WO2004073732A1 - Modulators of notch signalling and of immune cell costimulatory activity for immunotherapy - Google Patents

Modulators of notch signalling and of immune cell costimulatory activity for immunotherapy Download PDF

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Publication number
WO2004073732A1
WO2004073732A1 PCT/GB2004/000668 GB2004000668W WO2004073732A1 WO 2004073732 A1 WO2004073732 A1 WO 2004073732A1 GB 2004000668 W GB2004000668 W GB 2004000668W WO 2004073732 A1 WO2004073732 A1 WO 2004073732A1
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WIPO (PCT)
Prior art keywords
notch
modulator
notch signalling
immune
protein
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PCT/GB2004/000668
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French (fr)
Inventor
Brian Robert Champion
Maria Lioumi
Grahame James Mckenzie
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Lorantis Limited
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Publication of WO2004073732A1 publication Critical patent/WO2004073732A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates inter alia to monitoring, detecting or measuring Notch signalling and to modulation of immune responses for therapeutic purposes.
  • 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.
  • T-cell mediated diseases include allergy, auto immunity, graft rejection, 5 tumour induced aberrations to the T-cell system and infectious diseases caused, for example, by Plasmodium species, Mcrofilariae, Helminths, Mycobacteria, HIV, Cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, Haemophilus influenza type B, measles, Hepatitis C or Toxicara, may be targeted.
  • regulatory T cells which are able to transmit antigen-specific tolerance to other T cells, a process termed infectious tolerance (W098/20142).
  • infectious tolerance W098/20142
  • the functional activity of these cells can be mimicked by over- expression of a Notch ligand protein on their cell surfaces or on the surface of antigen presenting cells.
  • regulatory T cells can be generated by over-expression of a 5 member of the Delta or Serrate family of Notch ligand proteins.
  • PCT/GB00/O4391 (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/GB02/05137 (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
  • a method for detecting, measuring or monitoring Notch signalling comprising determining the amount of an immune cell (preferably T-cell) costimulatory protein or polypeptide or determining the amount of a polynucleotide coding for such a protein or polypeptide.
  • an immune cell preferably T-cell
  • the amount of the costimulatory protein, polypeptide or polynucleotide in a biological sample taken from a subject is determined.
  • a biological sample may, for example comprise blood, serum, urine, lymphatic fluid, or tissue.
  • the sample may comprise an immune cell.
  • the sample comprises peripheral T-cells.
  • the sample may comprise a cancer or tumour cell or a stem cell.
  • 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 an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide.
  • a binding agent that binds to an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide.
  • 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 an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide; and iii) detecting in the sample an amount of costimulatory protein, polypeptide or polynucleotide that binds to the binding agent.
  • a binding agent that binds to an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide
  • detecting in the sample an amount of costimulatory protein, polypeptide or polynucleotide that binds to the binding agent.
  • 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 an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide; iii) detecting in the sample an amount of costimulatory protein, polypeptide or polynucleotide that binds to the binding agent; and iv) comparing the amount of costimulatory protein, polypeptide or polynucleotide to a reference value and therefrom determining the degree of
  • a binding agent that binds to an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide
  • detecting in the sample an amount of costimulatory protein, polypeptide or polynucleotide that binds to the binding agent
  • iv) comparing the amount of costimulatory protein, polypeptide or polynucleotide to a reference value and therefrom
  • the binding agent may be a protein or polypeptide.
  • the binding agent may be an antibody or antibody fragment which binds to a human immune cell/ T- cell costimulatory molecule such as CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L.
  • the binding agent may comprise a binding domain from an immune cell, preferably a T-cell costimulatory molecule such as CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD- 1, PD-L1, PD-L2, OX40 or OX40L.
  • the binding agent may be a polynucleotide, for example a polynucleotide "probe".
  • a polynucleotide may be a sequence of at least 10, preferably at least 20 nucleotide residues which hybridises to a nucleotide sequence of human T-cell costimulatory molecule mRNA.
  • a polynucleotide hybridises to a nucleotide sequence of human T-cell costimulatory polynucleotide (eg mRNA) under stringent hybridisation conditions as described herein.
  • a probe may be labelled as described infra.
  • the method may comprise the further step of amplifying an immune cell, (preferably T-cell) costimulatory polynucleotide in a sample and detecting the amplified polynucleotide.
  • an immune cell preferably T-cell
  • the amplification may be by PCR, for example by real-time PCR.
  • an immune cell preferably T-cell
  • the immune cell may be a T-cell, a B-cell or an antigen presenting cell (APC).
  • the method further comprises a step of comparing the amount of costimulatory protein or polypeptide or a polynucleotide coding for such a protein or polypeptide with a reference amount.
  • a diagnostic kit for monitoring or detecting Notch signalling comprising a binding agent that binds to an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide for detecting, measuring or monitoring Notch signalling.
  • an immune cell preferably T-cell
  • a method of modulating the immune system comprising administering an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide or a modulator of immune cell (preferably T-cell) costimulatory activity in combination with a modulator of the Notch signalling pathway.
  • an immune cell preferably T-cell
  • a modulator of immune cell preferably T-cell
  • an agonist of immune cell costimulation in combination with an agent capable of inhibiting Notch signalling.
  • an antagonist of immune cell costimulation in combination with an agent capable of activating Notch signalling.
  • a product comprising an immune cell costimulatory protein, polypeptide or polynucleotide or a modulator of immune cell costimulatory activity in combination with a modulator of the Notch signalling pathway.
  • a product comprising an inhibitor of immune cell costimulatory activity in combination with an inhibitor of the Notch signalling pathway.
  • a product comprising an immune cell costimulatory protein, polypeptide or polynucleotide or an activator of immune cell costimulatory activity in combination with an activator of the Notch signalling pathway.
  • kits comprising in one or more containers (a) a modulator of the Notch signalling pathway and (b) an immune cell costimulatory protein, polypeptide or polynucleotide or a modulator of immune cell costimulatory activity.
  • a method for increasing immune cell costimulation by administering a modulator of Notch signalling According to a further aspect of the invention there is provided a method for decreasing immune cell (preferably T-cell) costimulation by administering a modulator of Notch signalling.
  • T-cell costimulation is mediated by CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L.
  • a method for increasing expression of an immune cell costimulatory protein, polypeptide or polynucleotide by administering a modulator of Notch signalling.
  • a method for decreasing expression of an immune cell costimulatory protein, polypeptide or polynucleotide by administering a modulator of Notch signalling.
  • the costimulatory protein, polypeptide or polynucleotide is CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L or a polynucleotide coding for CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L.
  • a modulator of Notch signalling to increase immune cell (preferably T-cell) costimulation.
  • a modulator of Notch signalling to decrease immune cell (preferably T-cell) costimulation.
  • a modulator of Notch signalling to increase expression of an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide.
  • a modulator of Notch signalling to decrease expression of an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide.
  • the costimulatory protein, polypeptide or polynucleotide may be CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L or a polynucleotide coding therefor.
  • costimulatory protein, polypeptide or polynucleotide is modulated in activated T-cells (suitably stimulated by antigen presentation).
  • a product comprising: i) a modulator of the Notch signalling pathway; and ii) a modulator of immune cell (preferably T-cell) costimulation; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
  • Such a product may be used for modulation of immune cell activity, for example in the treatment of inflammation, asthma, allergy, graft rejection, graft-versus-host disease or autoimmune disease.
  • a method for modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering: i) an effective amount of a modulator of the Notch signalling pathway; and ii) an effective amount of a modulator of immune cell (preferably T-cell) costimulation.
  • a modulator of the Notch signalling pathway i) a modulator of the Notch signalling pathway; and ii) a modulator of immune cell (preferably T-cell) costimulation; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway ii) a modulator of immune cell (preferably T-cell) costimulation; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a modulator of immune cell (preferably T-cell) costimulation.
  • a modulator of the Notch signalling pathway i) a modulator of the Notch signalling pathway; and ii) a modulator of immune cell (preferably T-cell) costimulation; in the manufacture of a medicament for modulation of the immune system.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of the immune system in simultaneous, contemporaneous, separate or sequential combination with a modulator of immune cell (preferably T-cell) costimulation.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a modulator of immune cell (preferably T-cell) costimulation.
  • a pharmaceutical composition comprising a modulator of the Notch signalling pathway and a modulator of immune cell (preferably T-cell) costimulation and optionally a pharmaceutically acceptable carrier.
  • an activator of Notch signalling i) an activator of Notch signalling; and ii) an activator of negative immune costimulation; for simultaneous, contemporaneous, separate or sequential use in reducing an immune response.
  • an activator of Notch signalling ii) an inhibitor of positive immune costimulation; for simultaneous, contemporaneous, separate or sequential use in reducing an immune response.
  • an inhibitor of Notch signalling i) an inhibitor of Notch signalling; and ii) an inhibitor of negative immune costimulation; for simultaneous, contemporaneous, separate or sequential use in increasing an immune response.
  • an inhibitor of Notch signalling i) an inhibitor of Notch signalling; and ii) an activator of positive immune costimulation; for simultaneous, contemporaneous, separate or sequential use in increasing an immune response.
  • a method for reducing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; and ii) an activator of negative immune costimulation.
  • a method for reducing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; and ii) an inhibitor of positive immune costimulation.
  • a method for increasing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; and ii) an inhibitor of negative immune costimulation.
  • a method for increasing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; and ii) an activator of positive immune costimulation.
  • an activator of Notch signalling ii) an activator of negative immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in reducing an immune response to the antigen or antigenic determinant.
  • an activator of Notch signalling ii) an inhibitor of positive immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in reducing an immune response to the antigen or antigenic determinant.
  • an inhibitor of Notch signalling i) an inhibitor of negative immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in increasing an immune response to the antigen or antigenic dete ⁇ ninant.
  • an inhibitor of Notch signalling ii) an activator of positive immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in increasing an immune response to the antigen or antigenic determinant.
  • a method for reducing an immune response to an antigen or antigenic determinant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; ii) an activator of negative immune costimulation; and iii) the antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant)
  • a method for reducing an immune response to an antigen or antigenic determinant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; ii) an inhibitor of positive immune costimulation; and iii) the antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in reducing an immune response to the antigen or antigenic determinant
  • a method for increasing an immune response to an antigen or antigenic determinant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; ii) an inhibitor of negative immune costimulation; and iii) the antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant).
  • a method for increasing an immune response to an antigen or antigenic dete ⁇ ninant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; ii) an activator of positive immune costimulation; and iii) the antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant).
  • the modulator of Notch signalling is a Notch receptor agonist.
  • the modulator of Notch signalling may be an activator of a Notch receptor (especially a human Notchl, Notch2, Notch3 or Notch4 receptor).
  • the modulator activates Notch signalling in immune cells, espeacially T-cells, B-cells and/or APCs.
  • the modulator of the Notch signalling patliway comprises Delta or Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide coding for Delta, Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • the modulator of the Notch signalling pathway may comprise a fusion protein comprising a segment of ' a Notch ligand extracellular domain and an immunoglobulin F c segment or a polynucleotide coding for such a fusion protein.
  • the modulator of the Notch signalling pathway comprises a protein or polypeptide comprising a DSL domain or a polynucleotide sequence coding for such a protein.
  • the modulator of the Notch signalling pathway comprises a protein or polypeptide comprising a Notch ligand DSL domain and from 1 to 16 or more EGF-like domains or a polynucleotide sequence coding for such a protein or polypeptide.
  • the modulator of the Notch signalling pathway may comprise Notch intracellular domain (Notch IC) or a fragment, derivative, homologue, analogue or allelic variant thereof, or a polynucleotide sequence which codes for Notch intracellular domain or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • the modulator of the Notch signalling pathway comprises Delta (preferably human Delta) or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide encoding Delta or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • the modulator of the Notch signalling pathway may comprise Serrate/Jagged (preferably human Jagged) or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide encoding Serrate/Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • the modulator of the Notch signalling pathway may comprise Notch (preferably humna Notch) or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide encoding Notch or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • Notch preferably humna Notch
  • the modulator of the Notch signalling pathway may comprise a dominant negative version of a Notch signalling repressor, or a polynucleotide which codes for a dominant negative version of a Notch signalling repressor.
  • the modulator of the Notch signalling pathway may comprise a polypeptide capable of upregulating the expression or activity of a Notch ligand or a downstream component of the Notch signalling pathway, or a polynucleotide which codes for such a polypeptide.
  • the modulator of the Notch signalling pathway may comprise an antibody, antibody fragment or antibody derivative or a polynucleotide which codes for an antibody, antibody fragment or antibody derivative, suitably an anti-Notch antibody.
  • the modulator of Notch signalling may be administered in a multimerised form.
  • the modulator of Notch signalling may be bound to a membrane or support.
  • a plurality or multiplicity of modulators for example at least 5 will be bound to the membrane or support.
  • the support is a particulate support matrix.
  • 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 Sepharose bead.
  • 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 suitably not more than 5) 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.
  • a modulator of Notch signalling may comprise a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 2-4 or more (or suitably 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.
  • one or more of the modulators of Notch signalling may comprise a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 2-3 or more (or suitably 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.
  • 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:
  • the modulator of costimulation may be a modulator of CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L signalling.
  • the modulator of costimulation may be a soluble fusion protein comprising at least an active part of a CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L extracellular domain.
  • the modulator of costimulation may be CTLA-4-Ig, ICOS-Ig, CD40-Ig or OX40-Ig.
  • an antigen or antigenic determinant may also be administered simultaneously, separately or sequentially with the modulator of Notch signalling and/or modulator of immune cell costimulation.
  • a method for generating an immune modulatory cytokine profile with increased EL- 10 expression by administering a combination of active agents according to the present invention is provided.
  • a method for generating an immune modulatory cytokine profile with increased EL-10 and TL-4 expression and reduced JL-5, LL-13 and TNF ⁇ expression by administering a combination of active agents according to the present invention.
  • a method for generating an immune modulatory cytokine profile with increased JX-10 and IL-4 expression and reduced IL-2, IFN ⁇ , EL-5, EL-13 and TNF ⁇ expression by administering a combination of active agents according to the present invention.
  • 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 aligned amino acid sequences of DSL domains from various Drosophila and mammalian Notch ligands
  • Figure 4 shows amino acid sequences of human Delta-1, Delta-2 and Delta-3;
  • Figure 5 shows amino acid sequences of human Jagged- 1 and Jagged-2
  • Figure 6 shows schematic representations of modulators of Notch signalling which may be used in the present invention
  • Figure 7 shows the results of Example 1 ;
  • Figure 8 shoes the results of Example 2; and
  • Figure 9 shoes the results of Example 3.
  • Drosophila and vertebrate names are used interchangeably and all homologues are included within the scope of the invention.
  • immune cell costimulation comprises signalling through a pathway involving a molecule on the surface of an immune cell and a molecule on the surface of a T-cell, B-cell or antigen presenting cell (APC).
  • An interaction between these two molecules in the context for example of antigen specific recognition by the T cell of an antigen presented by the antigen presenting cell, either promotes immune cell activation (positive immune costimulation) or reduces it (negative immune costimulation).
  • immune cell costimulation thus includes both upregulation (positive costimulation) and downregulation (negative costimulation) of immune cell (especailly T-cell) activation (or also modulation of other immune cells such as B-cell or APC activation).
  • T-lymphocyte activation and immune function are regulated by various co-stimulatory molecules.
  • CD28 a receptor for B7 gene products, has a chief role in initiating T-cell immune responses (positive costimulation).
  • CTLA4 binds B7 with a higher affinity, is induced after T-cell activation and is involved in downregulating T-cell responses (negative costimulation).
  • the inducible co-stimulatory molecule (ICOS) a third member of the CD28/CTLA4 family, is expressed on activated T cells. Its ligand (B7H/B7RP-1) is expressed on B cells.
  • agents that inhibit B7 interactions include, but are not limited to, molecules such as an antibody (or fragment or derivative thereof) that recognizes and binds to a CTLA4, CD28 or B7 (e.g. B7-1, B7-2) molecule; a soluble form (or portion or derivative thereof) of an extracellular domain of a costimulatory molecule such as soluble CTLA4; or a peptide fragment or other small molecule designed to interfere with the cell signal through the CTLA4/CD28/B7-mediated interaction.
  • molecules such as an antibody (or fragment or derivative thereof) that recognizes and binds to a CTLA4, CD28 or B7 (e.g. B7-1, B7-2) molecule; a soluble form (or portion or derivative thereof) of an extracellular domain of a costimulatory molecule such as soluble CTLA4; or a peptide fragment or other small molecule designed to interfere with the cell signal through the CTLA4/CD28/B7-mediated interaction.
  • an inhibitory agent may be a soluble CTLA4 molecule, such as CTLA4Ig (ATCC 68629) or L104EA29YIg (ATCC PTA-2104), a soluble CD28 molecule such as CD28Ig (ATCC 68628), a soluble B7 molecule such as B7Ig (ATCC 68627), an anti-B7 monoclonal antibody (e.g. ATCC HB-253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB-301, ATCC HB-11341 and monoclonal antibodies as described in by Anderson et al in U.S. Pat. No. 6,113,898 or Yokochi et al., 1982. J.
  • an anti-CTLA4 monoclonal antibody e.g. ATCC HB-304, and monoclonal antibodies as described in references 82-873
  • an anti-CD28 monoclonal antibody e.g. ATCC HB 11944 and mAb 9.3 as described by Hansen (Hansen et al., 1980. hnmunogenetics 10: 247-260) or Martin (Martin et al., 1984. J. Clin. Immun., 4(1): 18-22)).
  • a further class of costimulatory receptors comprises certain members of the TNF receptor (TNFR) family. These include CD40, the major B cell costimulatory molecule, as well as OX-40, 4-1BB, CD30, and CD27.
  • the ligands for these receptors are membrane-bound members of the TNF family.
  • a further negative costimulator is the Programmed Death-1 (PD-1) receptor.
  • PD-1 and PD-L2 are ligands for PD-1.
  • immune cell costimulatory protein or polypeptide means a protein or polypeptide involved in immune cell (especially T-cell) costimulation. It includes, for example, fragments, homologues and allelic variants.
  • immunoreactive polynucleotide means a polynucleotide coding for an immune cell (especially T-cell) costimulatory protein or polypeptide.
  • T-cell costimulatory 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 human T-cell costimulatory protein or polypeptide 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.
  • T-cell costimulatory activity means the biological activity of a T-cell costimulatory protein or polypeptide, including but not limited to transcription activation activity.
  • T-cell costimulatory proteins/polypeptides include inducible T-cell co-stimulator (ICOS) and its ligand B7-H2 (ICOS ligand); CD28 and its ligands CD80 and CD86 and CTLA-4 and its ligands CD80 and CD86.
  • a sequence for a human inducible T-cell co-stimulator (ICOS), mRNA is provided under GenBank Accession No NM_012092.
  • a sequence for a human transmembrane protein B7-H2 (ICOS ligand) mRNA is provided under GenBank Accession No AF289028.
  • a sequence for a human cytotoxic T-lymphocyte-associated protein 4 (CTLA4) mRNA is provided under GenBank Accession No NM_005214.
  • a sequence for a human CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) mRNA is provided under GenBank Accession No NM_006889.
  • a sequence for a human CD28 antigen (Tp44) (CD28) mRNA is provided under GenBank Accession No NM_006139.
  • a sequence for a human CD80 antigen (CD28 antigen ligand 1, B7-1 antigen) mRNA is provided under GenBank Accession No NM_005191.
  • amino acid and polynucleotide sequences for human CTLA-4 are provided as follows (GenBank Accession No AY209009):
  • CTLA-4 lg Fusion protein BMS 188667
  • US Bristol Myers Squibb
  • CTLA4Ig a Chinese Hamster Ovary (CHO) cell line expressing CTLA4Ig was deposited on May 31, 1991 with ATCC identification number CRL-10762.
  • B7 refers to the B7 family of molecules including, but not limited to, B7-1 (CD80) (Freeman et al, 1989, J. Immunol. 143:2714-2722, herein incorporated by reference), B7-2 (CD86) (Freeman et al, 1993, Science 262:909-911 herein incorporated by reference; Azuma et al, 1993, Nature 366:76-79 herein incorporated by reference) that may recognize and bind CTLA4 and/or CD28.
  • a B7 molecule can be expressed on an activated B cell.
  • Amino acid and polynucleotide sequences for a human inducible T-cell co-stimulator (ICOS) are provided as follows (GenBank Accession No AJ277832): MKSGL YFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIV QFKMQLLKGGQILCDLTKTKGSG NTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKF LP IGCAA.FVWCILGCILIC LTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL
  • CD40L human CD40 Ligand
  • modulation of immune cell costimulation refers to a change or alteration in the biological activity of immune cell (especially T-cell) costimulation.
  • modulator may refer to antagonists or inhibitors of immune cell costimulation, i.e. compounds which block, at least to some extent, the normal biological activity of immune cell (especially T-cell) costimulatory proteins, polypeptides or polynucleotides. Conveniently such compounds may be referred to herein as inhibitors or antagonists.
  • the term “modulator” may refer to agonists of immune cell (especially T- cell) costimulation, i.e.
  • upregulators compounds which activate, stimulate or upregulate, at least to some extent, the normal biological activity of immune cell (especially T-cell) costimulatory proteins, polypeptides or polynucleotides.
  • immune cell especially T-cell
  • upregulators or agonists.
  • an activator of immune cell (especially T-cell) costimulation may comprise all or part of a costimulatory protein, polypeptide or polynucleotide as described above.
  • a costimulatory protein/polypeptide may be administered or a polynucleotide coding for such a protein/polypeptide may be administered using a genetic vector as described herein.
  • An inhibitor of immune cell (especially T-cell) costimulation may be any agent which reduces the activity of an immune cell (especially T-cell) costimulatory protein, polypeptide or polynucleotide.
  • an inhibitor of immune cell (especially T- cell) costimulatory activity may be an immune cell (especially T-cell) costimulatory antisense polynucleotide or an antibody which binds to an immune cell (especially T-cell) costimulatory protein to reduce its activity.
  • Modulators of immune cell (especially T-cell) costimulation include for example, antibodies against costimulatory proteins/polypetides and proteins/polypeptides (preferably in substantially soluble forms) comprising extracellular domains of costimulatory proteins/polypeptides, such as fusion proteins fused to IgG domains as well known in the art, for example CTLA-4/Ig, OX40/Ig and ICOS/Ig.
  • Modulators of costimulation are suitably antibodies specific for costimulatory receptors or their ligands (eg antibodies specific for CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L), or soluble ligands or receptors (eg soluble forms of CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L such as lg fusion proteins).
  • Antibodies are avilable, for example, from suppliers such as Abeam (Cambridge UK, www.abcam.com) and R& D Systems, Inc (MN, USA; rndsystems.com) and many other suppliers.
  • Activators of costimulation may suitably be agonist antibodies, whilst conversely inhibitors of costimulation may suitably be antagonist antibodies.
  • the CD28-B7 interaction may be inhibited by administering a soluble ligand or receptor or antibody for CD28 or B7, e.g., a soluble CTLA4, e.g., a CTLA4 fusion protein, e.g., a CTLA4 immunoglobulin fusion, e.g, CTLA4/Ig.
  • a soluble ligand or receptor or antibody for CD28 or B7 e.g., a soluble CTLA4, e.g., a CTLA4 fusion protein, e.g., a CTLA4 immunoglobulin fusion, e.g, CTLA4/Ig.
  • the inhibitor binds B7.
  • anti-B7-l and/or anti-B7-2 antibodies may be administered.
  • the CD40 ligand-CD40 interaction may be inhibited by administering an antibody or soluble ligand or receptor for the CD40 ligand or CD40, e.g., by administering an anti-CD40L antibody, e.g., 5c8 or an antibody with similar efficacy or an antibody whose epitope overlaps that of 5c8, (see U.S. Pat No. 5,474,711, hereby incorporated by reference).
  • an antibody or soluble ligand or receptor for the CD40 ligand or CD40 e.g., by administering an anti-CD40L antibody, e.g., 5c8 or an antibody with similar efficacy or an antibody whose epitope overlaps that of 5c8, (see U.S. Pat No. 5,474,711, hereby incorporated by reference).
  • modulators of immune costimulation include, for example: soluble gp39 (also known as CD40 ligand (CD40L), CD154, T-BAM, TRAP), soluble CD40, soluble CD80 (e.g. ATCC 68627), soluble CD86, soluble CD28 (e.g. ATCC 68628), antibodies reactive with CD40L (e.g. ATCC HB-10916, ATCC HB-12055 and ATCC HB-12056), antibodies reactive with CD40 (e.g. ATCC HB-9110), antibodies reactive with B7 (e.g.
  • the modulator of immune costimulation may be a soluble CTLA-4 protein or polypeptide, suitably comprising at least an active fragment of a CTLA-4 extracellular domain, suitably fused to a heterologous amino acid sequence such as an IgFc domain, or a polynucleotide coding for any of the foregoing, or an antibody, preferably a monclonal antibody, specific for a CLTA-4 protein.
  • the modulator of immune costimulation may be a soluble ICOS or ICOS Ligand protein or polypeptide, suitably comprising at least an active fragment of an ICOS or ICOS Ligand extracellular domain, suitably fused to a heterologous amino acid sequence such as an IgFc domain, or a polynucleotide coding for any of the foregoing, or an antibody, preferably a monclonal antibody, specific for an ICOS or ICOS Ligand protein.
  • the modulator of immune costimulation may be a soluble CD40 or CD40 Ligand protein or polypeptide, suitably comprising at least an active fragment of a CD40 or CD40 Ligand extracellular domain, suitably fused to a heterologous amino acid sequence such as an IgFc domain, or a polynucleotide coding for any of the foregoing, or an antibody, preferably a monclonal antibody, specific for a CD40 or CD40 Ligand protein.
  • the modulator of immune costimulation may be a soluble OX40 or OX40 Ligand protein or polypeptide, suitably comprising at least an active fragment of a OX40 or OX40 Ligand extracellular domain, suitably fused to a heterologous amino acid sequence such as an IgFc domain, or a polynucleotide coding for any of the foregoing, or an antibody, preferably a monclonal antibody, specific for a OX40 or OX40 Ligand protein.
  • the modulator of immune costimulation may be a soluble CD28 or B7 protein or polypeptide, suitably comprising at least an active fragment of a CD28 or B7 extracellular domain, suitably fused to a heterologous amino acid sequence such as an IgFc domain, or a polynucleotide coding for any of the foregoing, or an antibody, preferably a monclonal antibody, specific for a CD28 or B7 protein.
  • Notch signalling may be a soluble CD28 or B7 protein or polypeptide, suitably comprising at least an active fragment of a CD28 or B7 extracellular domain, suitably fused to a heterologous amino acid sequence such as an IgFc domain, or a polynucleotide coding for any of the foregoing, or an antibody, preferably a monclonal antibody, specific for a CD28 or B7 protein.
  • 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.
  • Notch signalling we refer to any event directly upstream or downstream of Notch receptor activation or inhibition including activation or inhibition of Notch/Notch ligand interactions, upregulation or downregulation of Notch or Notch ligand 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.
  • 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.
  • 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.
  • a modulator of Notch signalling may be a protein or polypeptide for Notch signal activation, transduction, or inhibition, or a polynucleotide coding for any of the foregoing.
  • Notch signalling includes signalling events taking place extracellularly or at the cell membrane. In a further aspect, it includes signalling events taking place intracellularly, for example within the cell cytoplasm or within the cell nucleus.
  • an active agent/modulator used in the present invention may be an organic compound or other chemical.
  • a modulator may be an organic compound comprising two or more hydrocarbyl groups.
  • hydrocarbyl group means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc.
  • substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc.
  • a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other.
  • the carbons may be linked via a suitable element or group.
  • 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 cyclic group.
  • the cyclic group may be a polycyclic group, such as a non-fused polycyclic group.
  • the agent comprises at least the one of said cyclic groups linked to another hydrocarbyl group.
  • the modulator will be an amino acid sequence or a chemical derivative thereof, or a combination thereof.
  • 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.
  • antibody includes intact molecules as well as fragments tliereof, such as Fab, F(ab')2, Fv and scFv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and include, for example:
  • Fab 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 light chain and a portion of one heavy chain;
  • 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;
  • 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;
  • scFv including a genetically engineered fragment containing the variable region of a heavy and a light chain as a fused single chain molecule.
  • WO 0020576 discloses generation of antibodies against the human Notch-1 EGF-like repeats 11 and 12.
  • WO 0020576 discloses a monoclonal antibody secreted by a hybridoma designated A6 having the ATCC Accession No.
  • HB 12654 a monoclonal antibody secreted by a hybridoma designated Cll having the ATCC Accession No. HB12656 and a monoclonal antibody secreted by a hybridoma designated F3 having the ATCC Accession No. HB12655.
  • An anti-human- Jagged 1 antibody is available from R & D Systems, Inc, reference MAB12771 (Clone 188323).
  • an activator of Notch signalling may 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.
  • Modulators may be synthetic compounds or natural isolated compounds.
  • analogue of immune cell costimulation includes variants thereof which retain the activity of T-cell costimulatory proteins, polypeptides or polynucleotides.
  • analogue we include a protein which has immune cell costimulatory activity, but generally has a different evolutionary origin toT-cell costimulatory 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 immune cell costimulatory 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 solid support, borne on a cell surface, or located intracellularly.
  • Techniques for drug screening may be based, for example, on the method described in Geysen, European Patent No. 0138855, published on September 13, 1984.
  • large numbers of different small peptide candidate modulators or targeting molecules are synthesized on a solid 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. Cells can also be spread as "lawns".
  • non-neutralising 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 drug screening assays in which neutralising antibodies capable of binding a target specifically compete with a test compound for binding to a target.
  • a homologue of a known immune cell costimulatory protein or polynucleotide will be at least 20%, preferably at least 30%), identical at the amino acid level to the corresponding known costimulatory 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 preferably 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.nlm.nih.gov and Ausubel etal., 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 libraries with probes comprising all or part of a nucleic acid encoding a T-cell costimulatory 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).
  • medium to high stringency for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C.
  • 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.
  • nucleic acid constructs encoding the polypeptides may be used.
  • overexpression of T-cell costimulatory may be brought about by introduction of a nucleic acid construct capable of activating the endogenousT-cell costimulatory gene.
  • 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
  • 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 ligases) or glycosyltransferases.
  • the signalling may involve changes in expression, nature, amount or activity of DNA binding elements such as transcription factors.
  • Notch signalling preferably means specific signalling, meaning that the signalling results substantially or at least predominantly from the Notch signalling pathway, and preferably from Notch/Notch ligand interaction, rather than any other significant interfering or competing cause, such as cytokine signalling.
  • Notch signalling excludes cytokine signalling.
  • E[spl] Enhancer of split complex
  • these genes have been shown to be direct targets for binding by the Su(H) protein and to be transcriptionally activated in response to Notch signalling.
  • 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 o ⁇ E(spI) as well as other target genes.
  • Notch ligand 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 corresponding biological effects to the natural ligands.
  • 6121045 (Millennium), 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 (Genbank Accession No. AF029778 -Homo sapiens), and LAG-2. Homology between family members is extensive.
  • 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 ligands, 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 extracellular domain.
  • the proteolytic cleavage step of Notch to activate the receptor occurs in the Golgi apparatus and is mediated by a furin-like convertase.
  • EGF epidermal growth factor
  • L/N 3 Cysteine Rich Repeats
  • the cytoplasmic domain of Notch contains six ankyrin-like repeats, a polyglutamine stretch (OP A) and a PEST sequence.
  • a further domain termed RAM23 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 extracellular ligands, such as Delta, Serrate and Scabrous, to the EGF-like repeats of Notch's extracellular domain. Delta requires cleavage for activation. It is cleaved by the ADAM disintegrin metalloprotease
  • TAN-1 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.
  • Su(H) is t e Drosophila homologue of C-promoter binding factor-1 [CBF-1], a mammalian DNA binding protein involved in the Epstein-Barr virus-induced immortalization of B-cells. 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 ligand Delta on adjacent cells. 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.
  • NotchIC The intracellular 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 cdcl 0/ankyrin repeats of the Notch intracellular domain reach the nucleus and participate in transcriptional activation.
  • the site of proteolytic cleavage on the intracellular tail of Notch has been identified between gly 1743 and vail 744 (termed site 3, or S3) (Schroeter, E.H. et al. (1998) Nature 393j(6683 ⁇ :382-6 (Schroeter)). It is thought that the proteolytic cleavage step that releases the cdcl 0/ankyrin repeats for nuclear entry is dependent on Presenilin activity.
  • the intracellular 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) Cell 93 4):649-60 (Struhl)).
  • CSL family protein CBF1 suppressor of hairless, Su(H) in Drosophila, Lag-2 in C. elegans
  • the NotchlC-CBFl complexes then activate target genes, such as the bHLH proteins HES (hairy-enhancer of split like) 1 and 5 (Weinmaster G.
  • 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)).
  • Notch signalling pathway Other downstream components of the Notch signalling 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 tliereof, 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.
  • Notch IC proteolytic cleavage of the intracellular domain of Notch
  • CBF1 activator of Hairless, Su(H) in Drosophila, Lag-2 in C. elegans
  • NotchlC-CBFl complexes then activate target genes, such as the bHLH proteins HES (hairy-enhancer 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.
  • Target genes of the Notch signalling pathway include Deltex, genes of the Hes family (Hes-1 in particular), Enhancer of Split [E(spl)] complex genes, EL-10, CD-23, CD-4 and Dll-1.
  • Deltex an intracellular docking protein, replaces Su(H) as it leaves its site of interaction with the intracellular tail 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. Studies indicate that Deltex promotes Notch pathway activation by interacting with Grb2 and modulating the Ras-JNK signalling pathway (Matsuno etal.
  • Deltex also acts as a docking protein which prevents Su(H) from binding to the intracellular tail of Notch (Matsuno). Thus, Su(H) is released into the nucleus where it acts as a transcriptional modulator.
  • Notch the intracellular tail of Notch
  • DTX1 Homo sapiens Deltex
  • Hes-1 (Hairy-enhancer of Split-1) (Takebayashi K. etal. (1994) J Biol Chem 269(7): 150-6 (Takebayashi)) is a transcriptional factor with a basic helix-loop-helix 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-cell fate.
  • Other genes from the Hes family include Hes-5 (mammalian Enhancer of Split 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. D 16464.
  • 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 (EL-10) was first characterised in the mouse as a factor produced by Th2 cells which was able to suppress cytokine production by Thl cells. It was then shown that EL-10 was produced by many other cell types including macrophages, keratinocytes, B cells, ThO and Thl cells. It shows extensive homology with the Epstein-Barr bcrfl gene which is now designated viral EL-10. Although a few immunostimulatory effects have been reported, it is mainly considered as an immunosuppressive cytokine. Inhibition of T cell responses by IL-10 is mainly mediated through a reduction of accessory functions of antigen presenting cells.
  • 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. JJL-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 cells 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-cell 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.
  • FCE2 human leukocyte differentiation antigen CD23
  • the sequence for CD-23 may be found in GenBank ref. No. Gil 783344.
  • CTLA4 cytotoxic T-lymphocyte activated protein 4
  • CTLA4 is an accessory molecule found on the surface of T-cells which is thought to play a role in the regulation of airway inflammatory cell recruitment and T-helper cell 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 distalless-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 downregulated as a result of Notch activation.
  • a sequence for the CD-4 antigen may be found in GenBank Accession No. XM006966.
  • Notch receptor family participates in cell-cell signalling events that influence T cell fate decisions.
  • NotchIC localises to the nucleus and functions as an activated receptor.
  • Mammalian 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 & Cell Biology 16(3):952-959) suggests rather that the N-terminal 114 amino acid region of mouse NotchIC contains the CBFl interactive domain.
  • NotchIC acts by targeting DNA-bound CBFl within the nucleus and abolishing CBFl -mediated repression through masking of the repression domain.
  • Epstein Barr virus (EBV) immortalizing protein EBNA also utilises CBFl tethering and masking of repression to upregulate expression of CBFl -repressed B-cell genes.
  • EBV Epstein Barr virus
  • Strobl et al Strobl et al. (2000) J Virol 74 ⁇ 4): 1727-35
  • EBNA2 may hence be regarded as a functional equivalent of an activated Notch receptor.
  • Other EBV proteins which fall in this category include BARFO (Kusano and Raab-Truab (2001) J Virol 75(11:384-395 (Kusano and Raab-Traub)) and LMP2A.
  • a modulator of Notch signalling may comprise a protein/polypeptide comprising all or part of a Notch ligand or a polynucleotide coding for such a protein/polypeptide.
  • Examples of mammalian Notch ligands identified to date include the Delta family, for example Delta-1 (Genbank Accession No. AF003522 - Homo sapiens), Delta-3 (Genbank Accession No. AF084576 - Rattus noi egicus) and Delta-like 3 (Mus uscuhis), 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 ligands 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-like repeats on the extracellular surface. It is therefore preferred that homologues of Notch ligands also comprise a DSL domain at the N-terminus and up to 14 or more EGF- like repeats on the extracellular surface.
  • DSL domain D. Delta, S. Serrate, L. Lag2
  • homologues of Notch ligands also comprise a DSL domain at the N-terminus and up to 14 or more EGF- like repeats on the extracellular surface.
  • the modulator may comprise all or part of the extracellular domain of a Notch receptor (eg Notch 1, Notch2, Notch3, Notch4 or homologues thereof), which can bind to Notch ligands and so reduce interactions with endogenous Notch receptors.
  • a modulator may comprise at least the 11th and 12th domains of Notchl (EGF11 and EGF 12), as these are believed to be important for Notch ligand interaction.
  • rat Notch-1/Fc fusion protein is available from R& D Systems Inc.
  • Notch ligands typically comprise a number of distinctive domains. Some predicted/potential domain locations for various naturally occurring human Notch ligands (based on amino acid numbering in the precursor proteins) are shown below:
  • a typical DSL domain may include most or all of the following consensus amino acid sequence:
  • 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 Xa NOP Xaaa 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.
  • ACM is an acid or amide amino acid residue such as aspartic acid, glutamic acid, asparagine or glutamine.
  • 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 Cys Xaa Xaa Gly Trp Xaa Gly Xaa Xaa Cys
  • Xaa may be any amino acid and Asx is either aspartic acid or asparagine).
  • the DSL domain used may be derived from any suitable species, including for example Drosophila, Xenopus, rat, mouse or human.
  • the DSL domain is derived from a vertebrate, preferably a mammalian, preferably a human Notch ligand sequence.
  • DSL domain includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturally occurring domains.
  • 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.
  • 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.
  • 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.
  • 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 3.
  • 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.
  • the EGF-like motif has been found in a variety of proteins, as well as EGF and Notch and Notch ligands, including those involved in the blood clotting cascade (Furie and Furie, 1988, Cell 53: 505-518).
  • this motif has been found in extracellular proteins such as the blood clotting factors LX and X (Rees et al., 1988, EMBO J. 7:2053- 2061; Furie and Furie, 1988, Cell 53: 505-518), in other Drosophila genes (Knust et al., 1987 EMBO J.
  • 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 following schematic representation of a typical EGF-like domain:
  • I I I I I x (4) -C-x (0 , 48 ) -C-x (3 , 12 ) -C-x (l , 70) -C-x (l , 6) -C-x (2 ) -G-a-x ( 0 , 21 ) -G-x (2 ) -C-x I I
  • the region between the 5th and 6th cysteine contains two conserved glycines of which at least one is normally present in most EGF-like domains.
  • the EGF-like domain used may be derived from any suitable species, including for example Drosophila, Xenopus, rat, mouse or human.
  • the EGF-like domain is derived from a vertebrate, preferably a mammalian, preferably a human Notch ligand sequence.
  • EGF domain includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturally occurring domains.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • any particular amino acid sequence is at least X% identical to another sequence can be determined conventionally using known computer programs.
  • the best overall match between a query sequence and a subject sequence also referred to as a global sequence alignment
  • the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of the global sequence alignment is given as percent identity.
  • 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 will be appreciated that this term includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturally occurring domains.
  • a Notch ligand N-terminal 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 a Notch ligand N-terminal domain of human Jagged 1.
  • a Notch ligand N-terminal 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 a Notch ligand N-terminal domain of human Jagged 2.
  • a Notch ligand N-terminal 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 a Notch ligand N-terminal domain of human Delta 1.
  • a Notch ligand N-terminal 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 a Notch ligand N-terminal domain of human Delta 3.
  • a Notch ligand N-terminal 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 a Notch ligand N-terminal domain of human Delta 4.
  • heterologous amino acid sequence or “heterologous nucleotide sequence” as used herein means a sequence which is not found in the native sequence (eg in the case of a Notch ligand sequence is not found in the native Notch ligand sequence) or its coding sequence. Typically, for example, such a sequence may be an IgFc domain or a tag such as a V5His tag.
  • Whether a substance can be used for modulating Notch signalling or activating a Notch receptor may be determined using any suitable screening assay, for example, as described in our co-pending International Patent Application published as WO 03/012441, as described in the Examples herein, or in Varnum-Finney et al, Journal of Cell Science 113, 4313-4318 (2000).
  • Activation of Notch signalling may also be achieved by repressing inhibitors of the Notch signalling pathway.
  • polypeptides for Notch signalling activation will include molecules capable of repressing any Notch signalling inhibitors.
  • the molecule will 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.
  • the molecules will 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
  • BMP bone morphogenetic protein
  • BMP receptors BMP receptors and activins, derivatives, fragments, variants and homologues thereof
  • Suitable nucleic acid sequences may include for example, anti-sense constructs, for example nucleic acid sequences encoding antisense Notch ligand constructs as well as antisense constructs designed to reduce or inhibit the expression of upregulators of Notch ligand expression (see above).
  • the antisense nucleic acid may be an oligonucleotide such as a synthetic single-stranded DNA.
  • the antisense may be 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 cell.
  • the nucleic acid sequence for use in the present invention is capable of inhibiting Serrate and Delta, preferably Serrate 1 and Serrate 2 as well as Delta 1, Delta 3 and Delta 4 expression in APCs such as dendritic cells.
  • the nucleic acid sequence may be capable of inhibiting Serrate expression but not Delta expression, or Delta but not Serrate expression in APCs or T cells.
  • the nucleic acid sequence for use in the present invention is capable of inhibiting Delta expression in T cells such as CD4 + helper T cells or other cells of the immune system that express Delta (for example in response to stimulation of cell surface receptors).
  • the nucleic acid sequence may be capable of inhibiting Delta expression but not Serrate expression in T cells.
  • the nucleic acid sequence is capable of inhibiting Notch ligand expression in both T cells and APC, for example Serrate expression in APCs and Delta expression in T cells.
  • Molecules for inhibition of Notch signalling will also include polypeptides, or polynucleotides which encode therefore, capable of modifying Notch-protein expression or presentation on the cell membrane or signalling pathways.
  • Molecules that reduce or interfere with its presentation as a fully functional cell membrane protein may include MMP inhibitors such as hydroxymate-based inhibitors.
  • Notch ligands Other substances which may be used to reduce interaction between Notch and Notch ligands are exogenous Notch or Notch ligands or functional derivatives thereof.
  • Notch ligand derivatives would preferably have the DSL domain at the N-terminus and up to about 14 or more, for example between about 3 to 8 EGF-like repeats on the extracellular surface.
  • a peptide corresponding to the Delta/Serrate/LAG-2 domain of hJaggedl and supernatants from COS cells expressing a soluble form of the extracellular portion of hJaggedl was found to mimic the effect of Jaggedl in inhibiting Notchl (Li).
  • Notch signalling pathway antagonists include antibodies which inhibit interactions between components of the Notch signalling pathway, e.g. antibodies to Notch ligands.
  • 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”.
  • Protein typically refers to a short amino acid sequence that is 10 to 40 amino acids long, preferably 10 to 35 amino acids.
  • 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 sequence is synonymous with the term
  • 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.
  • PCR polymerase chain reaction
  • This will 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 cell, 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.
  • PCR polymerase chain reaction
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
  • primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing 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 10,000 bases or even more, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA.
  • the nucleic acid may be RNA or DNA and is preferably DNA. Where it is RNA, manipulations may be perfo ⁇ ned via cDNA intermediates. Generally, a nucleic acid sequence encoding the first region will 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 literature 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 available. Generally this may be done by reference to literature sources which describe the cloning of the gene in question.
  • nucleic acids can be characterised as those nucleotide sequences which hybridise to the nucleic acid sequences known in the art.
  • the nucleotide sequence is DNA.
  • the nucleotide sequence is prepared by use of recombinant DNA techniques (e.g. recombinant DNA).
  • the nucleotide sequence is cDNA.
  • the nucleotide sequence may be the same as the naturally occurring form.
  • the present invention also encompasses the use of variants, derivatives, analogues, homologues and fragments thereof.
  • 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.
  • derivative 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.
  • analogue 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.
  • 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 referred to as "variants".
  • a variant protein can be modified by addition, deletion and/or substitution of at least one amino acid present in the naturally-occurring protein.
  • amino 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-naturally occurring analogues.
  • Proteins of use 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.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the transport or modulation function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • G Gly Glycine GGT, GGC, GGA,GGG
  • protein includes single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means.
  • polypeptide and peptide refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds.
  • subunit and domain may also refer to polypeptides and peptides having biological function.
  • “Fragments” are also variants and the term typically 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 naturally-occurring sequence either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding 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 illustrative 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 stability and therefor gene expression.
  • the redundancy of the genetic code means that several different codons may encode the same amino-acid.
  • 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.
  • Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms.
  • at least part of the sequence is codon optimised. Even more preferably, the sequence is codon optimised in its entirety.
  • homologous sequence can be taken to include an amino acid sequence which may be at least 75, 85 or 90%o identical, preferably at least 95 or 98% identical.
  • 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.
  • 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 commercially 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 amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are perfo ⁇ ned only over a relatively short number of residues.
  • tblastn compares a protein query sequence against a nucleotide sequence database dynamically translated in all 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). DESCRJJPTIONS - 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 will 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). Typically, 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 limit 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 enor response.
  • 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 Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov). Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or pro line-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.
  • 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.
  • sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.
  • no gap penalties are used when determining sequence identity.
  • a scaled similarity score matrix is generally 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 public default values for the GCG package, or in the case of other sof Tware, the default matrix, such as BLOSUM62.
  • sof Tware Once the sof Tware has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The sof Tware typically does this as part of the sequence comparison and generates a numerical result.
  • sequence homologies similarities or identities are referred to according to preferred aspects of the present invention, these are preferably along substantially the full length of the reference sequence.
  • 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 libraries made from a range of sources.
  • other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells) may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of the reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic 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 will 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 alignments can be performed using computer sof Tware known in the art. For example the GCG Wisconsin PileUp 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.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which will 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 alignments can be performed using computer sof Tware known in the art. For example the GCG Wisconsin PileUp 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.
  • PCR technology as described e.g. in section 14 of Sambrook et al., 1989, requires the use of oligonucleotide probes that will hybridise to nucleic acid. Strategies for selection of oligonucleotides are described below.
  • 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 usually based on conserved or highly homologous nucleotide sequences or regions of polypeptides.
  • the nucleic acids used as probes may be degenerate at one or more positions.
  • nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridisation.
  • suitable label means is a radiolabel.
  • the preferred method of labelling a DNA fragment is by incorporating ⁇ 32 P dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art.
  • Oligonucleotides are usually end-labelled with ⁇ 32 P-labelled ATP and polynucleotide kinase.
  • other methods e.g. non-radioactive
  • nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell 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.
  • variant 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 signalling modulation.
  • 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 preferred 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.
  • the present invention also encompasses nucleotide sequences that are 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.
  • hybridization 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, will be generally at least 75%, preferably at least 85 or 90% and more preferably at least 95% or 98% homologous to the co ⁇ esponding 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.
  • Prefe ⁇ ed nucleotide sequences of the invention will 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 significantly above background.
  • the background hybridization may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened.
  • background implies 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 32 P.
  • 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 typically 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; inte ⁇ nediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
  • 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.
  • 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 skill in the field. As known to those of skill in the art, the stability 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 stability 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.
  • high stringency preferably refers to conditions that permit hybridisation of only those nucleic acid sequences that fo ⁇ n 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, 5x Denhardt's, 1 % SDS (sodium dodecyl sulphate), 0.1 Na+ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific competitor.
  • high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 - 0. lx SSC, 0.1 % SDS.
  • Nucleotide sequences which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of sources. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • mammalian cells e.g. rat, mouse, bovine and primate cells
  • Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of the reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic 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 will 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 can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer sof Tware l ⁇ iown in the art. For example the GCG Wisconsin PileUp 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.
  • nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell 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.
  • nucleotide sequences such as a DNA polynucleotides useful in the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • PCR polymerase chain reaction
  • This will 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 cell, 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.
  • PCR polymerase chain reaction
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified 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.
  • host cells can be genetically 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 etal and Sambrook etal, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, micro injection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection. It will be appreciated that such methods can be employed in vitro or in vivo as drug delivery systems.
  • bacterial cells such as streptococci, staphylococci, E. coli, streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, NSO, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as streptococci, staphylococci, E. coli, streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, NSO, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells
  • 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 baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, 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.
  • 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 baculoviruses
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • 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.
  • 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.
  • an additional amino acid sequence which contains secretory or leader sequences or pro-sequences (such as a HIS oligomer, immunoglobulin Fc, glutathione S-transferase, FLAG etc) to aid in purification.
  • secretory or leader sequences or pro-sequences such as a HIS oligomer, immunoglobulin Fc, glutathione S-transferase, FLAG etc
  • the additional sequence may sometimes be desirable to provide added stability during recombinant production.
  • the additional sequence may be cleaved (eg chemically or enzymatically) to yield the final product.
  • 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
  • Active agents for use in the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification. Anti-sense constructs
  • Suitable nucleic acid sequences may include anti-senseT-cell costimulatory constructs as well as antisense constructs designed to reduce or inhibit the expression of upregulators of T-cell costimulatory expression (see above).
  • the antisense nucleic acid may be an oligonucleotide such as a synthetic single-stranded DNA.
  • 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 cell.
  • 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.
  • inhibitory antisense or double stranded oligonucleotides can additionally 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-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine,
  • the antisense oligonucleotide 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.
  • a modified polymeric backbone such as a modified polypeptide backbone may be used (eg peptide nucleic acid: PNA).
  • the antisense oligonucleotide 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,
  • Oligonucleotides may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). Merely as examples, phosphorothioate oligonucleotides can be syntliesized by the method of Stein et al. (1988, Nucl. Acids Res.
  • 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.
  • Notch signalling can be 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" referred to herein may be any detectable manifestation attributable to the presence of the cleaved intracellular domain of Notch.
  • increased Notch signalling can be assessed at the protein level by measuring intracellular concentrations of the cleaved Notch domain.
  • the assay is a protein assay. In another preferred embodiment of the present invention, the assay is a nucleic acid assay.
  • nucleic acid assay The advantage of using a nucleic acid assay is that they are sensitive and that small samples can be analysed.
  • 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 a ⁇ ays and other hybridization methods.
  • gene presence, amplification and/or expression may be 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.
  • Southern blotting Northern blotting to quantitate the transcription of mRNA
  • dot blotting DNA or RNA analysis
  • in situ hybridisation using an appropriately labelled probe.
  • PCR was originally developed as a means of amplifying 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 fo ⁇ nation of duplicate daughter strands.
  • RT-PCR uses an RNA template for generation of a first strand cDNA with a reverse transcriptase. The cDNA is then amplified 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 amplification 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 mixture is diluted and treated with ribonuclease (RNase) to degrade all remaining single-stranded RNA.
  • RNase ribonuclease
  • the hybridized portion of the probe will 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 will be directly proportional to the amount of complementary RNA in the sample.
  • Gene expression may also be detected using a reporter system.
  • 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 preferred. Especially preferred are GFP and luciferase.
  • Another type of preferred reporter is cell surface markers, i.e. proteins expressed on the cell surface and therefore easily identifiable.
  • 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).
  • constructs 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 available commercially.
  • Sorting of cells may be performed by any technique known in the art, as exemplified above.
  • cells may be sorted by flow cytometry or FACS.
  • flow cytometry FACS
  • FACS Fluorescence Activated Cell Sorting
  • F.A.C.S. Fluorescence Activated Cell Sorting
  • flow cytometry Fluorescence Activated Cell Sorting
  • FACS can be used to measure gene expression in cells transfected with recombinant DNA encoding polypeptides. This can be achieved directly, by labelling of the protein product, or indirectly by using a reporter gene in the construct.
  • reporter genes are ⁇ -galactosidase and Green Fluorescent Protein (GFP).
  • ⁇ -galactosidase activity can be detected by FACS using fluorogenic substrates such as fluorescein digalactoside (FDG).
  • FDG fluorescein digalactoside
  • FDG fluorescein digalactoside
  • FDG fluorescein digalactoside
  • 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.
  • the invention comprises the use of nucleic acid probes complementary to mRNA.
  • Such probes can be used to identify cells expressing polypeptides individually, such that they may subsequently be sorted eitlier 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.
  • 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 cell sorting.
  • the advantage of using a protein assay is that Notch activation can be directly measured.
  • Assay techniques that can be used to determine 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
  • 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) Curr Biol 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 immune costimulatory activity therapeutically.
  • antigen-presenting cells may be "professional" antigen presenting cells or may be another cell that may be induced to present antigen to T cells.
  • 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 metliods of the invention is typically isolated from a tumour or peripheral blood found within the body of a patient.
  • the APC or precursor is of human origin.
  • APCs from any suitable source, such as a healthy patient, may be used.
  • APCs include dendritic cells (DCs) such as interdigitating DCs or follicular DCs, Langerhans cells, PBMCs, macrophages, B-lymphocytes, or other cell types such as epithelial cells, fibroblasts or endothelial cells, activated or engineered by transfection to express a MHC molecule (Class I or JJ) on their surfaces.
  • DCs dendritic cells
  • PBMCs macrophages
  • B-lymphocytes or other cell types
  • Precursors of APCs include CD34 + cells, monocytes, fibroblasts and endothelial 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, E -12, IFN- ⁇ , TNF- ⁇ , JX-18 etc.).
  • proteins include MHC molecules (Class I or Class II), CD80, CD86, or CD40.
  • DCs or DC-precursors are included as a source of APCs.
  • Dendritic cells 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 cells for example after mobilisation into peripheral blood by treatment with GM-CSF, or directly from bone ma ⁇ ow. 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 ma ⁇ ow, non-adherent CD34 + cells can be treated with GM-CSF and TNF-a (Caux C, et al. (1992) Nature 360: 258-261 (Caux)).
  • GM-CSF/IL-4 mixture Inaba K, et al. (1992) J. Exp. Med. 175: 1157-1167 (Inaba)
  • non-adherent CD34 + cells can be treated with GM-CSF and TNF-a (Caux C, et al. (1992)
  • DCs can also be routinely prepared from the peripheral blood of human volunteers, similarly to the method of Sallusto 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 may be depleted of CD19 + B cells and CD3 + , CD2 + T cells using magnetic beads (Coffin RS, et al. (1998) Gene Therapy 5: 718-722 (Coffin)).
  • PBMCs peripheral blood mononucleocytes
  • 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 cells.
  • cytokines such as GM-CSF or IL-4 for the maintenance and, or activity of the dendritic cells or other antigen presenting cells.
  • the term "antigen presenting cell or the like" are used herein is not intended to be limited to APCs.
  • suitable APCs include dendritic cells, L cells, hybridomas, fibroblasts, lymphomas, macrophages, B cells or synthetic APCs such as lipid membranes.
  • T cells 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 cells may be used in combination with other immune cells, obtained from the same or a different individual.
  • whole blood may be used or leukocyte enriched blood or purified white blood cells as a source of T cells and other cell types. It is particularly preferred to use helper T cells (CD4 4 ).
  • other T cells such as CD8 + cells may be used. It may also be convenient to use cell lines such as T cell hybridomas.
  • T cells/APCs may be cultured as described above.
  • the APCs/T cells may be incubated with or exposed to active substances in accordance with the invention. For example, they may be prepared for administration to a patient or incubated with T cells in vitro (ex vivo).
  • modified cells of the present invention are preferably administered to a host by direct injection into the lymph nodes of the patient Typically from 10 4 to 10 8 treated cells, preferably from 10 5 to 10 7 cells, more preferably about 10 6 cells are administered to the patient.
  • the cells will be taken from an enriched cell population.
  • the term "enriched" as applied to the cell populations of the invention refers to a more homogeneous population of cells which have fewer other cells with which they are naturally associated.
  • An enriched population of cells can be achieved by several methods l ⁇ iown 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 cells) or negative selection (removing the undesirable cells).
  • the technology for capturing specific cells on affinity materials is well known in the art (Wigzel, et al., J. Exp. Med., 128:23, 1969; Mage, et al., J. Imnmunol. 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-cells or malignant cells from allogeneic or autologous ma ⁇ ow grafts, respectively (Gee, et al., J.N.C.I. 80:154, 1988).
  • Purification of human hematopoietic cells 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 solid 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.
  • the therapeutic agents used in the present invention may be administered directly to patients in vivo.
  • the agents may be administered to cells such as T cells and/or APCs in an ex vivo manner.
  • leukocytes such as T cells or APCs may be obtained from a patient or donor in l ⁇ iown manner, treated/incubated ex vivo in the manner of the present invention, and then administered to a patient.
  • 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.
  • T-cells 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.
  • 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-cells and/or APCs by introducing nucleic acid constructs/viral vectors encoding the polypeptide into cells under conditions that allow for expression of the polypeptide in the T-cell and/or APC.
  • nucleic acid constructs encoding antisense constructs may be introduced into the T-cells and/or APCs by transfection, viral infection or viral transduction.
  • nucleotide sequences will be operably linked to control sequences, including promoters/enhancers and other expression regulation signals.
  • control sequences including promoters/enhancers and other expression regulation signals.
  • 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 cells, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used.
  • the promoter is typically derived from promoter sequences of viral or eukaryotic genes.
  • it may be a promoter derived from the genome of a cell in which expression is to occur.
  • eukaryotic promoters they may be promoters that function in a ubiquitous manner (such as promoters of a-actin, b-actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase).
  • Tissue-specific promoters specific for lymphocytes, dendritic cells, skin, brain cells and epithelial cells within the eye are particularly preferred, for example the CD2, CDl 1 c, keratin 14, Wnt-1 and Rhodopsin promoters respectively.
  • the epithelial cell promoter SPC is used.
  • Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RS V) LTR promoter or the human cytomegalovirus (CMV) IE promoter.
  • MMLV LTR Moloney murine leukaemia virus long terminal repeat
  • RS V rous sarcoma virus
  • CMV human cytomegalovirus
  • the promoters 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 life-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.
  • 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-cells. If required, a small aliquot of cells may be tested for up-regulation of Notch signalling activity as described above.
  • the cells may be prepared for administration to a patient or incubated with T-cells in viti'O (ex vivo).
  • any of the assays described above can be adapted to monitor or to detect reduced reactivity, modified immune responses and/or tolerisation in immune cells, and to detect suppression and enhancement of immune responses for use in clinical applications.
  • 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) cells will demonstrate enhanced cytotoxic activity after activation. Therefore any drop in or stabilisation of cytotoxicity will be an indication of reduced reactivity.
  • NK Natural killer
  • leukocytes Once activated, leukocytes express a variety of new cell surface antigens.
  • NK cells for example, will express transferrin receptor, HLA-DR and the CD25 IL-2 receptor after activation. Reduced reactivity may therefore be assayed by monitoring expression of these antigens.
  • EA-1 and MLR3 are glycoproteins having major components of 28kD and 32kD.
  • EA-1 and MLR3 are not HLA class II antigens and an MLR3 Mab will block IL-1 binding. These antigens appear on activated T-cells within 18 hours and can therefore be used to monitor immune cell reactivity.
  • leukocyte reactivity may be monitored as described in EP 0325489, which is incorporated herein by reference. Briefly this is accomplished using a monoclonal antibody ("Anti-Leu23”) which interacts with a cellular antigen recognised by the monoclonal antibody produced by the hybridoma designated as ATCC No. HB-9627.
  • Anti-Leu23 a monoclonal antibody
  • ATCC No. HB-9627 a monoclonal antibody
  • 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.
  • Anti-Leu 23 is useful in monitoring the reactivity of leukocytes.
  • immune cells may be used to present antigens or allergens and/or may be treated to with active agents according to the present invention.
  • Antigen Presenting Cells 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.
  • a serum such as fetal calf serum.
  • Optimum cytokine concentrations may be determined by titration.
  • One or more active substances are then typically added to the culture medium together with the antigen of interest.
  • the antigen may be added before, after or at substantially the same time as the substance(s).
  • Cells are typically 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 cell activation by monitoring surface markers, cytokine secretion or proliferation as described in WO98/20142.
  • polypeptide substances may be administered to APCs by introducing nucleic acid constructs/viral vectors encoding the polypeptide into cells under conditions that allow for expression of the polypeptide in the APC.
  • nucleic acid constructs encoding antigens may be introduced into the APCs by transfection, viral infection or viral transduction.
  • T cells are generally co-cultured with the APCs.
  • the primed APCs may be pelleted 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 cell will not be brought into contact with the different substance(s) used with the APC.
  • Incubations will typically 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 signalling may be determined for a small aliquot of cells using the methods described above.
  • T cells transfected with a nucleic acid construct directing the expression of, for example Delta may be used as a control.
  • Induction of immunotolerance may be determined, for example, by subsequently challenging T cells with antigen and measuring IL-2 production compared with control cells not exposed to APCs.
  • Primed T cells or B cells may also be used to induce immunotolerance in other T cells or B cells in the absence of APCs using similar culture techniques and incubation times.
  • T cells 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 cells may be activated with MHC-peptide complexes (e.g. tetramers).
  • 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 cells may be activated with MHC-peptide complexes (e.g. tetramers).
  • Induction of immunotolerance may be determined by subsequently challenging T cells with antigen and measuring D -2 production compared with control cells not exposed to APCs.
  • T cells or B cells which have been primed in this way may be used according to the invention to promote or increase immunotolerance in other T cells or B cells.
  • the therapeutic effect results from a protein for Notch signalling.
  • T cells A detailed description of the Notch signalling pathway and conditions affected by it may be found in our WO98/20142, WO00/36089 and PCT/GBOO/04391.
  • Diseased or infectious states that may be described as being mediated by T cells include, but are not limited to, any one or more of asthma, allergy, graft rejection, autoimmunity, tumour induced abe ⁇ ations to the T cell system and infectious diseases such as those caused by Plasmodium species, Microfilariae, Helminths, Mycobacteria, HTV, Cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, Haemophilus influenza type B, measles, Hepatitis C or Toxicara.
  • infectious diseases such as those caused by Plasmodium species, Microfilariae, Helminths, Mycobacteria, HTV, Cytomegalovirus, Pseudomonas, To
  • the present invention is useful in treating immune disorders such as autoimmune diseases or graft rejection such as allograft rejection.
  • disorders that may be treated include a group commonly called autoimmune diseases.
  • the spectrum of autoimmune disorders ranges from organ specific diseases (such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis) to systemic illnesses such as rheumatoid arthritis or lupus erythematosus.
  • organ specific diseases such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis
  • Other disorders include immune hyperreactivity, such as allergic reactions.
  • Organ-specific autoimmune diseases include multiple sclerosis, insulin dependent diabetes mellitus, 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 colitis).
  • Systemic autoimmune diseases include: rheumatoid arthritis, juvenile arthritis, scleroderma and systemic sclerosis, sjogren's syndrom, undifferentiated connective tissue syndrome, antiphospholipid syndrome, different forms of vasculitis (polyarteritis nodosa, allergic granulomatosis and angiitis, Wegner's granulomatosis, Kawasaki disease, hypersensitivity vasculitis, Henoch-Schoenlein purpura, Behcet's Syndrome, Takayasu arteritis, Giant cell arteritis, Thrombangiitis obliterans), lupus erythematosus, polymyalgia rheumatica, correspondingl (mixed) cryoglobulinemia, Psoriasis vulgaris and psoriatic arthritis, diffus fasciitis with or without eosinophilia, polymyositis and other idiopathic inflammatory myopathie
  • a more extensive list of disorders includes: unwanted immune reactions and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac a ⁇ est, 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 test
  • retinitis or cystoid macular oedema 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.
  • monocyte or leukocyte proliferative diseases e.g. leukaemia
  • 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 ma ⁇ ow, organs, lenses, pacemakers, natural or artificial skin tissue.
  • the present invention is also useful in cancer therapy.
  • the present invention is especially useful in relation to adenocarcinomas such as: small cell lung cancer, and cancer of the kidney, uterus, prostrate, bladder, ovary, colon and breast.
  • adenocarcinomas such as: small cell lung cancer, and cancer of the kidney, uterus, prostrate, bladder, ovary, colon and breast.
  • the present invention may be used, for example, for the treatment of organ transplants (e.g. kidney, heart, lung, liver or pancreas transplants), tissue transplants (e.g. skin grafts) or cell transplants (e.g. bone ma ⁇ ow transplants or blood transfusions).
  • organ transplants e.g. kidney, heart, lung, liver or pancreas transplants
  • tissue transplants e.g. skin grafts
  • cell transplants e.g. bone ma ⁇ ow transplants or blood transfusions.
  • 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 cellular 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 complications such as infection and metabolic bone disease.
  • Heart transplantation is a very complex and high-risk procedure. Donor hearts must be maintained in such a manner that they will 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 of Ten impossible because of the limited supply- of hearts and the urgency of the procedure.
  • Lung transplantation is used (either by itself or in combination with heart transplantation) 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.
  • 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 controlled fashion. Indeed, transplantation of the islet cells alone could be sufficient. Because kidney failure is a frequent complication of advanced diabetes, kidney and pancreas transplants are of Ten carried out simultaneously.
  • 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 abnormalities.
  • the liver is a large and complicated 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 split and given to two recipients.
  • leukocytes within the donor organ together with anti-blood group antibodies can mediate antibody-dependent hemo lysis of recipient red blood cells if there is a mismatch of blood groups.
  • manifestations of GVHD have occurred in liver transplants even when donor and recipient are blood-group compatible.
  • the present invention is also useful in methods for altering the fate of a cell, tissue or organ type by altering Notch pathway function in the cell.
  • the present application has application in the treatment of malignant and pre-neoplastic disorders.
  • the present invention is especially useful in relation to adenocarcinomas such as: small cell lung cancer, and cancer of the kidney, uterus, prostrate, bladder, ovary, colon and breast.
  • malignancies 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 cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, choriocarcinoma,
  • 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 HIV, 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 neuropatliy, 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 juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
  • diabetes including diabetic neuropatliy, Bell's palsy
  • Certain combinations of active agents of the present invention may also for example be used in therapeutic and prophylactic vaccine compositions such as cancer and pathogen vaccines (eg where it is desired to increase an immune response to an antigen or antigenic dete ⁇ ninant).
  • Vaccine Compositions such as cancer and pathogen vaccines (eg where it is desired to increase an immune response to an antigen or antigenic dete ⁇ ninant).
  • Agents according to the present invention which inhibit Notch signalling may be employed in vaccine compositions (such as pathogen or cancer vaccines) to protect or treat a mammal susceptible to, or suffering from disease, by means of administering said vaccine via a mucosal route, such as the oral/bucal/intestinal/vaginal/rectal or nasal route.
  • vaccine compositions such as pathogen or cancer vaccines
  • a mucosal route such as the oral/bucal/intestinal/vaginal/rectal or nasal route.
  • Such administration may for example be in a droplet, spray, or dry powdered form.
  • Nebulised or aerosolised vaccine formulations may also be used where appropriate.
  • Enteric formulations such as gastro resistant capsules and granules for oral administration, suppositories for rectal or vaginal administration may also be used.
  • the present invention may also be used to enhance the immunogenicity of antigens applied to the skin, for example by intradermal, transdermal or transcutaneous delivery.
  • the adjuvants of the present invention may be parentally delivered, for example by intramuscular or subcutaneous administration.
  • a variety of administration devices may be used.
  • a spray device such as the commercially available Accuspray (Becton Dickinson) may be used.
  • Preferred spray devices for intranasal use are devices for which the performance of the device is not dependent upon the pressure applied by the user. These devices are known as pressure threshold devices. Liquid is released from the nozzle only when a threshold pressure is attained. These devices make it easier to achieve a spray with a regular droplet size. Pressure threshold devices suitable for use with the present invention are known in the art and are described for example in WO 91/13281 and EP 311 863 B. Such devices are commercially available from Pfeiffer GmbH.
  • the adjuvant formulations of the present invention may also comprise a bile acid or derivative of cholic acid.
  • the derivative of cholic acid is a salt thereof, for example a sodium salt thereof.
  • bile acids examples include cholic acid itself, deoxycholic acid, chenodeoxy colic acid, lithocholic acid, taurodeoxycholate ursodeoxycholic acid, hyodeoxycholic acid and derivatives like glyco-, tauro-, amidopropyl-1- propanesulfonic- and amidopropyl-2-hydroxy-l-propanesulfonic- derivatives of the above bile acids, or N, N-bis (3DGluconoamidopropyl) deoxycholamide.
  • an adjuvant formulation of the present invention may be in the form of an aqueous solution or a suspension of non-vesicular forms.
  • Such formulations are convenient to manufacture, and also to sterilise (for example by terminal filtration through a 450 or 220 nm pore membrane).
  • the route of administration may be via the skin, intramuscular or via a mucosal surface such as the nasal mucosa.
  • the admixture When the admixture is administered via the nasal mucosa, the admixture may for example be administered as a spray.
  • the methods to enhance an immune response may be either a priming or boosting dose of the vaccine.
  • adjuvant includes an agent having the ability to enhance the immune response of a vertebrate subject's immune system to an antigen or antigenic determinant.
  • Immune response includes any response to an antigen or antigenic determinant by the immune system of a subject.
  • Immune responses include for example humoral immune responses (e. g. production of antigen-specific antibodies) and cell- mediated immune responses (e. g. lymphocyte proliferation).
  • lymphocytes include the immunological defence provided by lymphocytes, such as the defence provided by T cell lymphocytes when they come into close proximity with their victim cells.
  • Lymphocyte proliferation When “lymphocyte proliferation” is measured, the ability of lymphocytes to proliferate in response to specific antigen may be measured. Lymphocyte proliferation includes B cell, T-helper cell or CTL cell proliferation.
  • compositions of the present invention may be used to formulate vaccines containing antigens derived from a wide variety of sources.
  • antigens may include human, bacterial, or viral nucleic acid, pathogen derived antigen or antigenic preparations, host-derived antigens, including GnRH and IgE peptides, recombinantly produced protein or peptides, and chimeric fusion proteins.
  • the vaccine formulations of the present invention contain an antigen or antigenic composition capable of eliciting an immune response against a human pathogen.
  • the antigen or antigens may, for example, be peptides/proteins, polysaccharides and lipids and may be derived from pathogens such as viruses, bacteria and parasites/fungi as follows:
  • Viral antigens or antigenic determinants may be derived, for example, from:
  • Cytomegalovirus especially Human, such as gB or derivatives thereof); Epstein Barr virus (such as gp350); flaviviruses (e. g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus); hepatitis virus such as hepatitis B virus (for example Hepatitis B Surface antigen such as the PreSl, PreS2 and S antigens described in EP-A-414 374; EP-A-0304 578, and EP-A-198474), hepatitis A virus, hepatitis C virus and hepatitis E virus; HTV-l, (such as tat, nef, gpl20 or gpl ⁇ O); human herpes viruses, such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HS VI or HS V2; human papilloma viruses (for example HPV6, 11 , 16, 18); Influenza virus (who
  • Bacterial antigens or antigenic determinants may be derived, for example, from: Bacillus spp., including B. anthracis (eg botulinum toxin); Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin, filamenteous hemagglutinin, adenylate cyclase, fimbriae); Borrelia spp., including B. burgdorferi (eg OspA, OspC, DbpA, DbpB), B. garinii (eg OspA, OspC, DbpA, DbpB), B.
  • B. anthracis eg botulinum toxin
  • Bordetella spp including B. pertussis (for example pertactin, pertussis toxin, filamenteous hemagglutinin, adenylate cyclase, fimbria
  • afzelii eg OspA, OspC, DbpA, DbpB
  • B. andersonii eg OspA, OspC, DbpA, DbpB
  • B. hermsii Campylobacter spp, including C. jejuni (for example toxins, adhesins and invasins) and C. coli;
  • Chlamydia spp. including C. trachomatis (eg MOMP, heparin-binding proteins), C. pneumonie (eg MOMP, heparin-binding proteins), C. psittaci; Clostridium spp., including C. tetani (such as tetanus toxin), C. botulinum (for example botulinum toxin), C. difficile (eg clostridium toxins A or B); Corynebacterium spp., including C. diphtheriae (eg diphtheria toxin); Ehrlichia spp., including E.
  • C. trachomatis eg MOMP, heparin-binding proteins
  • C. pneumonie eg MOMP, heparin-binding proteins
  • C. psittaci Clostridium spp., including C. tetani (such as tetanus toxin), C.
  • influenzae for example OMP26, high molecular weight adhesins, P5, P6, protein D and hpoprotein D, and fimbrin and fimbrin derived peptides (see for example US 5,843,464), Hehcobacter spp, including H pylori (for example urease, catalase, vacuolating toxin), Pseudomonas spp, including P aeruginosa, Legionella spp, including L pneumophila , Leptospira spp , including L inte ⁇ ogans, Listeria spp , including L monocytogenes, Moraxella spp, including M cata ⁇ halis, also known as Branhamella catarrhalis (for example high and low molecular weight adhesins and invasins), Morexella Catarrhalis (including outer membrane vesicles thereof, and OMP106 (see for example W097/41731)), Mycobactenum spp,
  • Parasitic/fungal antigens or antigenic determinants may be derived, for example, from
  • Babesia spp including B microti, Candida spp , including C albicans, Cryptococcus spp , including C neoformans, Entamoeba spp , including E histolytica, Giardia spp , including ,G lambha, Leshmama spp , including L major, Plasmodium.
  • Approved/licensed vaccines include, for example anthrax vaccines such as Biothrax (BioPort Corp); tuberculosis (BCG) vaccines such as TICE BCG (Organon Teknika Corp) and Mycobax (Aventis Pasteur, Ltd); diphtheria & tetanus toxoid and acellular pertussis (DTP) vaccines such as Tripedia (Aventis Pasteur, Inc), Infanrix (GlaxoSmithKline), and DAPTACEL (Aventis Pasteur, Ltd); Haemophilus b conjugate vaccines (eg diphtheria CRM197 protein conjugates such as HibTITER from Lederle Lab Div, American Cyanamid Co; meningococcal protein conjugates such as PedvaxHIB from Merck & Co, Inc; and tetanus toxoid conjugates such as ActHIB from Aventis Pasteur, SA); Hepatitis A vaccines such as Havrix (G
  • cancer antigen or antigenic determinant or “tumour antigen or antigenic determinant” as used herein preferably means an antigen or antigenic determinant which is present on (or associated with) a cancer cell and not typically on normal cells, or an antigen or antigenic determinant which is present on cancer cells in greater amounts than on normal (non-cancer) cells, or an antigen or antigenic determinant which is present on cancer cells in a different form than that found on normal (non-cancer) cells.
  • Cancer antigens include, for example (but without limitation): beta chain of human chorionic gonadotropin (hCGbeta) antigen, carcinoembryonic antigen, EGFRvHJ antigen, Globo H antigen, GM2 antigen, GP100 antigen, HER2/neu antigen, KS A antigen, Le (y) antigen, MUCI antigen, MAGE 1 antigen, MAGE 2 antigen, MUC2 antigen, MUC3 antigen, MUC4 antigen, MUC5AC antigen, MUC5B antigen, MUC7 antigen, PSA antigen, PSCA antigen, PSMA antigen, Thompson- Friedenreich antigen (TF), Tn antigen, sTn antigen, TRP 1 antigen, TRP 2 antigen, tumor-specific immunoglobulin variable region and tyrosinase antigen.
  • hCGbeta human chorionic gonadotropin
  • EGFRvHJ antigen Globo H antigen
  • antigens and antigenic determinants may be used in many different forms.
  • antigens or antigenic determinants may be present as isolated proteins or peptides (for example in so-called "subunit vaccines") or, for example, as cell-associated or virus-associated antigens or antigenic determinants (for example in either live or killed pathogen strains). Live pathogens will preferably be attenuated in known manner.
  • antigens or antigenic determinants may be generated in situ in the subject by use of a polynucleotide coding for an antigen or antigenic determinant (as in so-called "DNA vaccination", although it will be appreciated that the polynucleotides which may be used with this approach are not limited to DNA, and may also include RNA and modified polynucleotides as discussed above).
  • the active agents are administered in combination with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutically acceptable ca ⁇ ier or diluent 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 prefe ⁇ ed to formulate the compound in an orally active form.
  • compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • the choice of pharmaceutical ca ⁇ ier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • 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.
  • excipients such as starch or lactose
  • capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents.
  • active agents may be administered by inhalation, intranasally or in the form of aerosol, or in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder.
  • transdermal administration is by use of a skin patch.
  • they can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid 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 sof T paraffin base together with such stabilisers 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 delivery 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, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
  • the routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
  • Active agents may be adminstered by conventional DNA delivery techniques, such as DNA vaccination etc., or injected or otherwise delivered 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.
  • a therapeutically 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.
  • the physician will determine the actual dosage which will be most suitable for an individual patient and it will 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 intracavemosally, intravenously, intramuscularly or subcutaneously
  • active agents may be used in the form of a sterile aqueous solution which may suitably contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • agents may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • the dosage level of active agents and their pharmaceutically acceptable salts and solvates may typically be from 10 to 500 mg (in single or divided doses).
  • 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.
  • the physician will determine the actual dosage which will be most suitable for an individual patient and it will 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.
  • a modulator of immune cell costimulation is administered within from about one minute to within about one day before or after the modulator of the Notch signalling pathway is administered. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the modulator of the Notch signalling pathway and the modulator of immune cell costimulation will be administered within about one minute to within about eight hours, and preferably within less than about one to about four hours. When administered contemporaneously, the modulator of the Notch signalling pathway and the modulator of immune cell costimulation are suitably administered at the same site on the animal.
  • the term "same site” includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters.
  • the term "separately" as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months.
  • the active agents may be administered in either order. - I l l -
  • the modulator of the Notch signalling pathway may be administered more frequently than the modulator of immune cell costimulation or vice versa.
  • the term “sequentially” as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the active agents may be administered in a regular repeating cycle.
  • compositions of the present invention may be administered either in-vivo or ex-vivo.
  • routes of administration may be employed if desired.
  • one component such as the modulator of Notch signalling
  • the other may be administered in vivo, or vice versa.
  • treatment or therapy as used herein should be taken to encompass diagnostic and prophylatic applications.
  • the treatment of the present invention includes both human and veterinary applications.
  • antibodies for use to treat human patients will be chimeric or humanised antibodies.
  • Antibody "humanisation” techniques are well known in the art. These techniques typically involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule.
  • CDRs complementarity determining regions
  • a mouse MAb is grafted onto the framework regions of the variable domains of a human immunoglobulin by site directed mutagenesis using long oligonucleotides.
  • CDR-grafted humanised antibodies are much less likely to give rise to an anti-antibody response than humanised chimeric antibodies in view of the much lower proportion of non-human amino acid sequence which they contain.
  • the first criterion is to use as the human acceptor the framework from a particular human immunoglobulin that is unusually homologous to the non-human donor immunoglobulin to be humanised, or to use a consensus framework from many human antibodies.
  • the second criterion is to use the donor amino acid rather than the acceptor if the human acceptor residue is unusual and the donor residue is typical for human sequences at a specific residue of the framework.
  • the third criterion is to use the donor framework amino acid residue rather than the acceptor at positions immediately adjacent to the CDRs.
  • the fourth criterion is to use the donor amino acid residue at framework positions at which the amino acid is predicted to have a side chain atom within about 3 A of the CDRs in a three-dimensional immunoglobulin model and to be capable of interacting with the antigen or with the CDRs of the humanised immunoglobulin. It is proposed that criteria two, three or four may be applied in addition or alternatively to criterion one, and may be applied singly or in any combination.
  • the agents of the present invention may be administered in simultaneous, separate or sequential combination with antigens or antigenic determinants (or polynucleotides coding therefor), to modify (increase or decrease) the immune response to such antigens or antigenic determinants.
  • 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.
  • 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 generally either cell mediated (T cell mediated killing) or humoral (antibody production via recognition of whole antigen).
  • T cell mediated killing cell mediated killing
  • humoral antibody production via recognition of whole antigen.
  • TH1 cell mediated immunity
  • TH2 humoral immunity
  • 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 cells, then these cells will drive a cellular response, whereas TH2 cells will drive an antibody-dominated response.
  • the type of antibodies responsible for some allergic reactions is induced by TH2 cells.
  • the antigen or allergen (or antigenic determinant thereof) 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 cells or virus/viral component
  • allergens such as dust mite, pollen or food allergens to treat or prevent allergy
  • autoantigens associated with autoimmune diseases such as myelin basic protein (associated with multiple sclerosis), collagen (associated with rheumatoid arthritis), and insulin (diabetes) to treat or prevent autoimmune disease
  • graft antigens associated with rejection of non-self tissue such as MHC antigens or antigenic determinants thereof to treat or prevent graft rejection.
  • An increased immune response will typically be appropriate for use with vaccine antigens such as pathogen or cancer antigens or antigenic determinants thereof to treat or prevent
  • Polynucleotides coding for antigens or antigenic determinants which may be expessed in a subject may also be used.
  • 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 Genbank Accession No AF003522) into the expression vector pCON ⁇ (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:
  • the first underlined sequence is the signal peptide (cleaved from the mature protein) and the second underlined sequence is the IgG4 Fc sequence.
  • the protein normally exists as a dimer linked by disulphide bonds (see eg schematic representation in Figure 6).
  • Freshly-isolated murine CD4 + T-cells were cultured in RPMI 1640 (GibcoBRL) supplemented with 2mM Glutamine (GibcoBRL), Penicillin-Streptomycin 50 units/ml (GibcoBRL), 50 ⁇ M 2-mercaptoethanol and with 10% Fetal Bovine Serum (FBS) (Biochrom KG).
  • Anti-human IGg4 anti-CD3 (human), anti-CD28 (human) antibodies were plated at 5 ⁇ g/ml in phosphate buffer saline (Gibco BRL) in 6 well tissue culture dishes (1ml PBS/well) overnight. Anti-IgG4 antibody was applied to every well, while mouse IgGi K isotype control at 10 ⁇ g/ml was applied in wells that no anti-CD3 or anti-CD28 was used.
  • Microarrays were manufactured by spotting purified PCR products onto glass slides. Microa ⁇ ay probes were prepared by labelling 2 ⁇ g of ⁇ RNA by a reverse transcriptase reaction incorporating dCTP-Cy3 or dCTP-Cy5 labelled nucleotide. Probe labelling and purification were then performed generally as described in Hegde P, Qi R, Abernathy K, Gay C, Dharap S, Gaspard R, Hughes JE, Snesrud E, Lee N, Quackenbush J: A concise guide to cDNA microarray analysis (2000). Biotechniques 29:548-50, 552-4, 556 passim.
  • Ratio signal strength of gene in 'CD3/CD28 plus Delta'/ 'CD3/CD28 only'
  • T-cells from Example 1 were harvested at 4 and 16 hours.
  • Total cellular RNA was iissoollaatteedd uussiinngg tthhee R RNNeeaassyyTM RNA isolation kit (Qiagen, Crawley, UK) according to the manufacturer's guidelines.
  • RNA was reverse transcribed using SuperscriptTM IJ Reverse Transcriptase (Invitrogen, Paisley, UK) using Oligo dT( 12-1 8) or a random decamer mix according to the manufacturer's guidelines.
  • Oligo dT( 12 -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 LightcyclerTM system (Roche, UK) and SYBR green detection chemistry according to the manufacturer's guidelines.
  • the following HPLC-purified primer pairs were used for cDNA-specific amplification (5' to 3'):
  • mouse I8s rRNA Forward GTAACCCGTTGAACCCCATT Reverse CCATCCAATCGGTAGTAGCG
  • C p The endpoint used in real-time PCR quantification, the Crossing Point (C p ), 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 C p s from a set of serially-diluted gene-specific amplicons which had been previously cloned into a plasmid vector (pCR2.1, Invitrogen). These serial dilutions fall into a standard curve against which the C p s from the cDNA samples were compared. Using this system, expression levels of the 18S rRNA house-keeping gene were generated for each cDNA sample.
  • ICOS was then analysed by the same method using serially-diluted ICOS specific standards, and the ICOS value divided by the 18S rRNA value to generate a value, which represents the relative expression of ICOS in each cDNA sample. All Cp analysis was performed using the Second Derivative Maximum algorithm within the Lightcycler system software.
  • Jurkat cells were cultured in RPMI 1640 (GibcoBRL) supplemented with 2mM Glutamine (GibcoBRL), Penicillin-Streptomycin 50 units/ml (GibcoBRL) and with 10% Fetal Bovine Serum (FBS) (Biochrom KG).
  • RPMI 1640 GibcoBRL
  • 2mM Glutamine GibcoBRL
  • Penicillin-Streptomycin 50 units/ml GibcoBRL
  • FBS Fetal Bovine Serum
  • Anti-V5 (Invitrogen) and anti-CD3 (human), anti-CD28 (human) antibodies were plated at 5 ⁇ g/ml in phosphate buffer saline (Gibco BRL) in 6 well tissue culture dishes (1ml PBS/well) overnight.
  • Anti-V5 antibody was applied to every well, while mouse IgGt K isotype control at 10 ⁇ g/ml was applied in wells that no anti- CD3 or anti-CD28 was used. The next day the wells were washed 3 times with PBS, and Delta-V5-His protein was plated at 5 ⁇ g/ml PBS (lml/well).
  • ⁇ RNA antisense RNA
  • RNA from cells that were plated on wells treated only with V5 was labelled 'V5'
  • RNA from cells plated on wells treated with anti- V5 and Delta- V5-His was labelled 'Delta'
  • RNA from cells plated on wells treated with anti-V5, anti-CD3, anti-CD28 were labelled 'CD3CD28'
  • RNA from cells plated on wells treated with anti-V5, anti-CD3, anti-CD28, Delta-V5-His was labelled 'CD3CD28Delta'.
  • RNA from cells plated on anti-V5 and further treated with ionomycin was labelled 'ionomycin' while RNA from cells plated on anti-V5, Delta- V5- His and further treated with ionomycin were labelled 'ionomycin-Delta'.
  • Microarrays were manufactured by spotting purified PCR products onto glass slides. Microarray probes were prepared by labelling 2 ⁇ g of ⁇ RNA by a reverse transcriptase reaction incorporating dCTP-Cy3 or dCTP-Cy5 labelled nucleotide. Probe labelling and purification were then performed generally as described in Hegde P, Qi R, Abernathy K, Gay C, Dharap S, Gaspard R, Hughes JE, Snesrud E, Lee N, Quackenbush J: A concise guide to cDNA microa ⁇ ay analysis (2000). Biotechniques 29:548-50, 552-4, 556 passim.
  • CD3CD28 was labelled with dCTP-Cy3 and hybridized on the same slide as the sample named 'CD3CD28Delta' that was labelled with dCTP-Cy5.
  • 'ionomycin' was labelled with dCTP-Cy3 and hybridized on the same slide as the sample labelled 'ionomycinDelta' that was labelled with dCTP-Cy5 (see Table-1).
  • Ratio signal strength of gene in 'ionomycinDelta'/ control strength of gene in
  • Spleens were removed from mice (variously Balb/c females, 8-10 weeks, C57B/6 females, 8-10 weeks, D011.10 transgenic females, 8-10 weeks) and passed through a 0.2 ⁇ M cell strainer into 20ml R10F medium (R10F-RPMI 1640 media (Gibco Cat No 22409) plus 2mM L-glutamine, 50 ⁇ g/ml Penicillin, 50 ⁇ g/ml Streptomycin, 5 x 10 "5 M ⁇ -mercapto-ethanol in 10% fetal calf serum). The cell suspension was spun (1150rpm 5 min) and the media removed.
  • 96 well flat-bottomed plates were coated with DPBS plus 1 ⁇ g/ml anti-hamsterlgG antibody (Pharmingen Cat No 554007) plus 1 ⁇ g/ml anti-IgG4 antibody. lOO ⁇ l of coating mixture was added per well. Plates were incubated overnight at 4°C then washed with DPBS. Each well then received either lOO ⁇ l DPBS plus anti-CD3 antibody (1 ⁇ g/ml) or, lOO ⁇ l DPBS plus anti-CD3 antibody (1 ⁇ g/ml) plus hDeltal-IgG4Fc fusion protein (lO ⁇ g/ml; as described above). The plates were incubated for 2-3 hours at 37°C then washed again with DPBS before cells (prepared as described above) were added.
  • CD4+ cells were cultured in 96 well, flat-bottomed plates pre-coated according to (ii) above. Cells were re-suspended, following counting, at 2 x 10 6 /ml in R10F medium plus 4 ⁇ g/ml anti-CD28 antibody (Pharmingen, Cat No 553294, Clone No 37.51). lOO ⁇ l cell suspension was added per well. lOO ⁇ l of R10F medium was then added to each well to give a final volume of 200 ⁇ l (2 x 10 5 cells/well, anti-CD28 final concentration 2 ⁇ g/ml). The plates were then incubated at 37°C for 72 hours.
  • hDeltal-IgG4Fc fusion protein (Example 1) was immobilised on Streptavidin-Dynabeads (CELLection Biotin Binder Dynabeads [Cat. No. 115.21] at 4.0 x 10 8 beads/ml from Dynal (UK) Ltd; "beads”) in combination with biotinylated ⁇ -IgG-4 (clone JDC14 at 0.5 mg/ml from Pharmingen [Cat. No. 555879]) as follows:
  • the mixture was then spun down at 13,000 rpm for 1 minute and the beads were resupsended in 50 ⁇ l PBS per sample. 50 ⁇ l of biotinylated ⁇ -IgG-4 -coated beads were added to each sample and the mixture was incubated on a rotary shaker at 4 °C overnight.
  • the tube was then spun at 1000 rpm for 5 minutes at room temperature.
  • the beads then were washed with 10 ml of PBS, spun down, resupended in 1 ml of PBS, transferred to a sterile Eppendorf tube, washed with a further 2 x 1 ml of PBS, spun down and resuspended in a final volume of 100 ⁇ l of DMEM plus 10%(HI)FCS plus glutamine plus P/S, i.e. at 1.0 x 10 5 beads/ ⁇ l.
  • Stable N27#ll cells (CHO cells expressing full length human Notcl 2 and a CBF1- luciferase reporter construct; T 8 o flask; as described in WO 03/012441, Lorantis, eg see Example 7 therein) were removed using 0.02% EDTA solution (Sigma), spun down and resuspended in 10 ml DMEM plus 10%(ffl)FCS plus glutamine plus P/S. 10 ⁇ l of cells were counted and the cell density was adjusted to 1.0 x 10 5 cells/ml with fresh DMEM plus 10%(HI)FCS plus glutamine plus P/S. 1.0 x 10 5 of the cells were plated out per well of a 24- well plate in a 1 ml volume of DMEM plus 10%(HI)FCS plus glutamine plus P/S and cells were placed in an incubator to settle down for at least 30 minutes.
  • the mixture was then pipetted up and down 2 times to ensure cell lysis and the contents from each well were transferred to a 96 well plate (with V-shaped wells) and spun in a plate holder for 5 minutes at 1000 rpm at room temperature.
  • Luminescence was then read in a TopCount (Packard) counter.

Abstract

A method is described for detecting, measuring or monitoring Notch signalling by determining the amount of an immune cell costimulatory protein, polypeptide or polynucleotide or determining the amount of a polynucleotide coding for such a protein or polypeptide. Methods of modulating the immune system are also described.

Description

MODULATORS OF NOTCH SIGNALLING AND OF IMMUNE CELL COSTIMULATORY ACTIVITY FOR
IMMUNOTHERAPY
5 Field of the invention
The present invention relates inter alia to monitoring, detecting or measuring Notch signalling and to modulation of immune responses for therapeutic purposes.
0 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, auto immunity, graft rejection, 5 tumour induced aberrations to the T-cell system and infectious diseases caused, for example, by Plasmodium species, Mcrofilariae, Helminths, Mycobacteria, HIV, Cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, Haemophilus influenza type B, measles, Hepatitis C or Toxicara, may be targeted.
0 It has also been shown that it is possible to generate a class of regulatory T cells which are able to transmit antigen-specific tolerance to other T cells, a process termed infectious tolerance (W098/20142). The functional activity of these cells can be mimicked by over- expression of a Notch ligand protein on their cell surfaces or on the surface of antigen presenting cells. In particular, regulatory T cells can be generated by over-expression of a 5 member of the Delta or Serrate 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 0 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/O4391 (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 GBOl 18153.6 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 GBOl 18153.6 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. etal. (2001) Immunological Reviews 182:215-227). According to the present invention combinations of modulators of Notch signalling and modulators of immune cell costimulation may be used to provide unexpectedly improved activity or an advantageous spectrum of activity.
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 an immune cell (preferably T-cell) costimulatory protein or polypeptide or determining the amount of a polynucleotide coding for such a protein or polypeptide.
Suitably, the amount of the costimulatory 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, for example, the sample may comprise a cancer or tumour cell or 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 an immune cell (preferably T-cell) costimulatory 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 an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide; and iii) detecting in the sample an amount of costimulatory 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 an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide; iii) detecting in the sample an amount of costimulatory protein, polypeptide or polynucleotide that binds to the binding agent; and iv) comparing the amount of costimulatory 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 immune cell/ T- cell costimulatory molecule such as CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L. Alternatively the binding agent may comprise a binding domain from an immune cell, preferably a T-cell costimulatory molecule such as CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD- 1, PD-L1, PD-L2, OX40 or OX40L.
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 human T-cell costimulatory molecule mRNA. Preferably such a polynucleotide hybridises to a nucleotide sequence of human T-cell costimulatory polynucleotide (eg mRNA) 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 amplifying an immune cell, (preferably T-cell) costimulatory 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 for detecting, measuring or monitoring Notch signalling in an immune cell by determining the amount of an immune cell (preferably T-cell) costimulatory 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).
Suitably the method further comprises a step of comparing the amount of costimulatory 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 an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide for detecting, measuring or monitoring Notch signalling.
According to a further aspect of the invention there is provided a method of modulating the immune system comprising administering an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide or a modulator of immune cell (preferably T-cell) costimulatory 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 immune cell costimulation, in combination with an agent capable of inhibiting Notch signalling.
According to a further aspect of the invention there is provided an antagonist of immune cell costimulation, in combination with an agent capable of activating Notch signalling.
According to a further aspect of the invention there is provided a product comprising an immune cell costimulatory protein, polypeptide or polynucleotide or a modulator of immune cell costimulatory 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 immune cell costimulatory 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 an immune cell costimulatory protein, polypeptide or polynucleotide or an activator of immune cell costimulatory activity in combination with an activator of the Notch signalling pathway.
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) an immune cell costimulatory protein, polypeptide or polynucleotide or a modulator of immune cell costimulatory activity.
According to a further aspect of the invention there is provided a method for increasing immune cell costimulation by administering a modulator of Notch signalling. According to a further aspect of the invention there is provided a method for decreasing immune cell (preferably T-cell) costimulation by administering a modulator of Notch signalling.
Suitably such T-cell costimulation is mediated by CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L.
According to a further aspect of the invention there is provided a method for increasing expression of an immune cell costimulatory protein, polypeptide or polynucleotide by administering a modulator of Notch signalling.
According to a further aspect of the invention there is provided a method for decreasing expression of an immune cell costimulatory protein, polypeptide or polynucleotide by administering a modulator of Notch signalling.
Suitably the costimulatory protein, polypeptide or polynucleotide is CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L or a polynucleotide coding for CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L.
According to a further aspect of the invention there is provided the use of a modulator of Notch signalling to increase immune cell (preferably T-cell) costimulation.
According to a further aspect of the invention there is provided the use of a modulator of Notch signalling to decrease immune cell (preferably T-cell) costimulation.
According to a further aspect of the invention there is provided the use of a modulator of Notch signalling to increase expression of an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide. According to a further aspect of the invention there is provided the use of a modulator of Notch signalling to decrease expression of an immune cell (preferably T-cell) costimulatory protein, polypeptide or polynucleotide.
For example, the costimulatory protein, polypeptide or polynucleotide may be CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L or a polynucleotide coding therefor.
Suitably the expression or activity of costimulatory protein, polypeptide or polynucleotide is modulated in activated T-cells (suitably stimulated by antigen presentation).
According to a further aspect of the invention there is provided a product comprising: i) a modulator of the Notch signalling pathway; and ii) a modulator of immune cell (preferably T-cell) costimulation; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
Suitably such a product may be used for modulation of immune cell activity, for example in the treatment of inflammation, asthma, allergy, graft rejection, graft-versus-host disease or autoimmune disease.
According to a further aspect of the invention there is provided a method for preparing a product as described above by combining: i) a modulator of the Notch signalling pathway; and ii) a modulator of immune cell (preferably T-cell) costimulation. According to a further aspect of the invention there is provided a method for modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering: i) an effective amount of a modulator of the Notch signalling pathway; and ii) an effective amount of a modulator of immune cell (preferably T-cell) costimulation.
According to a further aspect of the invention there is provided a combination of: i) a modulator of the Notch signalling pathway; and ii) a modulator of immune cell (preferably T-cell) costimulation; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
According to a further aspect of the invention there is provided a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a modulator of immune cell (preferably T-cell) costimulation.
According to a further aspect of the invention there is provided the use of a combination of: i) a modulator of the Notch signalling pathway; and ii) a modulator of immune cell (preferably T-cell) costimulation; 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 modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of the immune system in simultaneous, contemporaneous, separate or sequential combination with a modulator of immune cell (preferably T-cell) costimulation. According to a further aspect of the invention there is provided a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a modulator of immune cell (preferably T-cell) costimulation.
According to a further aspect of the invention there is provided a pharmaceutical composition comprising a modulator of the Notch signalling pathway and a modulator of immune cell (preferably T-cell) costimulation and optionally a pharmaceutically acceptable carrier.
According to a further aspect of the invention there is provided a combination of: i) an activator of Notch signalling; and ii) an activator of negative immune costimulation; for simultaneous, contemporaneous, separate or sequential use in reducing an immune response.
According to a further aspect of the invention there is provided a combination of: i) an activator of Notch signalling; ii) an inhibitor of positive immune costimulation; for simultaneous, contemporaneous, separate or sequential use in reducing an immune response.
According to a further aspect of the invention there is provided a combination of: i) an inhibitor of Notch signalling; and ii) an inhibitor of negative immune costimulation; for simultaneous, contemporaneous, separate or sequential use in increasing an immune response.
According to a further aspect of the invention there is provided a combination of: i) an inhibitor of Notch signalling; and ii) an activator of positive immune costimulation; for simultaneous, contemporaneous, separate or sequential use in increasing an immune response.
According to a further aspect of the invention there is provided a method for reducing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; and ii) an activator of negative immune costimulation.
According to a further aspect of the invention there is provided a method for reducing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; and ii) an inhibitor of positive immune costimulation.
According to a further aspect of the invention there is provided a method for increasing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; and ii) an inhibitor of negative immune costimulation.
According to a further aspect of the invention there is provided a method for increasing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; and ii) an activator of positive immune costimulation.
According to a further aspect of the invention there is provided a combination of: i) an activator of Notch signalling; ii) an activator of negative immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in reducing an immune response to the antigen or antigenic determinant.
According to a further aspect of the invention there is provided a combination of: i) an activator of Notch signalling; ii) an inhibitor of positive immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in reducing an immune response to the antigen or antigenic determinant.
According to a further aspect of the invention there is provided a combination of: i) an inhibitor of Notch signalling; ii) an inhibitor of negative immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in increasing an immune response to the antigen or antigenic deteπninant.
According to a further aspect of the invention there is provided a combination of: i) an inhibitor of Notch signalling; ii) an activator of positive immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in increasing an immune response to the antigen or antigenic determinant. According to a further aspect of the invention there is provided a method for reducing an immune response to an antigen or antigenic determinant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; ii) an activator of negative immune costimulation; and iii) the 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 a method for reducing an immune response to an antigen or antigenic determinant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; ii) an inhibitor of positive immune costimulation; and iii) the antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in reducing an immune response to the antigen or antigenic determinant
According to a further aspect of the invention there is provided a method for increasing an immune response to an antigen or antigenic determinant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; ii) an inhibitor of negative immune costimulation; and iii) the 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 a method for increasing an immune response to an antigen or antigenic deteπninant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; ii) an activator of positive immune costimulation; and iii) the antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant).
Suitably the modulator of Notch signalling is a Notch receptor agonist. Suitably the modulator of Notch signalling may be an activator of a Notch receptor (especially a human Notchl, Notch2, Notch3 or Notch4 receptor). Preferably the modulator activates Notch signalling in immune cells, espeacially T-cells, B-cells and/or APCs.
Suitably the modulator of the Notch signalling patliway comprises Delta or Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide coding for Delta, Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof.
In one embodiment the modulator of the Notch signalling pathway may comprise 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.
Suitably the modulator of the Notch signalling pathway comprises a protein or polypeptide comprising a DSL domain or a polynucleotide sequence coding for such a protein. Preferably the modulator of the Notch signalling pathway comprises a protein or polypeptide comprising a Notch ligand DSL domain and from 1 to 16 or more EGF-like domains or a polynucleotide sequence coding for such a protein or polypeptide.
Alternatively, the modulator of the Notch signalling pathway may comprise Notch intracellular domain (Notch IC) or a fragment, derivative, homologue, analogue or allelic variant thereof, or a polynucleotide sequence which codes for Notch intracellular domain or a fragment, derivative, homologue, analogue or allelic variant thereof. Suitably the modulator of the Notch signalling pathway comprises Delta (preferably human Delta) or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide encoding Delta or a fragment, derivative, homologue, analogue or allelic variant thereof.
Alternatively or in addition the modulator of the Notch signalling pathway may comprise Serrate/Jagged (preferably human Jagged) or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide encoding Serrate/Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof.
Alternatively or in addition the modulator of the Notch signalling pathway may comprise Notch (preferably humna Notch) or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide encoding Notch or a fragment, derivative, homologue, analogue or allelic variant thereof.
Alternatively or in addition the modulator of the Notch signalling pathway may comprise a dominant negative version of a Notch signalling repressor, or a polynucleotide which codes for a dominant negative version of a Notch signalling repressor.
Alternatively or in addition the modulator of the Notch signalling pathway may comprise a polypeptide capable of upregulating the expression or activity of a Notch ligand or a downstream component of the Notch signalling pathway, or a polynucleotide which codes for such a polypeptide.
Suitably the modulator of the Notch signalling pathway may comprise an antibody, antibody fragment or antibody derivative or a polynucleotide which codes for an antibody, antibody fragment or antibody derivative, suitably an anti-Notch antibody. 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 Sepharose bead.
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 suitably not more than 5) 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) 2-4 or more (or suitably 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 one or more of the modulators of Notch signalling may comprise a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 2-3 or more (or suitably 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:
GVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFRVCLKHYQASVSPEPPCTYGSA VTPV GVDSFS PDGGGADSAFSNPIRFPFGFTWPGTFSLIIEALHTDSPDDLATENPER LISRLATQRHLTVGEEWSQD HSSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHF TCGERGEKVCNPG KGPYCTEPICLPGCDEQHGFCDKPGECKCRVG QGRYCDECIRYPG CLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCK GATCTNTGQGSYTCSCRPGYTG ATCELGIDEC
Suitably the modulator of costimulation may be a modulator of CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L signalling.
For example, the modulator of costimulation may be a soluble fusion protein comprising at least an active part of a CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L extracellular domain. Suitably, for example, the modulator of costimulation may be CTLA-4-Ig, ICOS-Ig, CD40-Ig or OX40-Ig.
Suitably an antigen or antigenic determinant may also be administered simultaneously, separately or sequentially with the modulator of Notch signalling and/or modulator of immune cell costimulation. According to a further aspect of the invention there is provided a method for generating an immune modulatory cytokine profile with increased EL- 10 expression by administering a combination of active agents according to the present invention.
According to a further aspect of the invention there is provided a method for generating an immune modulatory cytokine profile with increased EL-10 and TL-4 expression and reduced JL-5, LL-13 and TNFα expression by administering a combination of active agents according to the present invention.
According to a further aspect of the invention there is provided a method for generating an immune modulatory cytokine profile with increased JX-10 and IL-4 expression and reduced IL-2, IFNγ , EL-5, EL-13 and TNFα expression by administering a combination of active agents according to the present invention.
Detailed description
Various preferred features and embodiments of the present invention will now be described in more detail by way of 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 aligned amino acid sequences of DSL domains from various Drosophila and mammalian Notch ligands; Figure 4 shows amino acid sequences of human Delta-1, Delta-2 and Delta-3;
Figure 5 shows amino acid sequences of human Jagged- 1 and Jagged-2;
Figure 6 shows schematic representations of modulators of Notch signalling which may be used in the present invention;
Figure 7 shows the results of Example 1 ; Figure 8 shoes the results of Example 2; and Figure 9 shoes the results of Example 3.
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 literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratoiγ 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, NY.); 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 Wiley & 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 all homologues are included within the scope of the invention.
Immune cell costimulation
The term "immune cell costimulation" as used herein, comprises signalling through a pathway involving a molecule on the surface of an immune cell and a molecule on the surface of a T-cell, B-cell or antigen presenting cell (APC). An interaction between these two molecules, in the context for example of antigen specific recognition by the T cell of an antigen presented by the antigen presenting cell, either promotes immune cell activation (positive immune costimulation) or reduces it (negative immune costimulation).
The term "immune cell costimulation" as used herein thus includes both upregulation (positive costimulation) and downregulation (negative costimulation) of immune cell (especailly T-cell) activation (or also modulation of other immune cells such as B-cell or APC activation). As described for example in Dong et al (Nature 2001 Jan 4;409(6816):97-101) T-lymphocyte activation and immune function are regulated by various co-stimulatory molecules. For example, CD28, a receptor for B7 gene products, has a chief role in initiating T-cell immune responses (positive costimulation). CTLA4 binds B7 with a higher affinity, is induced after T-cell activation and is involved in downregulating T-cell responses (negative costimulation). The inducible co-stimulatory molecule (ICOS), a third member of the CD28/CTLA4 family, is expressed on activated T cells. Its ligand (B7H/B7RP-1) is expressed on B cells.
Examples of agents that inhibit B7 interactions include, but are not limited to, molecules such as an antibody (or fragment or derivative thereof) that recognizes and binds to a CTLA4, CD28 or B7 (e.g. B7-1, B7-2) molecule; a soluble form (or portion or derivative thereof) of an extracellular domain of a costimulatory molecule such as soluble CTLA4; or a peptide fragment or other small molecule designed to interfere with the cell signal through the CTLA4/CD28/B7-mediated interaction. In one embodiment, an inhibitory agent may be a soluble CTLA4 molecule, such as CTLA4Ig (ATCC 68629) or L104EA29YIg (ATCC PTA-2104), a soluble CD28 molecule such as CD28Ig (ATCC 68628), a soluble B7 molecule such as B7Ig (ATCC 68627), an anti-B7 monoclonal antibody (e.g. ATCC HB-253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB-301, ATCC HB-11341 and monoclonal antibodies as described in by Anderson et al in U.S. Pat. No. 6,113,898 or Yokochi et al., 1982. J. Itnmun., 128(2)823-827), an anti-CTLA4 monoclonal antibody (e.g. ATCC HB-304, and monoclonal antibodies as described in references 82-83) and/or an anti-CD28 monoclonal antibody (e.g. ATCC HB 11944 and mAb 9.3 as described by Hansen (Hansen et al., 1980. hnmunogenetics 10: 247-260) or Martin (Martin et al., 1984. J. Clin. Immun., 4(1): 18-22)).
As discussed, for example, in Frauwirth (J Clin Invest, February 2002, Volume 109, Number 3, 295-299) a further class of costimulatory receptors comprises certain members of the TNF receptor (TNFR) family. These include CD40, the major B cell costimulatory molecule, as well as OX-40, 4-1BB, CD30, and CD27. The ligands for these receptors are membrane-bound members of the TNF family.
A further negative costimulator is the Programmed Death-1 (PD-1) receptor. Members of the B7 family, PD-L1 and PD-L2, are ligands for PD-1.
The term "immune cell costimulatory protein or polypeptide" as used herein means a protein or polypeptide involved in immune cell (especially T-cell) costimulation. It includes, for example, fragments, homologues and allelic variants. The term "immune cell costimulatory polynucleotide" means a polynucleotide coding for an immune cell (especially T-cell) costimulatory protein or polypeptide.
Suitably the term "T-cell costimulatory 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 human T-cell costimulatory protein or polypeptide 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 "T-cell costimulatory activity" as used herein means the biological activity of a T-cell costimulatory protein or polypeptide, including but not limited to transcription activation activity. Further T-cell costimulatory proteins/polypeptides include inducible T-cell co-stimulator (ICOS) and its ligand B7-H2 (ICOS ligand); CD28 and its ligands CD80 and CD86 and CTLA-4 and its ligands CD80 and CD86.
A sequence for a human inducible T-cell co-stimulator (ICOS), mRNA is provided under GenBank Accession No NM_012092.
A sequence for a human transmembrane protein B7-H2 (ICOS ligand) mRNA is provided under GenBank Accession No AF289028.
A sequence for a human cytotoxic T-lymphocyte-associated protein 4 (CTLA4) mRNA is provided under GenBank Accession No NM_005214.
A sequence for a human CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) mRNA is provided under GenBank Accession No NM_006889.
A sequence for a human CD28 antigen (Tp44) (CD28) mRNA is provided under GenBank Accession No NM_006139.
A sequence for a human CD80 antigen (CD28 antigen ligand 1, B7-1 antigen) mRNA is provided under GenBank Accession No NM_005191.
For example, amino acid and polynucleotide sequences for human CTLA-4 are provided as follows (GenBank Accession No AY209009):
MACLGFQRHKAQLNIiAΑRT PCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVT VLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQλ LTIQGLRAMDTGLYICKVELMYPPPYYLGIG NGTQIYVIDPEPCPDSDFLL ILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQ PYFIPIN 1 atggcttgcc ttggatttca gcggcacaag gctcagctga acctggctgc caggacctgg 61 ccctgcactc tcctgttttt tcttctcttc atccctgtct tctgcaaagc aatgcacgtg 121 gcccagcctg ctgtggtact ggccagcagc cgaggcatcg ccagctttgt gtgtgagtat 181 gcatctccag gcaaagccac tgaggtccgg gtgacagtgc ttcggcaggc tgacagccag 241 gtgactgaag tctgtgcggc aacctacatg atggggaatg agttgacctt cctagatgat 301 tccatctgca cgggcacctc cagtggaaat caagtgaacc tcactatcca aggactgagg 361 gccatggaca cgggactcta catctgcaag gtggagctca tgtacccacc gccatactac 421 ctgggcatag gcaacggaac ccagatttat gtaattgatc cagaaccgtg cccagattct 481 gacttcctcc tctggatcct tgcagcagtt agttcggggt tgttttttta tagctttctc 541 ctcacagctg tttctttgag caaaatgcta aagaaaagaa gccctcttac aacaggggtc 601 tatgtgaaaa tgcccccaac agagccagaa tgtgaaaagc aatttcagcc ttattttatt 661 cccatcaatt ga
An example of a CTLA-4 lg Fusion protein (BMS 188667) is manufactured by Bristol Myers Squibb (US), also described in U.S. Pat. Nos. 5,434,131, 5,844,095, and 5,851,795 incorporated herein by reference.
As noted by BMS, (US 20030083246) DNA encoding CTLA4Ig was deposited on May 31 , 1991 with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 under the provisions of the Budapest Treaty, and has been accorded ATCC accession number ATCC 68629; (Linsley, P., et al., 1994 Immunity 1:793-80). CTLA4Ig-24, a Chinese Hamster Ovary (CHO) cell line expressing CTLA4Ig was deposited on May 31, 1991 with ATCC identification number CRL-10762.
As used herein, "B7" refers to the B7 family of molecules including, but not limited to, B7-1 (CD80) (Freeman et al, 1989, J. Immunol. 143:2714-2722, herein incorporated by reference), B7-2 (CD86) (Freeman et al, 1993, Science 262:909-911 herein incorporated by reference; Azuma et al, 1993, Nature 366:76-79 herein incorporated by reference) that may recognize and bind CTLA4 and/or CD28. A B7 molecule can be expressed on an activated B cell.
Amino acid and polynucleotide sequences for a human inducible T-cell co-stimulator (ICOS) are provided as follows (GenBank Accession No AJ277832): MKSGL YFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIV QFKMQLLKGGQILCDLTKTKGSG NTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKF LP IGCAA.FVWCILGCILIC LTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL
1 cgagagcctg aattcactgt cagctttgaa cactgaacgc gaggactgtt aactgtttct
61 ggcaaacatg aagtcaggcc tctggtattt ctttctcttc tgcttgcgca ttaaagtttt
121 aacaggagaa atcaatggtt ctgccaatta tgagatgttt atatttcaca acggaggtgt
181 acaaatttta tgcaaatatc ctgacattgt ccagcaattt aaaatgcagt tgctgaaagg
241 ggggcaaata ctctgcgatc tcactaagac aaaaggaagt ggaaacacag tgtccattaa
301 gagtctgaaa ttctgccatt ctcagttatc caacaacagt gtctcttttt ttctatacaa
361 cttggaccat tctcatgcca actattactt ctgcaaccta tcaatttttg atcctcctcc
421 ttttaaagta actcttacag gaggatattt gcatatttat gaatcacaac tttgttgcca
481 gctgaagttc tggttaccca taggatgtgc agcctttgtt gtagtctgca ttttgggatg
541 catacttatt tgttggctta caaaaaagaa gtattcatcc agtgtgcacg accctaacgg
601 tgaatacatg ttcatgagag cagtgaacac agccaaaaaa tctagactca cagatgtgac
661 cctataatat ggaactctgg cacccaggca tgaagcacgt tggccagttt tcctcaactt
721 gaagtgcaag attctcttat ttccgggacc acggagagtc tgacttaact acatacatct
781 tctgctggtg ttttgttcaa tctggaagaa tgactgtatc agtcaatggg gattttaaca
841 gactgccttg gtactgccga gtcctctcaa aacaaacacc ctcttgcaac cagctttgga
901 gaaagcccag ctcctgtgtg ctcactggga gtggaatccc tgtctccaca tctgctccta
961 gcagtgcatc agccagtaaa acaaacacat ttacaagaaa aatgttttaa agatgccagg
1021 ggtactgaat ctgcaaagca aatgagcagc caaggaccag catctgtccg catttcacta
1081 tcatactacc tcttctttct gtagggatga gaattcctct tttaatcagt caagggagat
1141 gcttcaaagc tggagctatt ttatttctga gatgttgatg tgaactgtac attagtacat
1201 actcagtact ctccttcaat tgctgaaccc cagttgacca ttttaccaag actttagatg
1261 ctttcttgtg ccctcaattt tctttttaaa aatacttcta catgactgct tgacagccca
1321 acagccactc tcaatagaga gctatgtctt acattctttc ctctgctgct caatagtttt
1381 atatatctat gcatacatat atacacacat atgtatataa aattcataat gaatatattt
1441 gcctatattc tccctacaag aatatttttg ctccagaaag acatgttctt ttctcaaatt
1501 cagttaaaat ggtttacttt gttcaagtta gtggtaggaa acattgcccg gaattgaaag
1561 caaatttatt ttattatcct attttctacc attatctatg ttttcatggt gctattaatt
1621 acaagtttag ttctttttgt agatcatatt aaaattgcaa acaaaatcat ctttaatggg
1681 ccagcattct catggggtag agcagaatat tcatttagcc tgaaagctgc agttactata
1741 ggttgctgtc agactatacc catggtgcct ctgggcttga caggtcaaaa tggtccccat
1801 cagcctggag cagccctcca gacctgggtg gaattccagg gttgagagac tcccctgagc
1861 cagaggccac taggtattct tgctcccaga ggctgaagtc accctgggaa tcacagtggt
1921 ctacctgcat tcataattcc aggatctgtg aagagcacat atgtgtcagg gcacaattcc
1981 ctctcataaa aaccacacag cctggaaatt ggccctggcc cttcaagata gccttcttta
2041 gaatatgatt tggctagaaa gattcttaaa tatgtggaat atgattattc ttagctggaa
2101 tattttctct acttcctgtc tgcatgccca aggcttctga agcagccaat gtcgatgcaa
2161 caacatttgt aactttaggt aaactgggat tatgttgtag tttaacattt tgtaactgtg
2221 tgcttatagt ttacaagtga gacccgatat gtcattatgc atacttatat tatcttaagc
2281 atgtgtaatg ctggatgtgt acagtacagt actgaacttg taatttgaat ctagtatggt
2341 gttctgtttt cagctgactt ggacaacctg actggctttg cacaggtgtt ccctgagttg
2401 tttgcaggtt tctgtgtgtg gggtggggta tggggaggag aaccttcatg gtggcccacc
2461 tggcctggtt gtccaagctg tgcctcgaca catcctcatc cccagcatgg gacacctcaa
2521 gatgaataat aattcacaaa atttctgtga aatcaaatcc agttttaaga ggagccactt
2581 atcaaagaga ttttaacagt agtaagaagg caaagaataa acatttgata ttcagcaact
2641 g Amino acid and polynucleotide sequences for a human ICOS Ligand are provided as follows (GenBank Accession No AF289028):
MRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVY QTSESKTWTYHIPQNSS LENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPλΛ/S APHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDWSVLRIARTPSVNIGCC IEIWLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAAT SILAVLCLLVVVAVAIGVCRDRCLQHSYAG AWAVSPETELTGHV 1 gagtagagcc gatctcccgc gccccgaggt tgctcctctc cgaggtctcc cgcggcccaa 61 gttctccgcg ccccgaggtc tccgcgcccc gaggtctccg cggcccgagg tctccgcccg 121 caccatgcgg ctgggcagtc ctggactgct cttcctgctc ttcagcagcc ttcgagctga 181 tactcaggag aaggaagtca gagcgatggt aggcagcgac gtggagctca gctgcgcttg 241 ccctgaagga agccgttttg atttaaatga tgtttacgta tattggcaaa ccagtgagtc 301 gaaaaccgtg gtgacctacc acatcccaca gaacagctcc ttggaaaacg tggacagccg 361 ctaccggaac cgagccctga tgtcaccggc cggcatgctg cggggcgact tctccctgcg 421 cttgttcaac gtcacccccc aggacgagca gaagtttcac tgcctggtgt tgagccaatc 481 cctgggattc caggaggttt tgagcgttga ggttacactg catgtggcag caaacttcag 541 cgtgcccgtc gtcagcgccc cccacagccc ctcccaggat gagctcacct tcacgtgtac 601 atccataaac ggctacccca ggcccaacgt gtactggatc aataagacgg acaacagcct 661 gctggaccag gctctgcaga atgacaccgt cttcttgaac atgcggggct tgtatgacgt 721 ggtcagcgtg ctgaggatcg cacggacccc cagcgtgaac attggctgct gcatagagaa 781 cgtgcttctg cagcagaacc tgactgtcgg cagccagaca ggaaatgaca tcggagagag 841 agacaagatc acagagaatc cagtcagtac cggcgagaaa aacgcggcca cgtggagcat 901 cctggctgtc ctgtgcctgc ttgtggtcgt ggcggtggcc ataggctggg tgtgcaggga 961 ccgatgcctc caacacagct atgcaggtgc ctgggctgtg agtccggaga cagagctcac 1021 tggccacgtt tgaccggagc tcaccgccca gagcgtggac agggcttcca tgagacgcca 1081 ccgtgagagg ccaggtggca gcttgagcat ggactcccag actgcagggg agcacttggg 1141 gcagccccca gaaggaccac tgctggatcc cagggagaac ctgctggcgt tggctgtgat 1201 cctggaatga ggccctttca aaagcgtcat ccacaccaaa ggcaaatgtc cccaagtgag 1261 tgggctcccc gctgtcactg ccagtcaccc acaggaaggg actggtgatg ggctgtctct 1321 acccggagcg tgcgggattc agcaccaggc tcttcccagt accccagacc cactgtgggt 1381 cttcccgtgg gatgcgggat cctgagaccg aagggtgttt ggtttaaaaa gaagactggg 1441 cgtccgctct tccaggacgg cctctgtgct gctggggtca cgcgaggctg tttgcagggg 1501 acacggtcac aggagctctt ctgccctgaa cgcttccaac ctgctccggc cggaagccac 1561 aggacccact ca
Amino acid and polynucleotide sequences for human CD40 Ligand (CD40L) are provided as follows (GenBank Accession No L07414):
MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRC NTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKG YYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAK PCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL 1 cttctctgcc agaagatacc atttcaactt taacacagca tgatcgaaac atacaaccaa
61 acttctcccc gatctgcggc cactggactg cccatcagca tgaaaatttt tatgtattta
121 cttactgttt ttcttatcac ccagatgatt gggtcagcac tttttgctgt gtatcttcat
181 agaaggttgg acaagataga agatgaaagg aatcttcatg aagattttgt attcatgaaa
241 acgatacaga gatgcaacac aggagaaaga tccttatcct tactgaactg tgaggagatt
301 aaaagccagt ttgaaggctt tgtgaaggat ataatgttaa acaaagagga gacgaagaaa
361 gaaaacagct ttgaaatgca aaaaggtgat cagaatcctc aaattgcggc acatgtcata
421 agtgaggcca gcagtaaaac aacatctgtg ttacagtggg ctgaaaaagg atactacacc
481 atgagcaaca acttggtaac cctggaaaat gggaaacagc tgaccgttaa aagacaagga
541 ctctattata tctatgccca agtcaccttc tgttccaatc gggaagcttc gagtcaagct
601 ccatttatag ccagcctctg cctaaagtcc cccggtagat tcgagagaat cttactcaga
661 gctgcaaata cccacagttc cgccaaacct tgcgggcaac aatccattca cttgggagga
721 gtatttgaat tgcaaccagg tgcttcggtg tttgtcaatg tgactgatcc aagccaagtg
781 agccatggca ctggcttcac gtcctttggc ttactcaaac tctgaacagt gtcaccttgc
841 aggctgtggt ggagctgacg ctgggagtct tcataataca gcacagcggt taagcccacc
901 ccctgttaac tgcctattta taaccctagg atcctcctta tggagaacta tttattatac
961 actccaaggc atgtagaact gtaataagtg aattacaggt cacatgaaac caaaacgggc
1021 cctgctccat aagagcttat atatctgaag cagcaacccc actgatgcag acatccagag
1081 agtcctatga aaagacaagg ccattatgca caggttgaat tctgagtaaa cagcagataa
1141 cttgccaagt tcagttttgt ttctttgcgt gcagtgtctt tccatggata atgcatttga
1201 tttatcagtg aagatgcaga agggaaatgg ggagcctcag ctcacattca gttatggttg
1261 actctgggtt cctatggcct tgttggaggg ggccaggctc tagaacgtct aacacagtgg
1321 agaaccgaaa cccccccccc cccccccgcc accctctcgg acagttattc attctctttc
1381 aatctctctc tctccatctc tctctttcag tctctctctc tcaacctctt tcttccaatc
1441 tctctttctc aatctctctg tttccctttg tcagtctctt ccctccccca gtctctcttc
1501 tcaatccccc tttctaacac acacacacac acacacacac acacacacac acacacacac
1561 acacacacac acacacacac agagtcaggc cgttgctagt cagttctctt ctttccaccc
1621 tgtccctatc tctaccacta tagatgaggg tgaggagtag ggagtgcagc cctgagcctg
1681 cccactcctc attacgaaat gactgtattt aaaggaaatc tattgtatct acctgcagtc
1741 tccattgttt ccagagtgaa cttgtaatta tcttgttatt tattttttga ataataaaga
1801 cctcttaaca ttaaaa
Modulators of immune cell costimulation
The term "modulation of immune cell costimulation" as used herein refers to a change or alteration in the biological activity of immune cell (especially T-cell) costimulation. The term "modulator" may refer to antagonists or inhibitors of immune cell costimulation, i.e. compounds which block, at least to some extent, the normal biological activity of immune cell (especially T-cell) costimulatory 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 immune cell (especially T- cell) costimulation, i.e. compounds which activate, stimulate or upregulate, at least to some extent, the normal biological activity of immune cell (especially T-cell) costimulatory proteins, polypeptides or polynucleotides. Conveniently such compounds may be referred to as upregulators or agonists.
Suitably an activator of immune cell (especially T-cell) costimulation may comprise all or part of a costimulatory protein, polypeptide or polynucleotide as described above. For example, all or part of a T-cell costimulatory protein/polypeptide may be administered or a polynucleotide coding for such a protein/polypeptide may be administered using a genetic vector as described herein.
An inhibitor of immune cell (especially T-cell) costimulation may be any agent which reduces the activity of an immune cell (especially T-cell) costimulatory protein, polypeptide or polynucleotide. For example, an inhibitor of immune cell (especially T- cell) costimulatory activity may be an immune cell (especially T-cell) costimulatory antisense polynucleotide or an antibody which binds to an immune cell (especially T-cell) costimulatory protein to reduce its activity.
Modulators of immune cell (especially T-cell) costimulation include for example, antibodies against costimulatory proteins/polypetides and proteins/polypeptides (preferably in substantially soluble forms) comprising extracellular domains of costimulatory proteins/polypeptides, such as fusion proteins fused to IgG domains as well known in the art, for example CTLA-4/Ig, OX40/Ig and ICOS/Ig.
Modulators of costimulation are suitably antibodies specific for costimulatory receptors or their ligands (eg antibodies specific for CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L), or soluble ligands or receptors (eg soluble forms of CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-L1, PD-L2, OX40 or OX40L such as lg fusion proteins). Antibodies are avilable, for example, from suppliers such as Abeam (Cambridge UK, www.abcam.com) and R& D Systems, Inc (MN, USA; rndsystems.com) and many other suppliers.
Activators of costimulation may suitably be agonist antibodies, whilst conversely inhibitors of costimulation may suitably be antagonist antibodies.
For example, the CD28-B7 interaction may be inhibited by administering a soluble ligand or receptor or antibody for CD28 or B7, e.g., a soluble CTLA4, e.g., a CTLA4 fusion protein, e.g., a CTLA4 immunoglobulin fusion, e.g, CTLA4/Ig. Preferably, the inhibitor binds B7. Alternatively anti-B7-l and/or anti-B7-2 antibodies may be administered.
In another example, the CD40 ligand-CD40 interaction may be inhibited by administering an antibody or soluble ligand or receptor for the CD40 ligand or CD40, e.g., by administering an anti-CD40L antibody, e.g., 5c8 or an antibody with similar efficacy or an antibody whose epitope overlaps that of 5c8, (see U.S. Pat No. 5,474,711, hereby incorporated by reference).
Particular examples of modulators of immune costimulation include, for example: soluble gp39 (also known as CD40 ligand (CD40L), CD154, T-BAM, TRAP), soluble CD40, soluble CD80 (e.g. ATCC 68627), soluble CD86, soluble CD28 (e.g. ATCC 68628), antibodies reactive with CD40L (e.g. ATCC HB-10916, ATCC HB-12055 and ATCC HB-12056), antibodies reactive with CD40 (e.g. ATCC HB-9110), antibodies reactive with B7 (e.g. ATCC HB-253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB-301, ATCC HB-11341, etc), and antibodies reactive with CD28 (e.g. ATCC HB-11944 or mAb 9.3 as described by Martin et al (J. Clin. Immun. 4(l):18-22, 1980).
Thus, in one preferred embodiment, the modulator of immune costimulation may be a soluble CTLA-4 protein or polypeptide, suitably comprising at least an active fragment of a CTLA-4 extracellular domain, suitably fused to a heterologous amino acid sequence such as an IgFc domain, or a polynucleotide coding for any of the foregoing, or an antibody, preferably a monclonal antibody, specific for a CLTA-4 protein.
In an alternative preferred embodiment, the modulator of immune costimulation may be a soluble ICOS or ICOS Ligand protein or polypeptide, suitably comprising at least an active fragment of an ICOS or ICOS Ligand extracellular domain, suitably fused to a heterologous amino acid sequence such as an IgFc domain, or a polynucleotide coding for any of the foregoing, or an antibody, preferably a monclonal antibody, specific for an ICOS or ICOS Ligand protein.
In an alternative preferred embodiment, the modulator of immune costimulation may be a soluble CD40 or CD40 Ligand protein or polypeptide, suitably comprising at least an active fragment of a CD40 or CD40 Ligand extracellular domain, suitably fused to a heterologous amino acid sequence such as an IgFc domain, or a polynucleotide coding for any of the foregoing, or an antibody, preferably a monclonal antibody, specific for a CD40 or CD40 Ligand protein.
In an alternative preferred embodiment, the modulator of immune costimulation may be a soluble OX40 or OX40 Ligand protein or polypeptide, suitably comprising at least an active fragment of a OX40 or OX40 Ligand extracellular domain, suitably fused to a heterologous amino acid sequence such as an IgFc domain, or a polynucleotide coding for any of the foregoing, or an antibody, preferably a monclonal antibody, specific for a OX40 or OX40 Ligand protein.
In an alternative preferred embodiment, the modulator of immune costimulation may be a soluble CD28 or B7 protein or polypeptide, suitably comprising at least an active fragment of a CD28 or B7 extracellular domain, suitably fused to a heterologous amino acid sequence such as an IgFc domain, or a polynucleotide coding for any of the foregoing, or an antibody, preferably a monclonal antibody, specific for a CD28 or B7 protein. 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 ligand interactions, upregulation or downregulation of Notch or Notch ligand 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. For example, a modulator of Notch signalling may be a protein or polypeptide for Notch signal activation, transduction, or inhibition, or a polynucleotide coding for any of the foregoing.
In one aspect, Notch signalling includes signalling events taking place extracellularly or at the cell membrane. In a further aspect, it includes signalling events taking place intracellularly, for example within the cell cytoplasm or within the cell nucleus. Modulators
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 optionally 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 substituents may form a cyclic 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 cyclic group. The cyclic group may be a polycyclic group, such as a non-fused polycyclic group. For some applications, the agent comprises at least the one of said cyclic 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 tliereof, such as Fab, F(ab')2, Fv and scFv which are capable of binding the epitopic determinant. These antibody fragments retain some ability 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 light 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 light 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).
For example, antibodies against Notch and Notch ligands are described in US 5648464, US 5849869 and US 6004924 (Yale University/Imperial Cancer Technology), the texts of which are herein incorporated by reference. Antibodies generated against the Notch receptor are also described in WO 0020576 (the text of which is also incorporated herein by reference). For example, this document discloses generation of antibodies against the human Notch-1 EGF-like repeats 11 and 12. For example, in particular embodiments, WO 0020576 discloses a monoclonal antibody secreted by a hybridoma designated A6 having the ATCC Accession No. HB 12654, a monoclonal antibody secreted by a hybridoma designated Cll having the ATCC Accession No. HB12656 and a monoclonal antibody secreted by a hybridoma designated F3 having the ATCC Accession No. HB12655. An anti-human- Jagged 1 antibody is available from R & D Systems, Inc, reference MAB12771 (Clone 188323). 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), the text of which is herein incorporated by reference (eg see in particular Example 5 therein disclosing a dextran conjugate).
Modulators may be synthetic compounds or natural isolated compounds.
As used herein the term "analogue of immune cell costimulation" includes variants thereof which retain the activity of T-cell costimulatory proteins, polypeptides or polynucleotides. By "analogue" we include a protein which has immune cell costimulatory activity, but generally has a different evolutionary origin toT-cell costimulatory 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 immune cell costimulatory 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 solid support, borne on a cell surface, or located intracellularly.
Techniques for drug 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 solid 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. Cells can also be spread as "lawns". Alternatively, non-neutralising 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 drug 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 toT-cell costimulatory 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).
Typically, a homologue of a known immune cell costimulatory protein or polynucleotide will be at least 20%, preferably at least 30%), identical at the amino acid level to the corresponding known costimulatory 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 preferably 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.nlm.nih.gov and Ausubel etal., 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 libraries with probes comprising all or part of a nucleic acid encoding a T-cell costimulatory 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 T-cell costimulatory may be brought about by introduction of a nucleic acid construct capable of activating the endogenousT-cell costimulatory 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 signalling pathway is Notch receptor/Notch ligand 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 ligases) 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 signalling preferably means specific signalling, meaning that the signalling results substantially or at least predominantly from the Notch signalling pathway, and preferably from Notch/Notch ligand interaction, rather than any other significant interfering or competing cause, such as cytokine signalling. Thus, in a preferred 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 transcriptionally activated in response to Notch signalling. 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 oϊE(spI) 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 ligand" 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 corresponding 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 (Genbank Accession No. AF003522 - Homo sapiens), Delta-3 (Genbank Accession No. AF084576 - Rattus norvegicus) and Delta-like 3 (Mus musculus) (Genbank Accession No. NM_016941 - Homo sapiens) and US
6121045 (Millennium), 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 (Genbank 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 ligands, 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 extracellular 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/N) 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 RAM23 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 extracellular ligands, such as Delta, Serrate and Scabrous, to the EGF-like repeats of Notch's extracellular domain. Delta requires cleavage for activation. It is cleaved by the ADAM disintegrin metalloprotease
Kuzbanian at the cell 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 intracellular-domain repeats mediate physical interaction with intracellular signal transduction proteins. Most notably, the cdclO/ankyrin repeats interact with Suppressor of Hairless [Su(H)]. Su(H) is t e Drosophila homologue of C-promoter binding factor-1 [CBF-1], a mammalian DNA binding protein involved in the Epstein-Barr virus-induced immortalization of B-cells. 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 ligand Delta on adjacent cells. 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 intracellular 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 cdcl 0/ankyrin repeats of the Notch intracellular domain reach the nucleus and participate in transcriptional activation. The site of proteolytic cleavage on the intracellular tail of Notch has been identified between gly 1743 and vail 744 (termed site 3, or S3) (Schroeter, E.H. et al. (1998) Nature 393j(6683}:382-6 (Schroeter)). It is thought that the proteolytic cleavage step that releases the cdcl 0/ankyrin repeats for nuclear entry is dependent on Presenilin activity.
The intracellular 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) Cell 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 mammalian Notch homologue (Lu, F. M. et al. (1996) Proc Natl Acad Sci 93(111:5663-7 (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 ligand is expressed on a cell 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 <3-linked fucose groups to the EGF-like repeats (Moloney DJ, et al. (2000) Nature 406:369-375 (Moloney), Brucker 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 Serrate/Jagged ligands but allow it to preferentially bind Delta (Panin VM, et al. (1997) Nature 387:908-912 (Panin), Hicks C, et al. (2000) Nat Cell. 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 signalling 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 tliereof, 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 intracellular 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 (hairy-enhancer 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 illustrated in Figure 1. Target genes of the Notch signalling pathway include Deltex, genes of the Hes family (Hes-1 in particular), Enhancer of Split [E(spl)] complex genes, EL-10, CD-23, CD-4 and Dll-1.
Deltex, an intracellular docking protein, replaces Su(H) as it leaves its site of interaction with the intracellular tail 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. Studies indicate that Deltex promotes Notch pathway activation by interacting with Grb2 and modulating the Ras-JNK signalling pathway (Matsuno etal. (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 intracellular 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 CBFl, is responsible for inhibiting E47 function (Ordentlich etal. (1998) Mol. Cell. Biol.18:2230-2239 (Ordentlich)). 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 Split-1) (Takebayashi K. etal. (1994) J Biol Chem 269(7): 150-6 (Takebayashi)) is a transcriptional factor with a basic helix-loop-helix 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-cell fate. Other genes from the Hes family include Hes-5 (mammalian Enhancer of Split 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. D 16464.
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 Split 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 (EL-10) was first characterised in the mouse as a factor produced by Th2 cells which was able to suppress cytokine production by Thl cells. It was then shown that EL-10 was produced by many other cell types including macrophages, keratinocytes, B cells, ThO and Thl cells. It shows extensive homology with the Epstein-Barr bcrfl gene which is now designated viral EL-10. Although a few immunostimulatory effects have been reported, it is mainly considered as an immunosuppressive cytokine. Inhibition of T cell responses by IL-10 is mainly mediated through a reduction of accessory functions of antigen presenting cells. 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. JJL-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 cells 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-cell 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. Gil 783344.
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 cell recruitment and T-helper cell 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 (distalless-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 downregulated 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 all 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 cell-cell signalling events that influence T cell fate decisions. In this signalling NotchIC localises to the nucleus and functions as an activated receptor. Mammalian 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 & Cell 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 abolishing 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-cell genes. Thus, mimicry of Notch signal transduction is involved in EBV-driven immortalization. Strobl et al (Strobl et al. (2000) J Virol 74{4): 1727-35) similarly reports that "EBNA2 may hence be regarded as a functional equivalent of an activated Notch receptor". Other EBV proteins which fall in this category include BARFO (Kusano and Raab-Truab (2001) J Virol 75(11:384-395 (Kusano and Raab-Traub)) and LMP2A.
Modulators of Notch Signalling
In one embodiment a modulator of Notch signalling may comprise a protein/polypeptide comprising all or part of a Notch ligand or a polynucleotide coding for such a protein/polypeptide.
Examples of mammalian Notch ligands identified to date include the Delta family, for example Delta-1 (Genbank Accession No. AF003522 - Homo sapiens), Delta-3 (Genbank Accession No. AF084576 - Rattus noi egicus) and Delta-like 3 (Mus uscuhis), 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 ligands 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-like repeats on the extracellular surface. It is therefore preferred that homologues of Notch ligands also comprise a DSL domain at the N-terminus and up to 14 or more EGF- like repeats on the extracellular surface.
Alternatively, the modulator may comprise all or part of the extracellular domain of a Notch receptor (eg Notch 1, Notch2, Notch3, Notch4 or homologues thereof), which can bind to Notch ligands and so reduce interactions with endogenous Notch receptors. For example, such a modulator may comprise at least the 11th and 12th domains of Notchl (EGF11 and EGF 12), as these are believed to be important for Notch ligand interaction.
For example, a rat Notch-1/Fc fusion protein is available from R& D Systems Inc
(Minneapolis, USA and Abingdon, Oxon, UK: Catalog No 1057-TK). This comprises the 12 amino teπninal EGF domains of rat Notch-1 (amino acid residues Met 1 to Glu 488) fused to the Fc region of human IgG (Pro 100 to Lys 330) via a polypeptide linker (IEGRMD).
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 ligands (based on amino acid numbering in the precursor proteins) are shown below:
Human Delta 1
Component Amino acids Proposed function/domain
SIGNAL 1-17 SIGNAL
CHAIN 18-723 DELTA-LIKE 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 function/domain
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/doi
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
DOMAIN 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 flinetion/domain
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 funcfcion/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 mammalian, preferably a human Notch ligand sequence.
It will be appreciated that the term "DSL domain" as used herein includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturally 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% amino 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 well as EGF and Notch and Notch ligands, including those involved in the blood clotting cascade (Furie and Furie, 1988, Cell 53: 505-518). For example, this motif has been found in extracellular proteins such as the blood clotting factors LX and X (Rees et al., 1988, EMBO J. 7:2053- 2061; Furie and Furie, 1988, Cell 53: 505-518), in other Drosophila genes (Knust et al., 1987 EMBO J. 761-766; Rothberg et al., 1988, Cell 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; Appella 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 following schematic representation of a typical EGF-like domain:
+ + + +
I I I I x (4) -C-x (0 , 48 ) -C-x (3 , 12 ) -C-x (l , 70) -C-x (l , 6) -C-x (2 ) -G-a-x ( 0 , 21 ) -G-x (2 ) -C-x I I
+ + wherein: 'C: conserved cysteine involved in a disulfide bond. 'G': of Ten conserved glycine 'a': of Ten conserved aromatic amino acid 'x': any residue
The region between the 5th and 6th cysteine contains two conserved glycines of which at least one is normally present in most 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 naturally 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 conventionally using known computer programs. For example, the best overall 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 algoritiim of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of the global sequence alignment 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 will be appreciated that this term includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturally occurring domains.
Suitably, for example, a Notch ligand N-terminal 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 a Notch ligand N-terminal domain of human Jagged 1. Alternatively a Notch ligand N-terminal 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 a Notch ligand N-terminal domain of human Jagged 2.
Alternatively a Notch ligand N-terminal 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 a Notch ligand N-terminal domain of human Delta 1.
Alternatively a Notch ligand N-terminal 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 a Notch ligand N-terminal domain of human Delta 3.
Alternatively a Notch ligand N-terminal 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 a Notch ligand N-terminal domain of human Delta 4.
The term "heterologous amino acid sequence" or "heterologous nucleotide sequence" as used herein means a sequence which is not found in the native sequence (eg in the case of a Notch ligand sequence is not found in the native Notch ligand sequence) or its coding sequence. Typically, for example, such a sequence may be an IgFc domain or a tag such as a V5His tag.
Whether a substance can be used for modulating Notch signalling or activating a Notch receptor may be determined using any suitable screening assay, for example, as described in our co-pending International Patent Application published as WO 03/012441, as described in the Examples herein, or in Varnum-Finney et al, Journal of Cell Science 113, 4313-4318 (2000).
Activation of Notch signalling may also be achieved by repressing inhibitors of the Notch signalling pathway. As such, polypeptides for Notch signalling activation will include molecules capable of repressing any Notch signalling inhibitors. Preferably the molecule will 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 will 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 and Polynucleotides for Notch Signalling Inhibition
Suitable nucleic acid sequences may include for example, anti-sense constructs, for example nucleic acid sequences encoding antisense Notch ligand constructs as well as antisense constructs designed to reduce or inhibit the expression of upregulators of Notch ligand expression (see above). The antisense nucleic acid may be an oligonucleotide such as a synthetic single-stranded DNA. However, more preferably, the antisense may be 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 cell.
Preferably, the nucleic acid sequence for use in the present invention is capable of inhibiting Serrate and Delta, preferably Serrate 1 and Serrate 2 as well as Delta 1, Delta 3 and Delta 4 expression in APCs such as dendritic cells. In particular, the nucleic acid sequence may be capable of inhibiting Serrate expression but not Delta expression, or Delta but not Serrate expression in APCs or T cells. Alternatively, the nucleic acid sequence for use in the present invention is capable of inhibiting Delta expression in T cells such as CD4+ helper T cells or other cells of the immune system that express Delta (for example in response to stimulation of cell surface receptors). In particular, the nucleic acid sequence may be capable of inhibiting Delta expression but not Serrate expression in T cells. In a particularly preferred embodiment, the nucleic acid sequence is capable of inhibiting Notch ligand expression in both T cells and APC, for example Serrate expression in APCs and Delta expression in T cells.
Molecules for inhibition of Notch signalling will also include polypeptides, or polynucleotides which encode therefore, capable of modifying Notch-protein expression or presentation on the cell membrane or signalling pathways. Molecules that reduce or interfere with its presentation as a fully functional cell membrane protein may include MMP inhibitors such as hydroxymate-based inhibitors.
Other substances which may be used to reduce interaction between Notch and Notch ligands are exogenous Notch or Notch ligands or functional derivatives thereof. Such Notch ligand derivatives would preferably have the DSL domain at the N-terminus and up to about 14 or more, for example between about 3 to 8 EGF-like repeats on the extracellular surface. A peptide corresponding to the Delta/Serrate/LAG-2 domain of hJaggedl and supernatants from COS cells expressing a soluble form of the extracellular portion of hJaggedl was found to mimic the effect of Jaggedl in inhibiting Notchl (Li).
Other Notch signalling pathway antagonists include antibodies which inhibit interactions between components of the Notch signalling pathway, e.g. antibodies to Notch ligands.
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" typically 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 will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will 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 cell, 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 amplified DNA can be cloned into a suitable cloning vector. Li general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing 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 10,000 bases or even more, either ribonucleotides or deoxyribonucleotides 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 perfoπned via cDNA intermediates. Generally, a nucleic acid sequence encoding the first region will 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 literature 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 available. 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 applications, preferably, the nucleotide sequence is DNA. For some applications, preferably, the nucleotide sequence is prepared by use of recombinant DNA techniques (e.g. recombinant DNA). For some applications, 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 referred to as "variants". A variant protein can be modified by addition, deletion and/or substitution of at least one amino acid present in the naturally-occurring protein.
Typically, amino 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-naturally occurring analogues.
Proteins of use 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. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, 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 amino acids with uncharged polar head groups having similar hydrophilicity 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 naturally occurring amino acids (and their associated codons) are set out below: Symbol 3-letter Meaning Codons
A Ala Alanine GCT,GCC,GCA,GCG
B As ,Asn Aspartic,
Asparagine GAT, GAC,AAT,AAC
C Cys Cysteine TGT,TGC
D Asp Aspartic GAT, GAC
E Glu Glutamic GAA,GAG
F Phe Phenylalanine TTT,TTC
G Gly Glycine GGT, GGC, GGA,GGG
H His Histidine CAT,CAC
I He Isoleucine ATT,ATC,ATA
K Lys Lysine AAA,AAG
L Leu Leucine TTG, TTA,CT ,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,AG ,AGG
S Ser Serine TCT, CC, TC , CG, GT, GC
T Thr Threonine ACT, CC, C ,ACG
V Val Valine GTT,GTC,GTA,GTG
Trp Tryptophan TGG
X Unknown
Y Tyr Tyrosine TAT, TAG z Gl , Gin Glutamic,
Glutamine GAA, AG, CAA,CAG
* End Terminator TAA,TAG,TGA
Conservative substitutions may be 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 substituted for each other:
Figure imgf000062_0001
As used herein, the term "protein" includes single-chain polypeptide molecules as well 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 typically 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 naturally-occurring sequence either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding 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 illustrative 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 stability 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 cells, as well 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%o 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 commercially 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 amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are perfoπned 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 will cause the following amino acid residues to be put out of alignment, 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 alignment 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 - will 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 smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such sof Tware 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 alignment, 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 sof Tware 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.nlm.nih.gov perform the following tasks:
blast]? - compares an amino acid query sequence against a protein sequence database.
blastn - compares a nucleotide query sequence against a nucleotide sequence database.
blasts - compares the six-frame conceptual 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 all 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). DESCRJJPTIONS - 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 will 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). Typically, 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 limit 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 enor 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 Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov). Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or pro line-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 substituted using the letter "N" in nucleotide sequence (e.g., "NNNNNNNNNNNNN") 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 all 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 all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally 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 public default values for the GCG package, or in the case of other sof Tware, the default matrix, such as BLOSUM62.
Once the sof Tware has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The sof Tware typically does this as part of the sequence comparison and generates a numerical result.
Where sequence homologies, similarities or identities are referred to according to preferred aspects of the present invention, these are preferably along substantially the full length of the reference sequence.
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 libraries made from a range of sources. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of the reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic 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 will 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 alignments can be performed using computer sof Tware known in the art. For example the GCG Wisconsin PileUp 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.
Variants and strain/species homologues may also be obtained using degenerate PCR which will 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 alignments can be performed using computer sof Tware known in the art. For example the GCG Wisconsin PileUp 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. PCR technology as described e.g. in section 14 of Sambrook et al., 1989, requires the use of oligonucleotide probes that will 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 usually based on conserved or highly homologous nucleotide sequences or regions of polypeptides. The nucleic acids used as probes may be 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 preferred 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-labelled with γ32P-labelled ATP and polynucleotide kinase. However, other methods (e.g. non-radioactive) may also be used to label the fragment or oligonucleotide, 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 silent codon changes are required to sequences to optimise codon preferences for a particular host cell 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 signalling 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 preferred 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 are 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, will be generally at least 75%, preferably at least 85 or 90% and more preferably at least 95% or 98% homologous to the coπesponding 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. Prefeπed nucleotide sequences of the invention will 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 significantly 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 implies 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 typically 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; inteπnediate 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 preferred 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 individually 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 skill in the field. As known to those of skill in the art, the stability 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 stability 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 foπn 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, 5x Denhardt'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 - 0. 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 skill 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 fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of sources. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of the reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic 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 will 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 can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer sof Tware lαiown in the art. For example the GCG Wisconsin PileUp 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 silent codon changes are required to sequences to optimise codon preferences for a particular host cell 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 recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
Longer nucleotide sequences will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will 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 cell, 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 amplified 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 genetically 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 etal and Sambrook etal, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, micro injection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection. It will be appreciated that such methods can be employed in vitro or in vivo as drug delivery systems.
Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; 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 baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, 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 of Ten advantageous to include an additional amino acid sequence which contains secretory or leader sequences or pro-sequences (such as a HIS oligomer, immunoglobulin Fc, glutathione S-transferase, FLAG etc) to aid in purification. Likewise such an additional sequence may sometimes be desirable to provide added stability during recombinant production. In such cases the additional sequence may be cleaved (eg chemically 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 cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification. Anti-sense constructs
Suitable nucleic acid sequences may include anti-senseT-cell costimulatory constructs as well as antisense constructs designed to reduce or inhibit the expression of upregulators of T-cell costimulatory 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 cell.
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 additionally 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-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluraci- 1, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -metliylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyamoinomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. 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 oligonucleotide 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 oligonucleotide 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). Oligonucleotides may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). Merely as examples, phosphorothioate oligonucleotides can be syntliesized 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 can be used for modulating Notch signalling may be determined using suitable screening assays, for example as described in the Examples herein. For example, Notch signalling can be 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" referred to herein may be any detectable manifestation attributable to the presence of the cleaved intracellular domain of Notch. Thus, increased Notch signalling can be assessed at the protein level by measuring intracellular 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 intracellular concentrations of specific mRNAs. In one preferred embodiment of the present invention, the assay is a protein assay. In another preferred 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 small samples can be analysed.
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 aπays and other hybridization methods.
In particular, gene presence, amplification and/or expression may be 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 will readily envisage how these methods may be modified, if desired.
PCR was originally developed as a means of amplifying 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 foπnation of duplicate daughter strands. RT-PCR uses an RNA template for generation of a first strand cDNA with a reverse transcriptase. The cDNA is then amplified 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 amplification 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 mixture is diluted and treated with ribonuclease (RNase) to degrade all remaining single-stranded RNA. The hybridized portion of the probe will 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 will 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 preferred. Especially preferred are GFP and luciferase. Another type of preferred reporter is cell surface markers, i.e. proteins expressed on the cell surface and therefore easily 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, constructs 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 available commercially.
Sorting of cells, based upon detection of expression of genes, may be performed by any technique known in the art, as exemplified above. For example, cells may be sorted by flow cytometry or FACS. For a general reference, see Flow Cytometry and Cell Sorting: A Laboratoiy Manual (1992) A. Radbruch (Ed.), Springer Laboratory, New York.
Flow cytometry is a powerful method for studying and purifying cells. It has found wide application, particularly in immunology and cell biology: however, the capabilities of the FACS can be applied in many other fields of biology. The acronym F.A.C.S. stands for Fluorescence Activated Cell 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 light to be scattered and fluorescent dyes to emit light at various frequencies. Photomultiplier tubes (PMT) convert light to electrical signals, which are interpreted by sof Tware to generate data about the cells. Sub- populations of cells with defined characteristics can be 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 can be 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 cells 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. Cells expressing GFP constructs will 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 can be used to identify cells expressing polypeptides individually, such that they may subsequently be sorted eitlier 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 cell sorting.
Methods have also been described for obtaining information about gene expression and identity using so-called gene chip aπays or high density DNA aπays (Chee M. et al. (1996) Science 274:601-614 (Chee)). These high density aπays are particularly useful for diagnostic and prognostic purposes. Use may also be made of In Vivo Expression Technology (TVET) (Camilli et al. (1994) Proc Natl Acad Sci US A 9J_:2634-2638 (Camilli)). 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 determine 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) Curr Biol 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 immune costimulatory 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 cells. 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 metliods 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 cells (DCs) such as interdigitating DCs or follicular DCs, Langerhans cells, PBMCs, macrophages, B-lymphocytes, or other cell types such as epithelial cells, fibroblasts or endothelial cells, activated or engineered by transfection to express a MHC molecule (Class I or JJ) on their surfaces. Precursors of APCs include CD34+ cells, monocytes, fibroblasts and endothelial 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, E -12, IFN-γ, TNF-α, JX-18 etc.). Such proteins include MHC molecules (Class I or Class II), CD80, CD86, or CD40. Most preferably DCs or DC-precursors are included as a source of APCs.
Dendritic cells (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 cells for example after mobilisation into peripheral blood by treatment with GM-CSF, or directly from bone maπow. 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) Nature 360: 258-261 (Caux)). DCs can also be routinely prepared from the peripheral blood of human volunteers, similarly to the method of Sallusto 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 may be 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 cells. Thus, it will be understood that the term "antigen presenting cell or the like" are used herein is not intended to be limited to APCs. The skilled man will understand that any vehicle capable of presenting to the T cell population may be used, for the sake of convenience the term APCs is used to refer to all these. As indicated above, preferred examples of suitable APCs include dendritic cells, L cells, hybridomas, fibroblasts, lymphomas, macrophages, B cells or synthetic APCs such as lipid membranes.
T cells
Where required, T cells 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 cells 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 cells and other cell types. It is particularly preferred to use helper T cells (CD44). Alternatively other T cells such as CD8+ cells may be used. It may also be convenient to use cell lines such as T cell hybridomas.
Exposure of agent to APCs and T cells
T cells/APCs may be cultured as described above. The APCs/T cells may be incubated with or exposed to active substances in accordance with the invention. For example, they may be prepared for administration to a patient or incubated with T cells in vitro (ex vivo).
Where treated ex-vivo, modified cells of the present invention are preferably administered to a host by direct injection into the lymph nodes of the patient Typically from 104 to 108 treated cells, 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 cell population. As used herein, the term "enriched" as applied to the cell populations of the invention refers to a more homogeneous population of cells which have fewer other cells with which they are naturally associated. An enriched population of cells can be achieved by several methods lαiown 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 cells) or negative selection (removing the undesirable cells). The technology for capturing specific cells on affinity materials is well known in the art (Wigzel, et al., J. Exp. Med., 128:23, 1969; Mage, et al., J. Imnmunol. 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-cells or malignant cells from allogeneic or autologous maπow grafts, respectively (Gee, et al., J.N.C.I. 80:154, 1988). Purification of human hematopoietic cells 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 solid 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 will 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 cells or APCs may be obtained from a patient or donor in lαiown 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-cells 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-cells and/or APCs by introducing nucleic acid constructs/viral vectors encoding the polypeptide into cells under conditions that allow for expression of the polypeptide in the T-cell and/or APC. Similarly, nucleic acid constructs encoding antisense constructs may be introduced into the T-cells and/or APCs by transfection, viral infection or viral transduction.
In a preferred embodiment, nucleotide sequences will 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 cells, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used. The promoter is typically derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell 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, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). Tissue-specific promoters specific for lymphocytes, dendritic cells, skin, brain cells and epithelial cells within the eye are particularly preferred, for example the CD2, CDl 1 c, keratin 14, Wnt-1 and Rhodopsin promoters respectively. Preferably the epithelial cell 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 (MMLV LTR) promoter, the rous sarcoma virus (RS V) 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 life-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-cells. If required, a small aliquot of cells may be tested for up-regulation of Notch signalling activity as described above. The cells may be prepared for administration to a patient or incubated with T-cells in viti'O (ex vivo).
Assays of immune response and tolerisation
Any of the assays described above (see "Assays") can be adapted to monitor or to detect reduced reactivity, modified immune responses and/or tolerisation in immune cells, and to detect suppression and enhancement of immune responses for use in clinical applications.
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) cells will demonstrate enhanced cytotoxic activity after activation. Therefore any drop in or stabilisation of cytotoxicity will be an indication of reduced reactivity.
Once activated, leukocytes express a variety of new cell surface antigens. NK cells, for example, will express transferrin 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-cell Activation: HI, 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 II antigens and an MLR3 Mab will block IL-1 binding. These antigens appear on activated T-cells within 18 hours and can therefore be used to monitor immune cell reactivity.
Additionally, leukocyte reactivity may be monitored as described in EP 0325489, which is incorporated herein by reference. Briefly this is accomplished using a monoclonal antibody ("Anti-Leu23") which interacts with a cellular 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 correlates with stimulation of the T-cell antigen receptor complex, Anti-Leu 23 is useful in monitoring the reactivity of leukocytes.
Further details of techniques for the monitoring of immune cell 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 Natural Killer Cells' Adv. Immunol. 1989 vol 47 pp 187-376; '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 with active agents according to the present invention. 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 titration. One or more active substances are then typically added to the culture medium together with the antigen of interest. The antigen may be added before, after or at substantially the same time as the substance(s). Cells are typically 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 cell 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 constructs/viral vectors encoding the polypeptide into cells under conditions that allow 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.
Preparation of Regulatory T cells (and B cells) ex vivo
The techniques described below are described in relation to T cells, but are equally applicable to B cells. The techniques employed are essentially 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 cells. For example, once the primed APCs have been prepared, they may be pelleted 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 cell will not be brought into contact with the different substance(s) used with the APC. Incubations will typically 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 signalling may be determined for a small aliquot of cells using the methods described above. T cells transfected with a nucleic acid construct directing the expression of, for example Delta, may be used as a control. Induction of immunotolerance may be determined, for example, by subsequently challenging T cells with antigen and measuring IL-2 production compared with control cells not exposed to APCs.
Primed T cells or B cells may also be used to induce immunotolerance in other T cells or B cells in the absence of APCs using similar culture techniques and incubation times.
Alternatively, T cells 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 cells may be activated with MHC-peptide complexes (e.g. tetramers).
Induction of immunotolerance may be determined by subsequently challenging T cells with antigen and measuring D -2 production compared with control cells not exposed to APCs.
T cells or B cells which have been primed in this way may be used according to the invention to promote or increase immunotolerance in other T cells or B cells.
Therapeutic Uses
Immunological indications
In the preferred embodiment the therapeutic effect results from a protein for Notch signalling. A detailed description of the Notch signalling pathway and conditions affected by it may be found in our WO98/20142, WO00/36089 and PCT/GBOO/04391. Diseased or infectious states that may be described as being mediated by T cells include, but are not limited to, any one or more of asthma, allergy, graft rejection, autoimmunity, tumour induced abeπations to the T cell system and infectious diseases such as those caused by Plasmodium species, Microfilariae, Helminths, Mycobacteria, HTV, 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 cells 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 called autoimmune diseases. The spectrum of autoimmune disorders ranges from organ specific diseases (such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis) to systemic illnesses such as rheumatoid arthritis or lupus erythematosus. Other disorders include immune hyperreactivity, such as allergic reactions.
In more detail: Organ-specific autoimmune diseases include multiple sclerosis, insulin dependent diabetes mellitus, 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 colitis).
Systemic autoimmune diseases include: rheumatoid arthritis, juvenile arthritis, scleroderma and systemic sclerosis, sjogren's syndrom, undifferentiated connective tissue syndrome, antiphospholipid syndrome, different forms of vasculitis (polyarteritis nodosa, allergic granulomatosis and angiitis, Wegner's granulomatosis, Kawasaki disease, hypersensitivity vasculitis, Henoch-Schoenlein purpura, Behcet's Syndrome, Takayasu arteritis, Giant cell arteritis, Thrombangiitis obliterans), lupus erythematosus, polymyalgia rheumatica, essentiell (mixed) cryoglobulinemia, 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, 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, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune 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, 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 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. 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 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, myelitis, encephalitis, subacute sclerosiiig pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre 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 complications 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 caπier, or inflammation associated with ADDS, to suppress or inhibit a humoral and/or cellular 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 especially useful in relation to adenocarcinomas such as: small cell 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, liver or pancreas transplants), tissue transplants (e.g. skin grafts) or cell transplants (e.g. bone maπow 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 cellular 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 complications 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 will 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 of Ten impossible because of the limited supply- of hearts and the urgency of the procedure.
3. Lung Transplantation
Lung transplantation is used (either by itself or in combination with heart transplantation) 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 controlled fashion. Indeed, transplantation of the islet cells alone could be sufficient. Because kidney failure is a frequent complication of advanced diabetes, kidney and pancreas transplants are of Ten 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 will not grow on the host and will 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 bum 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 abnormalities. The liver is a large and complicated 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 split 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 hemo lysis of recipient red blood cells if there is a mismatch of blood groups. In addition, manifestations of GVHD have occurred in liver transplants even when donor and recipient are blood-group compatible.
Cell fate/cancer indications
The present invention is also useful in methods for altering the fate of a cell, tissue or organ type by altering Notch pathway function in the cell. Thus, the present application has application in the treatment of malignant and pre-neoplastic disorders.. The present invention is especially useful in relation to adenocarcinomas such as: small cell lung cancer, and cancer of the kidney, uterus, prostrate, bladder, ovary, colon and breast. For example, malignancies 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 cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, choriocarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma seminoma, embryonal carcinoma, cervical cancer, testicular tumour, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, medulloblastoma, craniopharyngioma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.
The present invention may also have application 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 HIV, 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 neuropatliy, 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 juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
Vaccines and Cancer Vaccines
Certain combinations of active agents of the present invention may also for example be used in therapeutic and prophylactic vaccine compositions such as cancer and pathogen vaccines (eg where it is desired to increase an immune response to an antigen or antigenic deteπninant). Vaccine Compositions
Agents according to the present invention which inhibit Notch signalling (eg as determined by use of assays as described herein) may be employed in vaccine compositions (such as pathogen or cancer vaccines) to protect or treat a mammal susceptible to, or suffering from disease, by means of administering said vaccine via a mucosal route, such as the oral/bucal/intestinal/vaginal/rectal or nasal route. Such administration may for example be in a droplet, spray, or dry powdered form. Nebulised or aerosolised vaccine formulations may also be used where appropriate.
Enteric formulations such as gastro resistant capsules and granules for oral administration, suppositories for rectal or vaginal administration may also be used. The present invention may also be used to enhance the immunogenicity of antigens applied to the skin, for example by intradermal, transdermal or transcutaneous delivery. In addition, the adjuvants of the present invention may be parentally delivered, for example by intramuscular or subcutaneous administration.
Depending on the route of administration, a variety of administration devices may be used. For example, for intranasal administration a spray device such as the commercially available Accuspray (Becton Dickinson) may be used.
Preferred spray devices for intranasal use are devices for which the performance of the device is not dependent upon the pressure applied by the user. These devices are known as pressure threshold devices. Liquid is released from the nozzle only when a threshold pressure is attained. These devices make it easier to achieve a spray with a regular droplet size. Pressure threshold devices suitable for use with the present invention are known in the art and are described for example in WO 91/13281 and EP 311 863 B. Such devices are commercially available from Pfeiffer GmbH.
For certain vaccine formulations, other vaccine components may be included in the formulation. For example the adjuvant formulations of the present invention may also comprise a bile acid or derivative of cholic acid. Suitably the derivative of cholic acid is a salt thereof, for example a sodium salt thereof. Examples of bile acids include cholic acid itself, deoxycholic acid, chenodeoxy colic acid, lithocholic acid, taurodeoxycholate ursodeoxycholic acid, hyodeoxycholic acid and derivatives like glyco-, tauro-, amidopropyl-1- propanesulfonic- and amidopropyl-2-hydroxy-l-propanesulfonic- derivatives of the above bile acids, or N, N-bis (3DGluconoamidopropyl) deoxycholamide.
Suitably, an adjuvant formulation of the present invention may be in the form of an aqueous solution or a suspension of non-vesicular forms. Such formulations are convenient to manufacture, and also to sterilise (for example by terminal filtration through a 450 or 220 nm pore membrane).
Suitably, the route of administration may be via the skin, intramuscular or via a mucosal surface such as the nasal mucosa. When the admixture is administered via the nasal mucosa, the admixture may for example be administered as a spray. The methods to enhance an immune response may be either a priming or boosting dose of the vaccine.
The term "adjuvant" as used herein includes an agent having the ability to enhance the immune response of a vertebrate subject's immune system to an antigen or antigenic determinant.
The term "immune response" includes any response to an antigen or antigenic determinant by the immune system of a subject. Immune responses include for example humoral immune responses (e. g. production of antigen-specific antibodies) and cell- mediated immune responses (e. g. lymphocyte proliferation).
The term "cell-mediated immune response" includes the immunological defence provided by lymphocytes, such as the defence provided by T cell lymphocytes when they come into close proximity with their victim cells.
When "lymphocyte proliferation" is measured, the ability of lymphocytes to proliferate in response to specific antigen may be measured. Lymphocyte proliferation includes B cell, T-helper cell or CTL cell proliferation.
Compositions of the present invention may be used to formulate vaccines containing antigens derived from a wide variety of sources. For example, antigens may include human, bacterial, or viral nucleic acid, pathogen derived antigen or antigenic preparations, host-derived antigens, including GnRH and IgE peptides, recombinantly produced protein or peptides, and chimeric fusion proteins.
Preferably the vaccine formulations of the present invention contain an antigen or antigenic composition capable of eliciting an immune response against a human pathogen. The antigen or antigens may, for example, be peptides/proteins, polysaccharides and lipids and may be derived from pathogens such as viruses, bacteria and parasites/fungi as follows:
Viral antigens
Viral antigens or antigenic determinants may be derived, for example, from:
Cytomegalovirus ( especially Human, such as gB or derivatives thereof); Epstein Barr virus (such as gp350); flaviviruses (e. g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus); hepatitis virus such as hepatitis B virus (for example Hepatitis B Surface antigen such as the PreSl, PreS2 and S antigens described in EP-A-414 374; EP-A-0304 578, and EP-A-198474), hepatitis A virus, hepatitis C virus and hepatitis E virus; HTV-l, (such as tat, nef, gpl20 or gplδO); human herpes viruses, such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HS VI or HS V2; human papilloma viruses (for example HPV6, 11 , 16, 18); Influenza virus (whole live or inactivated virus, split influenza virus, grown in eggs or MDCK cells, or Vero cells or whole flu virosomes (as described by Gluck, Vaccine, 1992,10, 915-920) or purified or recombinant proteins thereof, such as NP, NA, HA, or M proteins); measles virus; mumps virus; parainfluenza virus; rabies virus; Respiratory Syncytial virus (such as F and G proteins); rotavirus (including live attenuated viruses); smallpox virus; Varicella Zoster Virus (such as gpl, II and IE63); and the HPV viruses responsible for cervical cancer (for example the early proteins E6 or E7 in fusion with a protein D carrier to form Protein D-E6 or E7 fusions from HPV 16, or combinations thereof; or combinations of E6 or E7 with L2 (see for example WO 96/26277).
Bacterial antigens
Bacterial antigens or antigenic determinants may be derived, for example, from: Bacillus spp., including B. anthracis (eg botulinum toxin); Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin, filamenteous hemagglutinin, adenylate cyclase, fimbriae); Borrelia spp., including B. burgdorferi (eg OspA, OspC, DbpA, DbpB), B. garinii (eg OspA, OspC, DbpA, DbpB), B. afzelii (eg OspA, OspC, DbpA, DbpB), B. andersonii (eg OspA, OspC, DbpA, DbpB), B. hermsii; Campylobacter spp, including C. jejuni (for example toxins, adhesins and invasins) and C. coli;
Chlamydia spp., including C. trachomatis (eg MOMP, heparin-binding proteins), C. pneumonie (eg MOMP, heparin-binding proteins), C. psittaci; Clostridium spp., including C. tetani (such as tetanus toxin), C. botulinum (for example botulinum toxin), C. difficile (eg clostridium toxins A or B); Corynebacterium spp., including C. diphtheriae (eg diphtheria toxin); Ehrlichia spp., including E. equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including Rrickettsii; Enterococcus spp., including E. faecalis, E. faecium; Escherichia spp, including enterotoxic E. coli (for example colonization factors, heat-labile toxin or derivatives thereof, or heat-stable toxin), enterohemoπagic E. coli, enteropathogenic E. coli (for example shiga toxin-like toxin); Haemophilus spp., includingΗ. influenzae type B (eg PRP), non-typable H. influenzae, for example OMP26, high molecular weight adhesins, P5, P6, protein D and hpoprotein D, and fimbrin and fimbrin derived peptides (see for example US 5,843,464), Hehcobacter spp, including H pylori (for example urease, catalase, vacuolating toxin), Pseudomonas spp, including P aeruginosa, Legionella spp, including L pneumophila , Leptospira spp , including L inteπogans, Listeria spp , including L monocytogenes, Moraxella spp, including M cataπhalis, also known as Branhamella catarrhalis (for example high and low molecular weight adhesins and invasins), Morexella Catarrhalis (including outer membrane vesicles thereof, and OMP106 (see for example W097/41731)), Mycobactenum spp , including M tuberculosis (for example ES AT6, Antigen 85A, -B or -C), M bovis, M leprae, M avium, M paratuberculosis, M smegmatis, Neisseria spp, including N gonorrhea and N menmgitidis (for example capsular polysacchaπdes and conjugates thereof, transferπn- bmding proteins, lactoferπn binding proteins, PilC, adhesins), Neisseria mengitidis B (including outer membrane vesicles thereof, and NspA ( see for example WO 96/29412), Salmonella spp, including S typhi, S paratyphi, S choleraesuis, S enteπtidis, Shigella spp, including S sonnei, S dysenteπae, S flexneπi, Staphylococcus spp , including S aureus, S epidermidis, Streptococcus spp, including S pneumome (eg capsular polysacchaπdes and conjugates thereof, PsaA, PspA, streptolysin, chohne-binding proteins) and the protein antigen Pneumolysin (Biochem Biophys Acta, 1989,67,1007, Rubins et al , Microbial Pathogenesis, 25,337-342), and mutant detoxified derivatives thereof (see for example WO 90/06951 , WO 99/03884), Treponema spp , including T palhdum (eg the outer membrane proteins), T denticola, T hyodysenteπae, Vibrio spp, including V cholera (for example cholera toxin), and Yersinia spp, including Y enterocolitica (for example a Yop protein), Y pestis, Y pseudotuberculosis
Parasite/Fungal antigens
Parasitic/fungal antigens or antigenic determinants may be derived, for example, from
Babesia spp , including B microti, Candida spp , including C albicans, Cryptococcus spp , including C neoformans, Entamoeba spp , including E histolytica, Giardia spp , including ,G lambha, Leshmama spp , including L major, Plasmodium. faciparum (MSP1, AMAl, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin, PfEMPl, Pf332, LSA1, LSA3, STARP, SALSA, PfEXPl, Pfs25, Pfs28, PFS27/25, Pfsl6, Pfs48/45, Pfs230 and their analogues in Plasmodium spp.); Pneumocystis spp., including P. ;carinii; Schisostoma spp., including S. mansoni; Trichomonas spp., including T. vaginalis; Toxoplasma spp., including T. gondii (for example SAG2, SAG3, Tg34); Trypanosoma spp., including T. cruzi.
Approved/licensed vaccines include, for example anthrax vaccines such as Biothrax (BioPort Corp); tuberculosis (BCG) vaccines such as TICE BCG (Organon Teknika Corp) and Mycobax (Aventis Pasteur, Ltd); diphtheria & tetanus toxoid and acellular pertussis (DTP) vaccines such as Tripedia (Aventis Pasteur, Inc), Infanrix (GlaxoSmithKline), and DAPTACEL (Aventis Pasteur, Ltd); Haemophilus b conjugate vaccines (eg diphtheria CRM197 protein conjugates such as HibTITER from Lederle Lab Div, American Cyanamid Co; meningococcal protein conjugates such as PedvaxHIB from Merck & Co, Inc; and tetanus toxoid conjugates such as ActHIB from Aventis Pasteur, SA); Hepatitis A vaccines such as Havrix (GlaxoSmithKline) and VAQTA (Merck & Co, Inc); combined Hepatitis A and Hepatitis B (recombinant) vaccines such as Twinrix (GlaxoSmithKline); recombinant Hepatitis B vaccines such as Recombivax HB (Merck & Co, Inc) and Engerix-B (GlaxoSmithKline); influenza virus vaccines such as Fluvirin (Evans Vaccine), FluShield (Wyeth Laboratories, Inc) and Fluzone (Aventis Pasteur, Inc); Japanese Encephalitis virus vaccine such as JE-Vax (Research Foundation for Microbial Diseases of Osaka University); Measles virus vaccines such as Attenuvax (Merck & Co, Inc); measles and mumps virus vaccines such as M-M-Vax (Merck & Co, Inc); measles, mumps, and rubella virus vaccines such as M-M-R II (Merck & Co, Inc); meningococcal polysaccharide vaccines (Groups A, C, Y and W-135 combined) such as Menomune-A C/Y/W-135 (Aventis Pasteur, Inc); mumps virus vaccines such as Mumpsvax (Merck & Co, Inc); pneumococcal vaccines such as Pneumovax (Merck & Co, Inc) and Pnu-hnune (Lederle Lab Div, American Cyanamid Co); Pneumococcal 7- valent conjugate vaccines (eg diphtheria CRMl 97 Protein conjugates such as Prevnar from Lederle Lab Div, American Cyanamid Co); poliovims vaccines such as Poliovax (Aventis Pasteur, Ltd); poliovirus vaccines such as IPOL (Aventis Pasteur, SA); rabies vaccines such as Imovax (Aventis Pasteur, S A) and RabAvert (Chiron Behring GmbH & Co); rubella virus vaccines such as Meruvax JJ (Merck & Co, Inc); Typhoid Vi polysaccharide vaccines such as TYPHLM Vi (Aventis Pasteur, S A); Varicella virus vaccines such as Varivax (Merck & Co, L e) and Yellow Fever vaccines such as YF-Vax (Aventis Pasteur, Inc).
Cancer/Tumour antigens
The term "cancer antigen or antigenic determinant" or "tumour antigen or antigenic determinant" as used herein preferably means an antigen or antigenic determinant which is present on (or associated with) a cancer cell and not typically on normal cells, or an antigen or antigenic determinant which is present on cancer cells in greater amounts than on normal (non-cancer) cells, or an antigen or antigenic determinant which is present on cancer cells in a different form than that found on normal (non-cancer) cells.
Cancer antigens include, for example (but without limitation): beta chain of human chorionic gonadotropin (hCGbeta) antigen, carcinoembryonic antigen, EGFRvHJ antigen, Globo H antigen, GM2 antigen, GP100 antigen, HER2/neu antigen, KS A antigen, Le (y) antigen, MUCI antigen, MAGE 1 antigen, MAGE 2 antigen, MUC2 antigen, MUC3 antigen, MUC4 antigen, MUC5AC antigen, MUC5B antigen, MUC7 antigen, PSA antigen, PSCA antigen, PSMA antigen, Thompson- Friedenreich antigen (TF), Tn antigen, sTn antigen, TRP 1 antigen, TRP 2 antigen, tumor-specific immunoglobulin variable region and tyrosinase antigen.
It will be appreciated that in accordance with this aspect of the present invention antigens and antigenic determinants may be used in many different forms. For example, antigens or antigenic determinants may be present as isolated proteins or peptides (for example in so-called "subunit vaccines") or, for example, as cell-associated or virus-associated antigens or antigenic determinants (for example in either live or killed pathogen strains). Live pathogens will preferably be attenuated in known manner. Alternatively, antigens or antigenic determinants may be generated in situ in the subject by use of a polynucleotide coding for an antigen or antigenic determinant (as in so-called "DNA vaccination", although it will be appreciated that the polynucleotides which may be used with this approach are not limited to DNA, and may also include RNA and modified polynucleotides as discussed above).
Administration
Suitably the active agents are administered in combination with a pharmaceutically acceptable carrier or diluent. The pharmaceutically acceptable caπier or diluent 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 will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical caπier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
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, intranasally or in the form of aerosol, or in the form of a suppository or pessary, or they may be applied topically 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 can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid 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 sof T paraffin base together with such stabilisers 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 delivery 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, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof. The routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes. Active agents may be adminstered by conventional DNA delivery techniques, such as DNA vaccination etc., or injected or otherwise delivered 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 therapeutically 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.
Typically, the physician will determine the actual dosage which will be most suitable for an individual patient and it will 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 intracavemosally, intravenously, intramuscularly or subcutaneously
For parenteral administration, active agents may be used in the form of a sterile aqueous solution which may suitably 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 typically 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 will determine the actual dosage which will be most suitable for an individual patient and it will 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 skilled practitioner will 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.
By "simultaneously" is meant that the agents are administered at substantially the same time, and preferably together in the same formulation.
By "contemporaneously" it is meant that the agents are administered closely in time, e.g., a modulator of immune cell costimulation is administered within from about one minute to within about one day before or after the modulator of the Notch signalling pathway is administered. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the modulator of the Notch signalling pathway and the modulator of immune cell costimulation will be administered within about one minute to within about eight hours, and preferably within less than about one to about four hours. When administered contemporaneously, the modulator of the Notch signalling pathway and the modulator of immune cell costimulation are suitably administered at the same site on the animal. The term "same site" includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters.
The term "separately" as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The active agents may be administered in either order. - I l l -
Likewise, the modulator of the Notch signalling pathway may be administered more frequently than the modulator of immune cell costimulation or vice versa.
The term "sequentially" as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the active agents may be administered in a regular repeating cycle.
It will be appreciated that the methods, uses and compositions of the present invention may be administered either in-vivo or ex-vivo. 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.
The term treatment or therapy as used herein should be taken to encompass diagnostic and prophylatic applications.
The treatment of the present invention includes both human and veterinary applications.
Modified/Humanised antibodies
Preferably, antibodies for use to treat human patients will be chimeric or humanised antibodies. Antibody "humanisation" techniques are well known in the art. These techniques typically involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule.
As described in US5859205 early methods for humanising monoclonal antibodies (Mabs) involved production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody is linked to constant domains derived from another antibody. Such chimerisation procedures are described in EP-A-0120694 (Celltech Limited), EP-A-0125023 (Genentech Inc. and City of Hope), EP-A-0 171496 (Res. Dev. Corp. Japan), EP-A-0 173 494 (Stanford University), and WO 86/01533 (Celltech Limited). For example, WO 86/01533 discloses a process for preparing an antibody molecule having the variable domains from a mouse MAb and the constant domains from a human immunoglobulin.
In an alternative approach, described in EP-A-0239400 (Winter), the complementarity determining regions (CDRs) of a mouse MAb are grafted onto the framework regions of the variable domains of a human immunoglobulin by site directed mutagenesis using long oligonucleotides. Such CDR-grafted humanised antibodies are much less likely to give rise to an anti-antibody response than humanised chimeric antibodies in view of the much lower proportion of non-human amino acid sequence which they contain. Examples in which a mouse MAb recognising lysozyme and a rat MAb recognising an antigen on human T-cells were humanised by CDR-grafting have been described by Verhoeyen et al (Science, 239, 1534-1536, 1988) and Riechmann etal (Nature, 332, 323-324, 1988) respectively. The preparation of CDR-grafted antibody to the antigen on human T cells is also described in WO 89/07452 (Medical Research Council).
In WO 90/07861 Queen et al propose four criteria for designing humanised immunoglobulins. The first criterion is to use as the human acceptor the framework from a particular human immunoglobulin that is unusually homologous to the non-human donor immunoglobulin to be humanised, or to use a consensus framework from many human antibodies. The second criterion is to use the donor amino acid rather than the acceptor if the human acceptor residue is unusual and the donor residue is typical for human sequences at a specific residue of the framework. The third criterion is to use the donor framework amino acid residue rather than the acceptor at positions immediately adjacent to the CDRs. The fourth criterion is to use the donor amino acid residue at framework positions at which the amino acid is predicted to have a side chain atom within about 3 A of the CDRs in a three-dimensional immunoglobulin model and to be capable of interacting with the antigen or with the CDRs of the humanised immunoglobulin. It is proposed that criteria two, three or four may be applied in addition or alternatively to criterion one, and may be applied singly or in any combination.
Antigens and Allergens
In one embodiment, the agents of the present invention may be administered in simultaneous, separate or sequential combination with antigens or antigenic determinants (or polynucleotides coding therefor), to modify (increase or decrease) the immune response to such antigens or antigenic determinants.
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 generally either cell mediated (T cell mediated killing) or humoral (antibody production via recognition of whole antigen). The pattern of cytokine production by TH cells involved in an immune response can influence which of these response types predominates: cell mediated immunity (TH1) is characterised by high JX-2 and IFNγ but low EL-4 production, whereas in humoral immunity (TH2) the pattern is low IL-2 and IFNγ but high D -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 cells, then these cells will drive a cellular response, whereas TH2 cells will drive an antibody-dominated response. The type of antibodies responsible for some allergic reactions is induced by TH2 cells.
The antigen or allergen (or antigenic determinant thereof) 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 cells or virus/viral component In particular, for reduction of immune responses it may be preferred to use allergens such as dust mite, pollen or food allergens to treat or prevent allergy; autoantigens associated with autoimmune diseases such as myelin basic protein (associated with multiple sclerosis), collagen (associated with rheumatoid arthritis), and insulin (diabetes) to treat or prevent autoimmune disease; or graft antigens associated with rejection of non-self tissue such as MHC antigens or antigenic determinants thereof to treat or prevent graft rejection. An increased immune response will typically be appropriate for use with vaccine antigens such as pathogen or cancer antigens or antigenic determinants thereof to treat or prevent infectious disease or cancer.
Polynucleotides coding for antigens or antigenic determinants which may be expessed in a subject may also be used.
Various prefeπed features and embodiments of the present invention will now be described in more detail by way of non-limiting examples.
Example 1
Gene expression profiling
(ϊ) 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 Genbank 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 CACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADS AFSNPI
RFPFGFTWPGTFSLΠEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLH
SSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWK
GPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQP
WQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGA TCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNG GRCSDSPDGGYSCRCPVGYSGFNCEKKΓDYCSSSPCSNGAKCVDLGDAYLCRCQ AGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCE
HAPCHNGATCHERGHGYVCECARGYGGPNCOFLLPELPPGPAWDLTEKLEAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLO SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEF LGGPSWLFPPEJKDTLMISRTPEVTCVVVDVSOEDPEVOFNWYVDGVEVHNAK TKPREEOFNSTYRVVSVLTVLHOD^VLNGKΈYKCKVSNKGLPSSIEKTISKAKGOP REPOVYTLPPSOEEMTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWOEGNVFSCSVMHEALHNHYTOKSLSLSLGK
Wherein the first underlined sequence is the signal peptide (cleaved from the mature protein) and the second underlined sequence is the IgG4 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), Penicillin-Streptomycin 50 units/ml (GibcoBRL), 50μM 2-mercaptoethanol and with 10% Fetal Bovine Serum (FBS) (Biochrom KG).
Anti-human IGg4 anti-CD3 (human), anti-CD28 (human) antibodies (PharMingen) were plated at 5 μg/ml in phosphate buffer saline (Gibco BRL) in 6 well tissue culture dishes (1ml PBS/well) overnight. Anti-IgG4 antibody was applied to every well, while mouse IgGi K isotype control at 10 μg/ml was applied in wells 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 lOμg/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 lxl 06 cells /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 ligand stimulation, parallel cultures of cells 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 instructions. The optional DNase step recommended was also performed. A phenol extraction step was performed to ensure the removal of proteins in the RNA. RNA was then amplified using the MessageAmp aRNA Kit (Ambion) following the manufacturer's recommendations. Briefly, the procedure consists of reverse transcription with an oligo(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
Microarrays were manufactured by spotting purified PCR products onto glass slides. Microaπay probes were prepared by labelling 2μg of αRNA by a reverse transcriptase reaction incorporating dCTP-Cy3 or dCTP-Cy5 labelled nucleotide. Probe labelling and purification were then performed generally as described in Hegde P, Qi R, Abernathy K, Gay C, Dharap S, Gaspard R, Hughes JE, Snesrud E, Lee N, Quackenbush J: A concise guide to cDNA microarray 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. Slides 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 finally 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 slide 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-multiplier tube efficiency (depending on background). Slide images were processed as follows: Array spots representing the signal associated with individual spotted clones were identified and quantified using the Quantarray 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 all data analyses the GeneSpring package (Silicon 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 labelled 'CD3/CD28 plus Delta' were regarded as 'signals' while 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, while when the ratio was <0.5 the ratio the gene was considered to be downregulated.
Results for CD28 and ICOS 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 7.
Example 2
Real Time PCR analysis of primary stimulated CD4+ cells
T-cells from Example 1 were harvested at 4 and 16 hours. Total cellular RNA was iissoollaatteedd uussiinngg tthhee R RNNeeaassyy™ RNA isolation kit (Qiagen, Crawley, UK) according to the manufacturer's guidelines.
In each case lμg of total RNA was reverse transcribed using Superscript™ IJ Reverse Transcriptase (Invitrogen, Paisley, UK) using Oligo dT(12-18) or a random decamer mix according to the manufacturer's guidelines. After synthesis, Oligo dT(12-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 following HPLC-purified primer pairs were used for cDNA-specific amplification (5' to 3'):
mouse I8s rRNA: Forward GTAACCCGTTGAACCCCATT Reverse CCATCCAATCGGTAGTAGCG
ICOS; Forward TGCCAGACTACAGCCACACTTTG Reverse GTACTTCCCAGATCCCAGAGACCA
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 serially-diluted gene-specific amplicons which had been previously cloned into a plasmid vector (pCR2.1, Invitrogen). These serial dilutions fall 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. ICOS was then analysed by the same method using serially-diluted ICOS specific standards, and the ICOS value divided by the 18S rRNA value to generate a value, which represents the relative expression of ICOS in each cDNA sample. All Cp analysis was performed using the Second Derivative Maximum algorithm within the Lightcycler system software.
Results (ICOS expression relative to 18S rRNA expression at different concentrations of lιDeltal-IgG4Fc) are shown in Figure 8. Example 3
Gene expression profiling in Jurkat cells (CD80)
(i) Cell culture, treatments and RNA extraction
Jurkat cells were cultured in RPMI 1640 (GibcoBRL) supplemented with 2mM Glutamine (GibcoBRL), Penicillin-Streptomycin 50 units/ml (GibcoBRL) and with 10% Fetal Bovine Serum (FBS) (Biochrom KG).
Anti-V5 (Invitrogen) and anti-CD3 (human), anti-CD28 (human) antibodies (PharMingen) were plated at 5 μg/ml in phosphate buffer saline (Gibco BRL) in 6 well tissue culture dishes (1ml PBS/well) overnight. Anti-V5 antibody was applied to every well, while mouse IgGt K isotype control at 10 μg/ml was applied in wells that no anti- CD3 or anti-CD28 was used. The next day the wells were washed 3 times with PBS, and Delta-V5-His protein was plated at 5μg/ml PBS (lml/well). The plates were then incubated at 37°C for 2 hours and then washed with PBS three times. Jurkat cells were then plated out at a concentration of 2x10δ cells /ml and incubated at 37°C. Ionomycin was added to the appropriate wells at a concentration of 1 μg/ml (Sigma). Cells were taken out at 2, 4, 8, 18, 24, 36, 48 hrs, washed once with PBS at 4°C and collected at 300- 600 μl RLT lysis solution (Qiagen). hi order to ensure the efficacy of the stimulation, cells were tested for the correct expression of T cell activation markers using FACs analysis. The cells used in this experiment were all expressing CD69 (early activation marker) after 48h of anti-CD3, anti-CD28 activation.
RNA was extracted using an RNA Easy miniprep kit (Qiagen) according to the manufacturer's instructions. The optional DNase step recommended was also performed. A phenol extraction step was performed to ensure the complete lack of proteins in the RNA. RNA was then amplified using the MessageAmp aRNA Kit (Ambion) following the manufacturer's recommendations. Briefly, the procedure consists of reverse transcription with an oligo(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.
The nomenclature used was as follows: RNA from cells that were plated on wells treated only with V5 was labelled 'V5', while RNA from cells plated on wells treated with anti- V5 and Delta- V5-His was labelled 'Delta'. RNA from cells plated on wells treated with anti-V5, anti-CD3, anti-CD28 were labelled 'CD3CD28' while RNA from cells plated on wells treated with anti-V5, anti-CD3, anti-CD28, Delta-V5-His was labelled 'CD3CD28Delta'. Similarly RNA from cells plated on anti-V5 and further treated with ionomycin was labelled 'ionomycin' while RNA from cells plated on anti-V5, Delta- V5- His and further treated with ionomycin were labelled 'ionomycin-Delta'.
(ii) Gene Expression Profiling
Microarrays were manufactured by spotting purified PCR products onto glass slides. Microarray probes were prepared by labelling 2μg of αRNA by a reverse transcriptase reaction incorporating dCTP-Cy3 or dCTP-Cy5 labelled nucleotide. Probe labelling and purification were then performed generally as described in Hegde P, Qi R, Abernathy K, Gay C, Dharap S, Gaspard R, Hughes JE, Snesrud E, Lee N, Quackenbush 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. Slides 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 finally once in 0.2%SSC at room temperature. For each time point the sample named 'V5' was labelled with dCTP-Cy3 and hybridized on the same slide as the sample named 'Delta' that was labelled with dCTP-Cy5. Similarly the sample named CD3CD28 was labelled with dCTP-Cy3 and hybridized on the same slide as the sample named 'CD3CD28Delta' that was labelled with dCTP-Cy5. Finally the sample named 'ionomycin' was labelled with dCTP-Cy3 and hybridized on the same slide as the sample labelled 'ionomycinDelta' that was labelled with dCTP-Cy5 (see Table-1).
Table-1
Figure imgf000123_0001
Once dried the slides were scanned on a GSI Lumonics confocal scanner at 100% laser power and 65-75% photo-multiplier tube efficiency (depending on background). Slide images were processed as follows: 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).
(iii) Data Processing
For all data analyses the GeneSpring package (Silicon 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 labelled 'Delta' or 'CD3CD28Delta', or 'ionomycinDelta' were regarded as 'signals' while the intensities from genes from samples labelled either 'V5' or 'CD3CD28' or 'ionomycin' were regarded as 'controls'.
Ratio= signal strength of gene in 'Delta'/ control strength of gene in 'V5' Ratio= signal strength of gene in 'CD3CD28DeltaV control strength of gene in
'CD3CD28'
Ratio= signal strength of gene in 'ionomycinDelta'/ control strength of gene in
'ionomycin'
When this ratio was >2 the gene was considered to be upregulated, while when the ratio was <0.5 the ratio the gene was considered to be downregulated.
Results for CD80 expression (fold upregulation compared to control) at intervals from 2 to 72 hours with either 10 or 80 micrograms/ml hDeltal-IgG4Fc are shown in Figure 9.
Example 4
Pynabeads ELISA Assay Method For Detecting Notch Signalling Activity
(i) CD4+ cell purification
Spleens were removed from mice (variously Balb/c females, 8-10 weeks, C57B/6 females, 8-10 weeks, D011.10 transgenic 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 cell suspension was spun (1150rpm 5 min) and the media removed.
The cells were incubated for 4 minutes with 5ml ACK lysis buffer (0.15M NH4CI, 1.0M KHC03, O.lmM Na2EDTA in double distilled water) per spleen (to lyse red blood cells). The cells were then washed once with R10F medium and counted. CD4+ cells were purified from the suspensions by positive selection on a Magnetic Associated Cell 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) Antibody Coating
96 well flat-bottomed plates were coated with DPBS plus 1 μg/ml anti-hamsterlgG antibody (Pharmingen Cat No 554007) plus 1 μg/ml anti-IgG4 antibody. lOOμl of coating mixture was added per well. Plates were incubated overnight at 4°C then washed with DPBS. Each well then received either lOOμl DPBS plus anti-CD3 antibody (1 μg/ml) or, lOOμl DPBS plus anti-CD3 antibody (1 μg/ml) plus hDeltal-IgG4Fc fusion protein (lOμg/ml; as described above). The plates were incubated for 2-3 hours at 37°C then washed again with DPBS before cells (prepared as described above) were added.
(iii) Primary Polyclonal Stimulation and ELISA
CD4+ cells were cultured in 96 well, flat-bottomed plates pre-coated according to (ii) above. Cells 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.
125μl supernatant was then removed from each well and stored at -20°C until tested by ELISA for IL-2, IL-10, IFNg and IL-13 using antibody pairs from R & D Systems (Abingdon, UK). Example 5
Luciferase assay for detecting Notch signalling activity
hDeltal-IgG4Fc fusion protein (Example 1) was immobilised on Streptavidin-Dynabeads (CELLection Biotin Binder Dynabeads [Cat. No. 115.21] at 4.0 x 108 beads/ml from Dynal (UK) Ltd; "beads") in combination with biotinylated α-IgG-4 (clone JDC14 at 0.5 mg/ml from Pharmingen [Cat. No. 555879]) as follows:
1 x 107 beads (25 μl of beads at 4.0 x 108 beads/ml) and 2 μg biotinylated α-IgG-4 was used for each sample assayed. PBS was added to the beads to 1 ml and the mixture was spun down at 13,000 rpm for 1 minute. Following washing with a further 1 ml of PBS the mixture was spun down again. The beads were then resuspended in a final volume of 100 μl of PBS containing the biotinylated α-IgG-4 in a sterile Eppendorf tube and placed on a shaker at room temperature for 30 minutes. PBS to was added to 1 ml and the mixture was spun down at 13,000 rpm for 1 minute and then washed twice more with 1 ml of PBS.
The mixture was then spun down at 13,000 rpm for 1 minute and the beads were resupsended in 50 μl PBS per sample. 50 μl of biotinylated α-IgG-4 -coated beads were added to each sample and the mixture was incubated on a rotary shaker at 4 °C overnight.
The tube was then spun at 1000 rpm for 5 minutes at room temperature.
The beads then were washed with 10 ml of PBS, spun down, resupended in 1 ml of PBS, transferred to a sterile Eppendorf tube, washed with a further 2 x 1 ml of PBS, spun down and resuspended in a final volume of 100 μl of DMEM plus 10%(HI)FCS plus glutamine plus P/S, i.e. at 1.0 x 105 beads/μl.
Stable N27#ll cells (CHO cells expressing full length human Notcl 2 and a CBF1- luciferase reporter construct; T8o flask; as described in WO 03/012441, Lorantis, eg see Example 7 therein) were removed using 0.02% EDTA solution (Sigma), spun down and resuspended in 10 ml DMEM plus 10%(ffl)FCS plus glutamine plus P/S. 10 μl of cells were counted and the cell density was adjusted to 1.0 x 105 cells/ml with fresh DMEM plus 10%(HI)FCS plus glutamine plus P/S. 1.0 x 105 of the cells were plated out per well of a 24- well plate in a 1 ml volume of DMEM plus 10%(HI)FCS plus glutamine plus P/S and cells were placed in an incubator to settle down for at least 30 minutes.
20 μl of beads were then added in duplicate to a pair of wells to give 2.0 x 106 beads / well (100 beads / cell). The plate was left in a C02 incubator overnight. Supernatant was then removed from all the wells, 100 μl of SteadyGlo™ luciferase assay reagent (Promega) was added and the resulting mixture left at room temperature for 5 minutes.
The mixture was then pipetted up and down 2 times to ensure cell lysis and the contents from each well were transferred to a 96 well plate (with V-shaped wells) and spun in a plate holder for 5 minutes at 1000 rpm at room temperature.
175 μl of cleared supernatant was then transfeπed to a white 96-well plate (Nunc) leaving the beads pellet behind.
Luminescence was then read in a TopCount (Packard) counter.
References (incorporated herein by reference thereto)
Artavanis-Tsakonas S, et al. (1995) Science 268:225-232.
Artavanis-Tsakonas S, etal. (1999) Science 284:770-776. Brucker K, et al. (2000) Nature 406:411-415.
Camilli et al. (1994) Proc Natl Acad Sci USA 91:2634-2638.
Caux C, etal. (1992) Nature 360: 258-261.
CheeM. etal. (1996) Science 274:601-614.
Dkuz et al. (1997) Cell 90: 271-280. Hemmati-Brivanlou and Melton (1997) Cell 88: 13-17.
Hicks C, et al. (2000) Nat. Cell. Biol. 2:515-520.
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.
Hsieh et al. (1996) Molecular & Cell Biology 160}:952-959.
Iemura et al. (1998) PNAS 95:9337-9345.
Irvine KD (1999) Cuπ. Opin. Genet. Devel. 9:434-441.
Ju BJ, et al. (2000) Nature 405:191-195. Kusano and Raab-Truab (2001) J Virol 75(l):384-395.
Leimeister C. et al. (1999) Mech Dev 85(l-2):173-7.
Li et al. (1998) Immunity 8(l):43-55.
Lieber, T. et al. (1993) Genes Dev 7(10): 1949-65.
Lu, F. M. et al. (1996) Proc Natl Acad Sci 93(l l):5663-7. Matsuno K, et al. (1998) Nat. Genet. 19:74-78.
Matsuno, K. etal. (1995) Development 121(8 :2633-44.
McGuinness T. Et al (1996) Genomics 35(3):473-85.
Medhzhitov et al. (1997) Nature 388:394-397. Moloney DJ, et al. (2000) Nature 406:369-375. Munro S, Freeman M. (2000) Curr. Biol. 10:813-820.
Ordentlich et al. (1998) Mol. Cell. Biol. 18:2230-2239.
Osborne B, Miele L. (1999) Immunity 11:653-663.
Panin VM, et al. (1997) Nature 387:908-912. Sallusto F and Lanzavecchia A (1994) J. Exp. Med. 179: 1109-1118.
Sasai et al. (1994) Cell 79:779-790.
Schroeter, E.H. et al. (1998) Nature 393(6683):382-6.
Strobl et al. (2000) J Virol 74(4): 1727-35.
Struhl G, Adachi A. Cell 93(4):649-60. Takebayashi K. et al. (1994) J Biol Chem 269(7): 150-6.
Tamura K, et al. (1995) Curr. Biol. 5:1416-1423.
Valenzuela et al. (1995) J. Neurosci. 15:6077-6084.
Weinmaster G. (2000) Curr. Opin. Genet. Dev. 10:363-369.
Wilson and Hemmati-Brivanlou (1997) Neuron 18:699-710. Zhao et al. (1995) J. Immunol. 155:3904-3911.

Claims

1. A method of immunotherapy comprising administering a modulator of immune cell costimulatory activity in combination with a modulator of Notch signalling.
2. A method as claimed in claim 1 comprising administering an antagonist of positive immune cell costimulatory activity, in combination with an agent capable of activating Notch signalling.
3. A method as claimed in claim 1 comprising administering an agonist of positive immune cell costimulatory activity, in combination with an agent capable of inhibiting Notch signalling.
4. A product comprising a modulator of immune cell costimulatory activity in combination with a modulator of Notch signalling.
5. A product comprising an inhibitor of negative immune cell costimulatory activity in combination with an inhibitor of Notch signalling.
6. A product comprising an activator of negative immune cell costimulatory activity in combination with an activator of Notch signalling.
7. A kit comprising in one or more containers (a) a modulator of Notch signalling and (b) a modulator of immune cell costimulation.
8. A method for increasing immune cell costimulation by administering a modulator of Notch signalling.
9. A method for decreasing immune cell costimulation by administering a modulator of Notch signalling.
10. A method as claimed in any of the preceding claims wherein the costimulation is mediated by CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-Ll, PD-L2, OX40 or OX40L.
11. A method for increasing expression of an immune cell costimulatory protein, polypeptide or polynucleotide by administering a modulator of Notch signalling.
12. A method for decreasing expression of an immune cell costimulatory protein, polypeptide or polynucleotide by administering a modulator of Notch signalling.
13. A method as claimed in claim 11 or claim 12 wherein the immune cell costimulatory protein, polypeptide or polynucleotide is CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-Ll, PD-L2, OX40 or OX40L or a polynucleotide coding for CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-Ll, PD-L2, OX40 or OX40L.
14. A method as claimed in any of the preceding claims wherein the modulator of Notch signalling is a Notch receptor agonist.
15. A method as claimed in any of claims 1 to 13 wherein the modulator of Notch signalling is a Notch receptor antagonist.
16. A method as claimed in any of the preceding claims wherein the modulator of the Notch signalling pathway comprises Delta or Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide coding for Delta, Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof.
17. A method as claimed in any of claims 1 to 15 wherein the modulator of Notch signalling comprises a fusion protein comprising a segment of a Notch ligand extracellular domain and an immunoglobulin F0 segment or a polynucleotide coding for such a fusion protein.
18. A method as claimed in any of claims 1 to 15 wherein the modulator of Notch signalling comprises a protein or polypeptide comprising a DSL domain or a polynucleotide sequence coding for such a protein.
19. A method as claimed in claim 18 wherein the modulator of Notch signalling comprises a protein or polypeptide comprising a Notch ligand DSL domain and from 1 to 10 EGF-like domains or a polynucleotide sequence coding for such a protein or polypeptide.
20. A method as claimed in any of claims 1 to 15 wherein modulator of Notch signalling comprises Notch intracellular domain (Notch IC) or a fragment, derivative, homologue, analogue or allelic variant thereof, or a polynucleotide sequence which codes for Notch intracellular domain or a fragment, derivative, homologue, analogue or allelic variant thereof.
21. The use of a modulator of Notch signalling to increase immune cell costimulation.
22. The use of a modulator of Notch signalling to decrease immune cell costimulation.
23. The use of a modulator of Notch signalling to increase expression of an immune cell costimulatory protein, polypeptide or polynucleotide.
24. The use of a modulator of Notch signalling to decrease expression of an immune cell costimulatory protein, polypeptide or polynucleotide.
25. A use as claimed in any one of claims 21 to 24 wherein the costimulation is mediated by CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-Ll, PD-L2, OX40 or OX40L.
26. A use as claimed in any one of claims 21 to 25 wherein the modulator of Notch signalling is a Notch receptor agonist.
27. A use as claimed in any one of claims 21 to 25 wherein the modulator of Notch signalling is a Notch receptor antagonist.
28. A use as claimed in any one of claims 21 to 27 wherein the modulator of Notch signalling comprises Delta or Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide coding for Delta, Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof
29. A use as claimed in any one of claims 21 to 27 wherein the modulator of the Notch signalling pathway comprises a fusion protein comprising a segment of a Notch ligand extracellular domain and an immunoglobulin FG segment or a polynucleotide coding for such a fusion protein.
30. A use as claimed in any one of claims 21 to 27 wherein the modulator of the Notch signalling patliway comprises a protein or polypeptide comprising a DSL domain or a polynucleotide sequence coding for such a protein.
31. A use as claimed in claim 30 wherein the modulator of Notch signalling comprises a protein or polypeptide comprising a Notch ligand DSL domain and from 1 to 10 EGF-like domains; or a polynucleotide sequence coding for such a protein or polypeptide.
32. A use as claimed in any one of claims 21 to 27 wherein modulator of the Notch signalling pathway comprises Notch intracellular domain (Notch IC) or a fragment, derivative, homologue, analogue or allelic variant thereof, or a polynucleotide sequence which codes for Notch intracellular domain or a fragment, derivative, homologue, analogue or allelic variant thereof.
33. A product comprising: i) a modulator of the Notch signalling pathway; and ii) a modulator of immune cell costimulation; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
34. A product as claimed in claim 33 for modulation of T cell activity.
35. A product as claimed in claim 33 or claim 34 for the treatment of inflammation, asthma, allergy, graft rejection, graft-versus-host disease or autoimmune disease.
36. A product as claimed in any one of claims 33 to 35 wherein the modulator of Notch signalling comprises Delta or Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide coding for Delta, Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof.
37. A product as claimed in any one of claims 33 to 35 wherein the modulator of Notch signalling comprises a fusion protein comprising a segment of a Notch ligand extracellular domain and an immunoglobulin F0 segment or a polynucleotide coding for such a fusion protein.
38. A product as claimed in any one of claims 33 to 35 wherein the modulator of the Notch signalling pathway comprises a protein or polypeptide comprising a DSL domain or a polynucleotide sequence coding for such a protein.
39. A product as claimed in claim 38 wherein the modulator of Notch signalling comprises a protein or polypeptide comprising a Notch ligand DSL domain and from 1 to 10 EGF-like domains or a polynucleotide sequence coding for such a protein or polypeptide.
40. A product as claimed in any one of claims 33 to 35 wherein modulator of the Notch signalling pathway comprises Notch intracellular domain (Notch IC) or a fragment, derivative, homologue, analogue or allelic variant thereof, or a polynucleotide sequence which codes for Notch intracellular domain or a fragment, derivative, homologue, analogue or allelic variant thereof.
41. A product as claimed in any one of claims 33 to 35 wherein the modulator of the Notch signalling pathway comprises a dominant negative version of a Notch signalling repressor, or a polynucleotide which codes for a dominant negative version of a Notch signalling repressor.
42. A product as claimed in any one of claims 33 to 41 wherein the a modulator of immune cell costimulation is a modulator of CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-Ll, PD-L2, OX40 or OX40L signalling.
43. A product as claimed in any one of claims 33 to 41 wherein the modulator of immune cell costimulation is a soluble fusion protein comprising at least an active part of a CD28, CD80, CD86, CTLA-4, ICOS, ICOS ligand, CD40, CD40L, PD-1, PD-Ll, PD- L2, OX40 or OX40L extracellular domain.
44. A product as claimed in claim 43 wherein the modulator of immune cell costimulation is CTLA-4-Ig, ICOS-Ig, CD40-Ig or OX40-Ig.
45. A product as claimed in any one of claims 33 to 44 for the treatment of allergy, graft rejection, graft-versus host disease or autoimmune disease.
46. A method for preparing a product as claimed in any one of claims 33 to 45 by combining: i) a modulator of the Notch signalling pathway; and ii) a modulator of immune cell costimulation.
47. A method for modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering: i) an effective amount of a modulator of the Notch signalling pathway; and ii) an effective amount of a modulator of immune cell costimulation.
48. A combination of: i) a modulator of the Notch signalling pathway; and ii) a modulator of immune cell costimulation; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
49. A combination of: i) an activator of Notch signalling; and ii) an activator of negative immune costimulation; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
50. A modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a modulator of immune cell costimulation.
51. The use of a combination of: i) a modulator of the Notch signalling pathway; and ii) a modulator of immune cell costimulation; in the manufacture of a medicament for modulation of the immune system.
52. The use of a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of the immune system in simultaneous, contemporaneous, separate or sequential combination with a modulator of immune cell costimulation.
53. A pharmaceutical kit comprising a modulator of the Notch signalling pathway and a modulator of immune cell costimulation.
54. A pharmaceutical composition comprising a modulator of the Notch signallmg pathway and a modulator of immune cell costimulation and optionally a pharmaceutically acceptable caπier.
55. A combination of: i) an activator of Notch signalling; and ii) an activator of negative immune costimulation; for simultaneous, contemporaneous, separate or sequential use in reducing an immune response.
56. A combination of: i) an activator of Notch signalling; ii) an inhibitor of positive immune costimulation; for simultaneous, contemporaneous, separate or sequential use in reducing an immune response.
57. A combination of: i) an inhibitor of Notch signalling; and ii) an inhibitor of negative immune costimulation; for simultaneous, contemporaneous, separate or sequential use in increasing an immune response.
58. A combination of: i) an inhibitor of Notch signalling; and ii) an activator of positive immune costimulation; for simultaneous, contemporaneous, separate or sequential use in increasing an immune response.
59. A method for reducing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; and ii) an activator of negative immune costimulation.
60. A method for reducing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; and ii) an inhibitor of positive immune costimulation.
61. A method for increasing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; and ii) an inhibitor of negative immune costimulation.
62. A method for increasing an immune response by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; and ii) an activator of positive immune costimulation.
63. A combination of: i) an activator of Notch signalling; ii) an activator of negative immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in reducing an immune response to the antigen or antigenic determinant
64. A combination of: i) an activator of Notch signalling; ii) an inhibitor of positive immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in reducing an immune response to the antigen or antigenic determinant.
65. A combination of: i) an inhibitor of Notch signalling; ii) an inhibitor of negative immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in increasing an immune response to the antigen or antigenic determinant.
66. A combination of: i) an inhibitor of Notch signalling; ii) an activator of positive immune costimulation; and iii) an antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in increasing an immune response to the antigen or antigenic determinant.
67. A method for reducing an immune response to an antigen or antigenic determinant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; ii) an activator of negative immune costimulation; and iii) the antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant)
68. A method for reducing an immune response to an antigen or antigenic determinant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an activator of Notch signalling; ii) an inhibitor of positive immune costimulation; and iii) the antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant) for simultaneous, contemporaneous, separate or sequential use in reducing an immune response to the antigen or antigenic determinant.
69. A method for increasing an immune response to an antigen or antigenic determinant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; ii) an inhibitor of negative immune costimulation; and iii) the antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant).
70. A method for increasing an immune response to an antigen or antigenic determinant by simultaneously, contemporaneously, separately or sequentially administering (in any order) a combination of: i) an inhibitor of Notch signalling; ii) an activator of positive immune costimulation; and iii) the antigen or antigenic determinant (or a polynucleotide coding for an antigen or antigenic determinant).
71. A method for detecting, measuring or monitoring Notch signalling comprising determining the amount of an immune cell costimulatory protein, polypeptide or polynucleotide or determining the amount of a polynucleotide coding for such a protein or polypeptide.
72. A method as claimed in claim 71 wherein the amount of the costimulatory protein, polypeptide or polynucleotide in a biological sample taken from a subject is determined.
73. A method as claimed in claim 72 wherein the sample comprises blood, serum, urine, lymphatic fluid, or tissue.
74. A method as claimed in claim 72 or claim 73 wherein the sample comprises an immune cell.
75. A method as claimed in claim 72 or claim 73 wherein the sample comprises a cancer cell.
76. A method as claimed in claim 72 or claim 73 wherein the sample comprises a stem cell.
77. A method as claimed in claim 74 wherein the sample comprises a T-cell.
78. A method as claimed in any of claims 71 to 77, comprising the steps of: obtaining a biological sample from a subject; and contacting the biological sample with a binding agent that binds to an immune cell costimulatory protein, polypeptide or polynucleotide.
79. A method as claimed in claim 78 comprising the steps of: obtaining a biological sample from a subject; contacting the biological sample with a binding agent that binds to an immune cell costimulatory protein, polypeptide or polynucleotide; and detecting in the sample an amount of costimulatory protein, polypeptide or polynucleotide that binds to the binding agent.
80. A method as claimed in claim 79, comprising the steps of: obtaining a biological sample from the patient; contacting the biological sample with a binding agent that binds to an immune cell costimulatory protein, polypeptide or polynucleotide; detecting in the sample an amount of costimulatory protein, polypeptide or polynucleotide that binds to the binding agent; and comparing the amount of costimulatory protein, polypeptide or polynucleotide to a reference value and therefrom determining the degree of Notch signalling.
81. A method as claimed in any one of claims 78 to 80 wherein the binding agent is a protein or polypeptide
82. A method as claimed in claim 81 wherein the binding agent is an antibody or antibody fragment.
83. A method as claimed in claim 82 wherem the antibody is raised against ICOS, CD28, CD80 or CD86.
84. A method as claimed in claim 82 or claim 83 wherein the antibody or antibody fragment is specific for a human T-cell costimulatory protein or polypeptide.
85. A method as claimed in any one of claims 78 to 80 wherein the binding agent is a polynucleotide.
86. A method as claimed in claim 85 comprising the further step of amplifying an immune cell costimulatory polynucleotide in a sample and detecting the amplified polynucleotide.
87. A method as claimed in claim 86 wherein the amplification is by PCR.
88. A method as claimed in claim 87 wherein the amplification is by real-time PCR.
89. A method of detecting, measuring or monitoring Notch signalling in an immune cell by determining the amount of an immune cell costimulatory protein, polypeptide or polynucleotide or a polynucleotide coding for such a protein or polypeptide in the cell.
90. A method as claimed in claim 89 wherein the immune cell is a T-cell.
91. A method as claimed in claim 89 wherein the immune cell is a B-cell.
92. A method as claimed in claim 89 wherein the immune cell is an antigen presenting cell.
93. A method according to any one of claims 71 to 92 further comprising a step of comparing the amount of a T-cell costimulatory protein, polypeptide or polynucleotide or a polynucleotide coding for such a protein or polypeptide with a reference amount.
94. A method according to any one of claims 71 to 93 wherein the amount of costimulatory protein, polypeptide or polynucleotide or a polynucleotide coding for such a protein or polypeptide is detected using a nucleic acid assay.
95. A method according to any of claims 71 to 93 wherein the amount of costimulatory protein, polypeptide or polynucleotide or a polynucleotide coding for such a protein or polypeptide is detected using a protein assay.
96. A diagnostic kit comprising a binding agent that binds to an immune cell costimulatory protein, polypeptide or polynucleotide for detecting, measuring or monitoring Notch signalling.
PCT/GB2004/000668 2003-02-18 2004-02-18 Modulators of notch signalling and of immune cell costimulatory activity for immunotherapy WO2004073732A1 (en)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005073250A2 (en) * 2004-01-28 2005-08-11 Lorantis Limited Medical treatment using an rna1 targeting a human notch signalling pathway member
WO2014022759A1 (en) * 2012-08-03 2014-02-06 Dana-Farber Cancer Institute, Inc. Agents that modulate immune cell activation and methods of use thereof
WO2017076360A1 (en) * 2015-11-04 2017-05-11 Taipei Veterans General Hospital Title of the invention combination therapy for malignant diseases
WO2017079448A1 (en) * 2015-11-06 2017-05-11 The Board Of Trustees Of The University Of Illinois Ox40l-jagged-1 chimeric polypeptides and uses thereof
US9944700B2 (en) 2013-03-13 2018-04-17 Novartis Ag Notch2 binding molecules for treating respiratory diseases
EP1810026B1 (en) 2004-10-06 2018-04-18 Mayo Foundation For Medical Education And Research B7-h1 and pd-1 in treatment of renal cell carcinoma
CN109475629A (en) * 2016-05-20 2019-03-15 伊莱利利公司 With the combined therapy of NOTCH and PD-1 or PD-L1 inhibitor
US10259882B2 (en) 2015-05-07 2019-04-16 Agenus Inc. Anti-OX40 antibodies
WO2019226922A1 (en) * 2018-05-24 2019-11-28 Rush University Medical Center Icosl for use as a renal therapeutic
US10836830B2 (en) 2015-12-02 2020-11-17 Agenus Inc. Antibodies and methods of use thereof
WO2021164709A1 (en) * 2020-02-18 2021-08-26 至本医疗科技（上海）有限公司 Application of notch family gene mutation in predicting sensitivity of patient suffering from solid tumor to immune checkpoint inhibitor therapy
US11359028B2 (en) 2016-11-09 2022-06-14 Agenus Inc. Anti-OX40 antibodies and anti-GITR antibodies
US11873510B2 (en) 2013-02-22 2024-01-16 Board Of Trustees Of The University Of Illinois T-reg cell expansion

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5434131A (en) * 1991-06-27 1995-07-18 Bristol Myers Squibb Co. Chimeric CTLA4 receptor and methods for its use
WO1998020142A1 (en) * 1996-11-07 1998-05-14 Lorantis Limited Notch
WO2003011317A1 (en) * 2001-07-25 2003-02-13 Lorantis Limited Modulators of notch signalling for use in immunotherapy

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5434131A (en) * 1991-06-27 1995-07-18 Bristol Myers Squibb Co. Chimeric CTLA4 receptor and methods for its use
WO1998020142A1 (en) * 1996-11-07 1998-05-14 Lorantis Limited Notch
WO2003011317A1 (en) * 2001-07-25 2003-02-13 Lorantis Limited Modulators of notch signalling for use in immunotherapy

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005073250A3 (en) * 2004-01-28 2006-08-03 Lorantis Ltd Medical treatment using an rna1 targeting a human notch signalling pathway member
WO2005073250A2 (en) * 2004-01-28 2005-08-11 Lorantis Limited Medical treatment using an rna1 targeting a human notch signalling pathway member
US11939378B2 (en) 2004-10-06 2024-03-26 Mayo Foundation For Medical Education And Research Costimulatory B7-H1 in renal cell carcinoma patients: indicator of tumor aggressiveness and potential therapeutic target
EP1810026B1 (en) 2004-10-06 2018-04-18 Mayo Foundation For Medical Education And Research B7-h1 and pd-1 in treatment of renal cell carcinoma
US11242387B2 (en) 2004-10-06 2022-02-08 Mayo Foundation For Medical Education And Research Costimulatory B7-H1 in renal cell carcinoma patients: indicator of tumor aggressiveness and potential therapeutic target
US10457733B2 (en) 2012-08-03 2019-10-29 Dana-Farber Cancer Institute, Inc. Agents that modulate immune cell activation and methods of use thereof
WO2014022759A1 (en) * 2012-08-03 2014-02-06 Dana-Farber Cancer Institute, Inc. Agents that modulate immune cell activation and methods of use thereof
US11359020B2 (en) 2012-08-03 2022-06-14 Dana-Farber Cancer Institute, Inc. Agents that modulate immune cell activation and methods of use thereof
US11873510B2 (en) 2013-02-22 2024-01-16 Board Of Trustees Of The University Of Illinois T-reg cell expansion
US9944700B2 (en) 2013-03-13 2018-04-17 Novartis Ag Notch2 binding molecules for treating respiratory diseases
US11472883B2 (en) 2015-05-07 2022-10-18 Agenus Inc. Methods of administering anti-OX40 antibodies
US10626181B2 (en) 2015-05-07 2020-04-21 Agenus Inc. Nucleic acids encoding anti-OX40 antibodies
US11136404B2 (en) 2015-05-07 2021-10-05 Agenus Inc. Anti-OX40 antibodies
US10259882B2 (en) 2015-05-07 2019-04-16 Agenus Inc. Anti-OX40 antibodies
US11332536B2 (en) 2015-05-07 2022-05-17 Agenus Inc. Vectors comprising nucleic acids encoding anti-OX40 antibodies
WO2017076360A1 (en) * 2015-11-04 2017-05-11 Taipei Veterans General Hospital Title of the invention combination therapy for malignant diseases
WO2017079448A1 (en) * 2015-11-06 2017-05-11 The Board Of Trustees Of The University Of Illinois Ox40l-jagged-1 chimeric polypeptides and uses thereof
US11447557B2 (en) 2015-12-02 2022-09-20 Agenus Inc. Antibodies and methods of use thereof
US10836830B2 (en) 2015-12-02 2020-11-17 Agenus Inc. Antibodies and methods of use thereof
CN109475629A (en) * 2016-05-20 2019-03-15 伊莱利利公司 With the combined therapy of NOTCH and PD-1 or PD-L1 inhibitor
US11359028B2 (en) 2016-11-09 2022-06-14 Agenus Inc. Anti-OX40 antibodies and anti-GITR antibodies
WO2019226922A1 (en) * 2018-05-24 2019-11-28 Rush University Medical Center Icosl for use as a renal therapeutic
WO2021164709A1 (en) * 2020-02-18 2021-08-26 至本医疗科技（上海）有限公司 Application of notch family gene mutation in predicting sensitivity of patient suffering from solid tumor to immune checkpoint inhibitor therapy

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