinating a subject against a tumour or pathogen by administering an inhibitor of KLF activity
According to a further aspect of the invention there is provided a method for increasing an immune response of a subject against a tumour or pathogen by adniinistering an inhibitor of KLF activity
According to a further aspect of the invention there is provided a method for increasing the immune response of a subject to a tumour or pathogen antigen or antigenic determinant comprising administering an effective amount of an inhibitor of KLF activity simultaneously, contemporaneously, separately or sequentially with said tumour or pathogen antigen or antigenic determinant or simultaneously, contemporaneously, separately or sequentially with a polynucleotide coding for said tumour or pathogen antigen or antigenic determinant.
According to a further aspect of the invention there is provided an adjuvant composition comprising an inhibitor of KLF activity
According to a further aspect of the invention there is provided a vaccine composition comprising an adjuvant composition as described above and a tumour or pathogen antigen or antigenic determinant or a polynucleotide coding for a tumour or pathogen antigen or antigenic determinant.
Suitably such a vaccine composition may comprise a pathogen antigen or antigenic determinant in the form of a viral, fungal, parasitic or bacterial antigen or antigenic determinant or a polynucleotide coding for a viral, fungal, parasitic or bacterial antigen or antigenic determinant.
According to a further aspect of the invention there is provided a product comprising: i) a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; and ii) a tumour or pathogen antigen or antigenic determinant or a polynucleotide coding for a tumour or pathogen antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
Such a product may suitably be used for increasing effector T cell activity in respect of a mmour or pathogen antigen or antigenic determinant.
According to a further aspect of the invention there is provided a method for detecting, measuring or monitoring peripheral immune cell (suitably T-cell) activation comprising determining the amount of a KLF protein, polypeptide or polynucleotide or determining the amount of a polynucleotide coding for such a protein or polypeptide.
According to a further aspect of the invention there is provided a method for modifying cellular quiescence, especially immune cell quiescence, by adniinistering a modulator of Notch signalling.
According to a further aspect of the invention there is provided a method for increasing quiescence by administering an activator of Notch signalling.
According to a further aspect of the invention there is provided a method for decreasing quiescence by administering an inhibitor of Notch signalling.
According to a further aspect of the invention there is provided a method for increasing quiescence by administering a Notch receptor agonist.
According to a further aspect of the invention there is provided a method for decreasing quiescence by administering a Notch receptor antagonist. According to a further aspect of the invention there is provided the use of a modulator of Notch signalling for modifying cehular quiescence, especially immune cell quiescence.
According to a further aspect of the invention there is provided the use of an activator of Notch signalling for increasing cellular quiescence, especially immune cell quiescence .
According to a further aspect of the invention there is provided the use of an inhibitor of Notch signalling for decreasing cellular quiescence, especially immune cell quiescence.
According to a further aspect of the invention there is provided the use of a Notch receptor agonist for increasing cellular quiescence, especially immune cell quiescence.
According to a further aspect of the invention there is provided the use of a Notch receptor antagonist for decreasing cehular quiescence, especially immune cell quiescence.
Suitably the modulator of Notch signalling modifies quiescence in lymphocytes, preferably T-cells.
Suitably such methods and uses may be employed to treat graft rejection, allergy or autoimmune disorders.
According to a further aspect of the invention there is provided a method for modifying immune cell (especially T-cell) prohferation by administering a modulator of Notch signalling.
According to one aspect of the invention there is provided a method for reducing such proliferation by administering an activator of Notch signalling. According to a further aspect of the invention there is provided a method for increasing such proliferation by adniinistering an inhibitor of Notch signalling.
According to a further aspect of the invention there is provided a method for reducing such proliferation b y administering a Notch receptor agonist.
According to a further aspect of the invention there is provided a method for increasing such proliferation by adrninistering a Notch receptor antagonist.
According to a further aspect of the invention there is provided the use of a modulator of Notch signalling for modifying cehular prohferation, especially immune cell proliferation.
According to a further aspect of the invention there is provided the use of an activator of Notch signalling for reducing prohferation, especially immune cell prohferation.
According to a further aspect of the invention there is provided the use of an inhibitor of Notch signalling for increasing proliferation, especially immune cell prohferation.
According to a further aspect of the invention there is provided the use of a Notch receptor agonist for reducing immune cell prohferation.
According to a further aspect of the invention there is provided the use of a Notch receptor antagonist for increasing immune cell prohferation.
Suitably the modulator of Notch signalling modifies proliferation in lymphocytes, preferably T-cells.
Suitably such methods and uses may be employed to treat graft rejection, allergy or autoimmune disorders. Suitably the autoimmune disorder may be selected from the group consisting of multiple sclerosis, insulin-dependent diabetes, sympathetic ophthalmia, uveitis and psoriasis.
Suitably the modulator of Notch signalling may be administered to a patient in vivo.
Alternatively the modulator of Notch signalling may be administered to a ceh ex-vivo, after which the ceh may be administered to a patient.
Suitably such methods and uses may be employed to treat a disorder selected from the group consisting of: thyroiditis, insulitis, multiple sclerosis, iridocychtis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis, rheumatoid arthritis, lupus erythematosus, immune hyperreactivity, insulin dependent diabetes mellitus, anemia (aplastic, hemolytic), autoimmune hepatitis, skleritis, idiopathic thrombocytopenic purpura, inflammatory bowel diseases (Crohn's disease, ulcerative colitis), juvenile arthritis, scleroderma and systemic sclerosis, sjogren's syndrom, undifferentiated connective tissue syndrome, antiphospholipid syndrome, vasculitis (polyarteritis nodosa, allergic granulomatosis and angiitis, Wegner's granulomatosis, Kawasaki disease, hypersensitivity vasculitis, Henoch-Schoenlein purpura, Behcet's Syndrome, Takayasu arteritis, Giant ceh arteritis, Thrombangutis obhterans), polymyalgia rheumatica, essentiell (mixed) ciyoglobulinemia, Psoriasis vulgaris and psoriatic arthritis, diffus fasciitis with or without eosinophilia, polymyositis and other idiopathic inflammatory myopathies, relapsing panniculitis, relapsing polychondritis, lymphomatoid granulomatosis, erythema nodosum, ankylosing spondylitis, Reiter's syndrome, inflammatory dermatitis, unwanted immune reactions and inflammation associated with arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity and allergic reactions, systemic lupus erythematosus, collagen diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfiision injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino- laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory- related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophmalmia, sclentis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HEV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of strokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Gumaim-Batre syndrome, Sydenham chora, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory comphcations or side effects of surgery or organ, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cehular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.
Suitably in such methods and uses the modulator of Notch signalling comprises a protein or polypeptide comprising a Notch ligand DSL domain or a polynucleotide sequence coding for such a protein or polypeptide.
Preferably in such methods and uses the modulator of Notch signalling comprises a protein or polypeptide comprising a Notch ligand DSL domain and at least one, suitably at least 2, for example 2-20 EGF-like domain or a polynucleotide sequence coding for such a protein or polypeptide.
Suitably the DSL or EGF domains are from Delta or Jagged, for example human Delta or Jagged.
Suitably the modulator of the Notch signalling pathway comprises a fusion protein comprising a segment of a Notch ligand extracellular domain and an immunoglobulin Fc segment or a polynucleotide coding for such a fusion protein.
Alternatively, the modulator of the Notch signalling pathway may comprise a Notch intracellular domain (Notch IC) or a polynucleotide sequence which codes for a Notch intracellular domain.
Suitably an activator of Notch signalling may be be in a multimerised form, and may preferably comprise a construct comprising at least 3, preferably at least 5, preferably at least 10, at least 20 or at least 30 modulators of Notch signalling, or in some embodiments as many as 50 or 100 or 1000 or more modulators of Notch signalling, which may each be the same or different.
For example, modulators of Notch signalling in the form of Notch ligand proteins/polypeptides coupled to particulate supports such as beads are described in WO 03/011317 (Lorantis; herein incorporated by reference) and in Lorantis' co-pending International (PCT) application PCT/GB2003/001525 (herein incorporated by reference; filed on 4 April 2003), the texts of which are hereby incorporated by reference (eg see in particular Examples 17, 18, 19 of PCT/GB2003/001525). Reference is made also to the applicant's co-pending International (PCT) Application No PCT/GB2004/000046 filed on 7 January 2004 (herein incorporated by reference), in particular Example 26 therein disclosing Notch ligand fragment/microbead constructs.
Modulators of Notch signalling in the form of Notch ligand proteins/polypeptides coupled to polymer supports are described in Lorantis Ltd's co-pending PCT application
PCT/GB2003/003285 (filed on 1 August 2003 claiming priority from GB 0218068.5), published as WO04/013179; the text of which is herein incorporated by reference (eg see in particular Example 5 therein disclosing a dextran conjugate).
Suitably the modulator of Notch signalling may be administered in a multimerised form.
For example, in one embodiment the modulator of Notch signalling may be bound to a membrane or support. Suitably a plurality or multiplicity of modulators (for example at least 5) will be bound to the membrane or support. Such a membrane or support can be selected from those known in the art. In a preferred embodiment, the support is a particulate support matrix. In an even more preferred embodiment, the support is a bead.
The bead may be, for example, a magnetic bead (e.g. as available under the trade name
"Dynal") or a polymeric bead such as a Sepharosebead.
Suitably a modulator of Notch signalling for use in the present invention may comprise a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 1-5 or more (or for example not more than 5) Notch ligand EGF domains; iii) optionally all or part of a Notch ligand N-temninal domain; and iv) optionally one or more heterologous amino acid sequences; or a polynucleotide coding therefor.
Suitably a modulator of Notch signalling may comprise a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 2-4 or more (or for example not more than 4) Notch ligand EGF domains ; iii) optionally all or part of a Notch ligand N-terminal domain; and iv) optionally one or more heterologous amino acid sequences; or a polynucleotide coding therefor.
Suitably a modulator of Notch signalling may comprise a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 2-3 or more (or for example not more than 3) Notch ligand EGF domains; iii) optionally all or part of a Notch ligand N-terminal domain; and iv) optionally one or more heterologous amino acid sequences ; or a polynucleotide coding therefor.
Suitably the protein or polypeptide may have at least 50%, preferably at least 70%, preferably at least 90%, for example at least 95% amino acid sequence similarity (or preferably sequence identity) to the following sequence, preferably along the entire length of the latter:
GVFE K QEI^VKTKKG GNRNCCRGGAGPPPCACRTFFRVC KHYQASVSPEPPCTYGSA VTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIlEALHTDSPDDLATENPER LISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHF TCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPG CLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTG ATCELGIDEC Where reference is made to % homology, similarity or identity, this preferably means that the relevant % homology, similarity or identity occurs over over a region of at least 50 nucleic acid bases or amino acids, preferably over a region of at least 100 nucleic acid bases or amino acids, and preferably over the entire length of the reference sequence.
Detailed description
Various preferred features and embodiments of the present invention will now be describ ed in more detail by way o f non-limiting example and with reference to the accompanying Figures, in which:
Figure 1 shows a schematic representation of the Notch signalling pathway;
Figure 2 shows schematic representations of the Notch ligands Jagged and Delta; Figure 3 shows ahgned amino acid sequences of DSL domains from various Drosophila and mammalian Notch ligands;
Figure 4 shows the amino acid sequences of human Delta-1, Delta-3 and Delta-4;
Figure 5 shows the amino acid sequences of human Jagged-1 and Jagged-2;
Figure 6 shows schematic representations of various Notch ligand domain/IgFc domain fusion proteins which may be used in the present invention;
Figure 7 shows the results of Example 4;
Figure 8 shows the results of Example 5 Figure 9 shows the results of Example 6 and
Figure 10 shows the results of Example 7.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the Hterature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology : DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; and J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober (1992 and periodic supplements; Current Protocols in Immunology, John Whey & Sons, New York, NY). Each of these general texts is hereby incorporated herein by reference.
For the avoidance of doubt, Drosophila and vertebrate names are used interchangeably and ah homologues are included within the scope of the invention.
KLF proteins, polypeptides and polynucleotides
The term "KLF protein or polypeptide" as used herein means a protein or polypeptide having the characteristics of KLF. It includes, for example, fragments, homologues and allelic variants. The term "KLF polynucleotide" means a polynucleotide coding for a KLF protein or polypeptide.
Typically, a KLF protein or polypeptide comprises Zinc finger domains. As reported by Interpro (www.ebi.ac.uk/interpro) theses are nucleic acid-binding protein stmctures first identified in the Xenopus laevis transcription factor TFHIA. These domains have since been found in numerous nucleic acid-binding proteins. A zinc finger domain is typically composed of 25 to 30 amino-acid residues including 2 conserved Cys and 2 conserved His residues in a C-2-C-12-H-3-H type motif. The 12 residues separating the second Cys and the first His are mainly polar and basic, imphcating this region in particular in nucleic acid binding. The zinc finger motif is an unusually small, self-folding domain in which Zh is a crucial component of its tertiary structure. Most usually bind 1 atom of Zn in a tetrahedral array to yield a finger-like projection, which interacts with nucleotides in the major groove of the nucleic acid. The Zh binds to the conserved Cys and His residues. Fingers have been found to bind to about 5 base pairs of nucleic acid containing short runs of guanine residues. They have the ability to bind to both RNA and DNA, a versatility not demonstrated by the helix-turn-hehx motif. The zinc finger may thus represent the original nucleic acid binding protein. It has also been suggested that a Zn- centred domain could be used in a protein interaction, e.g. in protein kinase C. Many classes of zinc fingers are characterized according to the number and positions of the histidine and cysteine residues involved in the zinc atom coordination. In the first class to be characterized, called C2H2, the first pair of zinc coordinating residues are cysteines, while the second pair are histidines.
Preferably the ter "KLF protein or polypeptide" means a protein or polypeptide which has at least 30%, preferably at least 50%, preferably at least 70%, preferably at least 90%, preferably at least 95% amino acid sequence similarity or, preferably, amino acid identity to a KLF (preferably a human KLF) over a region of at least 20 amino acids, preferably at least 50 amino acids, preferably over a region of at least 100 amino acids, and preferably over its entire length.
The term "KLF activity" as used herein means the biological activity of a KLF protein or polypeptide, including but not limited to transcription activation activity.
A preferred KLF is human KLF-2 (also known as LKLF; Swiss Prot Accession No Q9Y5W3).
For example, amino acid and nucleic acid sequences for a human KLF2 are provided as follows (GenBank Accession No NM_016270):
A SEPILPSFSTFASPCRERGLQERWPRAEPESGGTDDD NSV DFI S G DGLGAEAAPΞPPPPPPPPA FYYPEPGAPPPYSAPAGGLVSΞL RPE DAPLGPA HGRFLLAPPGR VKAEPPEADGGGGYGCAPG TRGP RGLKREGAPGPAASCMRGPGGRPPPPPDTPP SPDGPAR PAPGPRASFPPPFGGPGFGAPGPGLHYAPPAP PAFG FDDAAAAAAA GLAPPAARGLLTPPASP E EAKPKRGRRSWPRKRTATHTCSYAGCGKTYTKSSH LKAHLRTHTGEKPYHC VTOGCG KFARSDE TRHYRKHTGHRPFQCHLCDRAFSRSDHLA HiKRHM 1 gcccgcccgc gccccgacca gcccggcctc gggcagccac tcaccggtgt ccccgtccgc 61 gtccttcctc cσcgggtccc ggcσatggcg ctgagtgaac ccatcctgcc gtccttctcc 121 actttcgcca gcccgtgccg cgagcgcggc ctgcaggagc gctggccgcg σgccgaaccc 181 gagtccggcg gcaσcgacga cgacctcaac agcgtgctgg acttcatcct gtcσatgggg 241 ctggatggcc tgggcgccga ggccgcσccg gagccgccgc cgccgccccc gccgcctgcg 301 ttctattacc ccgaacccgg cgcgccσσcg ccctaσagσg cccccgcggg tggcσtggtg 361 tctgagctgc tgcgacccga gctggatgcg ccgctggggc ccgcactgca cggccgcttt 421 ctgctggcgc cgcccggccg cctggtcaag gcσgagcccc ctgaagcgga cggcggcggc 481 ggctacggct gcgcccccgg gctgacccgt ggaccgcgcg gcctcaagcg cgagggcgσc 541 ccgggcccgg cggcttcgtg catgcgaggt cccgggggcc gccccσσgcc gccgcσcgac 601 acaccgccgc tcagccccga cggccσcgcg cgcctgcccg cgcccggtcc gcgcgcσtcc 661 ttcccgcσgc ctttcggtgg ccctggtttc ggσgcgcccg ggcccggcct gcattacgcg 721 ccgcctgcgσ ccccagcctt cggtσtcttc gacgacgcgg ccgccgσcgc ggcagccctg 781 ggcctggcgc cccccgccgc ccgcggtctc ctσacgccgσ ctgcgtcccc gctggagσtg 841 ctggaggcca agccaaagcg cggccgccgc tcttggσccc gcaaacgcac cgccactcac 901 acctgcagct acgcgggctg cggcaagacc tacaccaaga gttcgcatct gaaggcgcat 961 ctgcgcacgc acacaggtga gaagσcσtac cactgcaact gggacggctg cggctggaag 1021 tttgcgcgct cagacgagct cacgcgccac taccgaaagc acacgggcca ccggccattc 1081 cagtgccatc tgtgcgatcg tgccttttcg σgctccgatc aσctggcgct gcacatgaaa 1141 cggcacatgt agccgggacg cccccgccca cctgσggcgg gccgtggcgg gtcccacgσg 1201 ccgggcgcgg ccccctccca aactgtgact ggtatttatt ggacccagag aaccgggccg 1261 ggcacagcgt ggctacagag ggtσtccctc gatgacgacg acgacgaσgc caccacccca 1321 gcccccgtct gtgactgaag gccσggtggg aaaagaccaα gatcctcctt gacgagtttt 1381 gtttttcaaa atggtgcaat aatttaagtg gcatcttctσ tcccaccggg tctaσaσtag 1441 aggatcgagg cttgtgatgc cttgtgagaa ataagggcct taatttgtac tgtctgcggσ 1501 attttttata atattgtata tagtgactga caaatattgt attactgtac atagagagac 1561 aggtgggcat ttttgggcta cσtggttcgt ttttataaga ttttgctggg ttggtttttt 1621 ttttaattaa aaagttttgc atctttt
Activators of KLF activity
Suitably an activator of KLF activity may be a KLF protein, polypeptide or polynucleotide as described above. For example, a KLF polynucleotide may be administered using a genetic vector as described herein.
Inhibitors of KLF activity
An inhibitor of KLF activity may be any agent which reduces the activity of a KLF protein, polypeptide or polynucleotide. For example, an inhibitor of KLF activity may be a KLF antisense polynucleotide or RNAi construct or an antibody which binds to a KLF protein to reduce its activity. Notch signalling
As used herein, the expression "Notch signalling" is synonymous with the expression "the Notch signalling pathway" and refers to any one or more of the upstream or downstream events that result in, or from, (and including) activation of the Notch receptor.
Preferably, by "Notch signalling" we refer to any event directly upstream or downstream of Notch receptor activation or inhibition including activation or inhibition of Notch/Notch Hgand interactions, upregulation or downiegulation of Notch or Notch Hgand expression or activity and activation or inhibition of Notch signalling transduction including, for example, proteolytic cleavage of Notch and upregulation or downregulation of the Ras-Jnk signalling pathway.
Thus, by "Notch signalling" we refer to the Notch signalling pathway as a signal tranducing pathway comprising elements which interact, genetically and/or molecularly, with the Notch receptor protein. For example, elements which interact with the Notch protein on both a molecular and genetic basis are, by way of example only, Delta, Serrate and Deltex. Elements which interact with the Notch protein genetically are, by way of example only, Mastermind, Hairless, Su(H) and Presenilin.
In one aspect, Notch signalling includes signalling events taking place extracehularly or at the cell membrane. In a further aspect, it includes signalling events taking place intracellularly, for example within the ceh cytoplasm or within the ceh nucleus.
Modulators
The term "modulate" as used herein refers to a change or alteration in the biological activity of KLF. The term "modulator" may refer to antagonists or inhibitors of KLF activity, i.e. compounds which block, at least to some extent, the normal biological activity of KLF proteins, polypeptides or polynucleotides. Conveniently such compounds may be referred to herein as inhibitors or antagonists. Alternatively, the term "modulator" may refer to agonists of KLF activity, i.e. compounds which stimulate or upregulate, at least to some extent, the normal biological activity of KLF. Conveniently such compounds may be referred to as upregulators or agonists.
In one embodiment, an active agent/modulator used in the present invention may be an organic compound or other chemical. In one embodiment, for example, a modulator may be an organic compound comprising two or more hydrocarbyl groups. Here, the term "hydrocarbyl group" means a group comprising at least C and H and may optionahy comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substitaents may form a cychc group . If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. The candidate modulator may comprise at least one cychc group. The cyclic group may be a polycychc group, sucl as a non-fused polycyclic group. For some applications, the agent comprises at least the one of said cychc groups linked to another hydrocarbyl group.
In one preferred embodiment, the modulator will be an amino acid sequence or a chemical derivative thereof, or a combination thereof. In another preferred embodiment, the modulator will be a nucleotide sequence - which may be a sense sequence or an anti- sense sequence. The modulator may also be an antibody.
The term "antibody" includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, Fv and scFv which are capable of binding the epitopic determinant. These antibody fragments retain some abihty to selectively bind with its antigen or receptor and include, for example:
(i) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact Hght chain and a portion of one heavy chain;
(ii) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
(iii) F(ab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds ;
(iv) scFv, including a genetically engineered fragment containing the variable region of a heavy and a Hght chain as a fused single chain molecule.
General methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), which is incorporated herein by reference).
Modulators may be synthetic compounds or natural isolated compounds.
As used herein the term "analogue of KLF" includes variants thereof which retain the activity of KLF proteins, polypeptides or polynucleotides. By "analogue" we include a protein which has KLF activity, but generally has a different evolutionary origin to KLF
Any one or more of appropriate targets - such as an amino acid sequence and/or nucleotide sequence - may be used for identifying a compound capable of modulating KLF activity in any of a variety of drug screening techniques. The target employed in such a test may be free in solution, affixed to a sohd support, borne on a cell surface, or located intracehularly.
Techniques for drag screening may be based, for example, on the method described in Geysen, European Patent No. 0138855, published on September 13, 1984. In summary, large numbers of different small peptide candidate modulators or targeting molecules are synthesized on a sohd substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with a suitable target or fragment thereof and washed. Bound entities are then detected - such as by appropriately adapting methods well known in the art. A purified target can also be coated directly onto plates for use in drug screening techniques. Plates of use for high throughput screening (HTS) will be multi-well plates, preferably having 96, 384 or over 384 wells/plate. Cehs can also be spread as 'lawns". Alternatively, non-neutrahsing antibodies can be used to capture the peptide and immobilise it on a solid support. High throughput screening, as described above for synthetic compounds, can also be used for identifying organic candidate modulators and targeting molecules.
This invention also contemplates the use of competitive drag screening assays in which neutralising antibodies capable of binding a target specifically compete with a test compound for binding to a target.
Techniques are well known in the art for the screening and development of agents such as antibodies, peptidomimetics and small organic molecules which are capable of binding to KLF proteins, polypeptides or polynucleotides. These include the use of phage display systems for expressing signalling proteins, and using a culture of transfected E. coli or other microorganism to produce the proteins for binding studies of potential binding compounds (see, for example, G. Cesarini, FEBS Letters, 307(l):66-70 (July 1992); H. Gram et al., J. Immunol. Meth., 161:169-176 (1993); and C. Summer et al., Proc. Natl. Acad. Sci., USA, 89:3756-3760 (May 1992)). Further library and screening techniques are described, for example, in US 6281344 (Phylos).
By a "homologue" is meant a gene product that exhibits sequence homology, either amino acid or nucleic acid sequence homology, to any one of the known KLF proteins, polypeptides or polynucleotides, for example as mentioned above. Typically, a homologue of a known KLF protein or polynucleotide will be at least 20%, preferably at least 30%, identical at the amino acid level to the corresponding known KLF protein or polynucleotide over a sequence of at least 10, preferably at least 20, preferably at least 50, suitably at least 100 amino acids, or over its entire length. Techniques and software for calculating sequence homology between two or more amino acid or nucleic acid sequences are well known in the art (see for example http://www.ncbi.nhn.nih.gov and Ausubel et al, Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.)
Homologues can be identified in a number of ways, for example by probing genomic or cDNA Hbraries with probes comprising aH or part of a nucleic acid encoding a KLF protein under conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C). Alternatively, homologues may also be obtained using degenerate PCR which will generally use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences. The primers will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
Polypeptide substances may be purified from mammalian cells, obtained by recombinant expression in suitable host cells or obtained commercially. Alternatively, nucleic acid constructs encoding the polypeptides may be used. As a further example, overexpression of KLF may be brought about by introduction of a nucleic acid construct capable of activating the endogenous KLF gene. In particular, gene activation can be achieved by the use of homologous recombination to insert a heterologous promoter in place of the natural promoter in the genome of the target cell.
Notch signalling
A very important component of the Notch signaUing pathway is Notch receptor/Notch Hgand interaction. Thus Notch signalling may involve changes in expression, nature, amount or activity of Notch signalling pathway membrane proteins or G-proteins or Notch signalling pathway enzymes such as proteases, kinases (e.g. serine/threonine kinases), phosphatases, ligases (e.g. ubiquitin Hgases) or glycosyltransferases. Alternatively the signalling may involve changes in expression, nature, amount or activity of DNA binding elements such as transcription factors.
In the present invention Notch signaUing preferably means specific signalling, meaning that the signalling results substantiahy or at least predominantly from the Notch signaUing pathway, and preferably from Notch/Notch ligand interaction, rather than any other significant interfering or competing cause, such as cytokine signalling. Thus, in a prefened embodiment, Notch signalling excludes cytokine signalling.
Key targets for Notch-dependent transcriptional activation are genes of the Enhancer of split complex (E[spl]). Moreover these genes have been shown to be direct targets for binding by the Su(H) protein and to be transcriptionahy activated in response to Notch signaUing. By analogy with EBNA2, a viral coactivator protein that interacts with a mammalian Su(H) homologue CBF1 to convert it from a transcriptional repressor to a transcriptional activator, the Notch intracellular domain, perhaps in association with other proteins may combine with Su(H) to contribute an activation domain that allows Su(H) to activate the transcription of E(spl) as well as other target genes. It should also be noted that Su(H) is not required for all Notch-dependent decisions, indicating that Notch mediates some cell fate choices by associating with other DNA-binding transcription factors or be employing other mechanisms to transduce extracellular signals . The term "Notch Hgand" as used herein means an agent capable of interacting with a Notch receptor to cause a biological effect. The term as used herein therefore includes naturally occurring protein ligands such as Delta and Serrate/Jagged as well as antibodies to the Notch receptor, peptidomimetics and small molecules which have conesponding biological effects to the natural ligands.
Particular examples of mammalian Notch ligands identified to date include the Delta family, for example Delta or Delta-like 1 (Gehbank Accession No. AF003522 - Homo sapiens), Delta-3 (Gehbank Accession No. AF084576 - Rattus norvegicus) and Delta-like 3 (Mus musculus) (Gehbank Accession No. NM_016941 - Homo sapiens) and US 6121045 (MUlennium), Delta-4 (Genbank Accession Nos. AB043894 and AF 253468 - Homo sapiens) and the Serrate family, for example Serrate-1 and Serrate-2 (WO97/01571, WO96/27610 and W092/19734), Jagged-1 (Genbank Accession No. U73936 - Homo sapiens) and Jagged-2 (Gehbank Accession No. AF029778 - Homo sapiens), and LAG-2. Homology between family members is extensive.
Notch Signalling Transduction
The Notch signalling pathway directs binary cell fate decisions in the embryo. Notch was first described in Drosophila as a transmembrane protein that functions as a receptor for two different Hgands, Delta and Serrate. Vertebrates express multiple Notch receptors and ligands (discussed below). At least four Notch receptors (Notch-1, Notch-2, Notch-3 and Notch-4) have been identified to date in human cells (see for example GenBank Accession Nos. AF308602, AF308601 and U95299 - Homo sapiens).
Notch proteins are synthesized as single polypeptide precursors that undergo cleavage via a Furin-like convertase that yields two polypeptide chains that are further processed to form the mature receptor. The Notch receptor present in the plasma membrane comprises a heterodimer of two Notch proteolytic cleavage products , one comprising an N-terminal fragment consisting of a portion of the extracellular domain, the transmembrane domain and the intracellular domain, and the other comprising the majority of the extraceUular domain. The proteolytic cleavage step of Notch to activate the receptor occurs in the Golgi apparatus and is mediated by a furin-like convertase.
Notch receptors are inserted into the membrane as heterodimeric molecules consisting of an extracellular domain containing up to 36 epidermal growth factor (EGF)-like repeats [Notch 1/2 = 36, Notch 3 = 34 and Notch 4 = 29], 3 Cysteine Rich Repeats (Lin-Notch (L/JN) repeats) and a transmembrane subunit that contains the cytoplasmic domain. The cytoplasmic domain of Notch contains six ankyrin-like repeats, a polyglutamine stretch (OP A) and a PEST sequence. A further domain termed R AM23 lies proximal to the ankyrin repeats and is involved in binding to a transcription factor, known as Suppressor of Hairless [Su(H)] in Drosophila and CBF1 in vertebrates (Tamura K, et al. (1995) Curr. Biol. 5:1416-1423 (Tamura)). The Notch ligands also display multiple EGF-like repeats in their extracellular domains together with a cysteine-rich DSL (Delta-Serrate Lag2) domain that is characteristic of all Notch ligands (Artavanis-Tsakomas et al. (1995) Science 268:225-232, Artavanis-Tsakomas et al. (1999) Science 284:770-776).
The Notch receptor is activated by binding of extraceUular ligands, such as Delta, Serrate and Scabrous, to the EGF-like repeats of Notch's extraceUular domain. Delta requires cleavage for activation. It is cleaved by the ADAM disintegrin metalloprotease Kuzbanian at the ceU surface, the cleavage event releasing a soluble and active form of Delta. An oncogenic variant of the human Notch-1 protein, also known as TAN-1 , which has a truncated extracellular domain, is constitutively active and has been found to be involved in T-cell lymphoblastic leukemias.
The cdclO/ankyrin intraceUular-domain repeats mediate physical interaction with intracehular signal transduction proteins. Most notably, the cdclO/ankyrin repeats interact with Suppressor of Hairless [Su(H)]. Su(H) is the Drosophila homologue of C-promoter binding factor-1 [CBF-1], a mammahan DNA binding protein involved in the Epstein-Barr vhus-induced immortalization of B-ceUs. It has been demonstrated that, at least in cultured cells, Su(H associates with the cdclO/ankyrin repeats in the cytoplasm and translocates into the nucleus upon the interaction of the Notch receptor with its Hgand Delta on adjacent ceUs. Su(H) includes responsive elements found in the promoters of several genes and has been found to be a critical downstream protein in the Notch signalling pathway. The involvement of Su(H) in transcription is thought to be modulated by Hairless.
The intraceUular domain of Notch (NotchIC) also has a direct nuclear function (Lieber et al. (1993) Genes Dev 7(10):1949-65 (Lieber)). Recent studies have indeed shown that Notch activation requires that the six cdclO/ankyrin repeats of the Notch intraceUular domain reach the nucleus and participate in transcriptional activation. The site of proteolytic cleavage on the intracellular tad of Notch has been identified between gly 1743 and vall744 (termed site 3, or S3) (Schroeter, E.H. et al. (1998) Nature 393^6683^:382-6 (Schroeter)). It is thought that the proteolytic cleavage step that releases the cdclO/ahkyiin repeats for nuclear entry is dependent on Presenilin activity.
The intraceUular domain has been shown to accumulate in the nucleus where it forms a transcriptional activator complex with the CSL family protein CBF1 (suppressor of hairless, Su(H) in Drosophila, Lag-2 in C. elegans) (Schroeter; Struhl, G. et al. (1998) CeU 93£4):649-60 (Struhl)). The NotchlC-CBFl complexes then activate target genes, such as the bHLH proteins HES (hairy-enhancer of split like) 1 and 5 (Weinmaster G. (2000) Curr. Opin. Genet. Dev. 10:363-369 (Weinmaster)). This nuclear function of Notch has also been shown for the mammahan Notch homologue (Lu, F. M. et al. (1996) Proc Natl Acad Sci 93(1^:5663-7 (Lu)).
S3 processing occurs only in response to binding of Notch ligands Delta or Serrate/Jagged. The post-translational modification of the nascent Notch receptor in the Golgi (Munro S, Freeman M. (2000) Curr. Biol. 10:813-820 (Munro); Ju BJ, et al. (2000) Nature 405:191-195 (Ju)) appears, at least in part, to control which of the two types of Hgand is expressed on a ceU surface. The Notch receptor is modified on its extracellular domain by Fringe, a glycosyl transferase enzyme that binds to the Lin/Notch motif. Fringe modifies Notch by adding O-linked fiicose groups to the EGF-like repeats (Moloney DJ, et al. (2000) Nature 406:369-375 (Moloney), Bracker K, et al. (2000) Nature 406:411-415 (Brucker)). This modification by Fringe does not prevent ligand binding, but may influence ligand induced conformational changes in Notch. Furthermore, recent studies suggest that the action of Fringe modifies Notch to prevent it from interacting functionally with Senate/Jagged Hgands but allow it to preferentially bind Delta (Panin VM, et al. (1997) Nature 387:908-912 (Panin), Hicks C, et al. (2000) Nat. CeU. Biol. 2:515-520 (Hicks)). Although Drosophila has a single Fringe gene, vertebrates are known to express multiple genes (Radical, Manic and Lunatic Fringes) (Irvine KD (1999) Curr. Opin. Genet. Devel. 9:434-441 (Irvine)).
Other downstream components of the Notch signaUing pathway include Deltex-1, Deltex- 2, Deltex-3, Suppressor of Deltex (SuDx), Numb and isoforms thereof, Numb associated Kinase (NAK), Notchless, Dishevelled (Dsh), emb5, Fringe genes (such as Radical, Lunatic and Manic), PON, LNX, Disabled, Numblike, Nur77, NFkB2, Mirror, Warthog, Engrailed- 1 and Engrailed-2, Lip-1 and homologues thereof, the polypeptides involved in the Ras/MAPK cascade modulated by Deltex, polypeptides involved in the proteolytic cleavage of Notch such as Presenilin and polypeptides involved in the transcriptional regulation of Notch target genes, preferably in a constitutively active form, and analogues, derivatives, variants and fragments thereof.
Signal transduction from the Notch receptor can occur via two different pathways (Figure 1). The better defined pathway involves proteolytic cleavage of the intraceUular domain of Notch (Notch IC) that translocates to the nucleus and forms a transcriptional activator complex with the CSL family protein CBF1 (suppressor of Hairless, Su(H) in Drosophila, Lag-2 in C. elegans). NotchlC-CBFl complexes then activate target genes, such as the bHLH proteins HES (haiiy-ehhancer of split like) 1 and 5. Notch can also signal in a CBF1 -independent manner that involves the cytoplasmic zinc finger containing protein Deltex. Unlike CBF1 , Deltex does not move to the nucleus following Notch activation but instead can interact with Grb2 and modulate the Ras-JNK signalling pathway.
Thus, signal transduction from the Notch receptor can occur via two different pathways both of which are Ulustrated in Figure 1. Target genes of the Notch signaUing pathway include Deltex, genes of the Hes family (Hes-1 in particular), Enhancer of Split [E(spl)] complex genes, IL-10, CD-23, CD-4 and DIM.
Deltex, an intraceUular docking protein, replaces Su(H) as it leaves its site of interaction with the intraceUular taU of Notch. Deltex is a cytoplasmic protein containing a zinc-finger (Artavanis-Tsakomas et al. (1995) Science 268:225-232; Artavanis-Tsakomas et al. (1999) Science 284:770-776; Osborne B, Miele L. (1999) Immunity 11 :653-663 (Osborne)). It interacts with the ankyrin repeats of the Notch intracellular domain. Stadies indicate that Deltex promotes Notch pathway activation by interacting with Grb2 and modulating the Ras-JNK signalling pathway (Matsuno et al. (1995) Development 121(8):2633-44; Matsuno K, et al. (1998) Nat. Genet. 19:74-78). Deltex also acts as a docking protein which prevents Su(H) from binding to the intracehular tail of Notch (Matsuno). Thus, Su(H) is released into the nucleus where it acts as a transcriptional modulator. Recent evidence also suggests that, in a vertebrate B-cell system, Deltex, rather than the Su(H) homologue CBF1, is responsible for inhibiting E47 function (Ordentlich et al. (1998) Mol. Cell. Biol. 18:2230-2239 (OrdentHch)). Expression of Deltex is upregulated as a result of Notch activation in a positive feedback loop. The sequence of Homo sapiens Deltex (DTX1) mRNA may be found in GenBank Accession No. AF053700.
Hes-1 (Hairy-enhancer of SρHt-1) (Takebayashi K. et al. (1994) J Biol Chem 269(7}:150-6 (Takebayashi)) is a transcriptional factor with a basic hehx-loop-hehx structure. It binds to an important functional site in the CD4 silencer leading to repression of CD4 gene expression. Thus, Hes-1 is strongly involved in the determination of T-ceU fate. Other genes from the Hes family include Hes-5 (mammahan Enhancer of SpHt homologue), the expression of which is also upregulated by Notch activation, and Hes-3. Expression of Hes- 1 is upregulated as a result of Notch activation. The sequence of Mus musculus Hes-1 can be found in GenBank Accession No. D16464.
The E(spl) gene complex [E(spl)-C] (Leimeister C. et al. (1999) Mech Dev 85(1-2^:173-7
(Leimeister)) comprises seven genes of which only E(spl) and Groucho show visible phenotypes when mutant. E(spl) was named after its ability to enhance SpHt mutations,
Split being another name for Notch. Indeed, E(spl)-C genes repress Delta through regulation of achaete-scute complex gene expression. Expression of E(spl) is upregulated as a result of Notch activation.
Interleukin-10 (IL-10) was first characterised in the mouse as a factor produced by Th2 cehs which was able to suppress cytokine production by Thl ceUs. It was then shown that IL-10 was produced by many other ceU types including macrophages, keratinocytes, B ceUs, ThO and Thl cehs. It shows extensive homology with the Epstein-Barr bcrfl gene which is now designated viral IL-10. Although a few immunostimulatory effects have been reported, it is mainly considered as an immunosuppressive cytokine. Inhibition of T ceU responses by IL-10 is mainly mediated through a reduction of accessory functions of antigen presenting ceUs. IL-10 has notably been reported to suppress the production of numerous pro-inflammatory cytokines by macrophages and to inhibit co-stimulatory molecules and MHC class II expression. IL-10 also exerts anti-inflammatory effects on other myeloid cells such as neutrophils and eosinophils. On B cells, IL-10 influences isotype switching and proliferation. More recently, IL-10 was reported to play a role in the induction of regulatory T ceUs and as a possible mediator of their suppressive effect. Although it is not clear whether it is a direct downstream target of the Notch signalling pathway, its expression has been found to be strongly up-regulated coincident with Notch activation. The mRNA sequence of IL-10 may be found in GenBank ref. No. GI1041812. CD-23 is the human leukocyte differentiation antigen CD23 (FCE2) which is a key molecule for B-ceU activation and growth. It is the low-affinity receptor for IgE. Furthermore, the truncated molecule can be secreted, then functioning as a potent mitogenic growth factor. The sequence for CD-23 may be found in GenBank ref. No. GI1783344.
CTLA4 (cytotoxic T-lymphocyte activated protein 4) is an accessory molecule found on the surface of T-cells which is thought to play a role in the regulation of airway inflammatory ceU recruitment and T-helper ceh differentiation after allergen inhalation. The promoter region of the gene encoding CTLA4 has CBFl response elements and its expression is upregulated as a result of Notch activation. The sequence of CTLA4 can be found in GenBank Accession No. L15006.
Dlx-1 (distaUess-1) (McGuinness T. Et al (1996) Genomics 35(3):473-85 (McGuiness)) expression is downregulated as a result of Notch activation. Sequences for Dlx genes may be found in GenBank Accession Nos. U51000-3.
CD-4 expression is dowmegulated as a result of Notch activation. A sequence for the CD-4 antigen may be found in GenBank Accession No. XM006966.
Other genes involved in the Notch signaling pathway, such as Numb, Mastermind and Dsh, and aU genes the expression of which is modulated by Notch activation, are included in the scope of this invention.
As described above the Notch receptor family participates in ceU-cell signalling events that influence T cell fate decisions. In this signalling NotchIC localises to the nucleus and functions as an activated receptor. Mammahan NotchIC interacts with the transcriptional repressor CBFl. It has been proposed that the NotchIC cdclO/ankyrin repeats are essential for this interaction. Hsieh et al (Hsieh et al. (1996) Molecular & CeU Biology 16(3):952-959') suggests rather that the N-terminal 114 amino acid region of mouse NotchIC contains the CBFl interactive domain. It is also proposed that NotchIC acts by targeting DNA-bound CBFl within the nucleus and aboHshing CBFl -mediated repression through masking of the repression domain. It is known that Epstein Barr virus (EBV) immortalizing protein EBNA" also utilises CBFl tethering and masking of repression to upregulate expression of CBFl-repressed B-ceU genes. Thus, mimicry of Notch signal transduction is involved in EB V-driven immortalization. Strobl et al (Strobl et al. (2000) J Virol 74(4)1727-35') similarly reports that 'ΕBNA2 may hence be regarded as a functional equivalent of an activated Notch receptor". Other EBV proteins which fall in this category include BARF0 (Kusano and Raab-Truab (2001) J Virol 75£Ti:384-395 (Kusano and Raab-Traub)) and LMP2A
Notch Signalling Activation
Examples of mammalian Notch Hgands identified to date include the Delta family, for example Delta-1 (Gehbank Accession No. AF003522 - Homo sapiens), Delta-3 (Gehbank Accession No. AF084576 - Rattus noiyegicus) and Delta-like 3 (Mus musculus), the Serrate family, for example Serrate-1 and Serrate-2 (WO97/01571, WO96/27610 and W092/19734), Jagged-1 and Jagged-2 (Genbank Accession No. AF029778 - Homo sapiens), and LAG-2. Homology between family members is extensive.
Notch Hgands identified to date have a diagnostic DSL domain (D. Delta, S. Serrate, L. Lag2) comprising 20 to 22 amino acids at the amino terminus of the protein and up to 14 or more EGF-Hke repeats on the extraceUular surface. It is therefore preferred that homologues of Notch Hgands also comprise a DSL domain at the N-terminus and up to 14 or more EGF- Hke repeats on the extraceUular surface. Notch ligand domains
As discussed above, Notch ligands typically comprise a number of distinctive domains. Some predicted/potential domain locations for various naturally occurring human Notch Hgands (based on amino acid numbering in the precursor proteins) are shown below:
Human Delta 1
Component Amino acids Proposed function/doi
SIGNAL 1-17 SIGNAL
CHAIN 18-723 DELTA- IKE PROTEIN 1
DOMAIN 18-545 EXTRACELLULAR
TRANSMEM 546- 568 TRANSMEMBRANE
DOMAIN 569-723 CYTOPLASMIC
DOMAIN 159-221 DSL
DOMAIN 226-254 EGF-LIKE 1
DOMAIN 257-285 EGF-LIKE 2
DOMAIN 292-325 EGF-LIKE 3
DOMAIN 332-363 EGF-LIKE 4
DOMAIN 370-402 EGF-LIKE 5
DOMAIN 409-440 EGF-LIKE 6
DOMAIN 447-478 EGF-LIKE 7
DOMAIN 485-516 EGF-LIKE 8
Human Delta 3
Component Amino acids Proposed f'
DOMAIN 158-248 DSL
DOMAIN 278-309 EGF-LIKE 1
DOMAIN 316-350 EGF-LIKE 2
DOMAIN 357-388 EGF-LIKE 3
DOMAIN 395-426 EGF-LIKE 4
DOMAIN 433-464 EGF-LIKE 5
Human Delta 4
Component Amino acids Proposed function/doo
SIGNAL 1-26 SIGNAL
CHAIN 27-685 DELTA-LIKE PROTEIN 4
DOMAIN 27-529 EXTRACELLULAR
TRANSMEM 530-550 TRANSMEMBRANE
DOMAIN 551-685 CYTOPLASMIC
DOMAIN 155-217 DSL
DOMAIN 218-251 EGF-LIKE 1
DOMAIN 252-282 EGF-LIKE 2
DOMAIN 284-322 EGF-LIKE 3 OMAIN 324-360 EGF-LIKE 4
DOMAIN 362-400 EGF-LIKE 5
DOMAIN 402-438 EGF-LIKE 6
DOMAIN 440-476 EGF-LIKE 7
DOMAIN 480-518 EGF-LIKE 8
Human Jagged 1
Component Amino acids Proposed function/do ai:
SIGNAL 1-33 SIGNAL
CHAIN 34-1218 JAGGED 1
DOMAIN 34-1067 EXTRACELLULAR
TRANSMEM 1068-1093 TRANSMEMBRANE
DOMAIN 1094-1218 CYTOPLASMIC
DOMAIN 167-229 DSL
DOMAIN 234-262 EGF-LIKE 1
DOMAIN 265-293 EGF-LIKE 2
DOMAIN 300-333 EGF-LIKE 3
DOMAIN 340-371 EGF-LIKE 4
DOMAIN 378-409 EGF-LIKE 5
DOMAIN 416-447 EGF-LIKE 6
DOMAIN 454-484 EGF-LIKE 7
DOMAIN 491-522 EGF-LIKE 8
DOMAIN 529-560 EGF-LIKE 9
DOMAIN 595-626 EGF-LIKE 10
DOMAIN 633-664 EGF-LIKE 11
DOMAIN 671-702 EGF-LIKE 12
DOMAIN 709-740 EGF-LIKE 13
DOMAIN 748-779 EGF-LIKE 14
DOMAIN 786-817 EGF-LIKE 15
DOMAIN 824-855 EGF-LIKE 16
DOMAIN 863-917 VON WILLEBRAND FACTOR C Human Jagged 2
Component Amino acids Proposed function/domain SIGNAL 1-26 SIGNAL
CHAIN 27-1238 JAGGED 2
DOMAIN 27-1080 EXTRACELLULAR
TRANSMEM 1081-1105 TRANSMEMBRANE
DOMAIN 1106-1238 CYTOPLASMIC DOMAIN 178-240 DSL
DOMAIN 249-273 EGF-LIKE 1
DOMAIN 276-304 EGF-LIKE 2
DOMAIN 311-344 EGF-LIKE 3
DOMAIN 351-382 EGF-LIKE 4 DOMAIN 389-420 EGF-LIKE 5
DOMAIN 427-458 EGF-LIKE 6
DOMAIN 465-495 EGF-LIKE 7
DOMAIN 502-533 EGF-LIKE 8
DOMAIN 540-571 EGF-LIKE 9 DOMAIN 602-633 EGF-LIKE 10
DOMAIN 640-671 EGF-LIKE 11
DOMAIN 678-709 EGF-LIKE 12
DOMAIN 716-747 EGF-LIKE 13
DOMAIN 755-786 EGF-LIKE 14 DOMAIN 793-824 EGF-LIKE 15
DOMAIN 831-862 EGF-LIKE 16
DOMAIN 872-949 VON WILLEBRAND FACTOR C
DSL domain
A typical DSL domain may include most or all of the following consensus amino acid sequence:
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
Preferably the DSL domain may include most or all of the following consensus amino acid sequence:
Cys Xaa Xaa Xaa ARO ARO Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys BAS NOP BAS ACM ACM Xaa ARO NOP ARO Xaa Xaa Cys Xaa Xaa Xaa NOP Xaa Xaa Xaa Cys Xaa Xaa NOP ARO Xaa NOP Xaa Xaa Cys wherein:
ARO is an aromatic amino acid residue, such as tyrosine, phenylalanine, tryptophan or histidine; NOP is a non-polar amino acid residue such as glycine, alanine, proline, leucine, isoleucine or valine;
BAS is a basic amino acid residue such as arginine or lysine; and
ACM is an acid or amide amino acid residue such as aspartic acid, glutamic acid, asparagine or glutamine.
Preferably the DSL domain may include most or all of the following consensus amino acid sequence:
Cys Xaa Xaa Xaa Tyr Tyr Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Arg Pro Arg Asx Asp Xaa Phe Gly His Xaa Xaa Cys Xaa Xaa Xaa Gly Xaa Xaa Xaa Cys Xaa Xaa Gly Trp Xaa Gly Xaa Xaa Cys
(wherein Xaa may be any amino acid and Asx is either aspartic acid or asparagine).
An alignment of DSL domains from Notch ligands from various sources is shown in Figure 3.
The DSL domain used may be derived from any suitable species, including for example Drosophila, Xenopus, rat, mouse or human. Preferably the DSL domain is derived from a vertebrate, preferably a mammaHan, preferably a human Notch Hgand sequence.
It wul be appreciated that the term "DSL domain" as used herein includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturaUy occurring domains.
Suitably, for example, a DSL domain for use in the present invention may have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Jagged 1.
Alternatively a DSL domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Jagged 2.
Alternatively a DSL domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Delta 1.
Alternatively a DSL domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% ammo acid sequence identity to the DSL domain of human Delta 3.
Alternatively a DSL domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Delta 4.
EGF-like domain
The EGF-like motif has been found in a variety of proteins, as weU as EGF and Notch and Notch Hgands, including those involved in the blood clotting cascade (Furie and Furie, 1988, Cell 53: 505-518). For example, this motif has been found in extraceUular proteins such as the blood clotting factors IX and X (Rees et al., 1988, EMBO J. 7:2053- 2061; Furie and Furie, 1988, CeU 53: 505-518), in other Drosophila genes (Knust et al., 1987 EMBO J. 761-766; Rothberg et al., 1988, CeU 55:1047-1059), and in some cell- surface receptor proteins, such as thrombomodulin (Suzuki et al., 1987, EMBO J. 6:1891- 1897) and LDL receptor (Sudhof et al., 1985, Science 228:815-822). A protein binding site has been mapped to the EGF repeat domain in thrombomodulin and urokinase (Kurosawa et al., 1988, J. Biol. Chem 263:5993-5996; AppeUa et al., 1987, J. Biol. Chem. 262:4437-4440). As reported by PROSITE a typical EGF domain may include six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds. The main structure is proposed, but not necessarily required, to be a two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet. Subdomains between the conserved cysteines strongly vary in length as shown in the foUowing schematic representation of a typical EGF-like domain: '
+ + + + x(4) - _-x(0, _8) -C I-x (3, 12) -C-x(l/70) - _ I-x(l, 6) -CI-x(2) ~3-a-x(0, 21) -G-x(2) -C I-x
+ I + I
wherein:
'C: conserved cysteine involved in a disulfide bond. 'G': often conserved glycine 'a': often conserved aromatic amino acid __': any residue
The region between the 5th and 6th cysteine contains two conserved glycines of which at least one is normaUy present inmost EGF-like domains.
The EGF-like domain used may be derived from any suitable species, including for example Drosophila, Xenopus, rat, mouse or human. Preferably the EGF-like domain is derived from a vertebrate, preferably a mammalian, preferably a human Notch ligand sequence.
It will be appreciated that the term "EGF domain" as used herein includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturaUy occurring domains.
Suitably, for example, an EGF-like domain for use in the present invention may have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Jagged 1.
Alternatively an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Jagged 2.
Alternatively an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least
70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 1.
Alternatively an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least
70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 3.
Alternatively an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least
70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 4.
As a practical matter, whether any particular amino acid sequence is at least X% identical to another sequence can be determined conventionaUy using known computer programs. For example, the best overaU match between a query sequence and a subject sequence, also referred to as a global sequence alignment, can be determined using a program such as the FASTDB computer program based on the algorithm of Biutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence ahgmnent the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of the global sequence ahgnment is given as percent identity.
The term "Notch ligand N-terminal domain" means the part of a Notch ligand sequence from the N-terminus to the start of the DSL domain. It wUl be appreciated that this term includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturally occurring domains.
The term "heterologous amino acid sequence" or "heterologous nucleotide sequence" as used herein means a sequence which is not found in the native Notch ligand or its coding sequence.
For example, a recombinant human Delta 4 protein is available from R &D Systems, Inc, US (eg see www.rndsystems.com) Recombinant Human DLL4 (Recombinant Human DLL4, Cat Nos 1506-D4-050 1506-D4-050/CF). A recombinant mouse DLL4 (Cat Nos 1389-D4-050, 1389-D4-050/CF) and a recombinant Rat Jagged 1/Fc chimera, (CF 599- JG-100) are also available.
Whether a substance can be used for activating (or inhibiting) Notch may be determined using suitable screening assays (eg reporter assays or T-ceU assays), for example, as described in our co-pending International Patent AppHcation (WO 03/012441) and the examples herein.
Activation of Notch signalling may also be achieved by repressing inhibitors of the Notch signaUing pathway. As such, polypeptides for Notch signaUing activation wiU include molecules capable of repressing any Notch signalling inhibitors. Preferably the molecule whl be a polypeptide, or a polynucleotide encoding such a polypeptide, that decreases or interferes with the production or activity of compounds that are capable of producing an decrease in the expression or activity of Notch, Notch ligands, or any downstream components of the Notch signalling pathway. In a preferred embodiment, the molecules whl be capable of repressing polypeptides of the Toll-like receptor protein family and growth factors such as the bone morphogenetic protein (BMP), BMP receptors and activins, derivatives, fragments, variants and homologues thereof.
Polypeptides. Proteins and Amino Acid Sequences
As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "protein". '
"Peptide" usually refers to a short amino acid sequence that is 10 to 40 amino acids long, preferably 10 to 35 amino acids.
The amino acid sequence may be prepared and isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
Nucleotide Sequences
As used herein, the term "nucleotide sequence" is synonymous with the term "polynucleotide".
The nucleotide sequence may be DNA or RNA of genomic or synthetic or of recombinant origin. They may also be cloned by standard techniques. The nucleotide sequence may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
Longer nucleotide sequences wih generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This wih involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human ceU, performing a polymerase chain reaction (PCR) under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the ampHfied DNA can be cloned into a suitable cloning vector. In general, primers wϋl be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accompHshing this using automated techniques are readily available in the art.
"Polynucleotide" refers to a polymeric form of nucleotides of at least 10 bases in length and up to 5,000 bases or even more, either ribonucleotides or deoxyrϊbonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.
These may be constructed using standard recombinant DNA methodologies. The nucleic acid may be RNA or DNA and is preferably DNA. Where it is RNA, manipulations may be performed via cDNA intermediates. GeneraUy, a nucleic acid sequence encoding the first region whl be prepared and suitable restriction sites provided at the 5' and/or 3' ends. Conveniently the sequence is manipulated in a standard laboratory vector, such as a plasmid vector based on pBR322 or pUC19 (see below). Reference may be made to Molecular Cloning by Sambrook et al. (Cold Spring Harbor, 1989) or similar standard reference books for exact details of the appropriate techniques.
Sources of nucleic acid may be ascertained by reference to published hteratare or databanks such as GenBank. Nucleic acid encoding the desired first or second sequences may be obtained from academic or commercial sources where such sources are willing to provide the material or by synthesising or cloning the appropriate sequence where only the sequence data are avaUable. Generally this may be done by reference to literature sources which describe the cloning of the gene in question. Alternatively, where limited sequence data is available or where it is desired to express a nucleic acid homologous or otherwise related to a known nucleic acid, exemplary nucleic acids can be characterised as those nucleotide sequences which hybridise to the nucleic acid sequences known in the art.
For some appHcations, preferably, the nucleotide sequence is DNA. For some appHcations, preferably, the nucleotide sequence is prepared by use of recombinant DNA techniques (e.g. recombinant DNA). For some appHcations, preferably, the nucleotide sequence is cDNA For some applications, preferably, the nucleotide sequence may be the same as the naturally occurring form.
Variants, Derivatives, Analogues, Homologues and Fragments
In addition to the specific amino acid sequences and nucleotide sequences mentioned herein, the present invention also encompasses the use of variants, derivatives, analogues, homologues and fragments thereof.
In the context of the present invention, a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. A variant sequence can be modified by addition, deletion, substitution modification replacement and/or variation of at least one residue present in the naturally-occurring protein.
The term "derivative" as used herein, in relation to proteins or polypeptides of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide retains at least one of its endogenous functions. The term "analogue" as used herein, in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.
Within the definitions of "proteins" useful in the present invention, the specific amino acid residues may be modified in such a manner that the protein in question retains at least one of its endogenous functions, such modified proteins are refened to as "variants". A variant protein can be modified by addition, deletion and/or substitution of at least one amino acid present in thenataraUy-occuiring protein.
Typically, aniino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturaUy occuπing analogues.
Proteins ofuse in the present invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. DeHberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues as long as the transport or modulation function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and aniino acids with uncharged polar head groups having similar hydrophihcity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
For ease of reference, the one and three letter codes for the main naturaUy occurring amino acids (and then associated codons) are set out below: Symbol 3-letter Meaning Codons
A Ala Alanine GCT,GCC,GCA,GCG
B Asp,Asn Aspartic,
Asparagine GAT,GAC,AAT,AAC
C Cys Cysteine TGT,TGC
D Asp Aspartic GAT,GAC
Ξ Glu Glutamic GAA,GAG
F Phe Phenylalanine TTT,TTC
G Gly Glycine GGT,GGC,GGA,GGG
H His Histidine CAT,CAC
I lie Isoleucine ATT,ATC,ATA
K Lys Lysine AAA,AAG
L Leu Leucine TTG,TTA,CTT,CTC,CTA,CTG
M Met Methionine ATG
N Asn Asparagine AAT,AAC
P Pro Proline CCT,CCC,CCA,CCG
Q Gin Glutamine CAA,CAG
R Arg Arginine CGT,CGC,CGA,CGG,AGA,AGG
S Ser Serine TCT,TCC,TCA,TCG,AGT,AGC
T Thr Threonine ACT,ACC,ACA,ACG
V Val Valine GTT,GTC,GTA,GTG
W Trp Tryptophan TGG
X Xxx Unknown
Y Tyr Tyrosine TAT, TAC z Glu,Gin Glutamic,
Glutamine GAA,GAG,CAA,CAG
* End Terminator TAA,TAG,TGA
Conservative substitations maybe made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substitated for each other:
Figure imgf000061_0001
As used herein, the term "protein" includes single-chain polypeptide molecules as weU as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the terms "polypeptide" and "peptide" refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. The terms subunit and domain may also refer to polypeptides and peptides having biological function.
"Fragments" are also variants and the term typicaUy refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. "Fragment" thus refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.
Such variants may be prepared using standard recombinant DNA techniques such as site- directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the nalurally-occurring sequence either side of the insertion site. The flanking regions wiU contain convenient restriction sites conesponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only iUustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.
Polynucleotide variants will preferably comprise codon optimised sequences. Codon optimisation is known in the art as a method of enhancing RNA stabUity and therefor gene expression. The redundancy of the genetic code means that several different codons may encode the same amino-acid. For example, Leucine, Arginine and Serine are each encoded by six different codons. Different organisms show preferences in their use of the different codons. Viruses such as HTV, for instance, use a large number of rare codons. By changing a nucleotide sequence such that rare codons are replaced by the corresponding commonly used mammalian codons, increased expression of the sequences in mammalian target cells can be achieved. Codon usage tables are known in the art for mammalian cehs, as weU as for a variety of other organisms. Preferably, at least part of the sequence is codon optimised. Even more preferably, the sequence is codon optimised in its entirety.
As used herein, the term "homology" can be equated with "identity". An homologous sequence will be taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical. In particular, homology should typically be considered with respect to those regions of the sequence (such as amino acids at positions 51, 56 and 57) known to be essential for an activity. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commerciaUy available computer programs can calculate % homology between two or more sequences.
Percent homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each aniino acid in one sequence is directly compared with the conesponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" ahgnment. Typically, suchungapped ahgnments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion whl cause the following amino acid residues to be put out of ahgnment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence ahgnment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - wih achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaUer penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties wiU of course produce optimised alignments with fewer gaps. Most ahgnment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
Calculation of maximum % homology therefor firstly requires the production of an optimal ahgnment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410 (Atschul)) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.
The five BLAST programs available at http://www.ncbi.n_m.nih.gov perform the foUowing tasks:
blastp - compares an amino acid query sequence against a protein sequence database.
blastn - compares a nucleotide query sequence against a nucleotide sequence database.
blastx - compares the six-frame conceptaal translation products of a nucleotide query sequence (both strands) against a protein sequence database.
tblastn - compares a protein query sequence against a nucleotide sequence database dynamically translated in aU six reading frames (both strands).
tblastx - compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
BLAST uses the following search parameters:
HISTOGRAM - Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual). DESCRPTIONS - Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page).
EXPECT - The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match wih not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).
CUTOFF - Cutoff score for reporting high-scoring segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual). TypicaUy, significance thresholds can be more intuitively managed using EXPECT.
ALIGNMENTS - Restricts database sequences to the number specified for which high- scoring segment pairs (HSPs) are reported; the default limi is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual).
MATRIX - Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992). The valid alternative choices include: PAM40, PAM120, PAM250 and IDENTITY. No alternate scoring matrices are available for BLASTN; specifying the MATRIX directive in BLASTN requests returns an error response.
STRAND - Restrict a TBLASTN search to just the top or bottom strand of the database sequences ; or restrict a BLASTN, BLASTX or TBLASTX search to just reading frames on the top or bottom strand of the query sequence.
FILTER - Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17:149-163, or segments consisting of short-periodicity internal repeats, as determined by the XNU program of Claverie & States (1993) Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of Tatasov and Lipman (see http://www.ncbi.nlm.nih.gov). FUtering can eliminate statisticaUy significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.
Low complexity sequence found by a filter program is substitated using the letter "N" in nucleotide sequence (e.g., "NNNNNNNN SINNNN") and the letter "X" in protein sequences (e.g., "XXXXXXXXX").
Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs.
It is not unusual for nothing at aU to be masked by SEG, XNU, or both, when applied to sequences in SWISS-PROT, so filtering should not be expected to always yield an effect. Furthermore, in some cases, sequences are masked in their entirety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect. NCBI-gi - Causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.
Most preferably, sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www .ncbi.nlm.nih.gov/BLAST.
In some aspects of the present invention, no gap penalties are used when determining sequence identity.
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an aU-or-nothing pair comparison. Instead, a scaled sinhlarity score matrix is generahy used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the pub He default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
Nucleotide sequences which are homologous to or variants of sequences of use in the present invention can be obtained in a number of ways, for example by probing DNA Hbraries made from a range of sources. In addition, other vhal/bacterial, or cehular homologues particularly cehular homologues found in mammalian ceUs (e.g. rat, mouse, bovine and primate ceUs), maybe obtained and such homologues and fragments thereof in general wiU be capable of selectively hybridising to the sequences shown in the sequence 61 -
Hsting herein. Such sequences may be obtained by probing cDNA Hbraries made from or genomic DNA Hbraries from other animal species, and probing such Hbraries with probes comprising aU or part of the reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and ahehc variants of the amino acid and/or nucleotide sequences useful in the present invention.
Variants and strain/species homologues may also be obtained using degenerate PCR which whl use primers designed to target sequences within the variants and homologues encoding conserved ammo acid sequences within the sequences of use in the present invention. Conserved sequences can be predicted, for example, by aligning the aniino acid sequences from several variants homologues. Sequence ahgnments can be performed using computer software known in the art. For example the GCG Wisconsin PUeUp program is widely used. The primers used in degenerate PCR wiU contain one or more degenerate positions and wiU be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
Variants and strain/species homologues may also be obtained using degenerate PCR which wiU use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of use in the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants homologues. Sequence ahgnments can be performed using computer software known in the art. For example the GCG Wisconsin PUeUp program is widely used. The primers used in degenerate PCR wih contain one or more degenerate positions and wiU be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
PCR technology as described e.g. in section 14 of Sambrook et al., 1989, requires the use of oligonucleotide probes that whl hybridise to nucleic acid. Strategies for selection of oligonucleotides are described below. As used herein, a probe is e.g. a single-stranded DNA or RNA that has a sequence of nucleotides that includes between 10 and 50, preferably between 15 and 30 and most preferably at least about 20 contiguous bases that are the same as (or the complement of) an equivalent or greater number of contiguous bases. The nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are minimised. The nucleotide sequences are usuahy based on conserved or highly homologous nucleotide sequences or regions of polypeptides. The nucleic acids used as probes maybe degenerate at one or more positions.
Preferred regions from which to construct probes include 5' and/or 3' coding sequences, sequences predicted to encode ligand binding sites, and the like. For example, either the full-length cDNA clone disclosed herein or fragments thereof can be used as probes. Preferably, nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridisation. For example, a suitable label means is a radiolabel. The prefened method of labelling a DNA fragment is by incorporating α32P dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art. Oligonucleotides are usually end-labehed with γ32P-labelled ATP and polynucleotide kinase. However, other methods (e.g. non-radioactive) may also be used to label the fragment or ohgonucleotide, including e.g. enzyme labelling, fluorescent labelling with suitable fluorophores and biotinylation.
Preferred are such sequences, probes which hybridise under high-stringency conditions.
Alternatively, such nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences . This may be useful where for example shent codon changes are required to sequences to optimise codon preferences for a particular host ceU in which the nucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the activity of the polynucleotide or encoded polypeptide. In general, the terms "variant", "homologue" or "derivative" in relation to the nucleotide sequence used in the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for a target protein or protein for T cell signaUing modulation.
As indicated above, with respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the reference sequences. More preferably there is at least 95%, more preferably at least 98%, homology. Nucleotide homology comparisons may be conducted as described above. A prefeπed sequence comparison program is the GCG Wisconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty is - 3 for each nucleotide.
Hybridisation
The present invention also encompasses nucleotide sequences that ate capable of hybridising selectively to the reference sequences, or any variant, fragment or derivative thereof, or to the complement of any of the above. Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
Nucleotide sequences useful in the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, wiU be generally at least 75%, preferably at least 85 or 90% and more preferably at least 95% or 98% homologous to the conesponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides. Preferred nucleotide sequences of the invention whl comprise regions homologous to the nucleotide sequence, preferably at least 80 or 90% and more preferably at least 95% homologous to the nucleotide sequence.
The term "selectively hybridizable" means that the nucleotide sequence used as a probe is used under conditions where a target nucleotide sequence of the invention is found to hybridize to the probe at a level significantiy above background. The background hybridization may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened. In this event, background imphes a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32P.
Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below.
Maximum stringency typicaUy occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.
In a prefeπed aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention under stringent conditions (e.g. 65°C and O.lxSSC (lxSSC = 0.15 M NaCl, 0.015 M Na3 Citrate pH 7.0). Where the nucleotide sequence of the invention is double-stranded, both strands of the duplex, either individuaUy or in combination, are encompassed by the present invention. Where the nucleotide sequence is single-stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also included within the scope of the present invention.
Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skiU in the field. As known to those of skill in the art, the stabiHty of hybrids is reflected in the melting temperature (Tm) of the hybrid which decreases approximately 1 to 1.5°C with every 1 % decrease in sequence homology. In general, the stabiHty of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridisation reaction is performed under conditions of higher stringency, followed by washes of varying stringency.
As used herein, high stringency preferably refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65-68 °C. High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6x SSC, 5xDenhardt's, 1 % SDS (sodium dodecyl sulphate), 0.1 Na+ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific competitor. Following hybridisation, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 - O.lx SSC, 0.1 % SDS.
It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g. formamide-based buffers, and temperatures. Denhardt's solution and SSC are well known to those of skiU in the art as are other suitable hybridisation buffers (see, e.g. Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds. (1990) Cuπent Protocols in Molecular Biology, John Wiley & Sons, Inc.). Optimal hybridisation conditions have to be determined empirically, as the length and the GC content of the hybridising pair also play a role. Cloning and Expression
Nucleotide sequences which are not 100% homologous to the sequences of the present invention but f aU within the scope of the invention can b e obtained in a number of ways . Other variants of the sequences described herein may be obtained for example by probing DNA hbraries made from a range of sources. In addition, other viral/bacteiial, or cehular homologues particularly ceUular homologues found in mammahan ceUs (e.g. rat, mouse, bovine and primate ceUs), may be obtained and such homologues and fragments thereof in general wiU be capable of selectively hybridising to the sequences shown in the sequence Hsting herein. Such sequences may be obtained by probing cDNA Hbraries made from or genomic DNA Hbraries from other animal species, and probing such Hbraries with probes comprising aU or part of the reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and ahehc variants of the amino acid and/or nucleotide sequences useful in the present invention.
Variants and strain/species homologues may also be obtained using degenerate PCR which wiU use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences canbe predicted, for example, by ahgning the amino acid sequences from several variants/homologues. Sequence ahgnments canbe performed using computer software known in the art. For example the GCG Wisconsin PheUp program is widely used. The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. ,
Alternatively, such nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example sUent codon changes are required to sequences to optimise codon preferences for a particular host ceU in which the nucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the activity of the target protein or protein for T cell signalling modulation encoded by the nucleotide sequences.
The nucleotide sequences such as a DNA polynucleotides useful in the invention may be produced recombinandy, syntheticaUy, or by any means avaUable to those of skill in the art. They may also be cloned by standard techniques.
In general, primers wiU be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accompHshing this using automated techniques are readfly avaUable in the art.
Longer nucleotide sequences will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This wiU involve making a pah of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human ceU, performing a polymerase chain reaction (PCR) under conditions which bring about amplification of the desired region, isolating the amphfied fragment (e.g. by pmifying the reaction mixtare on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amphfied DNA can be cloned into a suitable cloning vector
The present invention also relates to vectors which comprise a polynucleotide useful in the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides useful in the present invention by such techniques.
For recombinant production, host cells can be geneticaUy engineered to incorporate expression systems or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis et al and Sambrook et al, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading, baUistic introduction and infection. It wih be appreciated that such methods can be employed in vitro or in vivo as drug delivery systems.
Representative examples of appropriate hosts include bacterial ceUs, such as streptococci, staphylococci, E. coli, streptomyces and Bacillus subtilis cells; fungal ceUs, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cehs; animal cells such as CHO, COS, NSO, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells ; and plant cells .
A great variety of expression systems can be used to produce a polypeptide useful in the present invention. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculovirases, papova viruses, such as SV40, vaccinia viruses, adenovituses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals. Proteins or polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein or precursor. For example, it is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences or pro-sequences (such as a HIS ohgomer, immunoglobulin Fc, glutathione S- transferase, FLAG etc) to aid in purification. Likewise such an additional sequence may sometimes be desirable to provide added stabiHty during recombinant production. In such cases the additional sequence may be cleaved (eg chemicaUy or enzymatically) to yield the final product. In some cases, however, the additional sequence may also confer a desirable pharmacological profile (as in the case of IgFc fusion proteins) in which case it may be preferred that the additional sequence is not removed so that it is present in the final product as administered.
Active agents for use in the invention can be recovered and purified from recombinant ceU cultures by weU-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocehulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Weh known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
Diagnostic assays
The ability to monitor the immune system in a simple and efficient assay presents innumerable applications. By way of illustration only, the present invention may be used, for example, for monitoring efficacy of immunotherapy. Such an assay could, for example, be used to detect induced tolerance or anergy in patients being treated with drugs. For instance, transplant patients are prescribed dmgs, such as cyclosporin, azathioprine, basiliximab or sirolimus, in an attempt to prevent graft rejection. It would be desirable to design a rationale for withdrawing such immunosuppressive dmgs, such that patients would not be put at risk. An assay for measuring tolerance would enable patients to come off immunosuppressive medication in a more controlled and therefore less speculative manner.
In another example, aUergy sufferers may benefit from an assay which would allow the responsiveness of then immune systems to be compared before and after therapy, such as allergy immunotherapy. Allergy immunotherapy typicaUy involves admrnistering increasing doses of an allergen (for example pollen aUergens such as Phi p 1, House Dust Mite antigens such as Der pi or Der f 1 , dog and cat aUergens such as Fel d 1 and Can f 1 or food allergens) over a period of several months or years with the aim of increasing tolerance over time. This present method of ensuring reduced sensitivity could remove the risk of previous tests involving exposure, even if only in small quantities, to an aUergen which has the potential of causing inflammatory reactions or even anaphylactic shock. In addition, the present assay method would provide a much more objective measure of the effectiveness of therapy than the rather subjective symptom-based measures which are often used at present. Similarly, the abihty to detect an immune response could be used in identifying the cause of an ahergic reaction by monitoring the activity of the immune system in the presence of different potential aUergens.
In an alternative scenario, the assay of the invention could be used to check for successful iinmunization against a given disease antigen. In one scenario, the assay method could be used to determine the state of a patient's immune system after administration of a vaccine, such that expression of a KLF protein of polynucleotide could indicate the effectiveness of the vaccination (a decrease in Notch signaUing generaUy indicating increased vaccine effectiveness). As we described above, downregulation of Notch signalling in vivo in T-ceUs may be used to prevent tumour ceUs from inducing immunotolerance in those T-cehs that recognise tumour-specific antigens. Thus, in an alternative scenario, it would be useful to be able to check for increased reactivity of T- ceUs and therefore for successful anti-tumour treatment. Anti-sense constructs
Suitable nucleic acid sequences may include anti-sense KLF constructs as well as antisense constructs designed to reduce or inhibit the expression of upregulators of KLF expression (see above). The antisense nucleic acid may be an oligonucleotide such as a synthetic single-stranded DNA. However, more preferably, the antisense is an antisense RNA produced in the patient's own cells as a result of introduction of a genetic vector. The vector is responsible for production of antisense RNA of the desired specificity on introduction of the vector into a host ceh.
Antisense nucleic acids can be oligonucleotides that are double-stranded or single- stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences.
For example, as described in US-A-20020119540 inhibitory antisense or double stranded oligonucleotides can additionaUy comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxyhnethyl) uracil, 5-carboxymethylammomethyl-2-thiouridine,
5-carboxymethylaminomethyluraci- 1, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isoρentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimemylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-memylaminomethyluracU, 5-methoxyamoinomethyl-2-thiouracU, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracU, 2-methylthio- N6-isopentenyladenine, uracfl-5 -oxy acetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiourach, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, urach-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracU, (acp3)w, and 2,6-dian_ opurine. An antisense oligonucleotide may also comprise one or more modified sugar moieties such as, for example, arabinose, 2-fluoroarabinose, xylulose, or hexose.
In yet another embodiment, the antisense ohgonucleotide may if desired comprise at least one modified phosphate backbone such as, for example, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof. Alternatively another polymeric backbone such as a modified polypeptide backbone may be used (eg peptide nucleic acid: PNA).
In yet another embodiment, the antisense ohgonucleotide may be an alpha-anomeric oligonucleotide. An alpha-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide may for example be a 2,-0-methylribonucleotide (Inoue et al., 1987,
Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330). OHgonucleotides maybe synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commerciaUy avaUable from Biosearch, Applied Biosystems, etc.). Merely as examples, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
Assays
Whether a substance canbe used for modulating Notch signalling may be determined using suitable screening assays, for example, as described in our co-pending International Patent AppHcation (published as WO 03/012441) or for example as described in the Examples herein. For example, Notch signaUing canbe monitored either through protein assays or through nucleic acid assays. Activation of the Notch receptor leads to the proteolytic cleavage of its cytoplasmic domain and the translocation thereof into the cell nucleus. The "detectable signal" refeπed to herein may be any detectable manifestation attributable to the presence of the cleaved intracellular domain of Notch. Thus, increased Notch signaUing can be assessed at the protein level by measuring intraceUular concentrations of the cleaved Notch domain. Activation of the Notch receptor also catalyses a series of downstream reactions leading to changes in the levels of expression of certain well defined genes. Thus, increased Notch signalling can be assessed at the nucleic acid level by say measuring intraceUular concentrations of specific mRNAs. In one preferred embodiment of the present invention, the assay is a protein assay. In another prefeπed embodiment of the present invention, the assay is a nucleic acid assay.
The advantage of using a nucleic acid assay is that they are sensitive and that smaU samples canbe analysed.
The intracehular concentration of a particular mRNA, measured at any given time, reflects the level of expression of the corresponding gene at that time. Thus, levels of mRNA of downstream target genes of the Notch signalling pathway canbe measured in an indirect assay of the T-cells of the immune system. In particular, an increase in levels of Deltex, Hes-1 and/or IL-10 mRNA may, for instance, indicate induced anergy whUe an increase in levels of Dh-1 or IFN-γ mRNA, or in the levels of mRNA encoding cytokines such as IL-2, IL-5 and IL-13 , may indicate improved responsiveness.
Various nucleic acid assays are known. Any convention technique which is known or which is subsequently disclosed may be employed. Examples of suitable nucleic acid assay are mentioned below and include amplification, PCR, RT-PCR, RNase protection, blotting, spectrometry, reporter gene assays, gene chip arrays and other hybridization methods.
In particular, gene presence, amplification and/or expression maybe measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe. Those skilled in the art wiU readily envisage how these methods may be modified, if desired.
PCR was originally developed as a means of amphfying DNA from an impure sample. The technique is based on a temperature cycle which repeatedly heats and cools the reaction solution allowing primers to anneal to target sequences and extension of those primers for the formation of duphcate daughter strands. RT-PCR uses an RNA template for generation of a first strand cDNA with a reverse transcriptase. The cDNA is then amphfied according to standard PCR protocol. Repeated cycles of synthesis and denaturation result in an exponential increase in the number of copies of the target DNA produced. However, as reaction components become limiting, the rate of ampHfication decreases until a plateau is reached and there is little or no net increase in PCR product. The higher the starting copy number of the nucleic acid target, the sooner this "end-point" is reached.
Real-time PCR uses probes labeled with a fluorescent tag or fluorescent dyes and differs from end-point PCR for quantitative assays in that it is used to detect PCR products as they accumulate rather than for the measurement of product accumulation after a fixed number of cycles. The reactions are characterized by the point in time during cycling when amplification of a target sequence is first detected through a significant increase in fluorescence.
The ribonuclease protection (RNase protection) assay is an extremely sensitive technique for the quantitation of specific RNAs in solution . The ribonuclease protection assay can be performed on total cellular RNA or poly(A)-selected mRNA as a target. The sensitivity of the ribonuclease protection assay derives from the use of a complementary in vitro transcript probe which is radiolabeled to high specific activity. The probe and target RNA are hybridized in solution, after which the mixtare is diluted and treated with ribonuclease (RNase) to degrade all remaining single-stranded RNA. The hybridized portion of the probe wiU be protected from digestion and can be visualized via electrophoresis of the mixture on a denaturing polyacrylamide gel followed by autoradiography. Since the protected fragments are analyzed by high resolution polyacrylamide gel electrophoresis, the ribonuclease protection assay can be employed to accurately map mRNA features. If the probe is hybridized at a molar excess with respect to the target RNA, then the resulting signal wiU be directly proportional to the amount of complementary RNA in the sample.
Gene expression may also be detected using a reporter system. Such a reporter system may comprise a readily identifiable marker under the control of an expression system, e.g. of the gene being monitored. Fluorescent markers, which can be detected and sorted by FACS, are prefeπed. Especially prefeπed are GFP and luciferase. Another type of prefeπed reporter is cell surface markers, i.e. proteins expressed on the cell surface and therefore easUy identifiable.
In general, reporter constructs useful for detecting Notch signalling by expression of a reporter gene may be constructed according to the general teaching of Sambrook et al (1989). Typically, constracts according to the invention comprise a promoter by the gene of interest, and a coding sequence encoding the desired reporter constructs, for example of GFP or luciferase. Vectors encoding GFP and luciferase are known in the art and avaUable commercially.
Sorting of cehs, based upon detection of expression of genes, may be performed by any technique known in the art, as exemphfied above. For example, ceUs may be sorted by flow cytometry or FACS. For a general reference, see Flow Cytometry and Cell Sorting: A Laboratory Manual (1992) A. Radbruch (Ed.), Springer Laboratory, New York. Flow cytometry is a powerful method for studying and purifying ceUs. It has found wide apphcation, particularly in immunology and cell biology: however, the capabilities of the FACS canbe apphed in many other fields of biology. The acronym F.AC.S. stands for Fluorescence Activated CeU Sorting, and is used interchangeably with "flow cytometry". The principle of FACS is that individual cells, held in a thin stream of fluid, are passed through one or more laser beams, causing Hght to be scattered and fluorescent dyes to emit light at various frequencies. Photomultipher tabes (PMT) convert Hght to electrical signals, which are interpreted by software to generate data about the ceUs. Sub- populations of cells with defined characteristics canbe identified and automatically sorted from the suspension at very high purity (-100%).
FACS can be used to measure gene expression in cells transfected with recombinant DNA encoding polypeptides. This canbe achieved directly, by labelling of the protein product, or indirectly by using a reporter gene in the construct. Examples of reporter genes are β-galactosidase and Green Fluorescent Protein (GFP). β-galactosidase activity can be detected by FACS using fluorogenic substrates such as fluorescein digalactoside (FDG). FDG is introduced into cehs by hypotonic shock, and is cleaved by the enzyme to generate a fluorescent product, which is trapped within the cell. One enzyme can therefore generate a large amount of fluorescent product. CeUs expressing GFP constracts whl fluoresce without the addition of a substrate. Mutants of GFP are available which have different excitation frequencies, but which emit fluorescence in the same channel. In a two-laser FACS machine, it is possible to distinguish cells which are excited by the different lasers and therefore assay two transfections at the same time.
Alternative means of cell sorting may also be employed. For example, the invention comprises the use of nucleic acid probes complementary to mRNA. Such probes canbe used to identify cells expressing polypeptides individually, such that they may subsequently be sorted either manually, or using FACS sorting. Nucleic acid probes complementary to mRNA may be prepared according to the teaching set forth above, using the general procedures as described by Sambrook et al (1989) supra. In a preferred embodiment, the invention comprises the use of an antisense nucleic acid molecule, complementary to a mRNA, conjugated to a fluorophore which may be used in FACS ceU sorting.
Methods have also been described for obtaining information about gene expression and identity using so-caUed gene chip aπays or high density DNA aπays (Chee M. et al. (1996) Science 274:601-614 (Chee)). These high density arrays are particularly useful for diagnostic and prognostic purposes. Use may also be made of In Vivo Expression Technology ( ET) (CamilH et al. (1994) Proc Natl Acad Sci USA 91 :2634-2638 (CamiUi)). IVET identifies genes up-regulated during say treatment or disease when compared to laboratory culture.
The advantage of using a protein assay is that Notch activation can be directly measured. Assay techniques that can be used to deteπnine levels of a polypeptide are well known to those skilled in the art. Such assay methods include radioimmunoassays, competitive- binding assays, Western Blot analysis, antibody sandwich assays, antibody detection, FACS and ELISA assays.
"RNA interference", "post-transcriptional gene silencing" and "quelling" resulting for example from the overexpression or misexpression of transgenes, or from the deliberate introduction of double-stranded RNA into cells (reviewed in Fire A (1999) Trends Genet 15:358-363; Sharp PA (1999) Genes Dev 13:139-141; Hunter C (1999) CuπBiol 9:R440-R442; Baulcombe DC (1999) Curr Biol 9:R599-R601 ; Vaucheret et al. (1998) Plant J 16:651-659) may also be used to modulate KLF activity therapeutically.
Antigen Presenting Cells
Where required, antigen-presenting cells (APCs) may be "professional" antigen presenting cells or may be another cell that may be induced to present antigen to T ceUs.
Alternatively a APC precursor may be used which differentiates or is activated under the conditions of culture to produce an APC. An APC for use in the ex vivo methods of the invention is typically isolated from a tumour or peripheral blood found within the body of a patient. Preferably the APC or precursor is of human origin. However, where APCs are used in preliminary in vitro screening procedures to identify and test suitable nucleic acid sequences, APCs from any suitable source, such as a healthy patient, may be used.
APCs include dendritic ceUs (DCs) such as interdigitating DCs or foUicular DCs, Langerhans cells, PBMCs, macrophages, B -lymphocytes, or other cell types such as epithelial ceUs, fibroblasts or endothelial ceUs, activated or engineered by transfection to express a MHC molecule (Class I or II) on then surfaces. Precursors of APCs include CD34+ cells, monocytes, fibroblasts and endotheHal cells. The APCs or precursors may be modified by the culture conditions or may be genetically modified, for instance by transfection of one or more genes encoding proteins which play a role in antigen presentation and/or in combination of selected cytokine genes which would promote to immune potentiation (for example IL-2, IL-12, IFN-γ, TNF-α, IL-18 etc.). Such proteins include MHC molecules (Class I or Class H), CD80, CD86, or CD40. Most preferably DCs or DC-precursors are included as a source of APCs.
Dendritic ceUs (DCs) can be isolated/prepared by a number of means, for example they can either be purified directly from peripheral blood, or generated from CD34+ precursor ceUs for example after mobihsation into peripheral blood by treatment with GM-CSF, or directly from bone marrow. From peripheral blood, adherent precursors can be treated with a GM-CSF/IL-4 mixture (Inaba K, et al. (1992) J. Exp. Med. 175: 1157-1167
(Inaba)), or from bone maπow, non-adherent CD34+ cells can be treated with GM-CSF and TNF-a (Caux C, et al. (1992) Natare 360: 258-261 (Caux)). DCs can also be routinely prepared from the peripheral blood of human volunteers, similarly to the method of Sahusto and Lanzavecchia (Sallusto F and Lanzavecchia A (1994) J. Exp.
Med. 179: 1109-1118) using purified peripheral blood mononucleocytes (PBMCs) and treating 2 hour adherent cells with GM-CSF and IL-4. If required, these maybe depleted of CD19+ B cells and CD3+, CD2+ T cells using magnetic beads (Coffin RS, et al. (1998) Gene Therapy 5: 718-722 (Coffin)). Culture conditions may include other cytokines such as GM-CSF or IL-4 for the maintenance and, or activity of the dendritic cells or other antigen presenting ceUs.
Thus, it whl be understood that the term "antigen presenting cell or the like" are used herein is not intended to be limited to APCs. The skUled man wUl understand that any vehicle capable of presenting to the T ceU population may be used, for the sake of convenience the term APCs is used to refer to aU these. As indicated above, preferred examples of suitable APCs include dendritic ceUs, L cells, hybridomas, fibroblasts, lymphomas, macrophages, B ceUs or synthetic APCs such as lipid membranes.
T cells
Where required, T ceUs from any suitable source, such as a healthy patient, may be used and may be obtained from blood or another source (such as lymph nodes, spleen, or bone marrow). They may optionally be enriched or purified by standard procedures. The T ceUs may be used in combination with other immune cells, obtained from the same or a different individual. Alternatively whole blood may be used or leukocyte enriched blood or purified white blood cells as a source of T ceUs and other cell types. It is particularly preferred to use helper T cehs (CD4+). Alternatively other T cells such as CD8+ cells may be used. It may also be convenient to use ceU lines such as T ceh hybridomas.
Exposure of agent to APCs and T cells
T cehs/APCs may be cultured as described above. The APCs/T cells may be incubated/exposed to substances which are capable of modulating Notch signalling. For example, they may be prepared for administration to a patient or incubated with T ceUs in vitro (ex vivo). Where treated ex-vivo, modified cehs of the present invention are preferably adnύnistered to a host by direct injection into the lymph nodes of the patient. Typically from 104 to 108 treated cehs, preferably from 105 to 107 cells, more preferably about 106 cells are administered to the patient. Preferably, the cells will be taken from an enriched ceh population.
As used herein, the term "enriched" as applied to the ceU populations of the invention refers to a more homogeneous population of ceUs which have fewer other cells with which they are naturally associated. An enriched population of ceUs can be achieved by several methods known in the art. For example, an enriched population of T-cells can be obtained using immunoaffinity chromatography using monoclonal antibodies specific for determinants found only on T-cells.
Enriched populations can also be obtained from mixed cell suspensions by positive selection (collecting only the desired ceUs) or negative selection (removing the undesirable ceUs). The technology for capturing specific cells on affinity materials is weh known in the art (Wigzel, et al., J. Exp. Med., 128:23, 1969; Mage, et al., J. Tmnmunol.
Meth., 15:47, 1977; Wysocki, et al., Proc. Natl. Acad. Sci. U.S.A., 75:2844, 1978;
Schrempf-Decker, et al., J. Immunol Meth., 32:285, 1980; Muller-Sieburg, et al., Cell, 44:653, 1986).
Monoclonal antibodies against antigens specific for mature, differentiated cells have been used in a variety of negative selection strategies to remove undesired cells, for example, to deplete T-cehs or malignant cehs from ahogeneic or autologous maπow grafts, respectively (Gee, et al., J.N.C.I. 80:154, 1988). Purification of human hematopoietic cehs by negative selection with monoclonal antibodies and immunomagnetic microspheres can be accomplished using multiple monoclonal antibodies (Griffin, et al., Blood, 63:904, 1984).
Procedures for separation of cells may include magnetic separation, using antibodycoated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, for example, complement and cytotoxins, and "panning" with antibodies attached to a sohd matrix, for example, plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, for example, a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
It wUl be appreciated that in one embodiment the therapeutic agents used in the present invention may be administered directly to patients in vivo. Alternatively or in addition, the agents may be administered to cells such as T cells and/or APCs in an ex vivo manner.
For example, leukocytes such as T ceUs or APCs may be obtained from a patient or donor in known manner, treated/incubated ex vivo in the manner of the present invention, and then administered to a patient. In addition, it will be appreciated that a combination of routes of administration may be employed if desired. For example, where appropriate one component (such as the modulator of Notch signalling) may be administered ex-vivo and the other may be administered in vivo, or vice versa.
Introduction of nucleic acid sequences into APCs and T-cells
T-cehs and APCs as described above are cultured in a suitable culture medium such as DMEM or other defined media, optionally in the presence of fetal calf serum.
Polypeptide substances may be administered to T-ceUs and/or APCs by introducing nucleic acid constructs/viral vectors encoding the polypeptide into ceUs under conditions that aUow for expression of the polypeptide in the T-ceU and/or APC. Similarly, nucleic acid constructs encoding antisense constructs may be inhoduced into the T-cehs and/or APCs by transfection, viral infection or viral transduction. In a preferred embodiment, nucleotide sequences whl be operably linked to control sequences, including promoters/enhancers and other expression regulation signals. The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is peferably ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
The promoter is typically selected from promoters which are functional in mammalian ceUs, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used. The promoter is typicaUy derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a ceU in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of a-actin, b-actin, tabulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyravate kinase). Tissue-specific promoters specific for lymphocytes, dendritic cells, skin, brain cells and epithelial cells within the eye are particularly prefeπed, for example the CD2, CD1 lc, keratin 14, Wnt-1 and Rhodopsin promoters respectively. Preferably the epithelial ceU promoter SPC is used. They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat ( MLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.
It may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the Hfe-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated. Any of the above promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences. Chimeric promoters may also be used comprising sequence elements from two or more different promoters.
Alternatively (or in addition), the regulatory sequences may be cell specific such that the gene of interest is only expressed in cells of use in the present invention. Such cells include, for example, APCs and T-cehs.
If required, a smaU ahquot of ceUs may be tested for up-regulation of Notch signalling activity as described above. The ceUs may be prepared for administration to a patient or incubated with T-ceUs in vitro (ex vivo).
Tolerisation assays
Any of the assays described above (see "Assays") can be adapted to monitor or to detect reduced reactivity and promotion of tolerance in immune cells for use in cHnical appHcations. Such assays wiU involve, for example, detecting increased Notch-ligand expression or activity in host cells or monitoring Notch cleavage in donor cells. Further methods of monitoring immune cell activity are set out below.
Immune cell activity may be monitored by any suitable method known to those skilled in the art. For example, cytotoxic activity may be monitored. Natural killer (NK) ceUs wiU demonstrate enhanced cytotoxic activity after activation. Therefore any drop in or stabilisation of cytotoxicity wiUbe an indication of reduced reactivity.
Once activated, leukocytes express a variety of new cell surface antigens. NK ceUs, for example, wiU express tiansferrin receptor, HLA-DR and the CD25 IL-2 receptor after activation. Reduced reactivity may therefore be assayed by monitoring expression of these antigens. Hara et al. Human T-ceU Activation: IH, Rapid Induction of a Phosphorylated 28 kD/32kD Disulfide linked Early Activation Antigen (EA-1) by 12-0-tetradecanoyl Phorbol-13-Acetate, Mitogens and Antigens, J. Exp. Med., 164:1988 (1986), and Cosulich et al. Functional Characterization of an Antigen (MLR3) Involved in an Early Step of T-Cell Activation, PNAS, 84:4205 (1987), have described cell surface antigens that are expressed on T-cells shortly after activation. These antigens, EA-1 and MLR3 respectively, are glycoproteins having major components of 28kD and 32kD. EA-1 and MLR3 are not HLA class π antigens and an MLR3 Mab wUl block IL-1 binding. These antigens appear on activated T-ceUs within 18 hours and can therefore be used to monitor immune ceU reactivity.
Additionally, leukocyte reactivity may be monitored as described in EP 0325489, which is incoφorated herein by reference. Briefly this is accomplished using a monoclonal antibody ("Anti-Leu23") which interacts with a cehular antigen recognised by the monoclonal antibody produced by the hybridoma designated as ATCC No. HB-9627.
Anti-Leu 23 recognises a cell surface antigen on activated and antigen stimulated leukocytes. On activated NK cells, the antigen, Leu 23, is expressed within 4 hours after activation and continues to be expressed as late as 72 hours after activation. Leu 23 is a disulfide-linked homodimer composed of 24 kD subunits with at least two N-linked carbohydrates.
Because the appearance of Leu 23 on NK cells coπelates with the development of cytotoxicity and because the appearance of Leu 23 on certain T-cells coπelates with stimulation of the T-ceh antigen receptor complex, Anti-Leu 23 is useful in monitoring the reactivity of leukocytes.
Further details of techniques for the monitoring of immune ceh reactivity may be found in: 'The Natural Killer Cell' Lewis C. E. and J. O'D. McGee 1992. Oxford University Press; Trinchieri G. 'Biology of Nataral Killer CeUs' Adv. Immunol. 1989 vol 47 ppl 87-376; 'Cytokines of the Immune Response' Chapter 7 in "Handbook of Immune Response Genes". Mak T.W. and J.J.L. Simard 1998, which are incorporated herein by reference.
Preparation of Primed APCs and Lymphocytes
According to one aspect of the invention immune cells may be used to present antigens or allergens and/or may be treated to modulate the Notch signalling pathway. Thus, for example, Antigen Presenting Cells (APCs) may be cultured in a suitable culture medium such as DMEM or other defined media, optionally in the presence of a serum such as fetal calf serum. Optimum cytokine concentrations may be determined by tifration. One or more substances capable of up-regulating or down-regulating the Notch signalling pathway are then typically added to the culture medium together with the antigen of interest. The antigen may be added before, after or at substantiahy the same time as the substance(s). Cells are typicaUy incubated with the substance(s) and antigen for at least one hour, preferably at least 3 hours, suitably at least 9, 12, 24, 48 or 36 or more hours at 37°C. If required, a small aliquot of cells may be tested for modulated target gene expression as described above. Alternatively, cell activity may be measured by the inhibition of T ceU activation by monitoring surface markers, cytokine secretion or proliferation as described in WO98/20142.
As discussed above, polypeptide substances may be administered to APCs by introducing nucleic acid constmcts/viral vectors encoding the polypeptide into cells under conditions that aUow for expression of the polypeptide in the APC. Similarly, nucleic acid constructs encoding antigens may be introduced into the APCs by transfection, viral infection or viral transduction. The resulting APCs that show increased levels of a Notch signalling are now ready for use. Preparation of Regulatory T cells (and B cells) ex vivo
The techniques described below are described in relation to T cells, but are equaUy appHcable to B cells. The techniques employed are essentiaUy identical to that described for APCs alone except that T cells are generally co-cultured with the APCs. However, it may be preferred to prepare primed APCs first and then incubate them with T cehs. For example, once the primed APCs have been prepared, they may be peUeted and washed with PBS before being resuspended in fresh culture medium. This has the advantage that if, for example, it is desired to treat the T cells with a different substance(s), then the T ceh whl not be brought into contact with the different substance(s) used with the APC. Once primed APCs have been prepared, it is not always necessary to adnhnister any substances to the T ceh since the primed APC is itself capable of inducing immunotolerance leading to increased Notch or Notch Hgand expression in the T ceU, presumably via Notch/Notch Hgand interactions between the primed APC and T ceU.
Incubations will typicaUy be for at least 1 hour, preferably at least 3, 6 , 12, 24, 48 or 36 or more hours, in suitable culture medium at 37°C. The progress of Notch signaUing may be determined for a smaU aliquot of ceUs using the methods described above. Induction of immunotolerance maybe deteimined, for example, by subsequently chaUenging T cells with antigen and measuring U_-2 production compared with control ceUs not exposed to APCs.
Primed T cells or B ceUs may also be used to induce immunotolerance in other T cells or B ceUs in the absence of APCs using sinhlar culture techniques and incubation times.
Alternatively, T cehs may be cultured and primed in the absence of APCs by use of APC substitutes such as anti-TCR antibodies (e.g. anti-CD3) with or without antibodies to costimulatory molecules (e.g. anti-CD28) or alternatively T ceUs may be activated with JVIHC-peptide complexes (e.g. tetramers). Induction of immunotolerance may be determined by subsequently challenging T cells with antigen and measuring IL-2 production compared with control ceUs not exposed to APCs.
T cells or B ceUs which have been primed in this way may be used according to the invention to promote or increase immunotolerance in other T cehs or B cells.
Therapeutic Uses
Immunological indications
In the prefeπed embodiment the therapeutic effect results from a protein for Notch signaUing. A detailed description of the Notch signalling pathway and conditions affected by it may be found in our WO98/20142, WO00/36089 and PCT/GB00/04391.
Diseased or infectious states that may be described as being mediated by T cehs include, but ate not limited to, any one or more of asthma, aUergy, graft rejection, autohnmunity, tumour induced abeπations to the T ceU system and infectious diseases such as those caused by Plasmodium species, Microfilaiiae, Helminths, Mycobacteria, HIV, Cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, Haemophilus influenza type B, measles, Hepatitis C or Toxicara. Thus particular conditions that may be treated or prevented which are mediated by T ceUs include multiple sclerosis, rheumatoid arthritis and diabetes. The present invention may also be used in organ transplantation or bone marrow transplantation.
As indicated above, the present invention is useful in treating immune disorders such as autoimmune diseases or graft rejection such as allograft rejection.
Autoimmune disease
Examples of disorders that may be treated include a group commonly caUed autoimmune diseases. The spectrum of autoimmune disorders ranges from organ specific diseases (such as thyroiditis, insulitis, multiple sclerosis, iridocycHtis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis) to systemic illnesses such as rheumatoid arthritis or lupus erythematosus. Other disorders include immune hypeπeactivity, such as allergic reactions.
hi more detaU: Organ-specific autoimmune diseases include multiple sclerosis, insulin dependent diabetes melhtas, several forms of anemia (aplastic, hemolytic), autoimmune hepatitis, thyroiditis, insulitis, iridocyclitis, scleritis, uveitis, orchitis, myasthenia gravis, idiopathic thrombocytopenic purpura, inflammatory bowel diseases (Crohn's disease, ulcerative cohtis).
Systemic autoimmune diseases include: rheumatoid arthritis, juvenile arthritis, sclerøderma and systemic sclerosis, sjogren's syndrom, undifferentiated connective tissue syndrome, antiphosphoHpid syndrome, different forms of vascuhtis (polyarteritis nodosa, allergic granulomatosis and angiitis, Wegner's granulomatosis, Kawasaki disease, hypersensitivity vascuhtis, Henoch-Schoenlein purpura, Behcet's Syndrome, Takayasu arteritis, Giant ceU arteritis, Thrombangutis obhterans), lupus erythematosus, polymyalgia rheumatica, essentieh (mixed) cryoglobulinemia, Psoriasis vulgaris and psoriatic arthritis, diffiis fasciitis with or without eosinophUia, polymyositis and other idiopathic inflammatory myopathies, relapsing pannicuHtis, relapsing polychondiitis, lymphomatoid granulomatosis, erythema nodosum, ankylosing spondylitis, Reiter's syndrome, different forms of inflammatory dermatitis.
A more extensive list of disorders includes: unwanted immune reactions and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, aUergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfiision injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopuhnonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomeralonephritis or other renal and urologic diseases, otitis or other oto-rMno-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo- orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory-related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophmalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, compHcation and/or side effects from treatment of Parkinson's disease, AEDS -related dementia complex HTV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myehtis, encephalitis, subacute sclerosing pan-encephaHtis, encephalomyeHtis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Baπe syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory compHcations or side effects of surgery or organ, mflammatoiy and/or immune compHcations and side effects of gene therapy, e.g. due to infection with a viral carrier, or inflammation associated with ADDS, to suppress or inhibit a humoral and/or cehular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone maπow, organs, lenses, pacemakers, natural or artificial skin tissue.
The present invention is also useful in cancer therapy. The present invention is especiahy useful in relation to adenocarcinomas such as: small ceU lung cancer, and cancer of the kidney, uterus , prostrate, bladder, ovary, colon and breast.
Transplant rejection
The present invention may be used, for example, for the treatment of organ transplants (e.g. kidney, heart, lung, Hver or pancreas transplants), tissue transplants (e.g. skin grafts) or ceU transplants (e.g. bone marrow transplants or blood transfusions).
A brief overview of the most common types of organ and tissue transplants is set out below.
1. Kidney Transplants:
Kidneys are the most commonly transplanted organs. Kidneys can be donated by both cadavers and living donors and kidney transplants can be used to treat numerous clinical indications (including diabetes, various types of nephritis and kidney failure). Surgical procedure for kidney transplantation is relatively simple. However, matching blood types and histocompatibility groups is desirable to avoid graft rejection. It is indeed important that a graft is accepted as many patients can become "sensitised" after rejecting a first transplant. Sensitisation results in the formation of antibodies and the activation of cehular mechanisms directed against kidney antigens. Thus, any subsequent graft containing antigens in common with the first is likely to be rejected. As a result, many kidney transplant patients must remain on some form of immunosuppressive treatment for the rest of their lives, giving rise to compHcations such as infection and metabolic bone disease.
2. Heart Transplantation
Heart transplantation is a very complex and high-risk procedure. Donor hearts must be maintained in such a manner that they wUl begin beating when they are placed in the recipient and can therefore only be kept viable for a limited period under very specific conditions. They can also only be taken from brain-dead donors. Heart transplants can be used to treat various types of heart disease and/or damage. HLA matching is obviously desirable but often impossible because of the Hmited supply of hearts and the urgency of the procedure.
3. Lung Transplantation
Lung transplantation is used (either by itself or in combination with heart fransplantation) to treat diseases such as cystic fibrosis and acute damage to the lungs (e.g. caused by smoke inhalation). Lungs for use in transplants are normally recovered from brain-dead donors.
4. Pancreas Transplantation
Pancreas transplantation is mainly used to treat diabetes mellitus, a disease caused by malfunction of insulin-producing islet cells in the pancreas. Organs for transplantation can only be recovered from cadavers although it should be noted that transplantation of the complete pancreas is not necessary to restore the function needed to produce insulin in a continued fashion. Indeed, transplantation of the islet ceUs alone could be sufficient. Because kidney faUure is a frequent complication of advanced diabetes, kidney and pancreas transplants are often carried out simultaneously.
5. Skin Grafting
Most skin transplants are done with autologous tissue. However, in cases of severe burning (for example), grafts of foreign tissue may be required (although it should be noted that these grafts are generally used as biological dressings as the graft wUl not grow on the host and wiU have to be replaced at regular intervals). In cases of true allogenic skin grafting, rejection may be prevented by the use of immunosuppressive therapy. However, this leads to an increased risk of infection and is therefore a major drawback in burn victims.
6. Liver Transplantation
Liver transplants are used to treat organ damage caused by viral diseases such as hepatitis, or by exposure to harmful chemicals (e.g. by chronic alcoholism). Liver transplants are also used to treat congenital abnormahties. The Hver is a large and compHcated organ meaning that transplantation initially posed a technical problem. However, most transplants (65%) now survive for more than a year and it has been found that a liver from a single donor may be spHt and given to two recipients. Although there is a relatively low rate of graft rejection by liver transplant patients, leukocytes within the donor organ together with anti-blood group antibodies can mediate antibody-dependent hemolysis of recipient red blood ceUs if there is a mismatch of blood groups. In addition, manifestations of GVHD have occurred in Hver transplants even when donor and recipient are blood-group compatible.
Ceh fate/cancer indications
The present invention is also useful in methods for altering the fate of a ceU, tissue or organ type by altering Notch pathway function in the cell. Thus, the present application has apphcation in the treatment of mahgnant and pre-neoplastic disorders.. The present invention is especiaUy useful in relation to adenocarcinomas such as: small ceh lung cancer, and cancer of the kidney, uterus, prostrate, bladder, ovary, colon and breast. For example, mangnancies which may be treatable according to the present invention include acute and chronic leukemias, lymphomas, myelomas, sarcomas such as Fibrosarcoma, myxosarcoma, liposarcoma, lymphangioendotheliosarcoma, angiosarcoma, endotheliosarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, lymphangiosarcoma, synovioma, mesothelioma, leimyosarcoma, rhabdomyosarcoma, colon carcinoma, ovarian cancer, prostate cancer, pancreatic cancer, breast cancer, squamous ceU carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papUlary carcinoma, papUlary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, choriocarcinoma, renal cell carcinoma, hepatoma, bUe duct carcinoma seminoma, embryonal carcinoma, cervical cancer, testicular tumour, lung carcinoma, small ceU lung carcinoma, bladder carcinoma, epithelial carcinoma, glio a, astrocytoma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, meduhoblastoma, craniopharyngioma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.
The present invention may also have apphcation in the treatment of nervous system disorders. Nervous system disorders which may be treated according to the present invention include neurological lesions including traumatic lesions resulting from physical injuries; ischaemic lesions; malignant lesions; infectious lesions such as those caused by H V, herpes zoster or herpes simplex virus, Lyme disease, tuberculosis or syphilis; degenerative lesions and diseases and demyelinated lesions.
The present invention may be used to treat, for example, diabetes (including diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, sarcoidosis, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, central pontine myelinolysis, Parkinson's disease, Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis, cerebral infarction or ischemia, spinal cord infarction or ischemia, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvemle muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), pohomyeUtis and the post poho syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
The present invention may further be useful in the promotion of tissue regeneration and repair. The present invention, therefore, may also be used to treat diseases associated with defective tissue repair and regeneration such as, for example, cirrhosis of the liver, hypertrophic scar formation and psoriasis. The invention may also be useful in the treatment of neutropenia or anemia and in techniques of organ regeneration and tissue engineering.
Administration
Suitably the active agents are administered in combination with a pharmaceuticaUy acceptable carrier or dUuent. The pharmaceuticaUy acceptable carrier or dUuent may be, for example, sterile isotonic saline solutions, or other isotonic solutions such as phosphate- buffered saline. The conjugates of the present invention may be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). It is also prefeπed to formulate the compound in an orally active form.
Pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and wUl typically comprise any one or more of a pharmaceuticaUy acceptable diluent, carrier, or excipient. Acceptable carriers or dduents for therapeutic use are weU known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or dUuent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or dUuent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Preservatives, stabhizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzo ate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
For some applications, active agents may be administered orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents.
Alternatively or in addition, active agents may be administered by inhalation, intranasahy or in the form of aerosol, or in the form of a suppository or pessary, or they may be apphed topicaUy in the form of a lotion, solution, cream, ointment or dusting powder. An alternative means of transdermal administration is by use of a skin patch. For example, they canbe incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or Hquid paraffin. They can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabhisers and preservatives as may be required.
Active agents such as polynucleotides and proteins/polypeptides may also be administered by viral or non-viral techniques. Viral dehvery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors. Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoHpo somes, Hpofectin, cationic facial amphiphiles (CFAs) and combinations thereof. The routes for such dehvery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes. Active agents may be adminstered by conventional DNA dehvery techniques, such as DNA vaccination etc., or injected or otherwise dehvered with needleless systems, such as ballistic delivery on particles coated with the DNA for delivery to the epidermis or other sites such as mucosal surfaces.
In general, a therapeuticahy effective oral or intravenous dose is likely to range from 0.01 to 50 mg/kg body weight of the subject to be treated, preferably 0.1 to 20 mg/kg. The conjugate may also be administered by intravenous infusion, at a dose which is likely to range from 0.001-10 mg/kg/hr.
TypicaUy, the physician whl determine the actaal dosage which will be most suitable for an individual patient and it wiU vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
Tablets or capsules of the conjugates may be administered singly or two or more at a time, as appropriate. It is also possible to administer the conjugates in sustained release formulations.
Active agents may also be injected parenterally, for example intracavernosahy, intravenously, intramuscularly or subcutaneously
For parenteral administration, active agents may be used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
For buccal or sublingual administration, agents may be administered in the form of tablets or lozenges which can be formulated in a conventional manner. For oral, parenteral, buccal and sublingual administration to subjects (such as patients), the dosage level of active agents and their pharmaceutically acceptable salts and solvates may typicaUy be from 10 to 500 mg (in single or divided doses). Thus, and by way of example, tablets or capsules may contain from 5 to 100 mg of active agent for administration singly, or two or more at a time, as appropriate. As indicated above, the physician wiU determine the actaal dosage which whl be most suitable for an individual patient and it wiU vary with the age, weight and response of the particular patient. It is to be noted that whilst the above-mentioned dosages are exemplary of the average case there can, of course, be individual instances where higher or lower dosage ranges are merited and such dose ranges are within the scope of this invention.
The routes of administration and dosages described are intended only as a guide since a skUled practitioner wUl be able to determine readily the optimum route of administration and dosage for any particular patient depending on, for example, the age, weight and condition of the patient.
The term treatment or therapy as used herein should be taken to encompass diagnostic and prophylatic appHcations.
The treatment of the present invention includes both human and veterinary applications.
Antigens and Allergens
An antigen suitable for use in the present invention may be any substance that can be recognised by the immune system, and is generally recognised by an antigen receptor. Preferably the antigen used in the present invention is an immunogen. An allergic response occurs when the host is re-exposed to an antigen that it has encountered previously.
The immune response to antigen is generaUy either cell mediated (T ceh mediated kiUing) or humoral (antibody production via recognition of whole antigen). The pattern of cytokine production by TH ceUs involved in an immune response can influence which of these response types predominates: cell mediated immunity (TH1) is characterised by high IL-2 and IFNγ but low IL-4 production, whereas in humoral immunity (TH2) the pattern is low IL-2 and IFNγ but high IL-4, IL-5 and IL-13. Since the secretory pattern is modulated at the level of the secondary lymphoid organ or cells, then pharmacological manipulation of the specific TH cytokine pattern can influence the type and extent of the immune response generated.
The TH1-TH2 balance refers to the relative representation of the two different forms of helper T cells. The two forms have large scale and opposing effects on the immune system. If an immune response favours TH1 ceUs, then these cells wiU drive a ceUular response, whereas TH2 cells will drive an antibody-dominated response. The type of antibodies responsible for some allergic reactions is induced by TH2 ceUs.
The antigen or aUergen used in the present invention may be a peptide, polypeptide, carbohydrate, protein, glycoprotein, or more complex material containing multiple antigenic epitopes such as a protein complex, cell-membrane preparation, whole cells (viable or non-viable cells), bacterial ceUs or virus/viral component. The term also includes fragments of antigens or allergens such as antigenic determinants and epitopes. In particular, it is prefeπed to use antigens known to be associated with auto-immune diseases such as myelin basic protein (associated with multiple sclerosis), collagen (associated with rheumatoid arthritis), and insulin (diabetes), or antigens associated with rejection of non-self tissue such as MHC antigens. Where primed the APCs and/or T ceUs of the present invention are to be used in tissue transplantation procedures, antigens may be obtained from the tissue donor.
The antigen or aUergen moiety may be, for example, a synthetic MHC-peptide complex i.e. a fragment of the MHC molecule bearing the antigen groove bearing an element of the antigen. Such complexes have been described in Airman et al, 1996. Various prefeπed features and embodiments of the present invention will now be described in more detail by way of non-limiting examples.
Example 1
i) CD4+ cell purification
Spleens were removed from mice (Balb/c females, 8-10 weeks) and passed through a 0.2μM cell strainer into 20ml R10F medium (R10F-RPMI 1640 media (Gibco Cat No 22409) plus 2mM L-glutamine, 50μg/ml Penicillin, 50μg/ml Streptomycin, 5 x 10"5 M β-mercapto-ethanol in 10% fetal calf serum). The ceh suspension was spun (1150rpm 5min) and the media removed.
The cells were incubated for 4 minutes with 5ml ACK lysis buffer (0.15M NH4CI, l.OM KHC03, O.lmM Na^DTA in double distiUed water) per spleen (to lyse red blood cells). The cehs were then washed once with R10F medium and counted. CD4+ ceUs were purified from the suspensions by positive selection on a Magnetic Associated Ceh Sorter (MACS) column (Miltenyi Biotec, Bisley, UK: Cat No 130-042-401) using CD4 (L3T4) beads (Miltenyi Biotec Cat No 130-049-201), according to the manufacturer's directions.
ii) CSFE Labelling
Cells were counted then resuspended in PBS (1-2 X 107 per ml). The CSFE (carboxyfluorescein diacetate succinimidyl ester) stock (5mM in DMSO; Molecular Probes, Catalogue number C-1157) was diluted 1/100 in PBS. This was then added to the ceUs (1 lOul/ml of cells) and mixed rapidly. The cehs were incubated 5min at room temp then washed 3 times in PBS + 5%FCS. The ceUs were then recounted and plated out.
Example 2
Antibody Coating
The following protocol was used for coating 96 well flat-bottomed plates with antibodies. The plates were coated with DPBS plus lμg/ml anti-hamsterlgG antibody ( harmingen Cat No 554007) plus lμg/ml anti-IgG4 antibody. lOOμl of coating mixture was added per well. Plates were incubated overnight at 4°C then washed with DPBS. Each weU then received either lOOμl DPBS plus anti-CD3 antibody (lμg/ml) or, lOOμl DPBS plus anti- CD3 antibody (lμg/ml) plus Fc-Delta titrated from 80 μg/ml (variously 0, 5, 20, 80 μg/ml). Lower anti-CD3 concentrations were also used (0.08ug/ml and 0.04ug/ml). The plates were incubated for 2-3 hours at 37°C then washed again with DPBS before cells (prepared and labelled as in Example 1) were added.
The plates were incubated for 2-3 hours at 37°C then washed again with DPBS before ceUs (prepared as in Example 1) were added.
Example, 3
Primary Polyclonal Stimulation CD4+ ceUs from Example 1 were cultured in 96 weU, flat-bottomed plates pre-coated according to Example 2. CeUs were re-suspended, following counting, at 2 x 106 /ml in R10F medium plus 4μg/ml anti-CD28 antibody (Pharmingen, Cat No 553294, Clone No 37.51). lOOμl cell suspension was added per well. lOOμl of R10F medium was then added to each well to give a final volume of 200μl (2 x 105 cells/well, anti-CD28 final concentration 2μg/ml) The plates were then incubated at 37°C for 72 hours.
125 μl supernatant was then removed from each well and stored at -20°C.
Example 4
FACS analysis
Cells from Example 3 were harvested after 72 hours into a round-bottomed 96 well plate, spun at 1100 rpm for 4 minutes, resuspended in 300 μl PFN buffer (PBS+l%FCS+0.025% sodium azide) before being ran on Coulter FACS machine.
Results are shown in Figure 7.
Example 5
Gene expression profiling
ti) hDeltal-IgG4Fc Fusion Protein
A fusion protein comprising the extracellular domain of human Deltal fused to the Fc domain of human IgG4 ("hDeltal-IgG4Fc") was prepared by inserting a nucleotide sequence coding for the extracellular domain of human Deltal (see, eg Gehbank Accession No AF003522) into the expression vector pCONγ (Lonza Biologies, Slough, UK) and expressing the resulting construct in CHO cells. The amino acid sequence of the resulting expressed fusion protein was as follows:
MGSRCALALAVLSALLCOVWSSGVFELKLOEFVNKKGLLGNRNCCRGGAGPPP CACRTIΦRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPI RFPFGFTWPGTFSLΠEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLH SSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWK GPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQP WQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGA TCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNG GRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQ AGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCE HAPCHNGATCHERGHGYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLO SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEF LGGPSVFLFPPKPKDTLλllSRTPEVTCVVVDVSOEDPEVOFNWYVDGVEVHNAK TKPREEOFNSTYRVVSVLTVLHODWLNGKEYKCKVSNKGLPSSIEKTISKAKGOP REPOVYTLPPSOEEMTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWOEGNVFSCSVMHEALHNHYTOKSLSLSLGK
Wherein the first underlined sequence is the signal peptide (cleaved from the mature protein) and the second underlined sequence is the ϊgG4 Fc sequence. The protein normally exists as a dimer linked by disulphide bonds (see eg schematic representation in Figure 6).
(ii) Cell culture, treatments and RNA extraction
Freshly-isolated murine CD4+ T-cells were cultured in RPMI 1640 (GibcoBRL) supplemented with 2mM Glutamine (GibcoBRL), PenicUlin-Streptomycin 50 units/ml (GibcoBRL), 50μM 2-mercaptoethanol and with 10% Fetal Bovine Serum (FBS) (BiochiOmKG).
Anti-human IGg4 anti-CD3 (human), anti-CD28 (human) antibodies (PharMingen) were plated at 5 ig/ml in phosphate buffer saline (Gibco BRL) in 6 weU tissue culture dishes
(lml PBS/weU) overnight. Anti-IgG4 antibody was applied to every well, while mouse
IgGi K isotype control at 10 g/ml was applied in weUs that no anti-CD3 or anti-CD28 was used. The next day the wells were washed 3 times with PBS, and human Deltal -
IgG4Fc fusion protein (hDeltal-IgG4Fc; see above) was plated at 10μg/ml or 80 μ.g/ml in PBS (lml/well) upon ligand-treated wells: control wells were coated with PBS only. The plates were then incubated at 37°C for 2 hours and then washed with PBS three times.
CD4+ T-cells cells were then plated out at a concentration of lxlO6 ceUs /ml and incubated at 37°C. Cells were taken out after 4 hours and 16 hours (overnight), span down and lysed in 300-600μl RLT lysis solution (Qiagen). In order to ensure the efficacy of the Notch Hgand stimulation, parallel cultures of ceUs were tested for expression of cytokines by standard ELISA techniques after 3 days in culture. RNA was extracted using an RNA Easy miniprep kit (Qiagen) according to the manufacturer's instmctions. The optional DNase step recommended was also perfoimed. A phenol extraction step was performed to ensure the removal of proteins in the RNA. RNA was then amphfied using the MessageAmp aRNA Kit (Ambion) foUowing the manufacturer's recommendations. Briefly, the procedure consists of reverse transcription with an ohgo(dT) primer bearing a T7 promoter and in vitro transcription of the resulting DNA with T7 RNA polymerase to generate hundreds of thousands of antisense RNA (αRNA) copies of each mRNA in the sample.
(iii) Gene Expression Profiling
Microaπays were manufactured by spotting purified PCR products onto glass shdes. Microaπay probes were prepared by labelling 2μg of αRNA by a reverse transcriptase reaction incorporating dCTP-Cy3 or dCTP-Cy5 labehed nucleotide. Probe labelling and purification were then performed generaUy as described in Hegde P, Qi R, Abernathy K, Gay C, Dharap S, Gaspard R, Hughes JE, Snesrud E, Lee N, Quackehbush J: A concise guide to cDNA microaπay analysis (2000). Biotechniques 29:548-50, 552-4, 556 passim.
Purified probes were then hybridized on the arrays overnight at 42°C in 10 x SSC, 50% formamide, 0.2% SDS solution. Shdes were then washed twice in 2 x SSC, 0.2% SDS for 7 min at 42 °C, twice in 0.1 SSC/ 0.2% SDS for 5 minutes at room temperature, and finaUy once in 0.2%SSC at room temperature. For each time point the sample treated in the absence of Notch ligand ('CD3/CD28 only') was labelled with dCTP-Cy3 and hybridized on the same shde as the sample named 'CD3/CD28 plus Delta' that was labelled with dCTP-Cy5.
Once dried the slides were scanned on a GSI Lumonics confocal scanner at 100% laser power and 65-75% photo-multipher tube efficiency (depending on background). Slide images were processed as foUows: Array spots representing the signal associated with individual spotted clones were identified and quantified using the Quantaπay application (GSI Lumonics). Numeric values for the gene expression intensities were calculated using the histogram method implemented in the same application. Values were calculated as integrals of the pixel signal distribution associated to each spot and local background values subtracted (raw data).
(iv) Data Processing
For aU data analyses the GeneSpring package (SUicon Genetics) was used. Raw data from Quantarray was introduced in GeneSpring, and the ratio between the signal and control intensities was calculated for each gene at each time point. Intensities for genes from samples labehed 'CD3/CD28 plus Delta' were regarded as 'signals' whUe the intensities from genes from samples labelled 'CD3/CD28 only' were regarded as 'controls'.
Ratio= signal strength of gene in 'CD3/CD28 plus Delta'/ 'CD3/CD28 only'
When this ratio was >2 the gene was considered to be upregulated, whUe when the ratio was <0.5 the ratio the gene was considered to be downregulated.
Results for CD28 and KLF-2 expression (fold upregulation compared to control) at 4 and 16 hours and with either 10 or 80 micrograms/ml hDeltal-IgG4Fc are shown in Figure 8.
Example 6
Real Time PCR analysis of primary stimulated CD4+ cells
T-cells from Example 5 were harvested at 4 and 16 hours. Total cellular RNA was isolated using the RNeasy™ RNA isolation kit (Qiagen, Crawley, UK) according to the manufacturer's guidelines. In each case lμg of total RNA was reverse transcribed using SuperScri.pt™ II Reverse Transcriptase (Invitrogen, Paisley, UK) using OHgo dT χ2-ι8) or a random decamer mix according to the manufacturer's guidelines. After synthesis, OHgo dT(i2-i8)- and random decamer-primed cDNAs were mixed in equal proportions to provide the working cDNA sample for real-time quantitative PCR analysis.
Real-time quantitative PCR was performed using the Roche Lightcycler™ system (Roche, UK) and SYBR green detection chemistry according to the manufacturer's guidelines. The foUowing HPLC-purified primer pairs were used for cDNA-specific amplification (5 ' to 3 ') :
mouse 18s rRNA: Forward GTAACCCGTTGAACCCCATT Reverse CCATCCAATCGGTAGTAGCG
mouse Hes-1: Forward GGTGCTGATAACAGCGGAAT
Reverse ATTTTTGGAATCCTTCACGC
KLF-2; Forward GAAGGCCCCAGGAAAGAAGACAGG
Reverse AAAACGAAGCAGGCGGCAGAGATG
The endpoint used in real-time PCR quantification, the Crossing Point (Cp), is defined as the PCR cycle number that crosses an algorithm-defined signal threshold. Quantitative analysis of gene-specific cDNA was achieved firstly by generating a set of standards using the Cps from a set of seriaUy-diluted gene-specific amplicons which had been previously cloned into a plasmid vector (pCR2T, Invitrogen). These serial dilutions fah into a standard curve against which the Cps from the cDNA samples were compared. Using this system, expression levels of the 18S rRNA house-keeping gene were generated for each cDNA sample. HES-1 (a known target of Notch signalling) and KLF- 2 was then analysed by the same method using serially-diluted HES-1 and KLF-2 specific standards, and the HES-1 and KLF-2 values were divided by the 18S rRNA value to generate a value, which represents the relative expression of HES-1 or KLF-2 in each cDNA sample. AU Cp analysis was performed using the Second Derivative Maximum algorithm within the Lightcycler system software.
Results (HES-1 and KLF-2 expression relative to 18S rRNA expression at different concentrations of hDeltal-IgG4Fc) are shown in Figure 9.
Example 7
CD4+ were purified from spleens of female BALB/c mice, aged between 8 and 12 weeks using CD4/L3T4 MACS beads (Miltenyi), according to the manufacturer's directions. Purity was between 80% and 95% CD4+ by FACS analysis. To produce naive and memory CD4+ populations, CD4+ ceUs were isolated by negative selection using a rat antibody cocktail of a-CD8, a-B220, a-Grl and a-Ad foUowed by a-Rat Dynal beads (Dynal, UK) at a 1 ratio. The CD4 cells were then further sub -fractionated into CD62LH and CD62L10 using various amounts of CD62L MACS beads (according to the manufacturers instructions), purity was normally >90% for CD62LH and >80% for CD62L10
Cells were treated with Delta (Notch hgand) protein at 20μg/ml (20D) and anti-CD3 at 0.8μg/ml as described above. Supematants were harvested at 24, 48 and 72hrs. RNA analysis was carried out as described above. Results are shown in Figures lOA-C.
References (incorporated herein bv reference thereto)
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Claims

1. A method for modifying immune cell quiescence by administering a modulator of Notch signalling.
2. A method for increasing immune cell quiescence by administering an activator of Notch signalling.
3. A method for decreasing immune ceh quiescence by administering an inhibitor of Notch signalling.
4. A method for increasing immune cell quiescence by administering a Notch receptor agonist.
5. A method for decreasing immune ceU quiescence by adπώustering a Notch receptor antagonist.
6. A method as claimed in any one of the preceding claims wherein the modulator of Notch signalling modifies quiescence in lymphocytes.
7. A method as claimed in claim 6 wherein the modulator of Notch signaUing modifies quiescence in T-cells.
8. A method as claimed in any one of the preceding claims to treat graft rejection
9. A method as claimed in any one of the preceding claims to treat autoimmune disease
10. A method as claimed in claim 9, wherein the autoimmune disorder is selected from the group consisting of multiple sclerosis, insulin-dependent diabetes, sympathetic ophthalmia, uveitis and psoriasis.
11. A method as claimed in any one of the preceding claims wherein the modulator of Notch signalling is administered to a patient in vivo.
12. A method as claimed in any one of claims 1 to 10 wherein the modulator of Notch signaUing is administered to a cell ex-vivo, after which the ceU may be administered to a patient.
13. A method as claimed in any one of the preceding claims to treat a disorder selected from the group consisting of: thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis, rheumatoid arthritis, lupus erythematosus, immune hypeπeactivity, insulin dependent diabetes mellitus, anemia (aplastic, hemolytic), autoimmune hepatitis, sHeritis, idiopathic fhrombocytopenic purpura, inflammatory bowel diseases (Crohn's disease, ulcerative cohtis), juvenile arthritis, scleroderma and systemic sclerosis, sjogren's syndrom, undifferentiated connective tissue syndrome, antiphospholipid syndrome, vascuhtis (polyarteritis nodosa, allergic granulomatosis and angntis, Wegner's granulomatosis, Kawasaki disease, hypersensitivity vasculitis, Henoch-Schoenlein purpura, Behcet's Syndrome, Takayasu arteritis, Giant ceh arteritis, Thrombangutis obhterans), polymyalgia rheumatica, essentiell (mixed) cryoglobulinemia, Psoriasis vulgaris and psόriatic arthritis, diffus faschtis with or without eosinophhia, polymyositis and other idiopathic inflammatory myopathies, relapsing panniculitis, relapsing polychondritis, lymphomatoid granulomatosis, erythema nodosum, ankylosing spondylitis, Reiter's syndrome, inflammatory dermatitis, unwanted immune reactions and inflammation associated with arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity and aUergic reactions, systemic lupus erythematosus, collagen diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac aπest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, Hver ciπhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomeralonephritis or other renal and urologic diseases, otitis or other oto-rhino- laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo -orchitis, infertUity, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory- related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and irrflammatoiy components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central neivous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, compHcation and/or side effects from treatment of Parkinson's disease, AEDS -related dementia complex HTV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of strokes, post-polio syndrome, immune and inflammatory components of psychiatric disordeis, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, GuiUaim-Baπe syndrome, Sydenham chora, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory compHcations or side effects of surgery or organ, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a vhal carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or ceUular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.
14. The use of a modulator of Notch signalling for modifying immune cell quiescence.
15. The use of an activator of Notch signalling for increasing immune ceU quiescence.
16. The use of an inhibitor of Notch signaUing for decreasing immune cell quiescence.
17. The use of a Notch receptor agonist for increasing immune ceU quiescence.
18. The use of a Notch receptor antagonist for decreasing immune ceU quiescence.
19. A use as claimed in any one of claims 14 to 18 wherein the modulator of Notch signaUing modifies quiescence in lymphocytes.
20. A use as claimed in claim 19 wherein the modulator of Notch signalling modifies quiescence in T-cells.
21. Ause as claimed in any one of claims 14 to 20 to treat graft rejection
22. Ause as claimed in any one of claims 14 to 20 to treat autoimmune disease
23. Ause as claimed in claim 22, wherein the autoimmune disorder is selected from the group consisting of multiple sclerosis, insulin-dependent diabetes, sympathetic ophthalmia, uveitis and psoriasis.
24. A use as claimed in any one of claims 14 to 23 to wherein the modulator of Notch signaUing is administered to a patient in vivo.
25. A use as claimed in any one of claims 14 to 23 wherein the modulator of Notch signaUing is administered to a cell ex-vivo, after which the ceU may be administered to a patient.
26. A use as claimed in any one of the claims 14 to 25 to treat a disorder selected from the group consisting of: thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis, rheumatoid arthritis, lupus erythematosus, immune hypeπeactivity, insulin dependent diabetes mellitus, anemia (aplastic, hemolytic), autoimmune hepatitis, skleritis, idiopathic thrombocytopenic purpura, inflammatory bowel diseases (Crohn's disease, ulcerative cohtis), juvenile arthritis, scleroderma and systemic sclerosis, sjogren's syndrom, undifferentiated connective tissue syndrome, antiphospholipid syndrome, vascuhtis (polyarteritis nodosa, aUergic granulomatosis and angiitis, Wegner's granulomatosis, Kawasaki disease, hypersensitivity vascuhtis, Henoch-Schoenlein purpura, Behcet's Syndrome, Takayasu arteritis, Giant ceU arteritis, Thrombangutis obhterans), polymyalgia rheumatica, essentiell (mixed) cryoglobuhnemia, Psoriasis vulgaris and psoriatic arthritis, diffus fasciitis with or without eosinophilia, polymyositis and other idiopathic inflammatory myopathies, relapsing panniculitis, relapsing polychondiitis, lymphomatoid granulomatosis, erythema nodosum, ankylosing spondylitis, Reiter's syndrome, inflammatory dermatitis, unwanted immune reactions and inflammation associated with arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity and ahergic reactions, systemic lupus erythematosus, collagen diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac aπest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, Hver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino- laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididrmo-orchitis, inferthity, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory- related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g. foUowing glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, comphcation and/or side effects from treatment of Parkinson's disease, AEDS -related dementia complex HEV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of strokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guihaim-Baπe syndrome, Sydenham chora, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory compHcations or side effects of surgery or organ, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a vhal carrier, or inflammation associated with AEDS, to suppress or inhibit a humoral and/or ceUular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of nataral or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, nataral or artificial skin tissue.
27. A method or use as claimed in any one of the preceding claims wherein the modulator of Notch signalling comprises a protein or polypeptide comprising a Notch Hgand DSL domain or a polynucleotide sequence coding for such a protein or polypeptide.
28. A method or use as claimed in claim 27 wherein the modulator of Notch signalling comprises a protein or polypeptide comprising a Notch Hgand DSL domain and at least one EGF-Hke domain or a polynucleotide sequence coding for such a protein or polypeptide.
29. A method or use as claimed in claim 28 wherein the modulator of Notch signalling comprises a protein or polypeptide comprising a Notch ligand DSL domain and at least two EGF-like domains or a polynucleotide sequence coding for such a protein or polypeptide.
30. A method or use as claimed in claim 29 wherein the modulator of Notch signalling comprises a protein or polypeptide comprising a Notch ligand DSL domain and 3 to 20 EGF-like domains or a polynucleotide sequence coding for such a protein or polypeptide.
31. A method or use as claimed in any one of claims 27 to 30 wherein the DSL or EGF domains are from Delta or Jagged.
32. A method or use as claimed in claim 31 wherein the DSL or EGF domains are from human Delta or Jagged.
33. A method or use as claimed in any one of claims 27 to 32 wherein the modulator of the Notch signaUing pathway comprises a fusion protein comprising a segment of a Notch hgand extracehular domain and an immunoglobulin Fc segment or a polynucleotide coding for such a fusion protein.
34. A method or use as claimed in any one of claims 1 to 26 wherein modulator of the Notch signaUing pathway comprises a Notch mtracehular domain (Notch IC) or a polynucleotide sequence which codes for a Notch intracellular domain.
35. A method for detecting, measuring or monitoring peripheral immune cell activation comprising determining the amount of a KLF protein, polypeptide or polynucleotide or determining the amount of a polynucleotide coding for such a protein or polypeptide.
36. A method as claimed in claim 35 for detecting, measuring or monitoring peripheral T-ceU activation
37. A method for detecting, measuring or monitoring Notch signalling comprising deteπnining the amount of a KLF protein, polypeptide or polynucleotide or determining the amount of a polynucleotide coding for such a protein or polypeptide.
38. A method as claimed in claim 37 wherein the amount of the KLF protein, polypeptide or polynucleotide in a biological sample taken from a subject is determined.
39. A method as claimed in claim 38 wherein the sample comprises blood, serum, urine, lymphatic fluid, or tissue.
40. A method as claimed in claim 37 or claim 38 wherein the sample comprises an immune ceU.
41. Amethod as claimed in claim 37 or claim 38 wherein the sample comprises a cancer ceh.
42. A method as claimed in claim 37 or claim 38 wherein the sample comprises a stem ceh.
43. A method as claimed in claim 40 wherein the sample comprises peripheral T- cells.
44. Amethod as claimed in any of claims 35 to 43, comprising the steps of: obtaining a biological sample from a subject; and contacting the biological sample with a binding agent that binds to a KLF protein, polypeptide or polynucleotide.
45. A method as claimed in claim 44 comprising the steps of: obtaining a biological sample from a subject; contacting the biological sample with a binding agent that binds to a KLF protein, polypeptide or polynucleotide; and detecting in the sample an amount of KLF protein, polypeptide or polynucleotide that binds to the binding agent.
46. A method as claimed in claim 45, comprising the steps of: obtaining a biological sample from the patient; contacting the biological sample with a binding agent that binds to a KLF protein, polypeptide or polynucleotide; detecting in the sample an amount of KLF protein, polypeptide or polynucleotide that binds to the binding agent; and comparing the amount of KLF protein, polypeptide or polynucleotide to a reference value and therefrom deternhning the degree of Notch signalling.
47. A method as claimed in any one of claims 44 to 46 wherein the binding agent is a protein or polypeptide
48. A method as claimed in claim 47 wherein the binding agent is an antibody or antibody fragment.
49. A method as claimed in claim 48 wherein the antibody or antibody fragment is specific for a human KLF.
50. A method as claimed in claim 49 wherein the antibody is raised against a human KLF.
51. A method as claimed in any one of claims 44 to 46 wherein the binding agent is a polynucleotide.
52. A method as claimed in claim 51 comprising the further step of amplifying a KLF polynucleotide in a sample and detecting the amplified polynucleotide.
53. A method as claimed in claim 52 wherein the ampHfication is by PCR.
54. A method as claimed in claim 53 wherein the ampHfication is by real-time PCR.
55. A method of detecting, measuring or monitoring Notch signalling in an immune ceh by determining the amount of a KLF protein, polypeptide or polynucleotide or a polynucleotide coding for such a protein or polypeptide in the cell.
56. A method as claimed in claim 55 wherein the immune cell is a T-cell.
57. A method as claimed in claim 55 wherein the immune cell is a B-cell.
58. A method as claimed in claim 55 wherein the immune cell is an antigen presenting cell.
59. A method for detecting , measuring or monitoring immunological tolerance or activity comprising determining the amount of a KLF protein, polypeptide or polynucleotide or a polynucleotide coding for such a protein or polypeptide.
60. A method for detecting, measuring or monitoring T-ceU activity comprising determining the amount of a KLF protein, polypeptide or polynucleotide or a polynucleotide coding for such a protein or polypeptide.
61. Amethod for detecting, measuring or monitoring the reactivity of a T-ceU to an antigen comprising detennining the amount of a KLF protein, polypeptide or polynucleotide or a polynucleotide coding for such a protein or polypeptide.
62. A method according to any one of claims 35 to 61 further comprising a step of comparing the amount of a KLF protein, polypeptide or polynucleotide or a polynucleotide coding for such a protein or polypeptide with a reference amount.
63. A method according to any one of claims 35 to 61 wherein the amount of KLF protein, polypeptide or polynucleotide or a polynucleotide coding for such a protein or polypeptide is detected using a nucleic acid assay.
64. A method according to any one of claims 35 to 61 wherein the amount of KLF protein, polypeptide or polynucleotide or a polynucleotide coding for such a protein or polypeptide is detected using a protein assay.
65. A diagnostic kit comprising a binding agent that binds to a KLF protein, polypeptide or polynucleotide for detecting, measuring or monitoring Notch signalling.
66. Use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of an immune response in peripheral immune cells.
67. Use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of an immune response to a selected antigen or antigenic determinant in peripheral immune cells.
68. Use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufactare of a medicament for modulation of peripheral lymphocyte activity.
69. Use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of peripheral T-ceU activity.
70. Use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of effector T-cell activity.
71. Use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of helper (Th) T-ceh activity.
72. Use of an inhibitor of KLF activity for the manufacture of a medicament for increasing helper (Th) T-cell activity.
73. Use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity for the manufacture of a medicament for decreasing helper (Th) T-cell activity.
74. Use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of cytotoxic (Tc) T-ceh activity.
75. Use of an inhibitor of KLF activity for the manufacture of a medicament for increasing cytotoxic (Tc) T-ceU activity.
76. Use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity for the manufacture of a medicament for decreasing cytotoxic (Tc) T-ceU activity.
77. Use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of regulatory (T reg) T-ceU activity.
78. Use of an inhibitor of KLF activity for the manufacture of a medicament for inhibition of regulatory (T reg) T-cell activity.
79. Use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity for the manufactare of a medicament for enhancement of regulatory (T reg) T- ceU activity.
80. Use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufacture of a medicament for modulation of Tri regulatory T-ceh activity.
81. Use of an inhibitor of KLF activity for the manufacture of a medicament for inhibition of Tri regulatory T-cell activity.
82. Use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity for the manufacture of a medicament for enhancing Tri regulatory T-ceU activity.
83. Use of a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity for the manufactare of a medicament for modulation of Th3 regulatory T-ceh activity.
84. Use of an inhibitor of KLF activity for the manufacture of a medicament for inhibition of Th3 regulatory T-cell activity.
85. Use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity for the manufacture of a medicament for enhancing Th3 regulatory T-ceU activity.
86. Use of a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity in the manufacture of a medicament for treatment of inflammation or an inflammatory condition.
87. Use of a combination of: a KLF protein, polypeptide or polynucleotide or an enhancer or inhibitor of KLF activity; and an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant; n the manufacture of a medicament for modulation of the immune system.
88. Use of a KLF protein, polypeptide or polynucleotide or an enhancer or inhibitor of KLF activity in the manufacture of a medicament for modulation of the immune system in simultaneous, contemporaneous, separate or sequential combination with an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant.
89. A use as claimed in any one of claims 66 to 88 wherein the modulator of KLF activity is administered to a patient in vivo.
90. A use as claimed in any one of claims 66 to 88 wherein the modulator of KLF activity is administered to a ceh ex-vivo.
91. A use as claimed in any one of claims 66 to 90 for treatment of aUergy, graft rejection, graft-versus-host disease, cancer or infectious disease.
92. A use as claimed in any one of claims 66 to 91 wherein the modulator of KLF activity is selected from polypeptides and fragments thereof, linear peptides, cyclic peptides, and nucleic acids which encode therefor, synthetic and natural compounds including low molecular weight organic or inorganic compounds and antibodies.
93. A method of immunotherapy in the peripheral immune system comprising administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
94. A method of immunotherapy comprising administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity in combination with a modulator of the Notch signalling pathway.
95. A method as claimed in claim 94 wherein the modulator is an agonist of KLF, in combination with an agent capable of activating Notch signalling.
96. A method as claimed in claim 94 wherein the modulator is an antagonist of KLF, in combination with an agent capable of inhibiting Notch signalling.
97. A method for modulating an immune response in the peripheral immune system by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
98. A method for modulating an immune response to a selected antigen or antigenic determinant by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
99. A method for modulating peripheral lymphocyte activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
100. A method for modulating peripheral T-cell activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
101. A method for modulating effector T-cell activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
102. A method for modulating helper (Th) T-ceU activity by adnhnistering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
103. A method for increasing helper (Th) T-cell activity by administering an inhibitor of KLF activity.
104. A method for decreasing helper (Th) T-ceU activity by administering a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity.
105. A method for modulating cytotoxic (Tc) T-ceU activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
106. A method for increasing cytotoxic (Tc) T-ceU activity by administering an inhibitor of KLF activity.
107. A method for decreasing cytotoxic (Tc) T-cell activity by administering an a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity.
108. A method for modulating regulatory (T reg) T-cell activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
109. A method for decreasing regulatory (T reg) T-ceU activity by administering an inhibitor of KLF activity.
110. Amethod for increasing regulatory (T reg) T-cell activity by administering a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity.
111. A method for modulating Tri regulatory T-ceU activity by administering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
112. A method for inhibiting Tri regulatory T-ceU activity by administering an inhibitor of KLF activity.
113. A method for increasing Tri regulatory T-cell activity by administering a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity.
114. A method for modulating Th3 regulatory T-ceU activity by adnhnistering a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
115. A method for inhibiting Th3 regulatory T-ceU activity by administering an inhibitor of KLF activity.
116. A method for increasing Th3 regulatory T-cell activity by administering a KLF protein, polypeptide or polynucleotide or an enhancer of KLF activity.
117. A method for modulating the immune system by simultaneously, contemporaneously, separately or sequentially administering a combination of: a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; and an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant.
118. A method as claimed in any one of claims 93 to 117 wherein the KLF protein, polypeptide or polynucleotide or modulator of KLF activity is administered to a patient in vivo.
119. A method as claimed in any one of claims 93 to 117 wherein the KLF protein, polypeptide or polynucleotide or modulator of KLF activity is administered to a ceh ex- vivo.
120. A method as claimed in any one of claims 93 to 119 for the modification of peripheral T-ceh activity.
121. A method as claimed any one of claims 93 to 120 for the treatment of autoimmune disease.
122. A method as claimed any one of claims 93 to 120 for the treatment of ahergy, graft rejection or GvHD.
123. A method as claimed any one of claims 93 to 120 for the treatment of cancer.
124. A method as claimed in claim 123 for enhancing the immune response to cancer.
125. A method as claimed any one of claims 93 to 120 for enhancing the immune response to a pathogen.
126. A modulator of KLF activity for use in affecting linked suppression.
127. A modulator of KLF activity for use in affecting infectious tolerance.
128. A product comprising a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity in combination with a modulator of the Notch signalling pathway.
129. A product comprising an inhibitor of KLF activity in combination with an inhibitor of the Notch signalling pathway.
130. A product comprising a KLF protein, polypeptide or polynucleotide or an activator of KLF activity in combination with an activator of the Notch signalling pathway.
131. A method for producing a lymphocyte or antigen presenting ceU (APC) for promoting tolerance to an aUergen or antigen which method comprises incubating a lymphocyte or APC obtained from a human or animal patient with (i) a KLF protein, polypeptide or polynucleotide or an agonist of KLF and (ii) the aUergen or antigen.
132. A method as claimed in claim 131 for producing an APC capable of inducing T ceU tolerance.
133. A method according to claim 131 or claim 132 for producing ex vivo a T ceU having tolerance to an allergen or antigen which method comprises incubating a T ceU obtained from a human or animal patient with an antigen presenting ceU (APC) in the presence of (i) a KLF protein, polypeptide or polynucleotide or an agonist of KLF activity and (h) the aUergen or antigen.
134. A method for producing a lymphocyte or APC for promoting tolerance to an aUergen or antigen which method comprises incubating a lymphocyte or APC obtained from a human or animal patient with a lymphocyte or APC produced by the method of any one of claims 131 to 133.
135. A method of vaccinating a patient against a tumour which method comprises: administering a tumour antigen or antigenic determinant to a patient; and exposing APCs present in the patient to a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
136. An assay method for modulators of KLF activity comprising contacting a KLF protein, polypeptide or polynucleotide, in the presence of Notch and a modulator of the Notch signalling pathway, with a candidate compound and determining if the compound affects the Notch signaUing pathway.
137. An assay method for identifying substances that modulate the activity of a KLF protein comprising: providing a preparation containing: a KLF protein, polypeptide or polynucleotide and a candidate substance; and detecting whether said candidate substance affects activity of the KLF protein, polypeptide or polynucleotide.
138. An assay method according to claim 137 wherein the KLF-interacting protein is Notch or a member of the Notch signalling pathway.
139. An assay method according to any one of claims 136 to 138 wherein the assay is conducted using an immune cell.
140. A kit comprising in one or more containers (a) a modulator of the Notch signaUing pathway and (b) a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity.
141. A product comprising: a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; and an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
142. A pharmaceutical composition comprising: a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; and an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
143. A pharmaceutical composition comprising: a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant; and a pharmaceutically acceptable carrier.
144. A composition as claimed in claim 142 or 143 for increasing effector T ceU activity.
145. A pharmaceutical kit comprising a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity and an antigen or antigemc determinant or a polynucleotide coding for an antigen or antigenic determinant.
146. The use of an inhibitor of KLF activity in the manufacture of a medicament for use as an immunostimulant.
147. The use of an inhibitor of KLF activity in the manufactare of a medicament for use in vaccination against a pathogen.
148. The use of an inhibitor of KLF activity in the manufactare of a medicament for use in vaccination against a tumour or pathogen.
149. The use of an inhibitor of KLF activity in the manufactare of a medicament for increasing the immune response against a tumour or pathogen antigen or antigenic determinant.
150. A method for stimulating the peripheral immune system by administering an inhibitor of KLF activity.
151. A method for vaccinating a subject against a tumour or pathogen by administering an inhibitor of KLF activity.
152. A method for increasing an immune response of a subj ect against a tumour or pathogen by administering an inhibitor of KLF activity.
153. A method for increasing the immune response of a subject to a tumour or pathogen antigen or antigenic determinant comprising administering an effective amount of an inhibitor of KLF activity simultaneously, contemporaneously, separately or sequentially with said tumour or pathogen antigen or antigenic determinant or simultaneously, contemporaneously, separately or sequentially with a polynucleotide coding for said tumour or pathogen antigen or antigenic deteπninant.
154. An adjuvant composition comprising an inhibitor of KLF activity.
155. A vaccine composition comprising an adjuvant composition as claimed in claim 154 and a tumour or pathogen antigen or antigenic deteπninant or a polynucleotide coding for a tumour or pathogen antigen or antigenic determinant.
156. A vaccine composition as claimed in claim 155 comprising a pathogen antigen or antigenic determinant in the form of a viral, fungal, parasitic or bacterial antigen or antigenic determinant or a polynucleotide coding for a vhal, fungal, parasitic or bacterial antigen or antigenic determinant.
157. A product comprising: a KLF protein, polypeptide or polynucleotide or a modulator of KLF activity; and a tumour or pathogen antigen or antigenic determinant or a polynucleotide coding for a tumour or pathogen antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
158. A product as claimed in claim 157 for increasing effector T cell activity in respect of the tumour or pathogen antigen or antigenic determinant.
PCT/GB2004/001379 2003-04-01 2004-03-29 Therapeutic use of modulators of notch and/or kruppel-like factors WO2004087195A2 (en)

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WO2022253794A3 (en) * 2021-06-02 2023-01-12 Cellestia Biotech Ag Compounds for use in a method for treating an autoimmune and inflammatory disease
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