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WO2004060262A2 - Modulators of notch signalling for use in immunotherpapy - Google Patents

Modulators of notch signalling for use in immunotherpapy Download PDF

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Publication number
WO2004060262A2
WO2004060262A2 PCT/GB2004/000046 GB2004000046W WO2004060262A2 WO 2004060262 A2 WO2004060262 A2 WO 2004060262A2 GB 2004000046 W GB2004000046 W GB 2004000046W WO 2004060262 A2 WO2004060262 A2 WO 2004060262A2
Authority
WO
WIPO (PCT)
Prior art keywords
ofthe
notch
antigenic determinant
antigen
modulators
Prior art date
Application number
PCT/GB2004/000046
Other languages
French (fr)
Other versions
WO2004060262A3 (en
Inventor
Mark William Bodmer
Emmanuel Cyrille Pascal Briend
Brian Robert Champion
Andrew Christopher Lennard
Grahame James Mckenzie
Tamara Tugal
George Albert Ward
Lesley Lynn Young
Original Assignee
Lorantis Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0300234A external-priority patent/GB0300234D0/en
Priority claimed from GB0301510A external-priority patent/GB0301510D0/en
Priority claimed from GB0301515A external-priority patent/GB0301515D0/en
Priority claimed from GB0301522A external-priority patent/GB0301522D0/en
Priority claimed from GB0301524A external-priority patent/GB0301524D0/en
Priority claimed from GB0301513A external-priority patent/GB0301513D0/en
Priority claimed from GB0301527A external-priority patent/GB0301527D0/en
Priority claimed from GB0301518A external-priority patent/GB0301518D0/en
Priority claimed from GB0301526A external-priority patent/GB0301526D0/en
Priority claimed from GB0301519A external-priority patent/GB0301519D0/en
Priority claimed from GB0301529A external-priority patent/GB0301529D0/en
Priority claimed from GB0301512A external-priority patent/GB0301512D0/en
Priority claimed from GB0301521A external-priority patent/GB0301521D0/en
Priority claimed from GB0306621A external-priority patent/GB0306621D0/en
Priority claimed from PCT/GB2003/001525 external-priority patent/WO2003087159A2/en
Priority claimed from GB0312062A external-priority patent/GB0312062D0/en
Priority claimed from PCT/GB2003/003285 external-priority patent/WO2004013179A1/en
Priority claimed from GB0323130A external-priority patent/GB0323130D0/en
Application filed by Lorantis Limited filed Critical Lorantis Limited
Priority to JP2006500232A priority Critical patent/JP2006517533A/en
Priority to PCT/GB2004/000263 priority patent/WO2004064863A1/en
Priority to EP04722319A priority patent/EP1646400B1/en
Priority to PCT/GB2004/001252 priority patent/WO2004082710A1/en
Priority to AT04722319T priority patent/ATE474593T1/en
Publication of WO2004060262A2 publication Critical patent/WO2004060262A2/en
Publication of WO2004060262A3 publication Critical patent/WO2004060262A3/en
Priority to US11/188,417 priority patent/US20060204508A1/en
Priority to US11/231,494 priority patent/US20060205823A1/en
Priority to US12/766,738 priority patent/US20100303867A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/577—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 tolerising response
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

  • the present invention relates to therapeutic modulation of Notch signalling, particularly, but not exclusively, in immune cells.
  • Notch signal transduction plays a critical role in cell fate determination in vertebrate and invertebrate tissues. Notch is expressed at many stages of Drosophila embryonic and larval development and in many different cells implying a wide range of functions including an important role in neurogenesis and in the differentiation of mesodermal and endodermal cells.
  • Notch- 1, Notch-2, Notch-3 and Notch-4 There are at least four mammalian Notch genes (Notch- 1, Notch-2, Notch-3 and Notch-4).
  • Notch-1 which most closely resembles the proteins of invertebrates and lower vertebrates, is widely expressed and is essential for early development. Recent evidence suggests that Notch signalling contributes to lineage commitment of immature T-cells in the thymus.
  • T-cells During maturation in the thymus, T-cells acquire the ability to distinguish self-antigens from those that are non-self, a process termed "self tolerance". Tolerance to a non-self antigen, however, may be induced by immunisation under specific conditions with a peptide fragment comprising that antigen. In autoimmune diseases such as multiple sclerosis, rheumatoid arthritis or diabetes, there is a failure of the proper regulation of tolerance. Improved treatment methods for re-establishing tolerance are desirable for autoimmune diseases. Similarly in allergic conditions and for transplantation of an organ or tissue from a donor individual, induction of tolerance to particular foreign antigens or profiles of foreign antigens is desirable.
  • Notch and its ligands suggests a role for these proteins in T-cell acquired immunocompetence.
  • T-cells express Notch-1 mRNA constitutively. Delta expression is limited to only a subset of T-cells in the peripheral lymphoid tissues. Serrate expression is restricted to a subset of antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • manipulation ofthe Notch signalling pathway can be used in immunotherapy and in the prevention and/or treatment of T-cell mediated diseases.
  • T-cell mediated diseases For example, allergy, autoimmunity, graft rejection, cancer and infectious diseases may be targeted.
  • PCT/GB97/03058 (filed on 6 November 1997 and published as WO 98/20142; claiming priority from GB 9623236.8 filed on 7 November 1996, GB 9715674.9 filed on 24 July 1997 and GB 9719350.2 filed on 11 September 1997);
  • PCT/GB00/04391 (filed on 17 November 2000 and published as WO 0135990; claiming priority from GB 9927328.6 filed on 18 November 1999);
  • PCT/GBOl/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 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); PCT/GB2003/003285 (filed on 1 August 2003); and PCT/GB2003/003908 (filed on 8 September 2003).
  • PCT/GB97/03058 (WO 98/20142), PCT/GB99/04233 (WO 00/36089), PCT/GB00/04391 (WO 0135990), PCT/GBOl/03503 (WO 02/12890), PCT/GB02/02438 (WO 02/096952), PCT/GB02/03381 (WO 03/012111), PCT/GB02/03397 (WO 03/012441), PCT/GB02/03426 (WO 03/011317), PCT/GB02/04390 (WO 03/029293), PCT/GB02/05137 (WO 03/041735), PCT/GB02/05133 (WO 03/042246), PCT/GB2003/003285 and PCT/GB2003/003908 is hereby incorporated herein by reference,
  • Varnum-Finney at al (Blood 1998, Vol 91, No 11, pp4084-4091) describes how the effect of Jagged- 1 on murine marrow precursor cells was assessed by co-culturing sorted precursor cells with a 3T3 cell layer that expressed human Jagged- 1 or by incubating sorted precursors with beads coated with the purified extracellular domain of human Jagged- 1.
  • the present invention seeks to provide products, methods, constructs, uses and compositions for modulating the Notch signalling pathway in therapy, and particularly for modulation of immune cell activity in immunotherapy.
  • Administration of modulators of Notch signalling according to the present invention provides improved activity, especially improved Notch signalling agonist activity.
  • a product comprising i) a pharmaceutically acceptable support matrix or substrate (the terms "matrix” and substrate” are used interchangeably herein) suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling (these preferably being activators ofthe Notch receptor); and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation ofthe immune system.
  • Such a product may be used to modulate (preferably reduce) an immune response to said antigen or antigenic determinant.
  • the product comprises i) a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of an immune response to said antigen or antigenic determinant.
  • the modulators of Notch signalling are presented by the support matrix or substrate in an orientation suitable for activation of a Notch receptor, and are preferably predominantly orientated on the surface of the support matrix or substrate.
  • the modulators of Notch signalling may be coupled to the matrix or substrate by chemical coupling, affinity coupling or adsorption coupling.
  • the support matrix or substrate may be a particulate support matrix or substrate, ie a particle such as a bead, preferably a microbead or nanobead.
  • the antigen or antigenic determinant used in the product may be an allergen or antigenic determinant thereof.
  • the antigen or antigenic determinant used in the product may be an autoantigen or bystander antigen or antigenic determinant thereof.
  • the antigen or antigenic determinant used in the product may be a graft antigen such as an MHC (HLA) antigen or antigenic determinant thereof.
  • HLA MHC
  • the pharmaceutically acceptable support matrix or substrate bears at least 5, suitably at least 10, at least 20, or at least 50, or at least 100 modulators of Notch signalling which are bound/coupled thereto.
  • the pharmaceutically acceptable support matrix or substrate has a maximum linear dimension of from about 1 nanometre to about 1000 micrometres, for example from about 10 nanometres to about 100 micrometres.
  • the pharmaceutically acceptable support matrix or substrate has a maximum linear dimension of from about 10 nanometres to about 100 micrometres, preferably from about 40 nanometres to about 10 micrometres, preferably from about 40 nanometres to about 1000 nanometres.
  • the pharmaceutically acceptable support matrix or substrate comprises a polymeric material, preferably a substantially biodegradable material or materials.
  • the support matrix or substrate may be in the form of an implantable support matrix or substrate.
  • the pharmaceutically acceptable support matrix or substrate can take many different forms such as polymers, plastics, porous materials such as resin or modified cellulose, beads (such as microspheres and microbeads, nanospheres and nanoparticles) and liposomes.
  • the pharmaceutically acceptable support matrix or substrate may be a particulate matrix or substrate, such as a bead, sphere, particle or carrier, for example having a diameter (or, for example, within a collection of beads, a mean diameter) of from about 0.001 to about 1000 micrometres, for example from about 0.01 to about 100 micrometres, suitably from about 0.1 to 10 micrometres, for example about 1 to 10 micrometres.
  • Particulate materials such as beads have the advantages of being easier to handle in certain situations, and of potentially providing a larger surface area for interaction with cells. They may also be more suitable for in-vivo applications, especially when the substrate comprises a biodegradable material.
  • the pharmaceutically acceptable support matrix or substrate eg particle
  • the pharmaceutically acceptable support matrix or substrate will be dispersible in water but is preferably not readily soluble in water. If water soluble, the support matrix or substrate should preferably dissolve sufficiently slowly to exhibit biological activity before fully decomposing.
  • the pharmaceutically acceptable support matrix or substrate may be a particle such as a bead.
  • a bead may be, for example, a magnetic or paramagnetic bead (e.g. as available under the trade names "Dynal” or “MACS” (Miltenyi)).
  • the magnetic or paramagenetic properties of such beads may facilitate manufacture, purification and handling. However, magnetic or paramagenetic properties are not required, and many other (non-magnetic) particle types may also be used.
  • the bead comprises polystyrene, polyacrylamide, latex, cellulose, silica, dextran, agarose, cellulose, polylactide, or poly(methylmethacrylate) (PMMA) optionally in modified, crosslinked or derivatized form
  • the modulators of Notch signalling will be activators of a Notch receptor (ie Notch receptor agonists), preferably activators of a mammalian Notch receptor, preferably activators of a human Notch receptor.
  • the modulators of Notch signalling are activators of a human Notchl, Notch2, Notch3 or Notch4 receptor.
  • they activate Notch receptors in immune cells, preferably peripheral immune cells, preferably T-cells, B-cells or antigen presenting cells (APCs).
  • a modulator of Notch signalling may be a Notch ligand or a biologically active fragment (preferably all or part ofthe extracellular domain) or derivative of a Notch ligand, or a peptidomimetic of such a Notch ligand or fragment.
  • the product may be used for modulation of peripheral immune cell activity, especially modulation of T-cell activation, preferably providing a degree of specificity to the antigen or antigenic determinant used.
  • the product may be used for generation of regulatory T-cells (T regs).
  • T regs regulatory T-cells
  • the product may be used for reducing an immune response to an antigen or antigenic determinant.
  • the product may be used for promoting immune tolerance to an antigen or antigenic determinant.
  • the product may be used for the treatment of autoimmune disease, allergy or graft/transplant rejection.
  • the modulator of Notch signalling comprises a Notch ligand protein or a fragment, derivative, homologue, analogue or allelic variant thereof, as described above.
  • At least one ofthe modulators of Notch signalling comprises a protein or polypeptide which is preferably bound to the substrate, matrix or particle/bead through a terminal amino acid residue.
  • At least one of the modulators of Notch signalling comprises a protein or polypeptide which is bound to the substrate, matrix or particle/bead through a C-terminal amino acid residue.
  • At least one ofthe modulators of Notch signalling comprises a protein or polypeptide which is bound to the substrate, matrix or particle/bead through a cysteine residue.
  • At least one ofthe modulators of Notch signalling comprises a protein or polypeptide which is bound to the particle through a C-terminal cysteine residue.
  • the product is in the form of a pharmaceutical composition.
  • a method for promoting immune tolerance to an antigen or antigenic determinant in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering, in either order: i) an effective amount of a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling; and ii) an effective amount of an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant.
  • a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling for use to treat autoimmune disease in simultaneous, contemporaneous, separate or sequential combination with an autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutically acceptable support matrix or substrate for in vivo administration preferably a particle
  • a multiplicity of modulators of Notch signalling preferably a multiplicity of modulators of Notch signalling
  • an allergen or antigenic determinant thereof or a polynucleotide coding for an allergen or antigenic determinant thereof in the manufacture of a medicament for the treatment of allergy.
  • a pharmaceutically acceptable support matrix or substrate for in vivo administration preferably a particle
  • a multiplicity of modulators of Notch signalling preferably a multiplicity of modulators of Notch signalling
  • a graft antigen or antigenic determinant thereof or a polynucleotide coding for a graft antigen or antigenic determinant thereof in the manufacture of a medicament for the treatment of graft rejection.
  • a pharmaceutically acceptable support matrix or substrate for in vivo administration preferably a particle
  • a multiplicity of modulators of Notch signalling in the manufacture of a medicament for treatment of autoimmune disease in simultaneous, contemporaneous, separate or sequential combination with an autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutically acceptable support matrix or substrate for in vivo administration preferably a particle
  • a multiplicity of modulators of Notch signalling in the manufacture of a medicament for treatment of allergy in simultaneous, contemporaneous, separate or sequential combination with an allergen or antigenic determinant thereof or a polynucleotide coding for an allergen or antigenic determinant thereof.
  • a pharmaceutically acceptable support matrix or substrate for in vivo administration preferably a particle
  • a multiplicity of modulators of Notch signalling in the manufacture of a medicament for treatment of graft rejection in simultaneous, contemporaneous, separate or sequential combination with a graft antigen or antigenic determinant thereof or a polynucleotide coding for a graft antigen or antigenic determinant thereof.
  • a pharmaceutical or veterinary kit comprising i) a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant
  • a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) to which is bound i) a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant.
  • a matrix or substrate may take the form of a particle such as a bead bearing both i) a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant.
  • the particle may be in the form of a microparticle or microbead.
  • These particles are particularly suitable for administration with antigens/antigenic determinants as discussed above, but may also be used in their own right to modulate immune cell activity, for example by modifying immune cell activity and/or cytokine expression as described herein.
  • maximum linear dimension means a particle having no larger linear dimension than that stated. For example, where the particle has a generally or approximately spherical shape, the maximum linear dimension will be the diameter.
  • such a particle may have a maximum linear dimension (or mean maximum linear dimension) of less than about 300 nm, suitably less than about 200 nm, for example less than about 150 nm, for example less than about 100 nm.
  • such a particle may have a maximum linear dimension (or mean maximum linear dimension) of from about 0.1 to 300 nm, suitably from about 1 to 100 nm, suitably from about 10 to 100 nm, suitably from about 20 to 100 nm, suitably from about 10 to 80 nm, suitably from about 20 to 80 nm, suitably from about 30 to 70 nm.
  • such particles may have an individual or average/mean volume of from about 0.5 to about 50,000,000 cubic nanometres (nm 3 ), preferably from about 5,000 to about 500,000 cubic nanometres (nm 3 ).
  • the particle may be substantially biodegradable.
  • the particle may be in the form of a microbead or microsphere.
  • the modulators of Notch signalling are presented by the particle in an orientation suitable for activation of a Notch receptor, and at least a significant and effective number are preferably orientated on the surface ofthe particle.
  • the invention further provides a method for therapeutic modulation of Notch signalling by administering particles with a size as described herein.
  • the invention further provides a method for generating a regulatory T-cell or increasing regulatory T-cell activity by contacting a particle with a size as described herein with a T- cell or APC (such contact being either in vivo or ex vivo, preferably in vivo).
  • a method for therapeutic modulation of Notch signalling in immune cells eg T-cells, B-cells or APCs
  • administering a particle with a size as described herein in vivo.
  • a method for therapeutic modulation of immune cell activity by administering a particle with a size as described herein in vivo.
  • a method for therapeutic modulation of T-cell activity by administering a particle with a size as described herein in vivo.
  • a method for treating inflammation, asthma, allergy, graft rejection, graft- versus-host disease or autoimmune disease by administering a particle with a size as described herein in vivo.
  • a particle with a size as described herein for use in vivo in the treatment of disease there is provided a particle with a size as described herein for use in vivo in the treatment of an immune disorder.
  • a particle with a size as described herein in the manufacture of a medicament for modulation of immune cell activity in vivo.
  • the immune cells are not stem cells.
  • a particle with a size as described herein for the manufacture of a medicament for modulation of expression of a cytokine selected from IL-10, IL-5, IL-2, TNF-alpha, IFN-gamma or IL-13.
  • a particle with a size as described herein for the manufacture of a medicament for decrease of expression of a cytokine selected from IL-2, IL-5, TNF-alpha, IFN-gamma or IL-13.
  • a particle with a size as described herein for the manufacture of a medicament for generating an immune modulatory cytokine profile with increased IL-10 expression and reduced IL-5 expression.
  • a particle with a size as described herein for the manufacture of a medicament for generating an immune modulatory cytokine profile with increased IL-10 expression and reduced IL-2, IFN-gamma, IL-5, IL-13 and TNF-alpha expression.
  • the modulators of Notch signalling are activators of a Notch receptor (ie Notch receptor agonists), preferably activators of a mammalian Notch receptor, preferably activators of a human Notch receptor.
  • a Notch receptor ie Notch receptor agonists
  • the modulators of a mammalian Notch receptor preferably activators of a human Notch receptor.
  • Notch signalling are activators of a human Notchl, Notch2, Notch3 or Notch4 receptor.
  • they activate Notch receptors in immune cells, preferably peripheral immune cells, preferably T-cells, B-cells or antigen presenting cells (APCs).
  • immune cells preferably peripheral immune cells, preferably T-cells, B-cells or antigen presenting cells (APCs).
  • the particle may be a microbead.
  • the modulator of Notch signalling may comprise a Notch ligand or fragment, derivative or variant thereof.
  • a multiplicity of Notch ligands or fragments, derivatives or variants thereof are bound/coupled to the particle.
  • modulators of Notch signalling may be proteins or polypeptides bound to the particle (preferably chemically bound) through a terminal amino acid residue, suitably a C- terminal amino acid residue.
  • modulators of Notch signalling may be proteins or polypeptides bound to the particle (preferably chemically bound) through a cysteine residue, suitably a C-terminal cysteine residue.
  • the particle comprises a polymeric material.
  • the particle comprises a substantially biodegradable material or materials.
  • the particle may comprise one or more of polystyrene, polyacrylamide, latex, cellulose, silica, dextran, agarose, cellulose, polylactide, or poly(methylmethacrylate) (PMMA) optionally in modified, crosslinked or derivatized form, or an inorganic material such as a salt or oxide (eg iron oxide).
  • PMMA poly(methylmethacrylate)
  • multiplex means a number being at least three, and preferably at least five, suitably at least ten, for example at least twenty, or at least thirty, or at least forty, or at least fifty or at least a hundred or in some embodiments at least a thousand, or several thounsand or more.
  • the construct used in the various embodiments ofthe invention bears at least 3, preferably at least 5, suitably at least 10, suitably at least 20, for example 50, 100, 1000, 10,000 or more modulators of Notch signalling which may be the same or different.
  • modulators of Notch signalling which may be the same or different.
  • Such matrices, substrates, particles, beads etc may be administered either in vivo or ex-vivo as well known in the art (for example as described herein under the heading "Pharmaceutical Compositions") and used to modulate the Notch signalling pathway (for example to treat conditions as described herein under the heading "Therapy").
  • the modulator of Notch signalling is an agent capable of activating Notch signalling.
  • the agent is capable of activating Notch signalling in lymphocytes, preferably T-cells.
  • the agent may act to reduce activity of effector T-cells such as Th or Tc T-cells, and or increase activity of regulatory T-cells.
  • the cells used or acted on in the present invention are not stem cells.
  • At least one modulator of Notch signalling is an agent capable of activating a Notch receptor, such as a human Notch 1, Notch 2, Notch 3 or Notch 4 receptor.
  • the modulator may be a Notch ligand or a biologically active fragment or derivative of a Notch ligand, or a peptidomimetic of such a Notch ligand.
  • the agent is capable of activating Notch receptors in lymphocytes such as T-cells.
  • At least one modulator ofthe Notch signalling pathway may comprise or code for a fusion protein.
  • the modulator may comprise or code for a fusion protein comprising a segment of a Notch ligand extracellular domain and an immunoglobulin F c segment.
  • a modulator ofthe Notch signalling pathway may comprise a fusion protein comprising a segment of a Notch ligand extracellular domain and an immunoglobulin F c segment (eg IgGl Fc or IgG4 Fc) or a polynucleotide coding for such a fusion protein.
  • an immunoglobulin F c segment eg IgGl Fc or IgG4 Fc
  • IgG fusion proteins are described, for example in Example 2 of WO 98/20142.
  • IgG fusion proteins may be prepared as well known in the art, for example, as described in US
  • At least one modulator ofthe Notch signalling pathway comprises or codes for a protein or polypeptide comprising a Notch ligand DSL domain or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • At least one modulator of the Notch signalling pathway comprises or codes for a Notch ligand DSL domain and at least one EGF-like domain, suitably at least 1 to 20 or more, suitably at least 2 to 16 or more, for example at least 2 to 10, for example from 2 to 5 EGF-like domains.
  • the DSL and EGF domain sequences are or correspond to mammalian sequences.
  • Preferred sequences include human sequences.
  • the Notch ligand domains are fused to a heterologous amino acid sequence such as an IgFc sequence.
  • At least one modulator ofthe Notch signalling pathway comprises Delta (preferably a mammalian Delta, preferably human Delta, eg human Deltal, Delta3 or Delta4) 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.
  • Delta preferably a mammalian Delta, preferably human Delta, eg human Deltal, Delta3 or Delta4
  • a modulator ofthe Notch signalling pathway may comprise Serrate/Jagged (preferably mammalian, preferably human Jagged, eg human Jagged 1 or Jagged2) 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.
  • At least one 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.
  • the particle may be a bead.
  • the bead may be, for example, a magnetic or paramagnetic bead (e.g. as available under the trade names "Dynal” or “MACS" (Miltenyi)).
  • the magnetic or paramagenetic properties of such beads may facilitate manufaacture, purification and handling.
  • magnetic or paramagenetic properties are not required, and many other particles types may also be used.
  • the modulation ofthe immune system comprises reduction of T cell activity.
  • the modulation ofthe immune system may comprise reduction of effector T-cell activity, for example reduction of helper (T H ) and or cytotoxic (Tc) T-cell activity.
  • T H helper
  • Tc cytotoxic T-cell activity
  • the modulation ofthe immune system may comprise reduction of a Thl or Th2 immune response.
  • the modulation ofthe immune system provides an increase of regulatory T-cell (T reg) activity, such as an increase of Trl or Th3 regulatory T-cell activity.
  • T reg regulatory T-cell
  • the modulation ofthe immune system comprises generation of regulatory T cells (Tregs) and/or enhancement of Treg activity.
  • Tregs regulatory T cells
  • the modulation ofthe immune system comprises treatment of asthma, allergy, graft rejection, graft-versus-host disease or autoimmune disease.
  • a method for producing a lymphocyte or antigen presenting cell (APC) capable of promoting tolerance comprises incubating a lymphocyte or APC obtained from a human or animal patient with a particle as described herein.
  • APC antigen presenting cell
  • the method comprises incubating a lymphocyte or APC obtained from a human or animal patient with an APC in the presence of particle as described herein.
  • a method for producing an APC capable of inducing tolerance in a T cell which method comprises contacting an APC with a particle as described herein.
  • a method for producing a lymphocyte or APC capable of promoting tolerance which method comprises incubating a lymphocyte or APC obtained from a human or animal patient with a particle as described herein.
  • lymphocyte or APC may be incubated either in vivo or ex-vivo.
  • composition preferably for in vivo administration
  • a pharmaceutical composition comprising a particle as described herein, and optionally a pharmaceutically acceptable carrier.
  • the antigen or antigenic determinant may be administered on a pharmaceutically acceptable support matrix suitable for in vivo administration bearing said antigen or antigenic determinant, which matrix may be the same as or different to the pharmaceutically acceptable support matrix bearing the modulators of Notch signalling.
  • Such constructs may be administered in simutaneous, separate or sequential combination with modulators of Notch signalling or may be administered independently in their own right to modulate immune responses.
  • a method for reducing an immune response to an antigen or antigenic determinant by administering a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing said antigen or antigenic determinant.
  • a method for promoting immune tolerance to an antigen or antigenic determinant by administering a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing said antigen or antigenic determinant.
  • the antigen or antigenic determinant may be an autoantigen or bystander antigen or antigenic determinant thereof for the treatment of autoimmune disease.
  • the antigen or antigenic determinant may be an allergen or antigenic determinant thereof for the treatment of allergy.
  • the antigen or antigenic determinant may be a graft antigen or antigenic determinant thereof for the treatment of graft rejection.
  • a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing an antigen or antigenic determinant thereof to reduce an immune response to said antigen or antigenic determinant.
  • a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing an antigen or antigenic determinant thereof to promote immune tolerance to said antigen or antigenic determinant.
  • a construct comprising a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing an allergen or antigenic determinant thereof.
  • a construct comprising a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing an autoantigen or bystander antigen or antigenic determinant thereof.
  • a construct comprising a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing a graft antigen or antigenic determinant thereof.
  • the pharmaceutically acceptable support matrix or substrate is a particulate matrix such as a bead, suitably a a microbead or microsphere, which preferably has a maximum linear dimension (or mean maximum linear dimension) of from about 1 nanometres to about 1000 micrometres, suitably from from about 10 nanometres to about 100 micrometres, suitably from about 10 nanometres to about 10 micrometres, suitably from about 40 nanometres to about 10 micrometres, suitably from about 40 nanometres to about 1000 nanometres.
  • the modulators of Notch signalling may be inhibitors of a Notch receptor (ie Notch receptor antagonists), eg inhibitors of a mammalian Notch receptor, preferably inhibitors of a human Notch receptor.
  • the modulators of Notch signalling may, for example, be inhibitors of human Notchl, Notch2, Notch3 or Notch4 receptors.
  • such modulators inhibit Notch receptors in immune cells, preferably peripheral immune cells, preferably T-cells, B-cells or antigen presenting cells (APCs).
  • inhibitors of Notch signalling presented on matrices, substrates, particles, beads and the like may be used to enhance immune responses to antigens such as tumour antigens or pathogen antigens, for example, for use as adjuvants and in vaccine compositions. These may be used for example to treat or prevent cancers or infectious diseases.
  • a method for increasing an immune response to a tumour or pathogen antigen or antigenic determinant in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering in vivo, in either order: i) an effective amount of a pharmaceutically acceptable support matrix bearing a multiplicity of inhibitors of Notch signalling; and ii) an effective amount of a tumour or pathogen antigen or antigenic determinant, or a polynucleotide coding for a tumour or pathogen antigen or antigenic determinant.
  • a vaccine composition comprising i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of inhibitors of Notch signalling; and ii) a vaccine antigen or antigenic determinant thereof, or a polynucleotide coding for a vaccine antigen or antigenic determinant thereof; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation ofthe immune system.
  • a vaccine composition comprising i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of inhibitors of Notch signalling; and ii) a vaccine antigen or antigenic determinant, or a polynucleotide coding for a vaccine antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for enhancing an immune response to said vaccine antigen or antigenic determinant.
  • the vaccine composition may take the form of a tumour vaccine wherein the vaccine antigen or antigenic determinant is a tumour antigen or antigenic determinant thereof.
  • the vaccine antigen or antigenic determinant may be a pathogen antigen or antigenic determinant thereof.
  • the vaccine composition may be used either prophylactically or therapeutically (eg to treat tumours or acute or chronic infections).
  • a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of inhibitors of Notch signalling.
  • a construct comprising a pharmaceutically acceptable support matrix suitable for in vivo administration bearing both i) a multiplicity of inhibitors of Notch signalling; and ii) a vaccine antigen or antigenic determinant thereof, or a polynucleotide coding for a vaccine antigen or antigenic determinant thereof.
  • the vaccine antigen or antigenic determinant may be a tumour antigen or antigenic determinant thereof.
  • the vaccine antigen or antigenic determinant may be a pathogen antigen or antigenic determinant thereof.
  • Figure 1 shows a schematic representation of the Notch signalling pathway
  • Figure 2 shows schematic representations ofthe 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 the amino acid sequences of human Delta- 1 , Delta-3 and Delta-4;
  • Figure 5 shows the amino acid sequences of human Jagged- 1 and Jagged-2
  • Figure 6 shows schematic representations of various Notch ligand domain/IgFc domain fusion proteins which may be used in the present invention
  • Figure 7 shows a reaction scheme for covalently linking modulators of Notch signalling to beads.
  • Figure 8 shows schematic representations of non-covalent linking of modulators of Notch signalling to beads (using a streptavidin/biotin link).
  • Figure 9 shows the results of Example 3
  • Figure 10 shows the results of Example 4
  • FIG 11 shows the results of Example 5
  • Figure 12 shows the results of Example 6
  • Figure 15 shows the results of Example 10
  • Figures 16 to 18 show the results of Example 11 ;
  • Figure 22 shows the results of Example 16
  • Figure 23 shows the results of Example 17
  • Figure 34 shows the results of Example 23
  • Figure 35 shows the results of Example 24
  • Figure 37 shows a results from Example 26
  • Figure 38 shows results from Example 27.
  • Figure 39 shows results from Example 28.
  • 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 ofthe Notch receptor.
  • Notch was first described in Drosophila as a transmembrane protein that functions as a receptor for two different ligands, Delta and Senate. 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 ofthe extracellular domain, the transmembrane domain and the infracellular domain, and the other comprising the majority ofthe 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
  • 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) Cu ⁇ . 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-Senate 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 and Senate to the EGF-like repeats of Notch's extracellular domain. Delta may sometimes require cleavage for activation. It may be cleaved by the ADAM disintegrin metalloprotease Kuzbanian at the cell surface, the cleavage event releasing a soluble and active form of
  • TAN-1 an oncogenic variant ofthe 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 the Drosophila homologue of C-promoter binding factor- 1 [CBF- 1], a mammalian DNA binding protein involved in the Epstein-Ban 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 ofthe 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 cdclO/ankyrin repeats ofthe 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 val 1744 (termed site 3, or S3) (Schroeter, E.H. et al. (1998) Nature 393(6683):382-6 (Schroeter)). It is thought that the proteolytic cleavage step that releases the cdclO/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 CBFl (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 CBFl 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. (2000) Cun. 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
  • Fringe modifies Notch by adding O- 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 Senate/Jagged ligands but allow it to preferentially bind Delta (Panin VM, et al. (1997) Nature 387:908-912 (Panin), Hicks C, et al.
  • Drosophila has a single Fringe gene
  • vertebrates are known to express multiple genes (Radical, Manic and Lunatic Fringes) (Irvine KD (1999) Cun. Opin. Genet. Devel. 9:434-441 (Irvine)).
  • Notch IC proteolytic cleavage ofthe intracellular domain of Notch
  • CBFl CSL family protein
  • HES hairy-enhancer of split like
  • Notch can also signal in a CBFl -independent manner that involves the cytoplasmic zinc finger containing protein Deltex. Unlike CBFl, Deltex does not move to the nucleus following Notch activation but instead can interact with Grb2 and modulate the Ras-JNK signalling pathway.
  • modulation ofthe Notch signalling pathway refers to a change or alteration in the biological activity ofthe Notch signalling pathway or a target signalling pathway thereof.
  • modulator of the Notch signalling pathway may refer to antagonists or inhibitors of Notch signalling, i.e. compounds which block, at least to some extent, the normal biological activity ofthe Notch signalling pathway. Conveniently such compounds may be refened to herein as inhibitors or antagonists.
  • modulator ofthe Notch signalling pathway may refer to agonists of Notch signalling, i.e. compounds which stimulate or upregulate, at least to some extent, the normal biological activity ofthe Notch signalling pathway.
  • such compounds may be refened to as upregulators or agonists.
  • the modulator is an agonist of Notch signalling, and preferably an agonist ofthe Notch receptor (eg an agonist ofthe Notchl, Notch2, Notch3 and/or Notch4 receptor, preferably being a human Notch receptor).
  • an agonist binds to and activates a Notch receptor, preferably including human Notch recpetors such as human Notchl, Notch2, Notch3 and/or Notch4. Binding to and/or activation of a Notch receptor may be assessed by a variety of techniques known in the art including in vitro binding assays and activity assays for example as described herein.
  • any particular agent activates Notch signalling may be readily determined by use of any suitable assay, for example by use of a Notch/luciferase reporter assay ofthe type described in Example 3 herein.
  • antagonist activity may be readily determined for example by monitoring any effect ofthe agent in reducing signalling by known Notch signalling agonists such as CHO cells expressing human Deltal; CHO-Delta cells (ie in a so-called "antagonist” assay; see the Examples herein).
  • Target genes ofthe Notch signalling pathway include Deltex, genes ofthe Hes family (Hes-1 in particular), Enhancer of Split [E(spl)] complex genes, IL-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
  • 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 et al. (1998) Mol. Cell. Biol. 18:2230-2239
  • 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. et al. (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. D16464.
  • 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 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 IL-10 was produced by many other cell types including macrophages, keratinocytes, B cells, ThO and Thl cells. It shows extensive homology with the Epstein-Ban bcrfl gene which is now designated viral IL-10. Although a few immunostimulatory effects have been reported, it is mainly considered as an immunosuppressive cytokine. Inhibition of T 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. IL-10 also exerts anti-inflammatory effects on other myeloid cells such as neutrophils and eosinophils. On B cells, IL-10 influences isotype switching and proliferation. More recently, IL-10 was reported to play a role in the induction of regulatory T cells and as a possible mediator of their suppressive effect. Although it is not clear whether it is a direct downstream target ofthe 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 ofthe 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.
  • NotchlC localises to the nucleus and functions as an activated receptor.
  • Mammalian NotchlC interacts with the transcriptional repressor CBFl . It has been proposed that the NotchlC cdcl 0/ankyrin repeats are essential for this interaction.
  • Hsieh et al 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 NotchlC contains the CBFl interactive domain.
  • NotchlC acts by targeting DNA-bound CBFl within the nucleus and abolishing CBFl -mediated repression through masking ofthe repression domain.
  • Epstein Ban virus (EBV) immortalizing protein EBNA also utilises CBFl tethering and masking of repression to upregulate expression of CBFl - repressed B-cell genes.
  • EBV Epstein Ban 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 BARF0 (Kusano and Raab-Truab (2001) J Virol 75(l):384-395 (Kusano and Raab-Traub)) and LMP2A.
  • 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 (eg from Drosophila, verterbrates, mammals) such as Delta and Senate/Jagged (eg mammalian ligands Deltal, Delta 3, Delta4, Jaggedl and Jagged2 and homologues) and their biologically active fragments and sequence variants as well as antibodies to the Notch receptor, peptidomimetics and small molecules which have conesponding biological effects to the natural ligands.
  • the Notch ligand interacts with the Notch receptor by binding.
  • antibody as used herein includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, Fv and scFv which are capable of binding the epitopic determinant. These antibody fragments retain some 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 ofthe heavy chain; two Fab' fragments are obtained per antibody molecule;
  • F(ab') 2 the fragment ofthe 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.
  • a homologue is meant a gene product that exhibits sequence homology, either amino acid or nucleic acid sequence homology, to any one ofthe known Notch ligands, for example as mentioned above.
  • a homologue of a known Notch ligand will be at least 20%, preferably at least 30%, identical at the amino acid level to the conesponding known Notch ligand over a sequence of at least 10, preferably at least 20, preferably at least 50, suitably at least 100 amino acids, or over the entire length ofthe Notch ligand.
  • Homologues of Notch ligands 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 Notch ligand 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.
  • a modulator of Notch signalling for use in the present invention will be a Notch receptor agonist such as a Notch ligand capable of binding to and activating a Notch receptor, preferably a human Notch receptor such as human Notchl , Notch2, Notch3 or Notch4.
  • a Notch receptor agonist such as a Notch ligand capable of binding to and activating a Notch receptor, preferably a human Notch receptor such as human Notchl , Notch2, Notch3 or Notch4.
  • Such binding and activation may be assessed by a variety of techniques known in the art including activity assays for example as described herein.
  • 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.
  • modulate refers to a change or alteration in the biological activity ofthe Notch signalling pathway or a target signalling pathway thereof.
  • modulator may refer to antagonists or inhibitors of Notch signalling, i.e. compounds which block, at least to some extent, the normal biological activity ofthe Notch signalling pathway. Conveniently such compounds may be refened to herein as inhibitors or antagonists.
  • modulator may refer to agonists of Notch signalling, i.e. compounds which stimulate or upregulate, at least to some extent, the normal biological activity ofthe Notch signalling pathway. Conveniently such compounds may be refened to as upregulators or agonists.
  • candidate modulator is used to describe any one or more molecule(s) which may be, or is suspected of being, capable of functioning as a modulator of Notch signalling.
  • Said molecules may for example be organic "small molecules” or polypeptides.
  • candidate molecules comprise a plurality of, or a library of such molecules or polypeptides. These molecules may be derived from known modulators. "Derived from” means that the candidate modulator molecules preferably comprise polypeptides which have been fully or partially randomised from a starting sequence which is a known modulator of Notch signalling.
  • candidate molecules comprise polypeptides which are at least 40% homologous, more preferably at least 60% homologous, even more preferably at least 75% homologous or even more, for example 85 %, or 90 %, or even more than 95% homologous to one or more known Notch modulator molecules, using the BLAST algorithm with the parameters as defined herein.
  • the modulator ofthe present invention may for example be an organic compound or other chemical.
  • the modulator will 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.
  • substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc.
  • a combination of substituents may form a cyclic group.
  • the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other.
  • at least two ofthe 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 of Notch signalling will be an amino acid sequence or a chemical derivative thereof, or a combination thereof.
  • the candidate modulator may also be an antibody.
  • Candidate modulators may be synthetic compounds or natural isolated compounds. Various examples of such synthetic or natural modulators are listed below.
  • the modulator of Notch signalling may be a Notch ligand, or a polynucleotide encoding a Notch ligand.
  • Notch ligands will typically be capable of binding to a Notch receptor polypeptide present in the membrane of a variety of mammalian cells, for example hemapoietic stem cells.
  • Endogenous Notch ligands include polypeptides ofthe Delta family, for example Delta-1 (Genbank Accession No. AF003522 - Homo sapiens), Delta-3 (Genbank Accession No. AF084576 - Rattus norvegicus), Delta-like 3 (Mus musculus), Delta-4 (Genbank Accession No.
  • Candidate compounds ofthe present invention include fragments, derivatives, variants, mimetics, analogues and homologues of any ofthe above.
  • the modulator ofthe present invention will be a protein, polypeptide or peptide.
  • Whether or not any given agent acts as a modulator of Notch signalling may be readily determined by use of suitable assays or screens, for example, those described in the Examples herein.
  • 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 norvegicus) and Delta-like 3 (Mus musculus), the Senate family, for example Senate-1 and Senate-2 (WO97/01571, WO96/27610 and WO92/19734), Jagged- 1 and Jagged-2 (Genbank Accession No. AF029778 - Homo sapiens), and LAG-2. Homology between family members is extensive.
  • a homologue is meant a gene product that exhibits sequence homology, either amino acid or nucleic acid sequence homology, to any one ofthe known Notch ligands, for example as mentioned above.
  • a homologue of a known Notch ligand will be at least 20%, preferably at least 30%, identical at the amino acid level to the conesponding known Notch ligand over a sequnce of at least 10, preferably at least 20, preferably at least 50, suitably at least 100 amino acids, or over the entire length ofthe Notch ligand.
  • 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 ofthe protein and up to 14 or more EGF-like repeats on the extracellular surface. It is therefore prefened 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.
  • suitable homologues will preferably be capable of binding to a Notch receptor. Binding may be assessed by a variety of techniques known in the art including in vitro binding assays and activation ofthe receptor (in the case of an agonist or partial agonist) may be determined for example by use of assays as described in the Examples hereto and in WO 03/012441 (Lorantis) the text of which is hereby inco ⁇ orated herein by reference.
  • Homologues of Notch ligands 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 Notch ligand 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.
  • 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:
  • a typical DSL domain may include most or all ofthe following consensus amino acid sequence:
  • DSL domain may include most or all ofthe following consensus amino acid sequence:
  • 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.
  • DSL domain may include most or all ofthe following consensus amino acid sequence:
  • 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.
  • 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%o, 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.
  • Notch ligand N-terminal domain means the part of a Notch ligand sequence from the N-terminus to the start ofthe DSL domain. It will be appreciated that this term includes sequence variants, fragments, derivatives and mimetics having activity conesponding 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%o, 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.
  • any such heterologous amino acid sequence is not a TSST sequence, and preferably it is not a superantigen sequence.
  • 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 IX 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-like 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 (1 , 70) -C-x (1 , 6) -C-x (2) -G-a-x (0 , 21) -G-x (2) -C-x
  • 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.
  • 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%o amino acid sequence identity to an EGF-like domain of human Delta 4.
  • the modulator of Notch signalling may be an antibody, derivative or fragment which binds to and activates Notch.
  • the invention also provides a particle to which is coupled (eg chemically, by affinity or adsportion) an antibody capable of binding to and activating Notch.
  • Antibodies may be monoclonal or polyclonal but are preferably monoclonal.
  • the binding affinity (equilibrium association constant (Ka)) may be at least about 10° M "1 , at least about 10 7 M " ', at least about 10 8 M ' ⁇ or at least about IO 9 M "1 .
  • the antibody, derivative or fragment binds to one or more EGF or Lin/Notch (L/N) domains of Notch (for example to EGF repeats 11 and 12 of Notch).
  • L/N Lin/Notch
  • Suitable antibodies for use as blocking agents are obtained by immunizing a host animal with peptides comprising all or a portion of Notch.
  • the peptide used may comprise the complete protein or a fragment or derivatives thereof.
  • Prefened immunogens comprise all or a part of the extracellular domain of human Notch (eg Notchl, Notch2, Notch3 or Notch4, preferably Notchl or Notch2), where these residues contain any post-translation modifications, such as glycosylation, found in the native proteins.
  • Immunogens comprising the extracellular domain may be produced by a number of techniques which are well known in the art such as expression of cloned genes using conventional recombinant methods and/or isolation from T cells or cell populations expressing high levels of Notch.
  • Monoclonal antibodies may be produced by means well known in the art. Generally, the spleen and or lymph nodes of an immunized host animal provide a source of plasma cells.
  • the plasma cells are immortalized by fusion with myeloma cells to produce hybridoma cells.
  • Culture supernatant from individual hybridomas is screened using standard techniques to identify those producing antibodies with the desired specificity.
  • the antibody may be purified from the hybridoma cell supematants or ascites fluid by conventional techniques, such as affinity chromatography using Notch, Notch ligands or fragments thereof bound to an insoluble support, protein A sepharose, or the like.
  • 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. HB 12656 and a monoclonal antibody secreted by a hybridoma designated F3 having the ATCC Accession No. HB 12655.
  • antibodies for use to treat human patients in vivo 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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.
  • isotype will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity.
  • Suitable isotypes include IgG 1, IgG3 and IgG4.
  • IgG1 IgG1
  • IgG3 IgG4
  • IgG4 IgG4
  • kappa or lambda either ofthe human light chain constant regions, kappa or lambda, may be used.
  • the pharmaceutically acceptable matrix or substrate eg particle/bead to which modulators of Notch signalling are bound according to the present invention
  • Matrices, substrates and particles are preferably substantially non-immunogenic or have low immunogenicity, and may suitably comprise either organic or inorganic materials.
  • Inorganic materials include for example inorganic salts or oxides, such as iron oxide. Where an inorganic material is used, this is preferably at least partially (and preferably substantially completely) coated with a biocompatible polymer, for example as discussed below.
  • the particle may, for example comprise a polymer or combination of polymers alone.
  • a particle used may be biocompatible, and preferably biodegradable.
  • the particle may be a microbead or microsphere.
  • the term "diameter” normally applies to particles having a substantially spherical or other circular form.
  • particles used in the present invention do not need to have such a regular form, and may have a more inegular form, in which case the relevant dimension is suitably the maximum (largest) linear dimension (ie length, width or depth).
  • the dimension given for individual particles or beads it will be appreciated that these apply also to collections or populations of particles or beads, in which case the dimension given will relate to the average dimension of the collection or population, suitably the mean dimension.
  • a bead has a maximum linear dimension (or diameter) in a given range
  • this can also be considered in terms of a collection or population of beads having a mean maximum linear dimension (or diameter) in the same range.
  • Matrices/substrates and particles etc may, for example, comprise natural or synthetic polymers such as polystyrene, polyethylene glycol (PEG), polyglycollic acid (PGA), polycaprolactide, polyacrylamide, latex, silica, dextran, agarose, starch, cellulose, chitin/chitosan, polylactide, poly(methylmethacrylate) (PMMA); proteins/polypeptides such as albumins, for example human serum albumins; and modified, crosslinked and derivatized embodiments thereof.
  • natural or synthetic polymers such as polystyrene, polyethylene glycol (PEG), polyglycollic acid (PGA), polycaprolactide, polyacrylamide, latex, silica, dextran, agarose, starch, cellulose, chitin/chitosan, polylactide, poly(methylmethacrylate) (PMMA); proteins/polypeptides such as albumins, for example human serum albumins; and modified
  • Suitable materials include, for example polystyrene, cellulose, dextran crosslinked with epichlorohydrin (Sephadex.TM., Pharmacia, Uppsala, Sweden), polyacrylamide crosslinked with bisacrylamide (Biogel.TM., BioRad, USA), agar, glass beads, polylactide beads and latex beads.
  • Derivatized microparticles include microparticles derivatized with eg maleimide, aldehyde groups, allyl groups, carboxyalkyl groups such as carboxymethyl, phosphoryl and substituted phosphoryl groups, sulfate, sulfhydryl and sulfonyl groups, and amino and substituted amino groups. Beads may have either hydrophilic or hydrophobic properties.
  • the modulators of Notch signalling may be bound to the matrix/substrate eg particle etc (eg bead) by any suitable means.
  • binding may be by non-covalent linking such as surface adso ⁇ tion (eg by hydrophobic and/or electrostatic interactions) or by covalent linking such as chemical linking.
  • reactive groups such as amino, aldehyde, carboxy, epoxy or toluenesulfonyl (tosyl), thiol or maleimide groups
  • modulators of Notch signalling in the form of proteins or polypeptides may be linked to a particle (such as a bead) by incubation of a surface activated particle (such as a bead) with the protein or polypeptide, suitably by incubation for at least 12 to 24 hours suitably at neutral or neutral to high pH and suitably at a sufficiently high temperature such that a reactive group on the particle/bead (for example a tosyl, epoxy, amino, carboxy, aldehyde, thiol or maleimide group) reacts with a reactive group on the protein or polypeptide (for example a free amino or sulfhydryl group on the protein or polypeptide, or an aldehyde group) to form a covalent link.
  • a reactive group on the particle/bead for example a tosyl, epoxy, amino, carboxy, aldehyde, thiol or maleimide group
  • a reactive group on the protein or polypeptide for example a
  • linker groups may also be used to bind the matrix/substrate/particle/bead to the modulators of Notch signalling if required.
  • Suitable linkers are well known in the art and suitably comprise an acid, basic, aldehyde, ether or ester reactive group or a residue thereof.
  • Suitable linker moieties include, for example, succinimidyl propionate, succinimidyl butanoate, N-hydroxysuccinimide, benzotriazole carbonate, propionaldehyde, maleimide or forked maleimide, biotin, vinyl derivative and phospholipids.
  • modulators of Notch signalling such as Notch ligand proteins and polypeptides may be chemically coupled to beads using a coupling agent such as sulpho-SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate).
  • a coupling agent such as sulpho-SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate).
  • Antibodies may also be used to bind modulators of Notch signalling to a matrix/substrate such as a particle (such as a bead).
  • a matrix/substrate such as a particle (such as a bead).
  • beads coated with antibody-binding materials such as streptavidin may be used to bind a biotinylated anti- IgG antibody, which in turn may be bound to a modulator of Notch signalling in the form of an IgG fusion protein.
  • suitably functionalised agents such as biotinylated agents/ligands may be directly coupled to beads such as streptavidin beads.
  • particles or beads may be magnetic particles such as magnetic beads, such as for example those available under the trade name DYNABEAD TM from Dynal Biotech, Oslo, Norway. These have the advantage of being easy to separate from fluids magnetically.
  • Such particles or beads may be prepared, for example, by inco ⁇ orating a magnetic or paramagnetic material such as iron oxide into a polymeric matrix such as polystyrene.
  • DynabeadsTM are supe ⁇ aramagnetic polymer spheres, magnetic only when placed in a magnetic field and with no residual magnetism when the magnetic field is removed. They are composed of highly cross-linked polystyrene with magnetic material precipitated in pores evenly distributed throughout each bead. The pores are preferably filled with an additional polymer layer, which seals the iron material inside the beads. Chemical groups (such as amino, carboxy, epoxy or toluenesulfonyl (tosyl) groups) may then be introduced on the bead surface if appropriate.
  • Particles or beads may be either hydrophilic or hydrophobic. Hydrophilic particles or beads allow gentle coupling of ligands to the surface to maintain functional activity of labile proteins. The hydrophilic properties ofthe beads ensure optimal dispersity in aqueous solution. These particles or beads may be surface activated with, for example amine and carboxylic acid functional groups. The functional groups ofthe amine and carboxylic acid beads allow further introduction of a large variety of alternative reactive groups by coupling to commercially available cross-linkers.
  • Hydrophobic particles or beads are well suited to coupling of antibodies.
  • the hydrophobic Fc region ofthe antibody may be adsorbed to the hydrophobic particle or bead surface, followed by a rapid covalent bond formation.
  • the orientation ofthe antibody is thereby generally optimal with the Fab regions facing outwards.
  • particles or beads may also need to be taken into consideration.
  • Hydrophobic particles or beads facilitate hydrophobic-hydrophobic interactions between the particles or beads and the protein's hydrophobic parts, whereas the hydrophilic beads are suited when hydrophilic-hydrophilic interactions between the particles or beads and the protein's hydrophilic parts are desired.
  • the particles or beads may for example be latex microspheres such as those available from Interfacial Dynamics Co ⁇ oration (Portland, US).
  • latex microspheres/beads are available with either anionic (negative) or cationic (positive) surface charges.
  • Anionic latexes - such as those with sulfate, carboxyl, or carboxylate modified surface groups - are less likely to bind to negatively-charged cell surfaces and are therefore used frequently in biological applications.
  • Particles or beads may suitably be sterilized before use, for example by pasteurization, suitably for about 24 hours at 78-80° C; or by gamma inadiation, for example at 0.03 megarads suitably for about 24 hours.
  • the particles or beads may be coated with various proteins or polysaccharides that will greatly reduce their capacity to absorb biomolecules non-specifically.
  • Specific ineversible adso ⁇ tion of protein molecules such as avidin, streptavidin, and antibodies may be accomplished by simply mixing the latex and protein together for a specified period of time, then separating the bound from the unbound protein through centrifugation and removal ofthe supernatant.
  • particles or beads may be coated, for example with BSA or dextrans.
  • proteins, nucleic acids, and other biomolecules may if desired be covalently coupled to the particles or beads. Covalent coupling may require more effort than passive adso ⁇ tion, but can result in conjugates with greater specificity that remain active longer.
  • Carbodiimide-mediated coupling to CML latexes is a suitable method for conjugating low molecular weight peptides and oligonucleotides.
  • a wide range of suitable beads, micro/nanobeads and micro/nanospheres is also available for example from Polysciences, Inc. 400 Valley Road, Warrington, PA, US.
  • modulators of Notch signalling may be conjugated to the linear or cross-linked backbone of a liposome using conventional techniques (see, e.g. Ostro, M. J. (Ed.), Liposomes: from Biophysics to Therapeutics (Marcel Dekker, New York, 1987)).
  • Ostro M. J. (Ed.), Liposomes: from Biophysics to Therapeutics (Marcel Dekker, New York, 1987)
  • One prefened method of preparing liposomes and conjugating immunoglobulins to their surface is described by Ishimoto, Y. et al., J. Immunol. Met. 75, 351-360 (1984). For example, multilamillar liposomes composed of dipalmitoylphosphatidylcholine, cholesterol and phosphotidylethanolamine are prepared.
  • Modulators of Notch signalling may then be coupled to the phosphatidylethanolamine by the cross-linking agent N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate.
  • the coupling ofthe fragment to the liposome can be demonstrated by the release of a pre-trapped marker, e.g., carboxyfluorescence, from the liposomes upon the treatment of secondary antibody against the conjugated fragment and complement.
  • modulator of Notch signalling comprises an IgFc domain it may, for example, be coupled to a liposome or another carrier ofthe invention via carbohydrate moieties on the Fc domain.
  • Dextran In vitro biocompatibility of biodegradable dextran-based hydrogels tested with human fibroblasts, Biomaterials 22 (2001) 1197-1203 De Groot et al.. Methacrylate- derivatized dextran is biocompatible and a promising system for drug delivery.
  • PEG Copolymer with styrene; Polystyrene-poly (ethylene glycol) (PS-PEG2000) particles as model systems for site specific drug delivery. 2. The effect of PEG surface density on the in vitro cell interaction and in vivo biodistribution. Pharm. Res. 11 (1994) 1016-1022.
  • Albumin nanoparticles Preparation of surface modified protein nanoparticles by introduction of sulfhydryl groups. Int. J. Pharm. 211 (2000) 67-78 Weber et al. Thiol groups introduced to the surface of human serum albumin nanoparticles.
  • Stepth nanospheres Prolonging the circulation time and modifying the body distribution of intravenously injected polystyrene nanospheres by prior intravenous administration of poloxamine-908.
  • BBA 1336 (1997) 1-6 Moghimi, S. M. 60 or 250 nanometre polystyrene have extended half-life in vivo if coated with poloxamine 908 or poloxamine- protein conjugates. Chemical camouflage of nanospheres with a poorly reactive surface: towards development of stealth and target-specific nanocarriers.
  • BBA 1590 (2002) 131-139 Moghimi, S. M. Coating of polystyrene nanospheres with PEGylated BSA or IgG.
  • a prefened source of particles for use in the present invention is those from Miltenyi Biotec (Bergisch Gladbach, Germany, and Auburn, US) As described in US 5385707 (Miltenyi), US 4,452,773 describes the preparation of magnetic iron-dextran microspheres and provides a summary of art describing the various means of preparation of particles suitable for attachment to biological materials. As long ago as 1977, preparation of colloidal iron oxide, gamma-i ⁇ adiated in the presence of hydrophilic and hydrophobic methacrylate monomers, to provide particles for attachment to biological targets through coupling to immunoglobulin was described (Rembaum, A., et al., Nature (1977) 268:437-438.
  • US 4,230,685 describes an improvement in attaching specific binding agents to the magnetic particles wherein a particle coated with an acrylate polymer or a polysaccharide can be linked through, for example, glutaraldehyde to a preparation of protein A which can then selectively bind antibodies through the Fc portion, leaving the immunoreactive Fab regions exposed.
  • Albumin rather than polyacrylamide or polysaccharides, is the prefened matrix. A wide size range of particles is disclosed.
  • particles of 100-700 angstroms, particularly 300-400 angstroms are intended to be prepared; many ofthe particles are thus colloidal, and are fenomagnetic with a coating of dextran.
  • the resulting particles are described as discrete colloidal size particles having a fenomagnetic iron oxide core coated with a polysaccharide derivative having pendant functional groups provided by periodate oxidation.
  • These particles are prepared by mixing an aqueous solution of a fenous and ferric salt with a solution ofthe polysaccharide or polysaccharide derivative. After this mixing, alkali is added to cause the formation ofthe magnetic iron oxide particles to which the polysaccharide or derivative attaches.
  • the resulting particles are separated from excess dextran using gel filtration chromatography. A single peak containing the entire size range of particles is obtained.
  • the polysaccharide is then treated to provide the needed functional groups for conjugation to a protein or polypeptide.
  • fenomagnetic particles supe ⁇ aramagnetic particles
  • paramagnetic particles Several types of magnetic particles may be prepared: fenomagnetic particles, supe ⁇ aramagnetic particles and paramagnetic particles. Methods to prepare supe ⁇ aramagnetic particles are described in U.S. 4,770,183. With respect to terminology, as is the general usage in the art:
  • Magnetic refers to materials which do not acquire magnetic properties even in the presence of a magnetic field, i.e., they have no appreciable magnetic susceptibility.
  • Paramagnetic materials have only a weak magnetic susceptibility and when the field is removed quickly lose their weak magnetism. They are characterized by containing unpaired electrons which are not coupled to each other through an organized matrix. Paramagnetic materials can be ions in solution or gases, but can also exist in organized particulate form.
  • Fenomagnetic materials are strongly susceptible to magnetic fields and are capable of retaining magnetic properties when the field is removed. Fenomagnetism occurs only when unpaired electrons in the material are contained in a crystalline lattice thus permitting coupling ofthe unpaired electrons. Fenomagnetic particles with permanent magnetization have considerable disadvantages for application to biological material separation since suspension of these particles easily aggregate due to their high magnetic attraction for each other. " Supe ⁇ aramagnetic” materials are highly magnetically susceptible ⁇ i.e., they become strongly magnetic when placed in a magnetic field, but, like paramagnetic materials, rapidly lose their magnetism. Supe ⁇ aramagnetism occurs in fenomagnetic materials when the crystal diameter is decreased to less than a critical value.
  • the extent of magnetization which is acquired by a particle is a function of its magnetic susceptibility and the applied magnetic field.
  • the magnetization is a function ofthe resulting magnetic moment and ofthe volume ofthe particle. The higher the magnetic moment and the smaller the volume, the higher the magnetization.
  • Particles may be initially prepared in a range of sizes which result in variations in their abilities to acquire magnetic properties upon exposure to a magnetic field.
  • particles themselves may suitably be collections of iron oxide microcrystal of approx 50-600 angstroms which are aggregated into particles of colloidal size ⁇ i.e., approximately 100-2000 angstroms in diameter, preferably around 400-1000 angstroms in diameter.
  • particles of such homogeneity has the advantage of effecting a sha ⁇ separation peak in chromatographic procedure, as well as the potential for labeling various components in a mixture with sets of particles of differing magnetic susceptibilities to permit chromatographic separation of these components.
  • the particles can also be separated according to size.
  • such particles may for example be prepared by a modification ofthe method described in U.S. 4,452,773.
  • Solutions comprising ferric and fenous salts in a molar ratio of about 2:1-1.5:1 along with a suitable quantity of coating material, typically a weight of polysaccharide approximately 5-20 times the weight of iron salts in solution are stined and heated to about 40 degrees C.
  • Prefened coatings to be used in this process are polysaccharide or protein coatings.
  • a prefened polysaccharide coating is dextran.
  • the mixture is then typically titrated to basic pH eg with sodium hydroxide eg by dropwise addition over a period of about 1 hour.
  • the addition of base at elevated temperatures assists in the formation ofthe desired size particles.
  • a coating material can be included, as above, in the magnetic oxide forming preparation, or can be added after the colloidal oxide particles are formed. After neutralization with acid, aggregates are suitably removed by any convenient means such as filtration or centrifugation and the magnetic particles removed from the uncomplexed coating material eg by washing in a high gradient magnetic field.
  • the particles can be subjected to HGMS at any stage of this preparation process-before or after coating and before or after size separation.
  • the prepared particles may suitably be applied to any standard HGMS apparatus and fractionated according to magnetization (i.e., magnetic susceptibility).
  • magnetization i.e., magnetic susceptibility
  • the mixture is applied to the HGMS column, containing matrix, at a very high magnetic field strength so that virtually all ofthe particles are retained.
  • the particles are then eluted by gradual reduction ofthe magnetic field across the column. Fractions are collected at arbitrary intervals resulting in the preparation of a series of compositions each having a desired degree of homogeneity of magnetization.
  • the mixture of particles is segregated by overloading an
  • HGMS matrix with the preparation In this case, only the most highly magnetized particles - i.e., those having the highest magnetization- are retained, while the remainder ofthe preparation flows through the matrix. The retained materials can then be eluted by removing the imposed magnetic field.
  • any arbitrary fraction ofthe highest magnetization particles in the distribution resulting from the initial preparation can be retained.
  • a sufficiently small amount of matrix should be used so that the majority ofthe particles, about 80%, preferably about 90%), ofthe particles, pass through the matrix.
  • the polysaccharide or other coating is suitably derivatized to provide functional groups for conjugation to the specific binding moiety.
  • a variety of such modifications is known in the art.
  • polysaccharides are conveniently oxidized using periodate to provide aldehyde functional groups which can then be conjugated to amino substituents on a proteinaceous binding moiety, or can be reacted with CNBr to provide this functionality.
  • Protein coatings can be linked to targets for example through amino or sulfhydryl groups.
  • Supe ⁇ aramagnetic particles were prepared by mixing 10 g dextran T40 (Pharmacia, Uppsala), 1.5 g ferric chloride hexahydrate, and 0.64 g fenous chloride tetrahydrate in 20 ml water and heating to 40 degrees C. The solution was stined and 20 ml 4M NaOH was added dropwise with continued stirring. The resulting particle suspension was neutralized with acetic acid, and centrifuged for 10 min at 2,000.times.g followed by filtration through a 0.22 uM pore-size filter (Millex GV) to remove aggregates.
  • Millex GV 0.22 uM pore-size filter
  • Unbound dextran was removed by washing in a high gradient magnetic field (HGMF). This was performed by washing the magnetic particles in columns of ordinary household steel wool (average diameter about 30 um) which was placed in the chamber of a HGMS device shown in FIG. 1 at a strength of 0.6 Tesla. Ten ml of particle suspension were applied to a 15 times 40 mm column of 2 g of steel wool, and loaded column was washed with 30 ml buffer. About 90%) ofthe particles were washed through the column. The column was then removed from the external field and the remaining 10%> ofthe magnetic particles were eluted.
  • HGMF high gradient magnetic field
  • Molday (U.S. Pat. No. 4,452,773) describes the preparation of further magnetic iron-dextran microparticles and provides a summary describing the various means of preparing particles suitable for attachment to biological materials.
  • HGMS high gradient magnetic separation
  • a polysaccharide or other coating of a microparticle may be suitably derivatized to provide functional groups.
  • amino groups may be introduced before or after forming the particles, or an aldehyde function may be introduced by reacting the polysaccharide with, eg diimidazole or DCCD, and coupling hexane diamine to the sugar molecules.
  • aminodextran may be mixed with unsubstituted dextran to provide amino groups.
  • the polysaccharides may be conveniently oxidized using periodate to provide aldehyde functional groups that can be conjugated to amino substituents on a proteinaceous binding moiety, particularly under the conditions of reductive amination. Proteins and polypeptides can then be coupled to the particles through side chain amino or sufhydryl groups and heterofunctional cross-linking reagents.
  • a large number of heterofunctional compounds are available for linking to entities.
  • Illustrative entities include but are not limited to: azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3 , -[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N-.gamma- maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N- succinimidyl [4-azidophenyl]-l,3'-dithiopropionate, N-succinimidyl [4- iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl 4-[N- maleimidyl
  • a prefened linking group is 3-(2- pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) or 4-(N- maleimidomethyl)-cyclohexane-l -carboxylic acid N-hydroxysuccinimide ester (SMCC) with a reactive sulfhydryl group on the protein or polypeptide and a reactive amino group on the particle.
  • SPDP 3-(2- pyridyldithio)propionic acid N-hydroxysuccinimide ester
  • SMCC 4-(N- maleimidomethyl)-cyclohexane-l -carboxylic acid N-hydroxysuccinimide ester
  • the particle may comprise or be coated with a carbohydrate polymer, preferably a polysaccharide polymer.
  • a polysaccharide polymer Preferably such a polysaccharide is water-soluble.
  • polysaccharides are generally made up of a number of monosaccharide units typically joined by glycosidic bonds, such as 1-4 or 1-6 linkages.
  • the monosaccharide units may be, for example, aldoses (which may for example be trioses, tetroses such as erythrose or threose; pentoses such as ribose, arabinose, xylose or lyxose; hexoses such as allose, altrose, glucose, mannose, gulose, idose, galactose or tulose, or heptoses); or ketoses (which may for example be ketotrioses, ketotetroses such as erythulose; ketopentoses such as ribulose or xylulose; ketohexoses such as fructose, psicose, tagatose or sorbose, or ketoheptoses).
  • aldoses which may for example be trioses, tetro
  • Units may be in either D- or L- form, but the D form is generally prefened (eg D-glucose).
  • monosaccharide units may be in either alpha or beta forms, for example alpha-D-glucose.
  • the monosaccharides in a polysaccharide may be substantially the same (ie to provide a homopolysaccharide) or combinations of units may be used (ie to provide a heteropolysaccharide). Tens, hunreds or thousands of monosaccharide units may be present in such a polymer, and branching will commonly be present.
  • Suitable carbohydrate polymers include for example, glucans such as dextrans including aminodextrans and carboxymethyl-dextrans, heparins, celluloses (and derivatives thereof such as methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose and hydroxypropylcellulose), chitosan and hydrolysates of chitosan, starches (and derivatives thereof such as hydroxyethyl-starches and hydroxy propyl- starches), glycogens, heparins, alginates, agaroses and derivatives and activated versions thereof, guar gums, pullulans, inulins, xanthan gums, canageenans, pectins and alginic acid hydrolysates and derivatives and activated versions thereof.
  • glucans such as dextrans including aminodextrans and carboxymethyl-dextrans
  • heparins celluloses (and derivatives thereof such as
  • derivatised polymers may also be used in the present invention.
  • Such derivatised polymers may typically for example result from activation processes as described below.
  • a number of groups on the polymer may be converted into more reactive functional groups which facilitate conjugation. This process is frequently refened to as “activation” and the product is called an “activated” or “functionalized” polymer.
  • a polymeric molecule to be used as a support is not active (or is not considered sufficiently active) on its own it should preferably be activated by the use of a suitable technique.
  • Modulators of Notch signalling are preferably covalently attached to a polymer or activated polymer (either directly or via a linker) using chemical techniques. Reaction chemistries resulting in such linkages are well known in the art and may for example involve the use of complementary functional groups (eg on the linker, polymer and/or modulator of Notch signalling) for example as shown below: First Reactive Group Second Reactive Group Linkage carboxyl amine amide sulfonyl halide amine sulfonamide hydroxyl alkyl/aryl halide ether hydroxyl isocyanate urethane amine epoxide beta-hydroxyamine amine alkyl/aryl halide alkylamine hydroxyl carboxyl ester amine aldehyde amide/amine thiol/sulfhydryl maleimide amine succinimide
  • the functional groups on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which may typically comprise i) activation of polymer, ii) conjugation, and iii) if required, blocking of residual active groups.
  • coupling polymeric molecules to the free acid groups of polypeptides may be performed for example with the aid of diimide and for example amino-PEG or hydrazino- PEG (Pollak et al., (1976), J. Amr. Chem. Soc, 98, 289-291) or diazoacetate/amide (Wong et al., (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press).
  • Coupling to free sulfhydryl groups can be achieved for example with groups like maleimido or ortho-pyridyl disulfide.
  • vinylsulfone U.S. Pat. No. 5,414,135, (1995), Snow et al. has a preference for sulfhydryl groups.
  • Accessible arginine residues in a polypeptide chain may suitably be targeted by groups comprising two vicinal carbonyl groups.
  • Organic sulfonyl chlorides e.g. tresyl chloride, effectively convert hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in proteins or polypeptides allow stable linkages to be formed between polymer and polypeptide.
  • the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity).
  • Epoxides may also be used for creating amine bonds.
  • Converting PEG into a chloroformate with phosgene may facilitate carbamate linkages to lysines.
  • the many variations include substituting the chlorine with N-hydroxy succinimide (U.S. Pat. No. 5,122,614, (1992); Zalipsky et al., (1992), Biotechnol. Appl. Biochem., 15, p. 100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., (1991), Carbohydr. Res., 213, pp 309-319), with para-nitrophenol, DMAP (EP 632 082 Al, (1993), Looze, Y.) etc.
  • the derivatives are typically made for example by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide.
  • isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.
  • urethane (carbamate) linkages may be formed between an amino acid amino group (eg lysine, histidine, N-terminal residue), and an activated polymer.
  • an amino acid amino group eg lysine, histidine, N-terminal residue
  • an activated polymer eglysine, histidine, N-terminal residue
  • a urethane linkage is formed using a terminal oxycarbonyl-oxy-N- dicarboximide group such as a succinimidyl carbonate group.
  • Alternative activating groups include N-succinimide, N-phthalimide, N-glutarimide, N-tetrahydrophthalimide and N- norborene-2,3-dicarboxide. These urethane-forming groups are described for example in U.S. Pat. No.
  • Suitable starting materials and reagents for preparing the conjugates ofthe present invention are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis., USA), Bachem (Tonance, Calif, USA), Emka-Chemie, or Sigma (St.
  • protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions.
  • the choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, N.Y., 1991, and references cited therein.
  • a suitable linker reagent for use in the present invention may be a bifunctional reagent with a group for reacting with a modulator of Notch signalling (for example for reacting with a protein or polypeptide modulator of Notch signalling) and a group for reacting with a polymer support structure.
  • a modulator of Notch signalling for example for reacting with a protein or polypeptide modulator of Notch signalling
  • the linker reagent may typically remain in the resulting conjugate as a linker reagent residue (which may also be termed, for example, a "linker").
  • linker reagents are available for example from the Pierce Chemical
  • N-alpha-maleimidoacetoxy)-succinimide ester (AMAS)
  • BMDB ,4-R/5-Maleimidyl-2,3-dihydroxybutane
  • BMH ,6-Rts-maleimidohexane
  • BMP A N-beta-maleimidopropionic acid
  • BSOCOES Bis(sulfosuccinimidyl)-suberate
  • DMS Dimethylsuberimidate
  • DPDPB Dimethylsuberimidate
  • DST Disuccinimidyl tartarate
  • DTBP Dimethyl 3,3'-dithiobis-propionimidate
  • DTS SP 3 ,3 '-dithiobis(sulfosuccinimidylpropionate)
  • Ethylene glycol bis(succinimidylsuccinate) (EGS) Ethylene glycol bis(succinimidylsuccinate) (EGS)
  • ECMA N-epsilon-maleimidocaproic acid
  • EMCS N-epsilon-(maleimidocaprolyloxy)succinimide ester
  • GMBS N-gamma-maleimidobutyryloxy-succinimide ester
  • N-Hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA)
  • SAED Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido) ethyl l,3'-dithiopropionate
  • SAND N-succinimidyl 6-(4'-azido-2'-nitrophenylamino) hexanoate
  • SASD Sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl 1,3-dithiopropionate
  • SASD N-succinimidyl S-acetylthioacetate
  • SATA N-succinimidyl S-acetylthiopropionate
  • SATP Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido) ethyl l,3'-dithiopropionate
  • Sulfosuccinimidyl(perfluoroazidobenzamido) ethyl l,3'-dithiopropionate SFAD
  • N-succinimidyl iodoacetate SIA
  • N-succinimidyl(4-iodoacetyl)aminobenzoate SIAB
  • SPB Succinimidyl 4-(p-maleimidophenyl)butyrate
  • SMPH Succinimidyl-6-(beta-maleimido-propionamido) hexanoate
  • SPT 4-succinimidyloxy-carbonyl-methyl-alpha-(2-pyridylthio) toluene
  • SMPT Succinimidyl-(4-psoralen-8-yl
  • N-succinimidyl-(4-vinylsulfonyl) benzoate SBSB
  • TAEA 2-maleimidoethyl amine
  • TSAT Tris-(succinimidyl amino-triacetate
  • a linker used will be a bifunctional reagent, such as a heterobifunctional reagent (although it will be appreciated that homobifunctional reagents may also be used). Trifunctional and higher reagents may also be used if desired.
  • the modulators of Notch signalling are presented on the polymer in an orientation suitable for binding to and/or activation of a Notch receptor.
  • the matrix/substrate eg particle used comprises and/or is coated with, a polymeric structure which is preferably a pharmaceutically acceptable polymer.
  • Prefened polymers are water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • polymers include, for example, polyethylene glycol propionaldehyde, monomethoxy-polyethylene glycol, polyvinyl pynolidone (PVP), poly-l,3-dioxolane, poly- 1,3,6-trioxane, ethylene/maleic anhydride copolymer, (either homopolymers or random copolymers), poly(n-vinyl pynolidone)polyethylene glycol, polypropylene glycol homopolymers (PPG) and other polyalkylene oxides, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and other polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, colonic acids or other carbohydrate polymers, Ficoll or dextran and mixtures thereof. It will be appreciated that polymers may also be used in activated
  • Modulators of Notch signalling may be attached to the support structure at random positions within the molecule, or at predetermined positions within the molecule and may be attached to one, two, three or more chemical moieties.
  • Polymers may be either homopolymers or copolymers, eg random copolymers and may be either straight or branched.
  • the polymer may be a polysaccharide polymer, such as a glucan, for example a dextran or a dextran derivative such as amino-dextran.
  • a polymer where used may, if desired, have a branched structure.
  • branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Mo ⁇ urgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are inco ⁇ orated herein by reference.
  • the protein should preferably be attached to the particulate support structure with consideration of effects on functional or antigenic domains ofthe protein.
  • attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein inco ⁇ orated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride).
  • polymers such as polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group.
  • Reactive groups are those to which an activated polymer such as polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include, for example, lysine residues and N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue.
  • Sulfhydryl groups from cysteine residues may also be used as a reactive group for attaching polymers such as polyethylene glycol molecules.
  • attachment may be at an amino group, such as attachment at the N-terminus or a lysine group, or at a cysteine group, for example a C-terminal cysteine group.
  • Polymers such as polyethylene glycol may, for example, be attached to proteins and polypeptides via linkage to any of a number of amino acid residues ofthe protein or polypeptide.
  • polymers such as polyethylene glycol can be linked to a protein via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
  • reaction chemistries may be employed to attach polymers such as polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) ofthe protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) ofthe protein.
  • polymers such as polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) ofthe protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) ofthe protein.
  • proteins attached to the support structure through their N-termini may be desirable to have proteins attached to the support structure through their N-termini.
  • polyethylene glycol as an illustration, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization ofthe protein at the N-terminus with a carbonyl group containing polymer may be achieved.
  • proteins, polypeptides or peptides may be attached to the support structure through a suitably provided terminal residue, for example an C-terminal residue such as a terminal lysine, histidine, aspartic acid, glutamic acid or cysteine residue, which may be readily created or exposed by genetic manipulation techniques if not already present in the protein or peptide to be attached.
  • a terminal residue for example an C-terminal residue such as a terminal lysine, histidine, aspartic acid, glutamic acid or cysteine residue, which may be readily created or exposed by genetic manipulation techniques if not already present in the protein or peptide to be attached.
  • Attachment at a terminal residue, or at a point close to the protein/peptide terminus typically provides better presentation of ligands for binding to and/or activation of Notch receptors.
  • a multiplicity of protein/peptide modulators of Notch signalling (such as Notch ligand constructs comprising a DSL domain and 1-5, eg 3 EGF domains) are attached to a water-soluble polymeric support such as a polysaccharide, eg a dextran, by C-terminal residues (eg cysteine, lysine, histidine, glutamic or aspartic acid, preferably cysteine) via a linker such as sulfosuccinimidyl 4-[N- maleimidomethyl]-cyclohexane-l -carboxylate (sulfo-SMCC) or the like.
  • a water-soluble polymeric support such as a polysaccharide, eg a dextran
  • C-terminal residues eg cysteine, lysine, histidine, glutamic or aspartic acid, preferably cysteine
  • linker such as sulfosuccinimidy
  • polypeptide is synonymous with the term “amino acid sequence” and/or the term “protein”. In some instances, the term “polypeptide” is synonymous with the term “peptide”.
  • Protein usually refers to a short amino acid sequence that is 10 to 40 amino acids long, preferably 10 to 35 amino acids.
  • polypeptide 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.
  • polynucleotide sequence is synonymous with the term “polynucleotide” and/or the term “nucleotide sequence”.
  • the polynucleotide sequence may be DNA or RNA of genomic or synthetic or of recombinant origin. They may also be cloned by standard techniques. The polynucleotide sequence may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
  • Polynucleotide refers to a polymeric form of nucleotides of at least 10 bases in length and up to 1,000 to 10,000 bases or more. Longer polynucleotide 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 ofthe 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 ofthe 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 nucleic acid may be RNA or DNA and is preferably DNA. Where it is RNA, manipulations may be performed 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 ofthe 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 ofthe gene in question.
  • nucleic acids can be characterised as those nucleotide sequences which hybridise to the nucleic acid sequences known in the art.
  • the polynucleotide sequence may comprise, for example, a protein-encoding domain, an antisense sequence or a functional motif such as a protein-binding domain and includes variants, derivatives, analogues and fragments thereof.
  • the term also refers to polypeptides encoded by the nucleotide sequence. Variants, Derivatives, Analogues, Homologues and Fragments
  • the present invention also encompasses the use of variants, derivatives, analogues, homologues, mimetics 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 polypeptide or polynucleotide which retains at least one of the functions of the endogenous polypeptide or polynucleotide but generally has a different evolutionary origin thereto.
  • mimetic in relation to polypeptides or polynucleotides, refers to a chemical compound that possesses at least one of the endogenous functions of the polypeptide or polynucleotide which it mimics.
  • 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 transport activity or ability to modulate Notch signalling.
  • 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 ofthe 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 polynucleodtide.
  • 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 conesponding to the naturally-occurring sequence either side ofthe insertion site. The flanking regions will contain convenient restriction sites conesponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative ofthe 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 HIV, 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 ofthe 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%) identical, preferably at least 95 or 98%> identical.
  • homology should typically be considered with respect to those regions ofthe 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 ofthe present invention it is prefened 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 conesponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • 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 (Devereux).
  • Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA (Atschul) and the GENE WORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching. However it is prefened to use the GCG Bestfit program.
  • 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.
  • 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 prefened to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • %> homology preferably % sequence identity.
  • the software typically does this as part ofthe sequence comparison and generates a numerical result.
  • Nucleotide sequences which are homologous to or variants of sequences of use in the present invention can be obtained in a number of ways, for example by probing DNA 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 ofthe reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants ofthe 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.
  • conserveed sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software 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.
  • 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 ofthe polynucleotide or encoded polypeptide.
  • 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 ofthe 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 ofthe 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
  • host cells can be genetically engineered to inco ⁇ orate expression systems or polynucleotides ofthe invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis et al and Sambrook et al, such as calcium phosphate transfection, DEAE- dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, fransduction, 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.
  • 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,
  • 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 inco ⁇ orated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • 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.
  • any one or more ofthe above candidate modulators is brought into contact with a cell ofthe immune system.
  • Cells ofthe immune system of use in the present invention are described below.
  • Cells of use in the present invention are preferably cells ofthe immune system capable of transducing the Notch signalling pathway.
  • the cells of use in the present invention are T-cells. These include, but are not limited to, CD4 + and CD8 + mature T cells, immature T cells of peripheral or thymic origin and NK-T cells.
  • the cells may for example be antigen-presenting cells (APCs).
  • APCs include dendritic cells (DCs) such as interdigitating DCs or follicular DCs, Langerhans cells, PBMCs, macrophages, B-lymphocytes, T-lymphocytes, or other cell types such as epithelial cells, fibroblasts or endothelial cells, constitutively expressing or activated to express a MHC Class II molecules 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.
  • the T cells or APCs may be isolated from a patient, or from a donor individual or another individual.
  • the cells are preferably mammalian cells such as human or mouse cells. Preferably the cells are of human origin.
  • the APC or precursor APC may be provided by a cell proliferating in culture such as an established cell line or a primary cell culture. Examples include hybridoma cell lines, L-cells and human fibroblasts such as MRC-5.
  • Prefened cell lines for use in the present invention include Jurkat, H9, CEM and EL4 T- cells; long-term T-cell clones such as human HA1.7 or mouse D10 cells; T-cell hybridomas such as DO11.10 cells; macrophage-like cells such as U937 or THP1 cells; B-cell lines such as EBV-transformed cells such as Raji, A20 and Ml cells.
  • 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 manow. From peripheral blood, adherent precursors can be treated with a GM-CSF/IL-4 mixture (Inaba et al), or from bone manow, non-adherent CD34 + cells can be treated with GM-CSF and TNF- ⁇ (Caux et al).
  • DCs can also be routinely prepared from the peripheral blood of human volunteers, similarly to the method of Sallusto and Lanzavecchia J Exp Med (1994) 179(4) 1109-18 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 et al). Culture conditions may include other cytokines such as GM-CSF or IL-4 for the maintenance and, or activity ofthe dendritic cells or other antigen presenting cells.
  • PBMCs peripheral blood mononucleocytes
  • T cells and B cells for use in the invention are preferably obtained from cell lines such as lymphoma or leukemia cell lines, T cell hybridomas or B cell hybridomas but may also be isolated from an individual suffering from a disease of the immune system or a recipient for a transplant operation or from a related or unrelated donor individual.
  • T cells and B cells may be obtained from blood or another source (such as lymph nodes, spleen, or bone manow) and may be enriched or purified by standard procedures. 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 prefened to use helper T cells (CD4 + ). Alternatively other T cells such as CD8 + cells may be used.
  • Candidate modulators of use in the present invention are brought into contact with a cell of the immune system as described above.
  • modulation of Notch signalling by a candidate modulator is detected.
  • Assays for detecting modulation of Notch signalling will be described below. Many of these assays will involve monitoring the expression of a "target gene”. Assays
  • an assay may be set up to detect either inhibition or enhancement of Notch signalling in cells ofthe immune system by candidate modulators.
  • the method comprises mixing cells ofthe immune system, where necessary transformed or transfected, etc. with a synthetic reporter gene, in an appropriate buffer, with a sufficient amount of candidate modulator and monitoring Notch signalling.
  • the modulators may be small molecules, proteins, antibodies or other ligands as described above. Amounts or activity ofthe target gene (also described above) will be measured for each compound tested using standard assay techniques and appropriate controls.
  • the detected signal is compared with a reference signal and any modulation with respect to the reference signal measured.
  • the assay may also be run in the presence of a known antagonist ofthe Notch signalling pathway in order to identify compounds capable of rescuing the Notch signal.
  • 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 the Notch signalling pathway in cells ofthe immune system 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.
  • an assay for use with the present invention will be a cell based assay.
  • 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.
  • nucleic acid assays are also known. Any conventional 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 anays and other hybridization methods.
  • Target 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 target 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 target mRNA
  • dot blotting DNA or RNA analysis
  • in situ hybridisation using an appropriately labelled probe.
  • nucleic acids for analysis from samples generally requires nucleic acid amplification.
  • Many amplification methods rely on an enzymatic chain reaction (such as a polymerase chain reaction, a ligase chain reaction, or a self-sustained sequence replication) or from the replication of all or part of the vector into which it has been cloned.
  • the amplification according to the invention is an exponential amplification, as exhibited by for example the polymerase chain reaction.
  • amplification methods have been described in the literature, for example, general reviews of these methods in Landegren, U., et al., Science 242:229-237 (1988) and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990).
  • amplification methods may be used in the methods of our invention, and include polymerase chain reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase hybridisation, Qbeta bacteriophage replicase, transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based amplification (NASBA) and in situ hybridisation.
  • Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.
  • PCR is a nucleic acid amplification method described mter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202.
  • PCR consists of repeated cycles of DNA polymerase generated primer extension reactions.
  • 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 formation 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.
  • PCR can be used to amplify any known nucleic acid in a diagnostic context (Mok et al., (1994), Gynaecologic
  • Self-sustained sequence replication is a variation of TAS, which involves the isothermal amplification of a nucleic acid template via sequential rounds of reverse transcriptase (RT), polymerase and nuclease activities that are mediated by an enzyme cocktail and appropriate oligonucleotide primers (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874). Enzymatic degradation of the RNA of the RNA/DNA heteroduplex is used instead of heat denaturation. RNase H and all other enzymes are added to the reaction and all steps occur at the same temperature and without further reagent additions. Following this process, amplifications of 10 6 to 109 have been achieved in one hour at 42 °C.
  • Ligation amplification reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two per target strand. This technique is described by Wu, D. Y. and
  • rolling circle amplification (Lizardi et al, (1998) Nat Genet 19:225) is an amplification technology available commercially (RCATTM) which is driven by DNA polymerase and can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions.
  • RCATTM rolling circle amplification
  • a geometric amplification occurs via DNA strand displacement and hyperbranching to generate 10 12 or more copies of each circle in 1 hour.
  • RCAT generates in a few minutes a linear chain of thousands of tandemly linked DNA copies of a target covalently linked to that target.
  • SDA strand displacement amplification
  • SDA comprises both a target generation phase and an exponential amplification phase.
  • target generation double-stranded DNA is heat denatured creating two single-stranded copies.
  • a series of specially manufactured primers combine with DNA polymerase (amplification primers for copying the base sequence and bumper primers for displacing the newly created strands) to form altered targets capable of exponential amplification.
  • the exponential amplification process begins with altered targets (single-stranded partial DNA strands with restricted enzyme recognition sites) from the target generation phase.
  • DNA polymerase then uses the primer to identify a location to extend the primer from its 3' end, using the altered target as a template for adding individual nucleotides.
  • the extended primer thus forms a double-stranded DNA segment containing a complete restriction enzyme recognition site at each end.
  • a restriction enzyme is then bound to the double stranded DNA segment at its recognition site.
  • the restriction enzyme dissociates from the recognition site after having cleaved only one strand ofthe double-sided segment, forming a nick.
  • DNA polymerase recognises the nick and extends the strand from the site, displacing the previously created strand.
  • the recognition site is thus repeatedly nicked and restored by the restriction enzyme and DNA polymerase with continuous displacement of DNA strands containing the target segment.
  • Each displaced strand is then available to anneal with amplification primers as above. The process continues with repeated nicking, extension and displacement of new DNA strands, resulting in exponential amplification ofthe original DNA target.
  • PCR technology as described e.g. in section 14 of Sambrook et al., 1989, requires the use of oligonucleotide probes that will hybridise to target nucleic acid sequences. 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 ofthe invention are labelled with suitable label means for ready detection upon hybridisation.
  • suitable label means is a radiolabel.
  • the prefened method of labelling a DNA fragment is by inco ⁇ orating 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 P-labelled ATP and polynucleotide kinase.
  • other methods e.g. non-radioactive
  • Prefened are such sequences, probes which hybridise under high-stringency conditions.
  • 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) ofthe 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 refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65-68 °C.
  • High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6x SSC, 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 - O.lx SSC, 0.1 % SDS.
  • 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. ofthe gene being monitored.
  • Fluorescent markers which can be detected and sorted by FACS, are prefened. Especially prefened are GFP and luciferase.
  • Another type of prefened reporter is cell surface markers, i.e. proteins expressed on the cell surface and therefor easily identifiable.
  • cell-based screening assays can be designed by constructing cell lines in which the expression of a reporter protein, i.e.
  • an easily assayable protein such as ⁇ - galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase
  • CAT chloramphenicol acetyltransferase
  • luciferase is dependent on the activation of a Notch.
  • a reporter gene encoding one ofthe above polypeptides may be placed under the control of an response element which is specifically activated by Notch signalling.
  • Alternative assay formats include assays which directly assess responses in a biological system. If a cell-based assay system is employed, the test compound(s) indentified may then be subjected to in vivo testing to determine their effect on Notch signalling pathway.
  • 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
  • constructs according to the invention comprise a promoter ofthe gene of interest (i.e. of an endogenous target gene), and a coding sequence encoding the desired reporter constructs, for example of GFP or luciferase.
  • a promoter ofthe gene of interest i.e. of an endogenous target gene
  • 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
  • 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 inte ⁇ reted by software 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 target gene expression in cells transfected with recombinant DNA encoding polypeptides. This can be achieved directly, by labelling ofthe protein product, or indirectly by using a reporter gene in the construct.
  • reporter genes are ⁇ -galactosidase and Green Fluorescent Protein (GFP).
  • GFP Green Fluorescent Protein
  • ⁇ -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 therefor 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 therefor 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 either manually, or using FACS sorting.
  • Nucleic acid probes complementary to mRNA may be prepared according to the teaching set forth above, using the general procedures as described by Sambrook et al (1989).
  • the invention comprises the use of an antisense nucleic acid molecule, complementary to a target mRNA, conjugated to a fluorophore which may be used in FACS cell sorting.
  • the present invention also provides a method of detection of polypeptides.
  • 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, protein gel assay, Western Blot analysis, antibody sandwich assays, antibody detection, FACS and ELISA assays.
  • polypeptides can be detected by differential mobility on protein gels, or by other size analysis techniques, such as mass spectrometry.
  • the detection means may be sequence-specific.
  • polypeptide or RNA molecules can be developed which specifically recognise polypeptides in vivo or in vitro.
  • RNA aptamers can be produced by SELEX.
  • SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules. It is described, for example, in U.S. patents 5654151, 5503978, 5567588 and 5270163, as well as PCT publication WO 96/38579
  • the invention in certain embodiments, includes antibodies specifically recognising and binding to polypeptides.
  • Antibodies may be recovered from the serum of immunised animals. Monoclonal antibodies may be prepared from cells from immunised animals in the conventional manner.
  • the antibodies ofthe invention are useful for identifying cells expressing the genes being monitored.
  • Antibodies according to the invention may be whole antibodies of natural classes, such as IgE and IgM antibodies, but are preferably IgG antibodies. Moreover, the invention includes antibody fragments, such as Fab, F(ab')2, Fv and ScFv. Small fragments, such Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.
  • the antibodies may comprise a label.
  • labels which allow the imaging ofthe antibody in neural cells in vivo.
  • Such labels may be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within tissues.
  • they may be fluorescent labels or other labels which are visualisable in tissues and which may be used for cell sorting.
  • antibodies as used herein can be altered antibodies comprising an effector protein such as a label.
  • labels which allow the imaging ofthe distribution ofthe antibody in vivo.
  • Such labels can be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within the body of a patient.
  • they can be fluorescent labels or other labels which are visualisable on tissue
  • Antibodies as described herein can be produced in cell culture. Recombinant DNA technology can be used to produce the antibodies according to established procedure, in bacterial or preferably mammalian cell culture. The selected cell culture system optionally secretes the antibody product, although antibody products can be isolated from non-secreting cells.
  • Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, e.g. foetal calf serum, or trace elements and growth sustaining supplements, e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone manow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like.
  • suitable culture media which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium
  • a mammalian serum e.g. foetal calf serum
  • trace elements and growth sustaining supplements e.g. feeder cells
  • feeder cells such as normal mouse peritoneal exudate cells, spleen
  • Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
  • In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts ofthe desired antibodies.
  • Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stiner reactor, or immobilised or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges.
  • the desired antibodies can also be obtained by multiplying mammalian cells in vivo.
  • hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody-producing tumours.
  • the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection.
  • pristane tetramethyl-pentadecane
  • hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.
  • the cell culture supematants are screened for the desired antibodies, preferentially by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • an enzyme immunoassay e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • the immunoglobulins in the culture supematants or in the ascitic fluid can be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like.
  • the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinity chromatography with the target antigen, or with Protein-A.
  • the antibody is preferably provided together with means for detecting the antibody, which can be enzymatic, fluorescent, radioisotopic or other means.
  • the antibody and the detection means can be provided for simultaneous, simultaneous separate or sequential use, in a kit.
  • the antibodies for use with the invention are assayed for immunospecific binding by any method known in the art.
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA, sandwich immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.
  • Such assays are routine in the art (see, for example, Ausubel et al, eds, 1994, Cunent Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is inco ⁇ orated by reference herein in its entirety). Exemplary immunoassays are described briefly below.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2,1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e. g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2,1% Trasylol
  • protein phosphatase and/or protease inhibitors e. g., EDTA, PMSF, aprotin
  • Western blot analysis generally comprises preparing protein samples, electrophoresis ofthe protein samples in a polyacrylamide gel (e. g., 8%-20% SDS-PAGE depending on the molecular weight ofthe antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e. g., PBS with 3%> BSA or non-fat milk), washing the membrane in washing buffer (e. g., PBS-Tween 20), exposing the membrane to a primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, exposing the membrane to a secondary antibody (which recognises the primary antibody, e.
  • a polyacrylamide gel e. g., 8%-20% SDS-PAGE depending on the molecular weight ofthe antigen
  • a membrane such as nitrocellulose, PVDF or nylon
  • blocking solution e. g., PBS with 3%> BSA or non-fat milk
  • an antihuman antibody conjugated to an enzymatic substrate (e. g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e. g., 32 P or 125 I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence ofthe antigen.
  • an enzymatic substrate e. g., horseradish peroxidase or alkaline phosphatase
  • radioactive molecule e. g., 32 P or 125 I
  • ELISAs generally comprise preparing antigen, coating the well of a 96 well microtitre plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e. g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence ofthe antigen.
  • a detectable compound such as an enzymatic substrate (e. g., horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e. g., horseradish peroxidase or alkaline phosphatase)
  • Immobilisation approaches include covalent immobilisation, such as using amine coupling, surface thiol coupling, ligand thiol coupling and aldehyde coupling, and high affinity capture which relies on high affinity binding of a ligand to an immobilised capturing molecule.
  • capturing molecules include: streptavidin, anti-mouse Ig antibodies, ligand-specific antibodies, protian A, protein G and Tag-specific capture.
  • immobilisation 5 is achieved through binding to a support, particularly a particulate support which is preferably in the form of a bead.
  • the assay will generally involve removal of a sample from a patient prior to the step of detecting 0 a signal resulting from cleavage ofthe intracellular domain.
  • the matrices, substrates, constructs and particles of the present invention may be used to modify immune responses in the immune system of a mammal, such as a human.
  • modulation of the immune system is effected by control of immune cell, preferably T-cell, preferably peripheral T-cell, activity.
  • T-cell preferably peripheral T-cell
  • a detailed description ofthe Notch signalling pathway and conditions affected by it may be found for example in our WO98/20142, WO00/36089 and PCT/GBOO/04391.
  • T cells 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 abenations to the T cell system and infectious diseases such as those caused by Plasmodium species, Microfilariae, Helminths, Mycobacteria, HIV, 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, HIV, Cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, Haemophilus influenza type B, measles, Hepatitis C or Toxicara.
  • infectious diseases such as those caused by Plasmodium species, Microfilariae, Helminths, My
  • 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 hypeneactivity, 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 pu ⁇ ura, 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 pu ⁇ ura, 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 anest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases ofthe gastrointestinal tract, hepatic fibrosis, liver cinhosis 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
  • 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 manow, organs, lenses, pacemakers, natural or artificial skin tissue.
  • 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 manow 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 manow 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 often 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 often carried out simultaneously.
  • Liver transplants are used to treat organ damage caused by viral diseases such as hepititis, 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 hemolysis of recipient red blood cells if there is a mismatch of blood groups.
  • manifestations of GVHD have occuned in liver transplants even when donor and recipient are blood-group compatible.
  • the particles ofthe present invention may also be used in vaccine compositions such as cancer and pathogen vaccines.
  • vaccine compositions such as cancer and pathogen vaccines.
  • Particles 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 ofthe 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.
  • Prefened spray devices for intranasal use are devices for which the performance ofthe 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 ofthe 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 ofthe above bile acids, or N, N-bis (3DGluconoamidopropyl) deoxycholamide.
  • an adjuvant formulation ofthe 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 ofthe 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 ofthe 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 ofthe 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 Ban 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; HIV-1, (such as tat, nef, gpl20 or gpl60); human he ⁇ es viruses, such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSV1 or HSV2; human papilloma viruses (for example HPV6, 11, 16, 18); Influenza virus (whole live or in
  • 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); Bonelia spp., including B. burgdorferi (eg OspA, OspC, DbpA, DbpB), B. garinii (eg OspA, OspC, DbpA, DbpB), B. afzelii (eg OspA, OspC, DbpA, DbpB), B.
  • B. anthracis eg botulinum toxin
  • Bordetella spp including B. pertussis (for example pertactin, pertussis toxin, filamenteous hemagglutinin, adenylate cyclas
  • 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.
  • C. jejuni for example toxins, adhesins and invasins
  • Chlamydia spp. including C. trachomatis (eg MOMP, heparin-binding proteins), C. pneumonie (eg MOMP,
  • E. faecalis E. faecium
  • Escherichia spp including enterotoxic E. coli (for example colonization factors, heat-labile toxin or derivatives thereof, or heat- stable toxin), enterohemonagic E. coli, enteropathogenic E.
  • Haemophilus spp. including H. influenzae type B (eg PRP), non-typable H. influenzae, for example OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrin derived peptides (see for example US 5,843,464); Helicobacter spp, including H. pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa;
  • Legionella spp including L. pneumophila ; Leptospira spp., including L. intenogans; Listeria spp., including L. monocytogenes; Moraxella spp, including M catanhalis, also known as Branhamella catanhalis (for example high and low molecular weight adhesins and invasins); Morexella Catanhalis (including outer membrane vesicles thereof, and OMP106 (see for example W097/41731)); Mycobacterium spp., including M. tuberculosis (for example ESAT6, Antigen 85 A, -B or -C), M. bovis, M. leprae, M. avium, M.
  • ESAT6 Antigen 85 A, -B or -C for example ESAT6, Antigen 85 A, -B or -C
  • M. bovis for example ESAT6, Antigen 85 A, -B or -C
  • M. bovis for example ESAT6, Anti
  • Neisseria spp including N. gononhea and N. meningitidis (for example capsular polysaccharides and conjugates thereof, transferrin-binding proteins, lactoferrin 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. enteritidis; Shigella spp, including S. sonnei, S. dysenteriae, S.
  • Staphylococcus spp. including S. aureus, S. epidermidis
  • Streptococcus spp including S. pneumonie (eg capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline-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. pallidum (eg the outer membrane proteins), T.
  • T. pallidum eg the outer membrane proteins
  • V. cholera for example cholera toxin
  • Yersinia spp including Y. enterocolitica (for example a Yop protein), Y. pestis, Y. pseudotuberculosis.
  • 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. lamblia; Leshmania spp., including L. major;
  • PfEMPl PfEMPl, PD32, 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
  • Approved/licensed vaccines include, for example anthrax vaccines such as Biothrax (BioPort Co ⁇ ); tuberculosis (BCG) vaccines such as TICE BCG (Organon Teknika Co ⁇ ) 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
  • Pneumovax (Merck & Co, Inc) and Pnu-Imune (Lederle Lab Div, American Cyanamid Co); Pneumococcal 7-valent conjugate vaccines (eg diphtheria CRM197 Protein conjugates such as Prevnar from Lederle Lab Div, American Cyanamid Co); poliovirus vaccines such as Poliovax (Aventis Pasteur, Ltd); poliovirus vaccines such as IPOL (Aventis Pasteur, SA); rabies vaccines such as Imovax (Aventis Pasteur, SA) and RabAvert (Chiron Behring GmbH & Co); rubella virus vaccines such as Meruvax II (Merck & Co, Inc); Typhoid Vi polysaccharide vaccines such as TYPHIM Vi (Aventis Pasteur, SA); Varicella virus vaccines such as Varivax (Merck & Co, Inc) and Yellow Fever vaccines such as YF-Vax (Aventis Pasteur, Inc).
  • 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 (hCG beta) antigen, carcinoembryonic antigen, EGFRvIII antigen, Globo H antigen, GM2 antigen, GP100 antigen, HER2/neu antigen, KSA 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.
  • hCG beta human chorionic gonadotropin
  • EGFRvIII antigen Globo H antigen
  • GM2 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 particles of the present invention may also be used for altering the fate of a cell, tissue or organ type by altering Notch pathway function in a cell by a partially or fully non-immunological mode of action (eg by modifying general cell fate, differentiation or proliferation), as described, for example in WO 92/07474, WO 96/27610, WO 97/01571, US 5648464, US 5849869 and US 6004924 (Yale University/Imperial Cancer Technology), the texts of which are herein inco ⁇ orated by reference.
  • the particles ofthe present invention are also useful in methods for altering the fate of any cell, tissue or organ type by altering Notch pathway function in the cell.
  • the present constructs also have application in the treatment of malignant and pre- neoplastic disorders for example by an antiproliferative, rather than immunological mechanism.
  • the conjugates ofthe present invention are especially useful in relation to adenocarcinomas such as: small cell lung cancer, and cancer ofthe 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, breasy 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, he ⁇ es zoster or he ⁇ es simplex virus, Lyme disease, tuberculosis or syphilis; degenerative lesions and diseases and demyelinated lesions.
  • the present invention may be used to treat, for example, diabetes (including diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, sarcoidosis, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, central pontine myelinolysis, Parkinson's disease, Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis, cerebral infarction or ischemia, spinal cord infarction or ischemia, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and 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 neuropathy, Bell's palsy
  • the present invention may further be useful in the promotion of tissue regeneration and repair, for example by modification of differentiation processes.
  • the present invention may also be used to treat diseases associated with defective tissue repair and regeneration such as, for example, cinhosis ofthe liver, hypertrophic scar formation and psoriasis.
  • the invention may also be useful in the treatment of neutropenia or anemia and in techniques of organ regeneration and tissue engineering and stem cell treatments.
  • the active agents of the present invention are administered in the form of pharmaceutical compositions.
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and in addition to one or more active agents 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 carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • 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 also be provided in such a pharmaceutical composition. Examples of -I l l-
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
  • a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response ofthe particular patient.
  • the dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
  • 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 or stem or tissue cells) in an ex vivo manner.
  • cells such as T cells and/or APCs or stem or tissue cells
  • leukocytes such as T cells or APCs may be obtained from a patient or donor in known manner, treated incubated ex vivo in the manner of the present invention, and then administered to a patient.
  • a therapeutically effective daily dose may for example range from 0.01 to 500 mg/kg, for example 0.01 to 50 mg/kg body weight ofthe subject to be treated, for example 0.1 to 20 mg/kg.
  • the particles ofthe present invention may also be administered by intravenous infusion, at a dose which is likely to range from for example 0.001-10 mg/kg/hr.
  • compositions are in unit dosage form.
  • the agents ofthe present invention can be administered by any suitable means including, but not limited to, for example, oral, rectal, nasal, topical (including intradermal, transdermal, aerosol, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal) routes of administration. Subcutaneous, intramuscular or intradermal administration is generally prefened.
  • the active agents are administered in combination with a pharmaceutically acceptable carrier or diluent as described under the heading "Pharmaceutical compositions" above.
  • the pharmaceutically acceptable carrier or diluent may be, for example, sterile isotonic saline solutions, or other isotonic solutions such as phosphate-buffered saline (PBS).
  • Active agents ofthe present invention may suitably be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • 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.
  • Doses such as tablets or capsules comprising the conjugates may be administered singly or two or more at a time, as appropriate. It is also possible to administer the agents in sustained release formulations.
  • 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 inco ⁇ orated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. They can also be inco ⁇ orated, for example at a concentration of between 1 and 10%) by weight, into an ointment consisting of a white wax or white soft paraffin base together with such 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, he ⁇ es 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 also be adminstered by needleless systems, such as ballistic delivery on particles for delivery to the epidermis or dermis or other sites such as mucosal surfaces.
  • active agents may for example be used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • agents may for example 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 ofthe particular patient. It is to be noted that whilst the above- mentioned dosages are exemplary ofthe 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 ofthe patient.
  • treatment or therapy should be taken to encompass diagnostic and prophylactic applications , and treatment includes both human and veterinary applications.
  • modified cells of the present invention are preferably administered to a host by direct injection into the lymph nodes of 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 known in the art. For example, an enriched population of T-cells can be obtained using immunoaffmity 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 manow 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 constructs/particles 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.
  • THl 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 THl 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.
  • antigens known to be associated with auto-immune diseases such as myelin basic protein (associated with multiple sclerosis), collagen (associated with rheumatoid arthritis), and insulin (diabetes), or antigens associated with rejection of non-self tissue such as MHC antigens or antigenic determinants thereof.
  • antigens may be obtained from the tissue donor.
  • Polynucleotides coding for antigens or antigenic determinants which may be expessed in a subject may also be used.
  • antigens or antigenic determinants or polynucleotides coding for them may be included in or on the matrix/substrate eg particle.
  • autoantigen includes any substance or a component thereof normally found within a mammal that, in an autoimmune disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction, such as heatshock proteins (HSP), which although not necessarily specific to a particular tissue are normally shielded from the immune system.
  • HSP heatshock proteins
  • “Bystander suppression” is suppression at the locus of autoimmune attack of cells that contribute to autoimmune destruction; this suppression is mediated by the release of one or more immunosuppressive factors (including Th2-enhancing cytokines and Thl -inhibiting cytokines) from suppressor/regulatory T-cells elicited by a bystander antigen and recruited to the site where cells contributing to autoimmune destruction are found.
  • the result may be antigen-nonspecific but locally restricted downregulation ofthe autoimmune responses responsible for tissue destruction.
  • Autoimmune disease includes spontaneous or induced malfunction ofthe immune system of mammals, including humans, in which the immune system fails to distinguish between foreign immunogenic substances within the mammal and/or autologous substances and, as a result, treats autologous tissues and substances as if they were foreign and mounts an immune response against them.
  • T lymphocytes are specifically activated upon recognition of foreign and/or self antigens as a complex with self Major Histocompatibility Complex (MHC) gene products on the surface of antigen-presenting cells (APC).
  • MHC Histocompatibility Complex
  • Autoimmune disorders include organ specific diseases and systemic illnesses.
  • organ-specific autoimmune diseases include, for example, several forms of anemia (aplastic, hemolytic), autoimmune hepatitis, iridocyclitis, scleritis, uveitis, orchitis and idiopathic thrombocytopenic pu ⁇ ura.
  • Systemic autoimmune diseases include, for example: 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 pu ⁇ ura, Behcet's Syndrome, Takayasu arteritis, Giant cell arteritis, Thrombangiitis obliterans, polymyalgia rheumatica, essential (mixed) cryoglobulinemia, psoriasis vulgaris and psoriatic arthritis, diffuse fasciitis with or without eosinophilia, relapsing panniculitis, relapsing polychondritis, lymphomatoid granulomatosis, erythema nodosum, ankylosing spondylitis, Reiter's syndrome and different forms of inflammatory
  • Autoantigens may be derived from tissues, proteins etc associated with the disease which give rise to the relevant autoimmune response. For example:
  • Autoimmune hepatitis liver cell antigens Autoimmune parotitis parotid gland antigens Autoimmune haemolytic anemia red cell membrane proteins; 95-110 kDa membrane protein
  • Idiopathic leukopenia granulocyte antigens Idiopathic thrombocytopenia platelet membrane proteins; Glycoprotein Ila/IIIb
  • Spontaneous infertility Sperm antigens eg post-acrosomal sperm protein (PASP)
  • PASP post-acrosomal sperm protein
  • autoimmune antigens and autoimmune antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
  • 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 (T-cell) receptor.
  • the antigen used in the present invention is an immunogen.
  • the antigen 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.
  • the antigen moiety may be, for example, a synthetic MHC-peptide complex i.e. a fragment of the MHC molecule bearing the antigen groove bearing an element of the antigen.
  • a synthetic MHC-peptide complex i.e. a fragment of the MHC molecule bearing the antigen groove bearing an element of the antigen.
  • Such complexes have been described in Altman et al. (1996) Science 274: 94-96.
  • the autoantigen or bystander antigen may be a Goodpasture's autoantigen or bystander antigen for use to treat Goodpasture's disease/syndrome.
  • Goodpasture's autoantigen includes any substance or a component thereof normally found within a mammal that, in Goodpasture's disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of Goodpasture's disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • Goodpasture's bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in Goodpasture's disease.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • Examples of Goodpasture's autoantigens and Goodpasture's bystander antigens include, but are not limited to collagens in particular, type IV, alpha 3 collagens.
  • An amino acid sequence for a human collagen, type IV, alpha 3 (Goodpasture antigen) is reported as follows (GenBank Accession No NM_001723):
  • GenBank Accession Nos NM 031366.1, NM_031364.1, NM_031363.1, NM_031362.1 and NM_000091.2 (collagen, type IV, alpha 3 (Goodpasture antigen) (COL4A3)) and NM_130778.1 and NM_000494.2 (collagen, type XVII, alpha 1 (COL17A1)).
  • the autoantigen or bystander antigen may be a renal autoantigen or renal bystander antigen, for use to treat autoimmune disease ofthe kidney.
  • renal autoantigen includes any substance or a component thereof normally found within a mammal that, in autoimmune disease ofthe kidney, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease ofthe kidney when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • renal bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe kidney under autoimmune attack in an autoimmune disease ofthe kidney.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • renal autoantigens and renal bystander antigens include, but are not limited to glomerular basement membrane (GBM) antigens (Goodpasture's antigens as described further above) and tubular basement membrane (TBM) antigens associated with tubulointerstitial nephritis (TIN).
  • GBM glomerular basement membrane
  • TBM tubular basement membrane
  • the autoantigen or bystander antigen may be a Pemphigus autoantigen or bystander antigen for use to treat Pemphigus.
  • Pemphigus autoantigen includes any substance or a component thereof normally found within a mammal that, in Pemphigus, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of Pemphigus when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • Pemphigus bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in Pemphigus.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • Pemphigus includes, for example, pemphigus vulgaris, pemphigus foliaceus and bullous pemphigoid.
  • Pemphigus autoantigens and Pemphigus bystander antigens include, but are not limited to desmoglein 1 and desmoglein 3.
  • DSG1 desmoglein 1
  • one or more antigenic determinants may be used in place of a full antigen.
  • some specific class II MHC-associated autoantigen peptide sequences are as follows (see US 5783567):
  • the autoantigen or bystander antigen may be a thyroid autoantigen or bystander antigen for use to treat thyroid autoimmune disease.
  • thyroid autoimmune disease includes any condition in which there is an autoimmune reaction to the thyroid or a component thereof.
  • the best known autoimmune diseases ofthe thyroid include Graves' disease (also known as thyrotoxicosis), Hashimoto's thyroiditis and primary hypothyroidism. Further examples include atrophic autoimmune thyroiditis, primary myxoedema, asymptomatic thyroiditis, postpartal thyroiditis and neonatal hypothyroidism.
  • Diagnosis is typically based on the detection of autoantibodies in the patient.
  • the three main thyroid autoantigens are the TSH receptor, thyroperoxidase (TPO, also known as microsomal antigen) and thyroglobulin (Tg) (Dawe, K., Hutchings, P., Champion, B., Cooke, A., Roitt, I., "Autoantigens in Thyroid diseases", Springer Semin. Immunopathol. 14, 285-307, 1993).
  • thyroid autoantigen includes any substance or a component thereof normally found within a mammal that, in a thyroid autoimmune disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of a thyroid autoimmune disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ (usually the thyroid gland) under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • thyroid bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe thyroid gland under autoimmune attack.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • thyroid autoimmune/bystander antigens and thyroid autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
  • thyroid autoantigens and thyroid bystander antigens include, but are not limited to, the thyroid stimulatory hormone (TSH) receptor (associated in particular with Grave's disease), thyroperoxidase (TPO; associated with Hashimoto's thyroiditis) and thyroglobulin (Tg).
  • TSH thyroid stimulatory hormone
  • TPO thyroperoxidase
  • Tg thyroglobulin
  • an amino acid sequence for a human thyroid stimulatory hormone receptor (TSHR) is reported as follows (GenBank Accession No M32215):
  • the autoantigen or bystander antigen may be a Wegener's autoantigen or bystander antigen for use to treat Wegener's disease.
  • the term "Wegener's autoantigen” as used herein includes any substance or a component thereof normally found within a mammal that, in Wegener's disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of Wegener's disease when administered to mammals.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • Wegener's bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in Wegener's disease.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • the autoantigen or bystander antigen may be an autoimmune anemia autoantigen or bystander antigen fo use to treat autoimmune anemia.
  • autoimmune anemia includes any disease in which red blood cells (RBCs) or a component thereof come under autoimmune attack.
  • RBCs red blood cells
  • the term includes, for example, autoimmune haemolytic anemia, including both “warm autoantibody type” and “cold autoantibody type”.
  • autoimmune anemia autoantigen includes any substance or a component thereof normally found within a mammal that, in autoimmune anemia, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of autoimmune anemia when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • autoimmune anemia bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe red blood cells (RBCs) under autoimmune attack in autoimmune anemia.
  • RBCs red blood cells
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • Autoimmune anemia includes, in particular, autoimmune hemolytic anemia.
  • autoimmune hemolytic anemia autoantigens and bystander antigens include, but are not limited to Rhesus (Rh) antigens such as E, e or C, red cell proteins and glycoproteins such as red cell protein band 4.1 and red cell membrane band 3 glycoprotein.
  • Rhesus (Rh) antigens such as E, e or C
  • red cell proteins and glycoproteins such as red cell protein band 4.1 and red cell membrane band 3 glycoprotein.
  • Wr b En a
  • Ge, A, B antigens within the Kidd and Kell blood group systems.
  • the autoantigen or bystander antigen may be an autoimmune thrombocytopenia autoantigen or bystander antigen for use to treat autoimmune thrombocytopenia.
  • autoimmune thrombocytopenia autoantigen includes any substance or a component thereof normally found within a mammal that, in autoimmune thrombocytopenia, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of autoimmune thrombocytopenia when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • autoimmune thrombocytopenia bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe platelets under autoimmune attack in autoimmune thrombocytopenia.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • Autoimmune thrombocytopenia includes, in particular, autoimmune thrombocytopenia pu ⁇ ura.
  • autoimmune thrombocytopenia pu ⁇ ura autoantigens and bystander antigens include, but are not limited to platelet glycoproteins such as GPIIb/IIIa and/or GPIb/IX.
  • GPIIb human platelet glycoprotein lib
  • the autoantigen or bystander antigen may be an autoimmune gastritis autoantigen or bystander antigen for use to treat autoimmune gastritis.
  • autoimmune gastritis includes any disease in which gastric tissue or a component thereof comes under autoimmune attack.
  • the term includes, for example, pernicious anemia.
  • autoimmune gastritis autoantigen includes any substance or a component thereof normally found within a mammal that, in autoimmune gastritis, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of autoimmune gastritis when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • autoimmune gastritis bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe gastric tissue under autoimmune attack in autoimmune gastritis.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • Autoimmune gastritis includes, in particular, pernicious anemia.
  • autoimmune gastritis autoantigens and bystander antigens include, but are not limited to parietal cell antigens such as gastric H+/K+ ATPase, (lOOkDa alpha subunit and 60-90kDa beta subunit; especially the beta subunit) and intrinsic factor.
  • the autoantigen or bystander antigen may be an autoimmune hepatitis autoantigen or bystander antigen for use to treat autoimmune hepatitis.
  • autoimmune hepatitis includes any disease in which the liver or a component ofthe liver comes under autoimmune attack.
  • the term thus includes, for example, primary biliary cinhosis (PBC) and primary sclerosing cholangitis.
  • autoimmune hepatitis autoantigen includes any substance or a component thereof normally found within a mammal that, in autoimmune hepatitis, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of autoimmune hepatitis when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • autoimmune hepatitis bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in autoimmune gastritis.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • autoimmune hepatitis autoantigens and bystander antigens include, but are not limited to cytochrome P450s such as cytochrome P450 2D6, cytochrome P450 2C9 and cytochrome P450 1 A2, the asialoglycoprotein receptor (ASGP R) and UDP- glucuronosyltransferases (UGTs).
  • cytochrome P450s such as cytochrome P450 2D6, cytochrome P450 2C9 and cytochrome P450 1 A2
  • ASGP R asialoglycoprotein receptor
  • UDTs UDP- glucuronosyltransferases
  • cDNA encoding human cytochrome P450-2d6 (coding for antigen for AIH Type2a LKM1 antibody) is reported as follows (GenBank Accession No E15820): 1 atggggctag aagcactggt gcccctggcc atgatagtgg ccatcttcct gctcctggtg
  • CYP1A2 An amino acid sequence for a human cytochrome P450-1A2 (CYP1A2) is reported as follows (GenBank Accession No AF182274): MALSQSVPFSATELLLASAIFCLVFWVLKGLRPRVPKGLKSPPEP G PLLGHVLTLGKNPHLALSRMSQRYGD VLQIRIGSTPVLVLSRLDTIRQALVRQGDDFKGRPDLYTSTLITDGQSLTFSTDSGPVWAARRRLAQNALNTFS IASDPASSSSCYLEEHVSKEAMALISRLQELMAGPGHFDPYNQVVVSVSVANVIGAMCFGQHFPESSDEMLSLVKN THEFVETASSGNPLDFFPILRYLPNPALQRFKAFNQRFL FLQKTVQEHYQDFDKNSVRDITGALFKHSKKGPR ASGNLIPQEKIVNLVNDVFGAGFDTVTTAIS SLMYLVTKPEIQRKIQKELDTVIGRERRPRLSDRPQLPY
  • PBC primary biliary cinhosis
  • bystander antigens include, but are not limited to mitochondrial antigens such as pyruvate dehydrogenase (El -alpha decarboxylase, El -beta decarboxylase and E2 acetyltransferase), branched-chain 2-oxo-acid dehydrogenases and 2-oxoglutarate dehydrogenases.
  • mitochondrial antigens such as pyruvate dehydrogenase (El -alpha decarboxylase, El -beta decarboxylase and E2 acetyltransferase), branched-chain 2-oxo-acid dehydrogenases and 2-oxoglutarate dehydrogenases.
  • autoantigen or bystander antigen may be an autoimmune vasculitis autoantigen or bystander antigen for use to treat autoimmune vasculitis.
  • autoimmune vasculitis includes any disease in which blood vessels or a component thereof come under autoimmune attack and includes, for example, large vessel vasculitis such as giant cell arteritis and Takayasu's disease, medium-sized vessel vasculitis such as polyarteritis nodosa and Kawasaki disease and small vessel vasculitis such as Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis, Henoch Schonlein pu ⁇ ura, essential cryoglobulinaemic vasculitis and cutaneous leukocytoclastic angiitis.
  • large vessel vasculitis such as giant cell arteritis and Takayasu's disease
  • medium-sized vessel vasculitis such as polyarteritis nodosa and Kawasaki disease
  • small vessel vasculitis such as Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis, Henoch Schonlein pu ⁇ ura,
  • autoimmune vasculitis autoantigen includes any substance or a component thereof normally found within a mammal that, in autoimmune vasculitis, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of autoimmune vasculitis when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • autoimmune vasculitis bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe blood vessel tissue under autoimmune attack in autoimmune vasculitis.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction. Examples of vasculitis autoantigens and bystander antigens include, but are not limited to basement membrane antigens (especially the noncollagenous domain ofthe alpha 3 chain of type IV collagen) and endothelial cell antigens.
  • the autoantigen or bystander antigen may be an ocular autoantigen or bystander antigen for use to treat an autoimmune disease of the eye.
  • autoimmune disease ofthe eye includes any disease in which the eye or a component thereof comes under autoimmune attack.
  • the term thus includes, for example, cicatricial pemphigoid, uveitis, Mooren's ulcer, Reiter's syndrome, Behcet's syndrome, Vogt- Koyanagi-Harada Syndrome, scleritis, lens-induced uveitis, optic neuritis and giant-cell arteritis.
  • ocular autoantigen includes any substance or a component thereof normally found within the eye of a mammal that, in an autoimmune disease ofthe eye, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of autoimmune disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • ocular bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe eye under autoimmune attack.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • ocular autoantigens and bystander antigens include, but are not limited to retinal antigens such as ocular antigen, S-antigen, inte ⁇ hotoreceptor retinoid binding protein (see eg Exp. Eye Res. 56:463 (93)) in uveitis and alpha crystallin in lens-induced uveitis.
  • the autoantigen or bystander antigen may be an adrenal autoantigen or bystander antigen for use to treat adrenal autoimmune disease.
  • adrenal autoimmune disease includes any disease in which the adrenal gland or a component thereof comes under autoimmune attack.
  • the term includes, for example, Addison's disease.
  • adrenal autoantigen as used herein includes any substance or a component thereof normally found within a mammal that, in adrenal autoimmune disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of adrenal autoimmune disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • adrenal bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe adrenal gland under autoimmune attack in adrenal autoimmune disease.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • adrenal autoantigens and bystander antigens include, but are not limited to adrenal cell antigens such as the adrenocorticotropic hormone receptor (ACTH receptor) and enzymes such as 21-hydroxylase and 17-hydroxylase.
  • ACTH receptor adrenocorticotropic hormone receptor
  • the autoantigen or bystander antigen may be a cardiac autoantigen or bystander antigen for use to treat cardiac autoimmune disease.
  • cardiac autoimmune disease includes any disease in which the heart or a component thereof comes under autoimmune attack.
  • the term includes, for example, autoimmune myocarditis, dilated cardiomyopathy, autoimmune rheumatic fever and Chagas' disease.
  • cardiac autoantigen includes any substance or a component thereof normally found within a mammal that, in cardiac autoimmune disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of cardiac autoimmune disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • cardiac bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe heart tissue under autoimmune attack in cardiac autoimmune disease.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • cardiac autoantigens and bystander antigens include, but are not limited to heart muscle cell antigens such as mysosin, laminin, beta-1 adrenergic receptors, adenine nucleotide translocator (ANT) protein and branched-chain ketodehydrogenase (BCKD).
  • heart muscle cell antigens such as mysosin, laminin, beta-1 adrenergic receptors, adenine nucleotide translocator (ANT) protein and branched-chain ketodehydrogenase (BCKD).
  • the autoantigen or bystander antigen may be a scleroderma or myositis autoantigen or bystander antigen for use to treat scleroderma or myositis.
  • myositis/scleroderma autoantigen includes any substance or a component thereof normally found within a mammal that, in myositis (particularly in dermatomyositis or polymyositis) or scleroderma, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of myositis (particularly in dermatomyositis or polymyositis) or scleroderma when administered to mammals.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • myositis/scleroderma bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in myositis (particularly in dermatomyositis or polymyositis) or scleroderma.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • scleroderma As described, for example, in US 5862360, scleroderma, or systemic sclerosis, is characterized by deposition of fibrous connective tissue in the skin, and often in many other organ systems. It may be accompanied by vascular lesions, especially in the skin, lungs, and kidneys. The course of this disease is variable, but it is usually slowly progressive. Scleroderma may be limited in scope and compatible with a normal life span. Systemic involvement, however, can be fatal.
  • Scleroderma may be classified as either diffuse or limited, on the basis ofthe extent of skin and internal organ involvement.
  • the diffuse form is characterized by thickening and fibrosis of skin over the proximal extremities and trunk. The heart, lungs, kidneys, and gastrointestinal tract below the esophagus are often involved.
  • Limited scleroderma is characterized by cutaneous involvement ofthe hands and face. Visceral involvement occurs less commonly.
  • the limited form has a better prognosis than the diffuse form, except when pulmonary hypertension is present.
  • Antinuclear antibodies are found in over 95 percent of patients with scleroderma. Specific antinuclear antibodies have been shown to be directed to topoisomerase I, centromere proteins, RNA polymerases, or nucleolar components. Different antibodies are associated with particular clinical patterns of scleroderma. For example, antibodies to topoisomerase I (Scl-70) and to RNA polymerases (usually RNA polymerase III) are seen in patients with diffuse scleroderma. Antibodies to nuclear ribonucleoprotein (nRNP) are associated with diffuse and limited scleroderma.
  • topoisomerase I Scl-70
  • RNA polymerases usually RNA polymerase III
  • nRNP nuclear ribonucleoprotein
  • Centrosomes are essential structures that are highly conserved, from plants to mammals, and are important for various cellular processes. Centrosomes play a crucial role in cell division and its regulation. Centrosomes organize the mitotic spindle for separating chromosomes during cell division, thus ensuring genetic fidelity. In most cells, the centrosome includes a pair of centrioles that lie at the center of a dense, partially filamentous matrix, the pericentriolar material (PCM). The microtubule cytoskeleton is anchored to the centrosome or some other form of microtubule organizing center (MTOC), which is thought to serve as a site of microtubule nucleation.
  • PCM pericentriolar material
  • idiopathic inflammatory myopathies polymyositis, dermatomyositis and the related overlap syndromes disorder, such as polymyositis- sclerode ⁇ na overlap, are inflammatory myopathies that are characterized by chronic muscle inflammation and proximal muscle weakness.
  • the muscle inflammation causes muscle tenderness, muscle weakness, and ultimately muscle atrophy and fibrosis (see, for example, Plotz, et al. Annals of Internal Med. I l l : 143-157(1989)).
  • Other systems besides muscle can be affected by these conditions, resulting in arthritis, Raynaud's phenomenon, and interstitial lung disease.
  • polymyositis and dermatomyositis are distinguished by the presence of a characteristic rash in patients with dermatomyositis. Differences in the myositis of these conditions can be distinguished in some studies of muscle pathology.
  • Autoantibodies can be detected in about 90%> of patients with polymyositis and dermatomyositis (Reichlin and Arnett, Arthritis and Rheum. 27: 1150-1156 (1984)). Sera from about 60%> of these patients form precipitates with bovine thymus extracts on Ouchterlony immunodiffusion (ID), while sera from other patients stain tissue culture substrates, such as HEp-2 cells, by indirect immunofluorescence (IIF) (see, e.g., Targoff and Reichlin Arthritis and Rheum. 28: 796-803 (1985); Nishikai and Reichlin Arthritis and Rheum.
  • IIF indirect immunofluorescence
  • Anti-Ul RNP which is frequently found in patients with SLE, may also be found in mixed connective tissue disease, overlap syndromes involving myositis, or in some cases of myositis alone. This antibody reacts with proteins that are uniquely present on the UI small nuclear ribonucleoprotein, which is one ofthe nuclear RNPs that are involved in splicing mRNA.
  • Autoantibodies such as anti-Sm, anti-Ro/SSA, and anti-La/SSB, that are usually associated with other conditions, are sometimes found in patients with overlap syndromes.
  • Anti-Ku has been found in myositis-scleroderma overlap syndrome and in SLE.
  • the Ku antigen is a DNA binding protein complex with two polypeptide components, both of which have been cloned.
  • Anti Jo-1 and other anti-synthetases are disease specific.
  • Other myositis-associated antibodies are anti-PM-Scl, which is present in about 5-10% of myositis patients, many of whom have polymyositis-scleroderma overlap, and anti-Mi-2, which is present in about 8%> of myositis patients, almost exclusively in dermatomyositis.
  • Mi-2 is found in high titer in about 20%) of all dermatomyositis patients and in low titer in less than 5%> of polymyositis patients (see, e.g., Targoff and Reichlin, Mt. Sinai J. of Med. 55: 487-493 (1988)).
  • Anti-Mi-2 was found to be a myositis-specific autoantibody by Targoff, et al. Arthritis and Rheum. 28: 796-803 (1985). Furthermore, all patients with the antibody have the dermatomyositis rash.
  • Bovine thymus Mi-2 antigen was originally found to be a nuclear protein that separates in SDS polyacrylamide (SDS-PAGE) gels into two bands with apparent molecular weights of 53 kilodaltons (hereinafter kDa) and 61 KDa, respectively. Recently, additional higher molecular weight bands have been found.
  • SDS-PAGE SDS polyacrylamide
  • the bovine thymus antigenic activity is destroyed by SDS-PAGE and is trypsin sensitive, but not RNAse sensitive (Targroff et al. Arthritis and Rheum. 28: 796-803 (1985)).
  • Anti-PM-1 was first identified as an antibody found in 61% > of dermatomyositis/polymyositis patients, including patients; with polymyositis-scleroderma overlap (Wolfe, et al. J. Clin. Invest. 59: 176-178 (1977)). PM-1 was subsequently shown to be more than one antibody. The unique specificity component of PM-1 was later named PM-Scl (Reichlin, et al. J. Clin. Immunol. 4: 40-44 (1984)). Anti-PM-Scl is found in the sera of about 5-10% of myositis patients, but is most commonly associated with polymyositis-scleroderma overlap syndrome. It also occurs in patients with polymyositis or dermatomyositis alone or in patients with scleroderma without myositis.
  • Anti-PM-Scl antibody immunoprecipitates a complex from HeLa cell extracts of at least eleven polypeptides that have molecular weights ranging from about 20 to 110 kDa (see, Reimer, et al., J. Immunol. 137:3802-3808 (1986).
  • the antigen is trypsin-sensitive, occurs in nucleoli (see, e.g., Targoff and Reichlin Arthritis Rheum. 28: 226-230 (1985)) and is believed to be a preribosomal particle.
  • myositis/scleroderma autoimmune/bystander antigens and myositis/scleroderma autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
  • myositis/scleroderma autoantigens and myositis/scleroderma bystander antigens include, but are not limited to, Jo-1 (his-tRNA synthetase), PM-Scl, Mi-2, Ku, PL-7 (thr- tRNA synthetase), PL- 12 (ala-tRNA-synthetase), SRP (signal recognition particle), Anti- nRNP (UI small nuclear RNP), Ro/SS-A, and La SS-B.
  • PM/Scl-lOOa amino acid sequence for a human 100 kD Pm-Scl autoantigen protein
  • Jo-1 histidyl-tRNA synthetase autoantigen protein
  • GenBank Accession Nos AF241268.1, AF353396.1 (scleroderma-associated autoantigen); NM_005033.1 (polymyositis/scleroderma autoantigen 1 (75kDa) (PMSCL1)); XM_001527.4, NM_002685.1 (polymyositis/scleroderma autoantigen 2 (lOOkDa) (PMSCL2)).
  • the autoantigen or bystander antigen may be a nervous system autoantigen or bystander antigen for use to treat an autoimmune disease ofthe nervous system.
  • autoimmune disease ofthe nervous system includes any disease in which nervous tissue or a component thereof comes under autoimmune attack.
  • the term includes, for example central nervous system diseases having an autoimmune etiology such as multiple sclerosis (MS), perivenous encephalomyelitis, autoimmune myelopathies, paraneoplastic cerebellar degeneration, paraneoplastic limbic (cortical) degeneration, stiff man syndrome, choreas (such as Sydenham's chorea), stroke, focal epilepsy and migraine; and peripheral nervous system diseases having an autoimmune etiology such as Guillain-Bane syndrome, Miller Fisher syndrome, chronic inflammatory demyelinating neuropathy, multifocal motor neuropathy with conduction block, demyelinating neuropathy associated with anti-myelin-associated glycoprotein antibodies, paraneoplastyic sensory neuropathy, POEMS, dorsal root ganglion neuronitis, acute panautonomic neuropathy and brachial neutritis.
  • MS multiple sclerosis
  • POEMS dor
  • neural system autoantigen includes any nervous system substance or a component thereof normally found within a mammal that, in an autoimmune disease ofthe nervous system, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease ofthe nervous system when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • the term "nervous system bystander antigen” as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in an autoimmune disease ofthe nervous system.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • the nervous system autoantigen or nervous system bystander antigen is an MS autoantigen or MS bystander antigen.
  • MS autoantigen includes any nervous system substance or a component thereof normally found within a mammal that, in multiple sclerosis (MS), becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack.
  • the term also includes antigenic substances that induce conditions having the characteristics of MS when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens.
  • immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • MS bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component of nervous tissue under autoimmune attack in MS.
  • the term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack.
  • the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.

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Abstract

A product comprises i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.

Description

Medical Treatment
Field of the invention
The present invention relates to therapeutic modulation of Notch signalling, particularly, but not exclusively, in immune cells.
Background of the Invention
Notch signal transduction plays a critical role in cell fate determination in vertebrate and invertebrate tissues. Notch is expressed at many stages of Drosophila embryonic and larval development and in many different cells implying a wide range of functions including an important role in neurogenesis and in the differentiation of mesodermal and endodermal cells.
There are at least four mammalian Notch genes (Notch- 1, Notch-2, Notch-3 and Notch-4).
Notch-1, which most closely resembles the proteins of invertebrates and lower vertebrates, is widely expressed and is essential for early development. Recent evidence suggests that Notch signalling contributes to lineage commitment of immature T-cells in the thymus.
During maturation in the thymus, T-cells acquire the ability to distinguish self-antigens from those that are non-self, a process termed "self tolerance". Tolerance to a non-self antigen, however, may be induced by immunisation under specific conditions with a peptide fragment comprising that antigen. In autoimmune diseases such as multiple sclerosis, rheumatoid arthritis or diabetes, there is a failure of the proper regulation of tolerance. Improved treatment methods for re-establishing tolerance are desirable for autoimmune diseases. Similarly in allergic conditions and for transplantation of an organ or tissue from a donor individual, induction of tolerance to particular foreign antigens or profiles of foreign antigens is desirable.
The expression on the cell surface of normal adult cells of the peripheral immune system of
Notch and its ligands, Delta and Serrate, suggests a role for these proteins in T-cell acquired immunocompetence. T-cells express Notch-1 mRNA constitutively. Delta expression is limited to only a subset of T-cells in the peripheral lymphoid tissues. Serrate expression is restricted to a subset of antigen presenting cells (APCs). These observations reinforce the view that the Notch receptor ligand family continues to regulate cell fate decisions in the immune system beyond embryonic development with Notch signalling playing a central role in the induction of peripheral unresponsiveness (tolerance or anergy), linked suppression and infectious tolerance (Hoyne et al).
Thus, as described in WO 98/20142, manipulation ofthe Notch signalling pathway can be used in immunotherapy and in the prevention and/or treatment of T-cell mediated diseases. For example, allergy, autoimmunity, graft rejection, cancer and infectious diseases may be targeted.
A description of the Notch signalling pathway and conditions affected by it may be found, for example, in our published PCT Applications as follows:
PCT/GB97/03058 (filed on 6 November 1997 and published as WO 98/20142; claiming priority from GB 9623236.8 filed on 7 November 1996, GB 9715674.9 filed on 24 July 1997 and GB 9719350.2 filed on 11 September 1997);
PCT/GB99/04233 (filed on 15 December 1999 and published as WO 00/36089; claiming priority from GB 9827604.1 filed on 15 December 1999);
PCT/GB00/04391 (filed on 17 November 2000 and published as WO 0135990; claiming priority from GB 9927328.6 filed on 18 November 1999); PCT/GBOl/03503 (filed on 3 August 2001 and published as WO 02/12890; claiming priority from GB 0019242.7 filed on 4 August 2000);
PCT/GB02/02438 (filed on 24 May 2002 and published as WO 02/096952; claiming priority from GB 0112818.0 filed on 25 May 2001);
PCT/GB02/03381 (filed on 25 July 2002 and published as WO 03/012111; claiming priority from GB 0118155.1 filed on 25 July 2001);
PCT/GB02/03397 (filed on 25 July 2002 and published as WO 03/012441; claiming priority from GB0118153.6 filed on 25 July 2001, GB0207930.9 filed on 5 April 2002, GB
0212282.8 filed on 28 May 2002 and GB 0212283.6 filed on 28 May 2002);
PCT/GB02/03426 (filed on 25 July 2002 and published as WO 03/011317; claiming priority from GB0118153.6 filed on 25 July 2001, GB0207930.9 filed on 5 April 2002, GB
0212282.8 filed on 28 May 2002 and GB 0212283.6 filed on 28 May 2002); PCT/GB02/04390 (filed on 27 September 2002 and published as WO 03/029293; claiming priority from GB 0123379.0 filed on 28 September 2001);
PCT/GB02/05137 (filed on 13 November 2002 and published as WO 03/041735; claiming priority from GB 0127267.3 filed on 14 November 2001, PCT/GB02/03426 filed on 25 July 2002, GB 0220849.4 filed on 7 September 2002, GB 0220913.8 filed on 10 September 2002 and PCT/GB02/004390 filed on 27 September 2002);
PCT/GB02/05133 (filed on 13 November 2002 and published as WO 03/042246; claiming priority from GB 0127271.5 filed on 14 November 2001 and GB 0220913.8 filed on 10 September 2002); PCT/GB2003/003285 (filed on 1 August 2003); and PCT/GB2003/003908 (filed on 8 September 2003).
Each of PCT/GB97/03058 (WO 98/20142), PCT/GB99/04233 (WO 00/36089), PCT/GB00/04391 (WO 0135990), PCT/GBOl/03503 (WO 02/12890), PCT/GB02/02438 (WO 02/096952), PCT/GB02/03381 (WO 03/012111), PCT/GB02/03397 (WO 03/012441), PCT/GB02/03426 (WO 03/011317), PCT/GB02/04390 (WO 03/029293), PCT/GB02/05137 (WO 03/041735), PCT/GB02/05133 (WO 03/042246), PCT/GB2003/003285 and PCT/GB2003/003908 is hereby incorporated herein by reference,
Reference is made also to Hoyne G.F. et al (1999) Int Arch Allergy Immunol 118:122-124; Hoyne et al. (2000) Immunology 100:281-288; Hoyne G.F. et al (2000) Intl Immunol 12:177- 185; Hoyne, G. et al. (2001) Immunological Reviews 182:215-227; each of which is also incoφorated herein by reference.
Varnum-Finney at al (Blood 1998, Vol 91, No 11, pp4084-4091) describes how the effect of Jagged- 1 on murine marrow precursor cells was assessed by co-culturing sorted precursor cells with a 3T3 cell layer that expressed human Jagged- 1 or by incubating sorted precursors with beads coated with the purified extracellular domain of human Jagged- 1.
The present invention seeks to provide products, methods, constructs, uses and compositions for modulating the Notch signalling pathway in therapy, and particularly for modulation of immune cell activity in immunotherapy. Administration of modulators of Notch signalling according to the present invention provides improved activity, especially improved Notch signalling agonist activity.
Statements of the Invention
According to a first aspect ofthe present invention there is provided a product comprising i) a pharmaceutically acceptable support matrix or substrate (the terms "matrix" and substrate" are used interchangeably herein) suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling (these preferably being activators ofthe Notch receptor); and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation ofthe immune system.
Such a product may be used to modulate (preferably reduce) an immune response to said antigen or antigenic determinant.
Suitably the product comprises i) a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of an immune response to said antigen or antigenic determinant.
It will be appreciated that where there are a multiplicity of modulators of Notch signalling these may each be the same or different to any or all ofthe others.
Preferably the modulators of Notch signalling are presented by the support matrix or substrate in an orientation suitable for activation of a Notch receptor, and are preferably predominantly orientated on the surface of the support matrix or substrate. Suitably the modulators of Notch signalling may be coupled to the matrix or substrate by chemical coupling, affinity coupling or adsorption coupling. Suitably the support matrix or substrate may be a particulate support matrix or substrate, ie a particle such as a bead, preferably a microbead or nanobead.
In one embodiment the antigen or antigenic determinant used in the product may be an allergen or antigenic determinant thereof.
Alternatively the antigen or antigenic determinant used in the product may be an autoantigen or bystander antigen or antigenic determinant thereof.
Alternatively the antigen or antigenic determinant used in the product may be a graft antigen such as an MHC (HLA) antigen or antigenic determinant thereof.
Preferably the pharmaceutically acceptable support matrix or substrate bears at least 5, suitably at least 10, at least 20, or at least 50, or at least 100 modulators of Notch signalling which are bound/coupled thereto.
Suitably the pharmaceutically acceptable support matrix or substrate has a maximum linear dimension of from about 1 nanometre to about 1000 micrometres, for example from about 10 nanometres to about 100 micrometres.
Preferably the pharmaceutically acceptable support matrix or substrate has a maximum linear dimension of from about 10 nanometres to about 100 micrometres, preferably from about 40 nanometres to about 10 micrometres, preferably from about 40 nanometres to about 1000 nanometres.
Suitably the pharmaceutically acceptable support matrix or substrate comprises a polymeric material, preferably a substantially biodegradable material or materials. In one embodiment the support matrix or substrate may be in the form of an implantable support matrix or substrate.
It will be appreciated that the pharmaceutically acceptable support matrix or substrate can take many different forms such as polymers, plastics, porous materials such as resin or modified cellulose, beads (such as microspheres and microbeads, nanospheres and nanoparticles) and liposomes.
In one embodiment the pharmaceutically acceptable support matrix or substrate may be a particulate matrix or substrate, such as a bead, sphere, particle or carrier, for example having a diameter (or, for example, within a collection of beads, a mean diameter) of from about 0.001 to about 1000 micrometres, for example from about 0.01 to about 100 micrometres, suitably from about 0.1 to 10 micrometres, for example about 1 to 10 micrometres. Particulate materials such as beads have the advantages of being easier to handle in certain situations, and of potentially providing a larger surface area for interaction with cells. They may also be more suitable for in-vivo applications, especially when the substrate comprises a biodegradable material.
Typically the pharmaceutically acceptable support matrix or substrate (eg particle) will be dispersible in water but is preferably not readily soluble in water. If water soluble, the support matrix or substrate should preferably dissolve sufficiently slowly to exhibit biological activity before fully decomposing.
It will be appreciated that, where dimensions are given for individual particles or beads, these apply also to collections or populations of particles or beads, in which case the dimension given will relate to the average dimension ofthe collection or population, suitably the mean dimension. For example, where it is stated that a bead has a diameter in a given range, it will be appreciated that this can also be considered in terms of a collection or population of beads having a mean diameter in the same range.
As noted above, in one preferred embodiment, the pharmaceutically acceptable support matrix or substrate may be a particle such as a bead. Such a bead may be, for example, a magnetic or paramagnetic bead (e.g. as available under the trade names "Dynal" or "MACS" (Miltenyi)). The magnetic or paramagenetic properties of such beads may facilitate manufacture, purification and handling. However, magnetic or paramagenetic properties are not required, and many other (non-magnetic) particle types may also be used. Suitably, for example, the bead comprises polystyrene, polyacrylamide, latex, cellulose, silica, dextran, agarose, cellulose, polylactide, or poly(methylmethacrylate) (PMMA) optionally in modified, crosslinked or derivatized form
Preferably, in one embodiment the modulators of Notch signalling will be activators of a Notch receptor (ie Notch receptor agonists), preferably activators of a mammalian Notch receptor, preferably activators of a human Notch receptor. Preferably for example the modulators of Notch signalling are activators of a human Notchl, Notch2, Notch3 or Notch4 receptor. Preferably they activate Notch receptors in immune cells, preferably peripheral immune cells, preferably T-cells, B-cells or antigen presenting cells (APCs).
Suitably, for example, a modulator of Notch signalling may be a Notch ligand or a biologically active fragment (preferably all or part ofthe extracellular domain) or derivative of a Notch ligand, or a peptidomimetic of such a Notch ligand or fragment.
Suitably the product may be used for modulation of peripheral immune cell activity, especially modulation of T-cell activation, preferably providing a degree of specificity to the antigen or antigenic determinant used.
Suitably the product may be used for generation of regulatory T-cells (T regs).
Suitably the product may be used for reducing an immune response to an antigen or antigenic determinant.
Suitably the product may be used for promoting immune tolerance to an antigen or antigenic determinant.
Suitably the product may be used for the treatment of autoimmune disease, allergy or graft/transplant rejection. Suitably the modulator of Notch signalling comprises a Notch ligand protein or a fragment, derivative, homologue, analogue or allelic variant thereof, as described above.
Suitably at least one ofthe modulators of Notch signalling comprises a protein or polypeptide which is preferably bound to the substrate, matrix or particle/bead through a terminal amino acid residue.
Suitably at least one of the modulators of Notch signalling comprises a protein or polypeptide which is bound to the substrate, matrix or particle/bead through a C-terminal amino acid residue.
Suitably at least one ofthe modulators of Notch signalling comprises a protein or polypeptide which is bound to the substrate, matrix or particle/bead through a cysteine residue.
Suitably at least one ofthe modulators of Notch signalling comprises a protein or polypeptide which is bound to the particle through a C-terminal cysteine residue.
Suitably the product is in the form of a pharmaceutical composition.
According to a further aspect ofthe invention there is provided a method for promoting immune tolerance to an antigen or antigenic determinant in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering, in either order: i) an effective amount of a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling; and ii) an effective amount of an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant. According to a further aspect ofthe invention there is provided a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling for use to treat autoimmune disease in simultaneous, contemporaneous, separate or sequential combination with an autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoantigen or bystander antigen or antigenic determinant thereof.
According to a further aspect ofthe invention there is provided the use of a combination of i) a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling; and ii) an autoantigen or bystander antigen or antigenic determinant thereof; or a polynucleotide coding for an autoantigen or bystander antigen or antigenic determinant thereof; in the manufacture of a medicament for the treatment of autoimmune disease.
According to a further aspect ofthe invention there is provided the use of a combination of i) a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling; and ii) an allergen or antigenic determinant thereof or a polynucleotide coding for an allergen or antigenic determinant thereof, in the manufacture of a medicament for the treatment of allergy.
According to a further aspect ofthe invention there is provided the use of a combination of i) a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling; and ii) a graft antigen or antigenic determinant thereof or a polynucleotide coding for a graft antigen or antigenic determinant thereof, in the manufacture of a medicament for the treatment of graft rejection.
According to a further aspect ofthe invention there is provided the use of a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling in the manufacture of a medicament for treatment of autoimmune disease in simultaneous, contemporaneous, separate or sequential combination with an autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoantigen or bystander antigen or antigenic determinant thereof.
According to a further aspect ofthe invention there is provided the use of a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling in the manufacture of a medicament for treatment of allergy in simultaneous, contemporaneous, separate or sequential combination with an allergen or antigenic determinant thereof or a polynucleotide coding for an allergen or antigenic determinant thereof.
According to a further aspect ofthe invention there is provided the use of a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling in the manufacture of a medicament for treatment of graft rejection in simultaneous, contemporaneous, separate or sequential combination with a graft antigen or antigenic determinant thereof or a polynucleotide coding for a graft antigen or antigenic determinant thereof.
According to a further aspect ofthe invention there is provided a pharmaceutical or veterinary kit comprising i) a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) bearing a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant
According to a further aspect ofthe invention there is provided a pharmaceutically acceptable support matrix or substrate for in vivo administration (preferably a particle) to which is bound i) a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant. Suitably such a matrix or substrate may take the form of a particle such as a bead bearing both i) a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant. Suitably the particle may be in the form of a microparticle or microbead.
According to a further aspect ofthe invention there is provided a particle having a multiplicity of modulators of Notch signalling bound thereto, wherein the particle has a maximum linear dimension of less than about 500 nanometres (nm; 1 nm = 1 x IO"9 metres).
These particles are particularly suitable for administration with antigens/antigenic determinants as discussed above, but may also be used in their own right to modulate immune cell activity, for example by modifying immune cell activity and/or cytokine expression as described herein.
The term "maximum linear dimension" as used herein means a particle having no larger linear dimension than that stated. For example, where the particle has a generally or approximately spherical shape, the maximum linear dimension will be the diameter.
Preferably such a particle may have a maximum linear dimension (or mean maximum linear dimension) of less than about 300 nm, suitably less than about 200 nm, for example less than about 150 nm, for example less than about 100 nm.
Suitably such a particle may have a maximum linear dimension (or mean maximum linear dimension) of from about 0.1 to 300 nm, suitably from about 1 to 100 nm, suitably from about 10 to 100 nm, suitably from about 20 to 100 nm, suitably from about 10 to 80 nm, suitably from about 20 to 80 nm, suitably from about 30 to 70 nm.
In an alternative embodiment, regardless of linear dimensions, such particles may have an individual or average/mean volume of from about 0.5 to about 50,000,000 cubic nanometres (nm3), preferably from about 5,000 to about 500,000 cubic nanometres (nm3).
Suitably the particle may be substantially biodegradable. Suitably the particle may be in the form of a microbead or microsphere.
Preferably the modulators of Notch signalling are presented by the particle in an orientation suitable for activation of a Notch receptor, and at least a significant and effective number are preferably orientated on the surface ofthe particle.
The invention further provides a method for therapeutic modulation of Notch signalling by administering particles with a size as described herein.
The invention further provides a method for generating a regulatory T-cell or increasing regulatory T-cell activity by contacting a particle with a size as described herein with a T- cell or APC (such contact being either in vivo or ex vivo, preferably in vivo).
According to a further aspect ofthe invention there is provided a method for therapeutic modulation of Notch signalling in immune cells (eg T-cells, B-cells or APCs) by administering a particle with a size as described herein in vivo.
According to a further aspect ofthe invention there is provided a method for therapeutic modulation of immune cell activity by administering a particle with a size as described herein in vivo.
According to a further aspect ofthe invention there is provided a method for therapeutic modulation of T-cell activity by administering a particle with a size as described herein in vivo.
According to a further aspect ofthe invention there is provided a method for treating inflammation, asthma, allergy, graft rejection, graft- versus-host disease or autoimmune disease by administering a particle with a size as described herein in vivo.
According to a further aspect ofthe invention there is provided a particle with a size as described herein for use in vivo in the treatment of disease. According to a further aspect ofthe invention there is provided a particle with a size as described herein for use in vivo in the treatment of an immune disorder.
According to a further aspect ofthe invention there is provided the use of a particle with a size as described herein in the manufacture of a medicament for modulation of immune cell activity in vivo. Preferably the immune cells are not stem cells.
According to a further aspect of the invention there is provided the use of a particle with a size as described herein for the manufacture of a medicament for modulation of expression of a cytokine selected from IL-10, IL-5, IL-2, TNF-alpha, IFN-gamma or IL-13.
Thus in one aspect there is provided the use of a particle with a size as described herein for the manufacture of a medicament for increase of IL-10 expression.
Alternatively there is provided the use of a particle with a size as described herein for the manufacture of a medicament for decrease of expression of a cytokine selected from IL-2, IL-5, TNF-alpha, IFN-gamma or IL-13.
According to a further aspect of the invention there is provided the use of a particle with a size as described herein for the manufacture of a medicament for generating an immune modulatory cytokine profile with increased IL-10 expression and reduced IL-5 expression.
According to a further aspect of the invention there is provided the use of a particle with a size as described herein for the manufacture of a medicament for generating an immune modulatory cytokine profile with increased IL-10 expression and reduced IL-2, IFN-gamma, IL-5, IL-13 and TNF-alpha expression.
Preferably, in one embodiment the modulators of Notch signalling are activators of a Notch receptor (ie Notch receptor agonists), preferably activators of a mammalian Notch receptor, preferably activators of a human Notch receptor. Preferably for example the modulators of
Notch signalling are activators of a human Notchl, Notch2, Notch3 or Notch4 receptor. Preferably they activate Notch receptors in immune cells, preferably peripheral immune cells, preferably T-cells, B-cells or antigen presenting cells (APCs).
Suitably the particle may be a microbead. Suitably the modulator of Notch signalling may comprise a Notch ligand or fragment, derivative or variant thereof. Suitably a multiplicity of Notch ligands or fragments, derivatives or variants thereof are bound/coupled to the particle.
Suitably modulators of Notch signalling may be proteins or polypeptides bound to the particle (preferably chemically bound) through a terminal amino acid residue, suitably a C- terminal amino acid residue.
Suitably modulators of Notch signalling may be proteins or polypeptides bound to the particle (preferably chemically bound) through a cysteine residue, suitably a C-terminal cysteine residue.
Suitably the particle comprises a polymeric material. Preferably the particle comprises a substantially biodegradable material or materials.
Suitably, for example, the particle may comprise one or more of polystyrene, polyacrylamide, latex, cellulose, silica, dextran, agarose, cellulose, polylactide, or poly(methylmethacrylate) (PMMA) optionally in modified, crosslinked or derivatized form, or an inorganic material such as a salt or oxide (eg iron oxide).
The term "multiplicity" as used herein means a number being at least three, and preferably at least five, suitably at least ten, for example at least twenty, or at least thirty, or at least forty, or at least fifty or at least a hundred or in some embodiments at least a thousand, or several thounsand or more.
Suitably the construct used in the various embodiments ofthe invention bears at least 3, preferably at least 5, suitably at least 10, suitably at least 20, for example 50, 100, 1000, 10,000 or more modulators of Notch signalling which may be the same or different. Such matrices, substrates, particles, beads etc may be administered either in vivo or ex-vivo as well known in the art (for example as described herein under the heading "Pharmaceutical Compositions") and used to modulate the Notch signalling pathway (for example to treat conditions as described herein under the heading "Therapy").
Preferably the modulator of Notch signalling is an agent capable of activating Notch signalling. Preferably the agent is capable of activating Notch signalling in lymphocytes, preferably T-cells. For example, the agent may act to reduce activity of effector T-cells such as Th or Tc T-cells, and or increase activity of regulatory T-cells.
In one embodiment the cells used or acted on in the present invention are not stem cells.
Preferably at least one modulator of Notch signalling is an agent capable of activating a Notch receptor, such as a human Notch 1, Notch 2, Notch 3 or Notch 4 receptor. Suitably, for example, the modulator may be a Notch ligand or a biologically active fragment or derivative of a Notch ligand, or a peptidomimetic of such a Notch ligand. Preferably the agent is capable of activating Notch receptors in lymphocytes such as T-cells.
If desired at least one modulator ofthe Notch signalling pathway may comprise or code for a fusion protein. For example, the modulator may comprise or code for a fusion protein comprising a segment of a Notch ligand extracellular domain and an immunoglobulin Fc segment.
For example a modulator ofthe Notch signalling pathway may comprise a fusion protein comprising a segment of a Notch ligand extracellular domain and an immunoglobulin Fc segment (eg IgGl Fc or IgG4 Fc) or a polynucleotide coding for such a fusion protein.
Suitable such fusion proteins are described, for example in Example 2 of WO 98/20142. IgG fusion proteins may be prepared as well known in the art, for example, as described in US
5428130 (Genentech). Suitably at least one modulator ofthe Notch signalling pathway comprises or codes for a protein or polypeptide comprising a Notch ligand DSL domain or a fragment, derivative, homologue, analogue or allelic variant thereof.
Preferably at least one modulator of the Notch signalling pathway comprises or codes for a Notch ligand DSL domain and at least one EGF-like domain, suitably at least 1 to 20 or more, suitably at least 2 to 16 or more, for example at least 2 to 10, for example from 2 to 5 EGF-like domains. Suitably the DSL and EGF domain sequences are or correspond to mammalian sequences. Preferred sequences include human sequences. Suitably the Notch ligand domains are fused to a heterologous amino acid sequence such as an IgFc sequence.
Suitably at least one modulator ofthe Notch signalling pathway comprises Delta (preferably a mammalian Delta, preferably human Delta, eg human Deltal, Delta3 or Delta4) 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 a modulator ofthe Notch signalling pathway may comprise Serrate/Jagged (preferably mammalian, preferably human Jagged, eg human Jagged 1 or Jagged2) 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.
Suitably at least one 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.
In one embodiment, the particle may be a bead. The bead may be, for example, a magnetic or paramagnetic bead (e.g. as available under the trade names "Dynal" or "MACS" (Miltenyi)). The magnetic or paramagenetic properties of such beads may facilitate manufaacture, purification and handling. However, magnetic or paramagenetic properties are not required, and many other particles types may also be used. In one embodiment the modulation ofthe immune system comprises reduction of T cell activity. For example, the modulation ofthe immune system may comprise reduction of effector T-cell activity, for example reduction of helper (TH) and or cytotoxic (Tc) T-cell activity. Suitably the modulation ofthe immune system may comprise reduction of a Thl or Th2 immune response.
Alternatively or in addition, the modulation ofthe immune system provides an increase of regulatory T-cell (T reg) activity, such as an increase of Trl or Th3 regulatory T-cell activity.
Suitably the modulation ofthe immune system comprises generation of regulatory T cells (Tregs) and/or enhancement of Treg activity.
Suitably the modulation ofthe immune system comprises treatment of 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 producing a lymphocyte or antigen presenting cell (APC) capable of promoting tolerance which method comprises incubating a lymphocyte or APC obtained from a human or animal patient with a particle as described herein.
Suitably the method comprises incubating a lymphocyte or APC obtained from a human or animal patient with an APC in the presence of particle as described herein.
According to a further aspect of the invention there is provided a method for producing an APC capable of inducing tolerance in a T cell which method comprises contacting an APC with a particle as described herein. According to a further aspect of the invention there is provided a method for producing a lymphocyte or APC capable of promoting tolerance which method comprises incubating a lymphocyte or APC obtained from a human or animal patient with a particle as described herein.
Suitably in such methods the lymphocyte or APC may be incubated either in vivo or ex-vivo.
According to a further aspect ofthe invention there is provided a pharmaceutical composition (preferably for in vivo administration) comprising a particle as described herein, and optionally a pharmaceutically acceptable carrier.
In one embodiment the antigen or antigenic determinant may be administered on a pharmaceutically acceptable support matrix suitable for in vivo administration bearing said antigen or antigenic determinant, which matrix may be the same as or different to the pharmaceutically acceptable support matrix bearing the modulators of Notch signalling. Such constructs may be administered in simutaneous, separate or sequential combination with modulators of Notch signalling or may be administered independently in their own right to modulate immune responses.
Thus, according to a further aspect ofthe invention there is provided a method for reducing an immune response to an antigen or antigenic determinant by administering a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing said antigen or antigenic determinant.
According to a further aspect ofthe invention there is provided a method for promoting immune tolerance to an antigen or antigenic determinant by administering a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing said antigen or antigenic determinant.
Suitably the antigen or antigenic determinant may be an autoantigen or bystander antigen or antigenic determinant thereof for the treatment of autoimmune disease. Alternatively the antigen or antigenic determinant may be an allergen or antigenic determinant thereof for the treatment of allergy.
Alternatively the antigen or antigenic determinant may be a graft antigen or antigenic determinant thereof for the treatment of graft rejection.
According to a further aspect ofthe invention there is provided the use for immunotherapy of a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing an antigen or antigenic determinant thereof.
According to a further aspect ofthe invention there is provided the use of a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing an antigen or antigenic determinant thereof to reduce an immune response to said antigen or antigenic determinant.
According to a further aspect ofthe invention there is provided the use of a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing an antigen or antigenic determinant thereof to promote immune tolerance to said antigen or antigenic determinant.
According to a further aspect ofthe invention there is provided a construct comprising a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing an allergen or antigenic determinant thereof.
According to a further aspect ofthe invention there is provided a construct comprising a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing an autoantigen or bystander antigen or antigenic determinant thereof.
According to a further aspect ofthe invention there is provided a construct comprising a pharmaceutically acceptable support matrix or substrate suitable for in vivo administration bearing a graft antigen or antigenic determinant thereof. Suitably the pharmaceutically acceptable support matrix or substrate is a particulate matrix such as a bead, suitably a a microbead or microsphere, which preferably has a maximum linear dimension (or mean maximum linear dimension) of from about 1 nanometres to about 1000 micrometres, suitably from from about 10 nanometres to about 100 micrometres, suitably from about 10 nanometres to about 10 micrometres, suitably from about 40 nanometres to about 10 micrometres, suitably from about 40 nanometres to about 1000 nanometres.
In an alternative emdodiment the modulators of Notch signalling may be inhibitors of a Notch receptor (ie Notch receptor antagonists), eg inhibitors of a mammalian Notch receptor, preferably inhibitors of a human Notch receptor. In this embodiment the modulators of Notch signalling may, for example, be inhibitors of human Notchl, Notch2, Notch3 or Notch4 receptors. Preferably in this embodiment such modulators inhibit Notch receptors in immune cells, preferably peripheral immune cells, preferably T-cells, B-cells or antigen presenting cells (APCs).
Thus, in a further aspect ofthe invention, inhibitors of Notch signalling presented on matrices, substrates, particles, beads and the like may be used to enhance immune responses to antigens such as tumour antigens or pathogen antigens, for example, for use as adjuvants and in vaccine compositions. These may be used for example to treat or prevent cancers or infectious diseases.
Thus, according to a further aspect ofthe invention there is provided a method for increasing an immune response to a tumour or pathogen antigen or antigenic determinant in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering in vivo, in either order: i) an effective amount of a pharmaceutically acceptable support matrix bearing a multiplicity of inhibitors of Notch signalling; and ii) an effective amount of a tumour or pathogen antigen or antigenic determinant, or a polynucleotide coding for a tumour or pathogen antigen or antigenic determinant. According to a further aspect ofthe invention there is provided a vaccine composition comprising i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of inhibitors of Notch signalling; and ii) a vaccine antigen or antigenic determinant thereof, or a polynucleotide coding for a vaccine antigen or antigenic determinant thereof; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation ofthe immune system.
According to a further aspect ofthe invention there is provided a vaccine composition comprising i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of inhibitors of Notch signalling; and ii) a vaccine antigen or antigenic determinant, or a polynucleotide coding for a vaccine antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for enhancing an immune response to said vaccine antigen or antigenic determinant.
Suitably the vaccine composition may take the form of a tumour vaccine wherein the vaccine antigen or antigenic determinant is a tumour antigen or antigenic determinant thereof.
Alternatively the vaccine antigen or antigenic determinant may be a pathogen antigen or antigenic determinant thereof.
The vaccine composition may be used either prophylactically or therapeutically (eg to treat tumours or acute or chronic infections).
According to a further aspect ofthe invention there is provided a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of inhibitors of Notch signalling.
According to a further aspect ofthe invention there is provided a construct comprising a pharmaceutically acceptable support matrix suitable for in vivo administration bearing both i) a multiplicity of inhibitors of Notch signalling; and ii) a vaccine antigen or antigenic determinant thereof, or a polynucleotide coding for a vaccine antigen or antigenic determinant thereof.
Suitably the vaccine antigen or antigenic determinant may be a tumour antigen or antigenic determinant thereof.
Alternatively the vaccine antigen or antigenic determinant may be a pathogen antigen or antigenic determinant thereof.
Detailed Description
Various prefeπed features and embodiments ofthe present invention will now be described by way of non-limiting examples and with reference to the accompanying drawings in which:
Figure 1 shows a schematic representation ofthe Notch signalling pathway;
Figure 2 shows schematic representations ofthe 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 the amino acid sequences of human Delta- 1 , Delta-3 and Delta-4;
Figure 5 shows the amino acid sequences of human Jagged- 1 and Jagged-2;
Figure 6 shows schematic representations of various Notch ligand domain/IgFc domain fusion proteins which may be used in the present invention; Figure 7 shows a reaction scheme for covalently linking modulators of Notch signalling to beads.
Figure 8 shows schematic representations of non-covalent linking of modulators of Notch signalling to beads (using a streptavidin/biotin link).
Figure 9 shows the results of Example 3; Figure 10 shows the results of Example 4;
Figure 11 shows the results of Example 5; Figure 12 shows the results of Example 6;
Figure 13 shows the results of Example 7;
Figure 14 shows the results of Example 8;
Figure 15 shows the results of Example 10; Figures 16 to 18 show the results of Example 11 ;
Figure 19 shows the results of Example 12;
Figure 20 shows the results of Example 13;
Figure 21 shows the results of Example 14;
Figure 22 shows the results of Example 16; Figure 23 shows the results of Example 17;
Figure 24 shows the results of Example 18;
Figure 25 shows the results of Example 19;
Figures 26 to 33 show the results of Example 22;
Figure 34 shows the results of Example 23; Figure 35 shows the results of Example 24;
Figure 36 shows the results of Example 25;
Figure 37 shows a results from Example 26;
Figure 38 shows results from Example 27; and
Figure 39 shows results from Example 28.
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 Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, 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. Each of these general texts is herein incorporated by reference.
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 ofthe Notch receptor.
Notch was first described in Drosophila as a transmembrane protein that functions as a receptor for two different ligands, Delta and Senate. 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 ofthe extracellular domain, the transmembrane domain and the infracellular domain, and the other comprising the majority ofthe 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 comprising 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 (UN) 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) Cuπ. 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-Senate 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 and Senate to the EGF-like repeats of Notch's extracellular domain. Delta may sometimes require cleavage for activation. It may be 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 ofthe 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 cdc 10/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 the Drosophila homologue of C-promoter binding factor- 1 [CBF- 1], a mammalian DNA binding protein involved in the Epstein-Ban 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 ofthe 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 cdclO/ankyrin repeats ofthe 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 val 1744 (termed site 3, or S3) (Schroeter, E.H. et al. (1998) Nature 393(6683):382-6 (Schroeter)). It is thought that the proteolytic cleavage step that releases the cdclO/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 CBFl (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) Cun. 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(11):5663- 7 (Lu)).
S3 processing occurs only in response to binding of Notch ligands Delta or Senate/Jagged. The post-translational modification ofthe nascent Notch receptor in the Golgi (Munro S, Freeman M. (2000) Cun. Biol. 10:813-820 (Munro); Ju BJ, et al. (2000) Nature 405:191-195 (Ju)) appears, at least in part, to control which ofthe 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 O- 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 Senate/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) Cun. Opin. Genet. Devel. 9:434-441 (Irvine)).
Signal fransduction from the Notch receptor can occur via two different pathways (see eg Figure 1). The better defined pathway involves proteolytic cleavage ofthe intracellular domain of Notch (Notch IC) that translocates to the nucleus and forms a transcriptional activator complex with the CSL family protein CBFl (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 CBFl -independent manner that involves the cytoplasmic zinc finger containing protein Deltex. Unlike CBFl, Deltex does not move to the nucleus following Notch activation but instead can interact with Grb2 and modulate the Ras-JNK signalling pathway.
The term "modulation ofthe Notch signalling pathway" as used herein refers to a change or alteration in the biological activity ofthe Notch signalling pathway or a target signalling pathway thereof. The term "modulator of the Notch signalling pathway" may refer to antagonists or inhibitors of Notch signalling, i.e. compounds which block, at least to some extent, the normal biological activity ofthe Notch signalling pathway. Conveniently such compounds may be refened to herein as inhibitors or antagonists. Alternatively, the term "modulator ofthe Notch signalling pathway" may refer to agonists of Notch signalling, i.e. compounds which stimulate or upregulate, at least to some extent, the normal biological activity ofthe Notch signalling pathway. Conveniently such compounds may be refened to as upregulators or agonists. Preferably the modulator is an agonist of Notch signalling, and preferably an agonist ofthe Notch receptor (eg an agonist ofthe Notchl, Notch2, Notch3 and/or Notch4 receptor, preferably being a human Notch receptor). Preferably such an agonist ("activator of Notch") binds to and activates a Notch receptor, preferably including human Notch recpetors such as human Notchl, Notch2, Notch3 and/or Notch4. Binding to and/or activation of a Notch receptor may be assessed by a variety of techniques known in the art including in vitro binding assays and activity assays for example as described herein.
For example, whether any particular agent activates Notch signalling (eg is an activator of Notch signalling or a Notch agonist) may be readily determined by use of any suitable assay, for example by use of a Notch/luciferase reporter assay ofthe type described in Example 3 herein. Conversely, antagonist activity may be readily determined for example by monitoring any effect ofthe agent in reducing signalling by known Notch signalling agonists such as CHO cells expressing human Deltal; CHO-Delta cells (ie in a so-called "antagonist" assay; see the Examples herein). Target genes ofthe Notch signalling pathway include Deltex, genes ofthe Hes family (Hes-1 in particular), Enhancer of Split [E(spl)] complex genes, IL-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 ofthe Notch intracellular domain. Studies indicate that Deltex promotes Notch pathway activation by interacting with Grb2 and modulating the Ras- JNK signalling pathway (Matsuno et al. (1995) Development 121(8):2633-44; Matsuno K, et al. (1998) Nat. Genet. 19:74-78). Deltex also acts as a docking protein which prevents Su(H) from binding to the 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 et al. (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. et al. (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. D16464.
The E(spl) gene complex [E(spl)-C] (Leimeister C. et al. (1999) Mech Dev 85(1-2): 173-7 (Leimeister)) comprises seven genes of which only E(spl) and Groucho show visible phenotypes when mutant. E(spl) was named after its ability to enhance 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 (IL-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 IL-10 was produced by many other cell types including macrophages, keratinocytes, B cells, ThO and Thl cells. It shows extensive homology with the Epstein-Ban bcrfl gene which is now designated viral IL-10. Although a few immunostimulatory effects have been reported, it is mainly considered as an immunosuppressive cytokine. Inhibition of T 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. IL-10 also exerts anti-inflammatory effects on other myeloid cells such as neutrophils and eosinophils. On B cells, IL-10 influences isotype switching and proliferation. More recently, IL-10 was reported to play a role in the induction of regulatory T cells and as a possible mediator of their suppressive effect. Although it is not clear whether it is a direct downstream target ofthe 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 ofthe 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.
As described above the Notch receptor family participates in cell-cell signalling events that influence T cell fate decisions. In this signalling NotchlC localises to the nucleus and functions as an activated receptor. Mammalian NotchlC interacts with the transcriptional repressor CBFl . It has been proposed that the NotchlC cdcl 0/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 NotchlC contains the CBFl interactive domain. It is also proposed that NotchlC acts by targeting DNA-bound CBFl within the nucleus and abolishing CBFl -mediated repression through masking ofthe repression domain. It is known that Epstein Ban 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 fransduction 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 BARF0 (Kusano and Raab-Truab (2001) J Virol 75(l):384-395 (Kusano and Raab-Traub)) and LMP2A.
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 (eg from Drosophila, verterbrates, mammals) such as Delta and Senate/Jagged (eg mammalian ligands Deltal, Delta 3, Delta4, Jaggedl and Jagged2 and homologues) and their biologically active fragments and sequence variants as well as antibodies to the Notch receptor, peptidomimetics and small molecules which have conesponding biological effects to the natural ligands. Preferably the Notch ligand interacts with the Notch receptor by binding.
Particular examples of mammalian Notch ligands identified to date include the Delta family, for example Delta or Delta-like 1 (eg Genbank Accession No. AF003522 - Homo sapiens); Delta-3 (eg Genbank Accession No. AF084576 - Rattus norvegicus) and Delta-like 3 (Mus musculus) (eg Genbank Accession No. NM_016941 - Homo sapiens) and US 6121045 (Millennium); Delta-4 (Genbank Accession Nos. AB043894 and AF 253468 - Homo sapiens); and the Senate family, for example Senate-1 and Senate-2 (WO97/01571, WO96/27610 and WO92/19734); 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. Examples of sequences of human Deltal, Delta3, Delta4, Jaggedl and Jagged2 are shown in the Figures hereto.
The term "antibody" as used herein includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, Fv and scFv which are capable of binding the epitopic determinant. These antibody fragments retain some 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 ofthe heavy chain; two Fab' fragments are obtained per antibody molecule;
(iii) F(ab')2, the fragment ofthe 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.
By a "homologue" is meant a gene product that exhibits sequence homology, either amino acid or nucleic acid sequence homology, to any one ofthe known Notch ligands, for example as mentioned above. Typically, a homologue of a known Notch ligand will be at least 20%, preferably at least 30%, identical at the amino acid level to the conesponding known Notch ligand over a sequence of at least 10, preferably at least 20, preferably at least 50, suitably at least 100 amino acids, or over the entire length ofthe Notch ligand. 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 et al., Cunent Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.)
Homologues of Notch ligands 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 Notch ligand 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.
Techniques are well known in the art for the screening and development of agents such as antibodies, peptidomimetics and small organic molecules which are capable of binding to components ofthe Notch signalling pathway such as the Notch receptor. 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). Preferably a modulator of Notch signalling for use in the present invention will be a Notch receptor agonist such as a Notch ligand capable of binding to and activating a Notch receptor, preferably a human Notch receptor such as human Notchl , Notch2, Notch3 or Notch4. Such binding and activation may be assessed by a variety of techniques known in the art including activity assays for example as described herein.
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 prefened embodiment, Notch signalling excludes cytokine signalling.
Modulators of Notch Signalling
The term "modulate" as used herein refers to a change or alteration in the biological activity ofthe Notch signalling pathway or a target signalling pathway thereof. The term "modulator" may refer to antagonists or inhibitors of Notch signalling, i.e. compounds which block, at least to some extent, the normal biological activity ofthe Notch signalling pathway. Conveniently such compounds may be refened to herein as inhibitors or antagonists. Alternatively, the term "modulator" may refer to agonists of Notch signalling, i.e. compounds which stimulate or upregulate, at least to some extent, the normal biological activity ofthe Notch signalling pathway. Conveniently such compounds may be refened to as upregulators or agonists.
The term "candidate modulator" is used to describe any one or more molecule(s) which may be, or is suspected of being, capable of functioning as a modulator of Notch signalling. Said molecules may for example be organic "small molecules" or polypeptides. Suitably, candidate molecules comprise a plurality of, or a library of such molecules or polypeptides. These molecules may be derived from known modulators. "Derived from" means that the candidate modulator molecules preferably comprise polypeptides which have been fully or partially randomised from a starting sequence which is a known modulator of Notch signalling. Most preferably, candidate molecules comprise polypeptides which are at least 40% homologous, more preferably at least 60% homologous, even more preferably at least 75% homologous or even more, for example 85 %, or 90 %, or even more than 95% homologous to one or more known Notch modulator molecules, using the BLAST algorithm with the parameters as defined herein.
The modulator ofthe present invention may for example be an organic compound or other chemical. In this embodiment, the modulator will 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 ofthe 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 ofthe 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 prefened embodiment, the modulator of Notch signalling will be an amino acid sequence or a chemical derivative thereof, or a combination thereof. The candidate modulator may also be an antibody.
Candidate modulators may be synthetic compounds or natural isolated compounds. Various examples of such synthetic or natural modulators are listed below.
Suitably, the modulator of Notch signalling may be a Notch ligand, or a polynucleotide encoding a Notch ligand. Notch ligands will typically be capable of binding to a Notch receptor polypeptide present in the membrane of a variety of mammalian cells, for example hemapoietic stem cells. Endogenous Notch ligands include polypeptides ofthe Delta family, for example Delta-1 (Genbank Accession No. AF003522 - Homo sapiens), Delta-3 (Genbank Accession No. AF084576 - Rattus norvegicus), Delta-like 3 (Mus musculus), Delta-4 (Genbank Accession No. AB043894) and polypeptides ofthe Senate family, for example Senate-1 and Senate-2 (WO97/01571, WO96/27610 and WO92/19734), Jagged- 1 and Jagged-2 (Genbank Accession No. AF029778 - Homo sapiens), and LAG-2. Candidate compounds ofthe present invention include fragments, derivatives, variants, mimetics, analogues and homologues of any ofthe above.
Preferably, the modulator ofthe present invention will be a protein, polypeptide or peptide.
Whether or not any given agent acts as a modulator of Notch signalling (and if so whether it is an activator or inhibitor of such signalling) may be readily determined by use of suitable assays or screens, for example, those described in the Examples herein.
Notch Ligands and Homologues
As noted above, 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 norvegicus) and Delta-like 3 (Mus musculus), the Senate family, for example Senate-1 and Senate-2 (WO97/01571, WO96/27610 and WO92/19734), Jagged- 1 and Jagged-2 (Genbank Accession No. AF029778 - Homo sapiens), and LAG-2. Homology between family members is extensive.
By a "homologue" is meant a gene product that exhibits sequence homology, either amino acid or nucleic acid sequence homology, to any one ofthe known Notch ligands, for example as mentioned above. Typically, a homologue of a known Notch ligand will be at least 20%, preferably at least 30%, identical at the amino acid level to the conesponding known Notch ligand over a sequnce of at least 10, preferably at least 20, preferably at least 50, suitably at least 100 amino acids, or over the entire length ofthe Notch ligand. 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 et al, Cunent Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.)
As noted above, 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 ofthe protein and up to 14 or more EGF-like repeats on the extracellular surface. It is therefore prefened 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.
In addition, suitable homologues will preferably be capable of binding to a Notch receptor. Binding may be assessed by a variety of techniques known in the art including in vitro binding assays and activation ofthe receptor (in the case of an agonist or partial agonist) may be determined for example by use of assays as described in the Examples hereto and in WO 03/012441 (Lorantis) the text of which is hereby incoφorated herein by reference.
Homologues of Notch ligands 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 Notch ligand 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.
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/i
SIGNAL 1-17 SIGNAL
CHAIN 18-723 DELTA-LIKE PROTEIN
DOMAIN 18-545 EXTRACELLULAR
TRANSMEM 546- 568 TRANSMEMBRANE
DOMAIN 569-723 CYTOPLASMIC
DOMAIN 159-221 DSL
DOMAIN 226-254 EGF-LIKE 1
DOMAIN 257-285 EGF-LIKE 2
DOMAIN 292-325 EGF-LIKE 3
DOMAIN 332-363 EGF-LIKE 4
DOMAIN 370-402 EGF-LIKE 5
DOMAIN 409-440 EGF-LIKE 6
DOMAIN 447-478 EGF-LIKE 7
DOMAIN 485-516 EGF-LIKE 8
Human Delta 3
Component Amino acids Proposed f-
DOMAIN 158-248 DSL
DOMAIN 278-309 EGF-LIKE 1
DOMAIN 316-350 EGF-LIKE 2
DOMAIN 357-388 EGF-LIKE 3
DOMAIN 395-426 EGF-LIKE 4
DOMAIN 433-464 EGF-LIKE 5
Human Delta 4
Component Amino acids Proposed function/i
SIGNAL 1-26 SIGNAL
CHAIN 27-685 DELTA-LIKE PROTEIN
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 func
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 WILL! :BRANI
Human Jagged 2 Component Amino acids Proposed function/domain
SIGNAL 1-26 SIGNAL
CHAIN 27-1238 JAGGED 2
DOMAIN 27-1080 EXTRACELLULAR
TRANSMEM 1081-1105 TRANSMEMBRANE
DOMAIN 1106-1238 CYTOPLASMIC
DOMAIN 178-240 DSL
DOMAIN 249-273 EGF-LIKE 1
DOMAIN 276-304 EGF-LIKE 2
DOMAIN 311-344 EGF-LIKE 3
DOMAIN 351-382 EGF-LIKE 4
DOMAIN 389-420 EGF-LIKE 5
DOMAIN 427-458 EGF-LIKE 6
DOMAIN 465-495 EGF-LIKE 7
DOMAIN 502-533 EGF-LIKE 8
DOMAIN 540-571 EGF-LIKE 9
DOMAIN 602-633 EGF-LIKE 10
DOMAIN 640-671 EGF-LIKE 11
DOMAIN 678-709 EGF-LIKE 12
DOMAIN 716-747 EGF-LIKE 13 DOMAIN 755-786 EGF-LIKE 14
DOMAIN 793-824 EGF-LIKE 15
DOMAIN 831-862 EGF-LIKE 16
DOMAIN 872-949 VON ILLEBRAND FACTOR C
DSL domain
A typical DSL domain may include most or all ofthe 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 ofthe 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 ofthe 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.
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%o, 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.
The term "Notch ligand N-terminal domain" means the part of a Notch ligand sequence from the N-terminus to the start ofthe DSL domain. It will be appreciated that this term includes sequence variants, fragments, derivatives and mimetics having activity conesponding 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%o, 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.
Preferably any such heterologous amino acid sequence is not a TSST sequence, and preferably it is not a superantigen sequence.
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 IX 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-like 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 (1 , 70) -C-x (1 , 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': often conserved glycine 'a': often 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.
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%o amino acid sequence identity to an EGF-like domain of human Delta 4.
Antibodies
In one embodiment the modulator of Notch signalling may be an antibody, derivative or fragment which binds to and activates Notch. Thus the invention also provides a particle to which is coupled (eg chemically, by affinity or adsportion) an antibody capable of binding to and activating Notch.
General methods of making antibodies are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), the text of which is incoφorated herein by reference). Antibodies may be monoclonal or polyclonal but are preferably monoclonal.
Suitably, the binding affinity (equilibrium association constant (Ka)) may be at least about 10° M"1, at least about 107M"', at least about 108 M'^ or at least about IO9 M"1.
Suitably the antibody, derivative or fragment binds to one or more EGF or Lin/Notch (L/N) domains of Notch (for example to EGF repeats 11 and 12 of Notch).
Suitable antibodies for use as blocking agents are obtained by immunizing a host animal with peptides comprising all or a portion of Notch.
The peptide used may comprise the complete protein or a fragment or derivatives thereof.
Prefened immunogens comprise all or a part of the extracellular domain of human Notch (eg Notchl, Notch2, Notch3 or Notch4, preferably Notchl or Notch2), where these residues contain any post-translation modifications, such as glycosylation, found in the native proteins. Immunogens comprising the extracellular domain may be produced by a number of techniques which are well known in the art such as expression of cloned genes using conventional recombinant methods and/or isolation from T cells or cell populations expressing high levels of Notch. Monoclonal antibodies may be produced by means well known in the art. Generally, the spleen and or lymph nodes of an immunized host animal provide a source of plasma cells. The plasma cells are immortalized by fusion with myeloma cells to produce hybridoma cells. Culture supernatant from individual hybridomas is screened using standard techniques to identify those producing antibodies with the desired specificity. The antibody may be purified from the hybridoma cell supematants or ascites fluid by conventional techniques, such as affinity chromatography using Notch, Notch ligands or fragments thereof bound to an insoluble support, protein A sepharose, or the like.
For example, antibodies against Notch are described in US 5648464, US 5849869 and US 6004924 (Yale University/Imperial Cancer Technology), the texts of which are herein incoφorated by reference.
Antibodies generated against the Notch receptor are also described in WO 0020576 (the text of which is also incoφorated 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. HB 12656 and a monoclonal antibody secreted by a hybridoma designated F3 having the ATCC Accession No. HB 12655.
Suitably, antibodies for use to treat human patients in vivo 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 ofthe 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. Coφ. 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 ofthe 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 Riech ann et al (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 ofthe 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 ofthe 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 choice of isotype will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Suitable isotypes include IgG 1, IgG3 and IgG4. Suitably, either ofthe human light chain constant regions, kappa or lambda, may be used.
Matrices, Substrates and Particles
It will be appreciated that the pharmaceutically acceptable matrix or substrate, eg particle/bead to which modulators of Notch signalling are bound according to the present invention can take many different forms such as polymers, plastics, porous materials such as resin or modified cellulose, beads (such as microspheres and microbeads, nanospheres and nanoparticles), and liposomes.
Matrices, substrates and particles are preferably substantially non-immunogenic or have low immunogenicity, and may suitably comprise either organic or inorganic materials. Inorganic materials include for example inorganic salts or oxides, such as iron oxide. Where an inorganic material is used, this is preferably at least partially (and preferably substantially completely) coated with a biocompatible polymer, for example as discussed below.
Alternatively, the particle may, for example comprise a polymer or combination of polymers alone.
Preferably a particle used may be biocompatible, and preferably biodegradable.
In one embodiment the particle may be a microbead or microsphere.
It will be appreciated that the term "diameter" normally applies to particles having a substantially spherical or other circular form. However, it will be appreciated that particles used in the present invention do not need to have such a regular form, and may have a more inegular form, in which case the relevant dimension is suitably the maximum (largest) linear dimension (ie length, width or depth). In addition, where dimensions are given for individual particles or beads, it will be appreciated that these apply also to collections or populations of particles or beads, in which case the dimension given will relate to the average dimension of the collection or population, suitably the mean dimension. For example, where it is stated that a bead has a maximum linear dimension (or diameter) in a given range, it will be appreciated that this can also be considered in terms of a collection or population of beads having a mean maximum linear dimension (or diameter) in the same range.
Matrices/substrates and particles etc may, for example, comprise natural or synthetic polymers such as polystyrene, polyethylene glycol (PEG), polyglycollic acid (PGA), polycaprolactide, polyacrylamide, latex, silica, dextran, agarose, starch, cellulose, chitin/chitosan, polylactide, poly(methylmethacrylate) (PMMA); proteins/polypeptides such as albumins, for example human serum albumins; and modified, crosslinked and derivatized embodiments thereof. Suitable materials include, for example polystyrene, cellulose, dextran crosslinked with epichlorohydrin (Sephadex.TM., Pharmacia, Uppsala, Sweden), polyacrylamide crosslinked with bisacrylamide (Biogel.TM., BioRad, USA), agar, glass beads, polylactide beads and latex beads. Derivatized microparticles include microparticles derivatized with eg maleimide, aldehyde groups, allyl groups, carboxyalkyl groups such as carboxymethyl, phosphoryl and substituted phosphoryl groups, sulfate, sulfhydryl and sulfonyl groups, and amino and substituted amino groups. Beads may have either hydrophilic or hydrophobic properties.
The modulators of Notch signalling may be bound to the matrix/substrate eg particle etc (eg bead) by any suitable means. For example, binding may be by non-covalent linking such as surface adsoφtion (eg by hydrophobic and/or electrostatic interactions) or by covalent linking such as chemical linking. For example, reactive groups (such as amino, aldehyde, carboxy, epoxy or toluenesulfonyl (tosyl), thiol or maleimide groups) may be present on or introduced onto a particle (such as a bead) surface and these may be linked to modulators of Notch signalling. For example, modulators of Notch signalling in the form of proteins or polypeptides may be linked to a particle (such as a bead) by incubation of a surface activated particle (such as a bead) with the protein or polypeptide, suitably by incubation for at least 12 to 24 hours suitably at neutral or neutral to high pH and suitably at a sufficiently high temperature such that a reactive group on the particle/bead (for example a tosyl, epoxy, amino, carboxy, aldehyde, thiol or maleimide group) reacts with a reactive group on the protein or polypeptide (for example a free amino or sulfhydryl group on the protein or polypeptide, or an aldehyde group) to form a covalent link.
A variety of linker groups may also be used to bind the matrix/substrate/particle/bead to the modulators of Notch signalling if required. Suitable linkers are well known in the art and suitably comprise an acid, basic, aldehyde, ether or ester reactive group or a residue thereof. Suitable linker moieties include, for example, succinimidyl propionate, succinimidyl butanoate, N-hydroxysuccinimide, benzotriazole carbonate, propionaldehyde, maleimide or forked maleimide, biotin, vinyl derivative and phospholipids.
For example, modulators of Notch signalling such as Notch ligand proteins and polypeptides may be chemically coupled to beads using a coupling agent such as sulpho-SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate).
Antibodies may also be used to bind modulators of Notch signalling to a matrix/substrate such as a particle (such as a bead). For example, in one of many embodiments, beads coated with antibody-binding materials such as streptavidin may be used to bind a biotinylated anti- IgG antibody, which in turn may be bound to a modulator of Notch signalling in the form of an IgG fusion protein. Alternatively, for example, suitably functionalised agents such as biotinylated agents/ligands may be directly coupled to beads such as streptavidin beads.
In one embodiment, particles or beads may be magnetic particles such as magnetic beads, such as for example those available under the trade name DYNABEAD ™ from Dynal Biotech, Oslo, Norway. These have the advantage of being easy to separate from fluids magnetically. Such particles or beads may be prepared, for example, by incoφorating a magnetic or paramagnetic material such as iron oxide into a polymeric matrix such as polystyrene.
As described on the Dynal Biotech web-site (www.dynal.no), Dynabeads™ are supeφaramagnetic polymer spheres, magnetic only when placed in a magnetic field and with no residual magnetism when the magnetic field is removed. They are composed of highly cross-linked polystyrene with magnetic material precipitated in pores evenly distributed throughout each bead. The pores are preferably filled with an additional polymer layer, which seals the iron material inside the beads. Chemical groups (such as amino, carboxy, epoxy or toluenesulfonyl (tosyl) groups) may then be introduced on the bead surface if appropriate.
Particles or beads may be either hydrophilic or hydrophobic. Hydrophilic particles or beads allow gentle coupling of ligands to the surface to maintain functional activity of labile proteins. The hydrophilic properties ofthe beads ensure optimal dispersity in aqueous solution. These particles or beads may be surface activated with, for example amine and carboxylic acid functional groups. The functional groups ofthe amine and carboxylic acid beads allow further introduction of a large variety of alternative reactive groups by coupling to commercially available cross-linkers.
Hydrophobic particles or beads are well suited to coupling of antibodies. The hydrophobic Fc region ofthe antibody may be adsorbed to the hydrophobic particle or bead surface, followed by a rapid covalent bond formation. The orientation ofthe antibody is thereby generally optimal with the Fab regions facing outwards.
It will be appreciated that the orientation ofthe active site ofthe ligand or compound to be coupled to the matrix, particles or beads may also need to be taken into consideration. Hydrophobic particles or beads facilitate hydrophobic-hydrophobic interactions between the particles or beads and the protein's hydrophobic parts, whereas the hydrophilic beads are suited when hydrophilic-hydrophilic interactions between the particles or beads and the protein's hydrophilic parts are desired. In one embodiment, the particles or beads may for example be latex microspheres such as those available from Interfacial Dynamics Coφoration (Portland, US). As disclosed on the Interfacial Dynamics web-site (www.idclatex.com) latex microspheres/beads are available with either anionic (negative) or cationic (positive) surface charges. Anionic latexes - such as those with sulfate, carboxyl, or carboxylate modified surface groups - are less likely to bind to negatively-charged cell surfaces and are therefore used frequently in biological applications.
Particles or beads may suitably be sterilized before use, for example by pasteurization, suitably for about 24 hours at 78-80° C; or by gamma inadiation, for example at 0.03 megarads suitably for about 24 hours.
Suitably the particles or beads may be coated with various proteins or polysaccharides that will greatly reduce their capacity to absorb biomolecules non-specifically. Specific ineversible adsoφtion of protein molecules such as avidin, streptavidin, and antibodies may be accomplished by simply mixing the latex and protein together for a specified period of time, then separating the bound from the unbound protein through centrifugation and removal ofthe supernatant.
To reduce nonspecific binding, particles or beads may be coated, for example with BSA or dextrans. To further reduce nonspecific binding, proteins, nucleic acids, and other biomolecules may if desired be covalently coupled to the particles or beads. Covalent coupling may require more effort than passive adsoφtion, but can result in conjugates with greater specificity that remain active longer. Carbodiimide-mediated coupling to CML latexes is a suitable method for conjugating low molecular weight peptides and oligonucleotides.
A wide range of suitable beads, micro/nanobeads and micro/nanospheres is also available for example from Polysciences, Inc. 400 Valley Road, Warrington, PA, US.
In an alternative embodiment, modulators of Notch signalling may be conjugated to the linear or cross-linked backbone of a liposome using conventional techniques (see, e.g. Ostro, M. J. (Ed.), Liposomes: from Biophysics to Therapeutics (Marcel Dekker, New York, 1987)). One prefened method of preparing liposomes and conjugating immunoglobulins to their surface is described by Ishimoto, Y. et al., J. Immunol. Met. 75, 351-360 (1984). For example, multilamillar liposomes composed of dipalmitoylphosphatidylcholine, cholesterol and phosphotidylethanolamine are prepared. Modulators of Notch signalling may then be coupled to the phosphatidylethanolamine by the cross-linking agent N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate. The coupling ofthe fragment to the liposome can be demonstrated by the release of a pre-trapped marker, e.g., carboxyfluorescence, from the liposomes upon the treatment of secondary antibody against the conjugated fragment and complement.
Where the modulator of Notch signalling comprises an IgFc domain it may, for example, be coupled to a liposome or another carrier ofthe invention via carbohydrate moieties on the Fc domain.
Methods for derivatizing sugar ring moieties to create hydrazide groups for coupling with fragments (and antibodies) are described, for example by Rodwell, J. D. et al., Proc. Nat'l Acad. Sci. USA 83:2632-36 (1986).
The following papers describe various polymers suitable for use with the present invention, especially for formation coating of particles.
Dextran: In vitro biocompatibility of biodegradable dextran-based hydrogels tested with human fibroblasts, Biomaterials 22 (2001) 1197-1203 De Groot et al.. Methacrylate- derivatized dextran is biocompatible and a promising system for drug delivery.
A novel somatostatin conjugate with a high affinity to al five somatostatin receptor subtypes, Cancer 94 (2002) 1293-1297 Wulbrand et al.. Somatostatin coupled to periodate-activated dextran has extended half-life, in clinical trials for hormone-refractory prostate cancer. Synthesis and inverse emulsion polymerization of aminated acrylamidodextran J. Pharm. Pharmacol. 45 (1993) 1018-1023 Daubresse et al.. Derivatized dextran with functional amine groups for drug conjugates.
PEG: Copolymer with styrene; Polystyrene-poly (ethylene glycol) (PS-PEG2000) particles as model systems for site specific drug delivery. 2. The effect of PEG surface density on the in vitro cell interaction and in vivo biodistribution. Pharm. Res. 11 (1994) 1016-1022.
PEG coating of beads; PEGylation of microspheres generates a heterogenous population of particles with different surface characteristics and biological performance. FEBS Letts 532 (2002)338-344 Ghadamosi et al. 1 micron polystyrene beads coated with BSA then PEGylated, a population resistant to phagocytosis and reduced complement activation.
Surface characterization of functionalized polylactide through the coating with heterobifunctional poly(ethylene glycol)/polylactide block copolymers. Biomacromolecules 1 (2000) 39-48 Otsuka et al. Surface reactive aldehyde PEG coated onto polylactide
Design of biodegradable particles for protein delivery. J. Control Release 78 (2002) 15-24 Vila et al. PEG-coated poly(lactide), chitosan-coated poly(lactic acid-glycolic acid), chitosan.
Albumin nanoparticles; Preparation of surface modified protein nanoparticles by introduction of sulfhydryl groups. Int. J. Pharm. 211 (2000) 67-78 Weber et al. Thiol groups introduced to the surface of human serum albumin nanoparticles.
"Stealth nanospheres"; Prolonging the circulation time and modifying the body distribution of intravenously injected polystyrene nanospheres by prior intravenous administration of poloxamine-908. A 'hepatic-blockade' event or manipulation of nanosphere surface in vivo? BBA 1336 (1997) 1-6 Moghimi, S. M. 60 or 250 nanometre polystyrene have extended half-life in vivo if coated with poloxamine 908 or poloxamine- protein conjugates. Chemical camouflage of nanospheres with a poorly reactive surface: towards development of stealth and target-specific nanocarriers. BBA 1590 (2002) 131-139 Moghimi, S. M. Coating of polystyrene nanospheres with PEGylated BSA or IgG.
Capture of Stealth Nanoparticles by the Body's Defenses Crit. Rev. Ther. Drug Car. Syst. 18 (2001) 527-550 Moghimi S. M. and Hunter, A. C. Review.
A prefened source of particles for use in the present invention is those from Miltenyi Biotec (Bergisch Gladbach, Germany, and Auburn, US) As described in US 5385707 (Miltenyi), US 4,452,773 describes the preparation of magnetic iron-dextran microspheres and provides a summary of art describing the various means of preparation of particles suitable for attachment to biological materials. As long ago as 1977, preparation of colloidal iron oxide, gamma-iπadiated in the presence of hydrophilic and hydrophobic methacrylate monomers, to provide particles for attachment to biological targets through coupling to immunoglobulin was described (Rembaum, A., et al., Nature (1977) 268:437-438. Various other preparations of magnetic microspheres of various sizes were described by Kronick, P. L., et al, Science (1978) 200:1074-1076 and Widder, K, et al, J Pharm Sci (1979) 68:79-82 and in U.S. 3,970,518 and 4,018,886.
US 4,230,685 describes an improvement in attaching specific binding agents to the magnetic particles wherein a particle coated with an acrylate polymer or a polysaccharide can be linked through, for example, glutaraldehyde to a preparation of protein A which can then selectively bind antibodies through the Fc portion, leaving the immunoreactive Fab regions exposed. Albumin, rather than polyacrylamide or polysaccharides, is the prefened matrix. A wide size range of particles is disclosed.
In the case ofthe particles prepared as described in US 4,452,773, particles of 100-700 angstroms, particularly 300-400 angstroms are intended to be prepared; many ofthe particles are thus colloidal, and are fenomagnetic with a coating of dextran. The resulting particles are described as discrete colloidal size particles having a fenomagnetic iron oxide core coated with a polysaccharide derivative having pendant functional groups provided by periodate oxidation. These particles are prepared by mixing an aqueous solution of a fenous and ferric salt with a solution ofthe polysaccharide or polysaccharide derivative. After this mixing, alkali is added to cause the formation ofthe magnetic iron oxide particles to which the polysaccharide or derivative attaches. The resulting particles are separated from excess dextran using gel filtration chromatography. A single peak containing the entire size range of particles is obtained. The polysaccharide is then treated to provide the needed functional groups for conjugation to a protein or polypeptide.
Other polymeric coatings for magnetic particles used in HGMS are described in PCT publication WO85/04330.
Several types of magnetic particles may be prepared: fenomagnetic particles, supeφaramagnetic particles and paramagnetic particles. Methods to prepare supeφaramagnetic particles are described in U.S. 4,770,183. With respect to terminology, as is the general usage in the art:
"Diamagnetic" as used herein, and as a first approximation, refers to materials which do not acquire magnetic properties even in the presence of a magnetic field, i.e., they have no appreciable magnetic susceptibility.
"Paramagnetic" materials have only a weak magnetic susceptibility and when the field is removed quickly lose their weak magnetism. They are characterized by containing unpaired electrons which are not coupled to each other through an organized matrix. Paramagnetic materials can be ions in solution or gases, but can also exist in organized particulate form.
"Fenomagnetic" materials are strongly susceptible to magnetic fields and are capable of retaining magnetic properties when the field is removed. Fenomagnetism occurs only when unpaired electrons in the material are contained in a crystalline lattice thus permitting coupling ofthe unpaired electrons. Fenomagnetic particles with permanent magnetization have considerable disadvantages for application to biological material separation since suspension of these particles easily aggregate due to their high magnetic attraction for each other. " Supeφaramagnetic" materials are highly magnetically susceptible~i.e., they become strongly magnetic when placed in a magnetic field, but, like paramagnetic materials, rapidly lose their magnetism. Supeφaramagnetism occurs in fenomagnetic materials when the crystal diameter is decreased to less than a critical value.
Although the above-mentioned definitions are used for convenience, it will immediately be apparent that there is a continuum of properties between paramagnetic, supeφaramagnetic, and fenomagnetic, depending on crystal size and particle composition. Thus, these terms are used only for convenience, and "supeφaramagnetic" is intended to include a range of magnetic properties between the two designated extremes.
The extent of magnetization which is acquired by a particle is a function of its magnetic susceptibility and the applied magnetic field. The magnetization is a function ofthe resulting magnetic moment and ofthe volume ofthe particle. The higher the magnetic moment and the smaller the volume, the higher the magnetization.
Particles may be initially prepared in a range of sizes which result in variations in their abilities to acquire magnetic properties upon exposure to a magnetic field. In one embodiment particles themselves may suitably be collections of iron oxide microcrystal of approx 50-600 angstroms which are aggregated into particles of colloidal size~i.e., approximately 100-2000 angstroms in diameter, preferably around 400-1000 angstroms in diameter. By sorting the mixture of particles into sub fractions which have uniform magnetization, compositions having homogeneous properties with respect to ability to be retained in the magnetic field can be obtained. Use of particles of such homogeneity has the advantage of effecting a shaφ separation peak in chromatographic procedure, as well as the potential for labeling various components in a mixture with sets of particles of differing magnetic susceptibilities to permit chromatographic separation of these components. The particles can also be separated according to size.
In general, such particles may for example be prepared by a modification ofthe method described in U.S. 4,452,773. Solutions comprising ferric and fenous salts in a molar ratio of about 2:1-1.5:1 along with a suitable quantity of coating material, typically a weight of polysaccharide approximately 5-20 times the weight of iron salts in solution are stined and heated to about 40 degrees C. Prefened coatings to be used in this process are polysaccharide or protein coatings. A prefened polysaccharide coating is dextran. The mixture is then typically titrated to basic pH eg with sodium hydroxide eg by dropwise addition over a period of about 1 hour. Furthermore, the addition of base at elevated temperatures assists in the formation ofthe desired size particles. A coating material can be included, as above, in the magnetic oxide forming preparation, or can be added after the colloidal oxide particles are formed. After neutralization with acid, aggregates are suitably removed by any convenient means such as filtration or centrifugation and the magnetic particles removed from the uncomplexed coating material eg by washing in a high gradient magnetic field.
The particles can be subjected to HGMS at any stage of this preparation process-before or after coating and before or after size separation. The prepared particles may suitably be applied to any standard HGMS apparatus and fractionated according to magnetization (i.e., magnetic susceptibility). In one procedure, the mixture is applied to the HGMS column, containing matrix, at a very high magnetic field strength so that virtually all ofthe particles are retained. The particles are then eluted by gradual reduction ofthe magnetic field across the column. Fractions are collected at arbitrary intervals resulting in the preparation of a series of compositions each having a desired degree of homogeneity of magnetization. In a prefened procedure, however, the mixture of particles is segregated by overloading an
HGMS matrix with the preparation. In this case, only the most highly magnetized particles - i.e., those having the highest magnetization- are retained, while the remainder ofthe preparation flows through the matrix. The retained materials can then be eluted by removing the imposed magnetic field. By properly balancing the amount of surface area ofthe matrix and quantity of particles, any arbitrary fraction ofthe highest magnetization particles in the distribution resulting from the initial preparation can be retained. Desirably, in order to obtain good homogeneity and high magnetization and susceptibility, a sufficiently small amount of matrix should be used so that the majority ofthe particles, about 80%, preferably about 90%), ofthe particles, pass through the matrix.
Thus, in a typical preparation resulting in a Gaussian distribution of magnetization, suitably only a small portion ofthe upper "tail" is retained and recovered. However, additional fractions of lower magnetization can be obtained by recycling the flow-through through a clean matrix. Elution from this matrix can be had by removing the magnetic field, or by displacement with the higher magnetization fraction.
In conjugating the coated particles to a protein or polypeptide, the polysaccharide or other coating is suitably derivatized to provide functional groups for conjugation to the specific binding moiety. A variety of such modifications is known in the art. For example, polysaccharides are conveniently oxidized using periodate to provide aldehyde functional groups which can then be conjugated to amino substituents on a proteinaceous binding moiety, or can be reacted with CNBr to provide this functionality. Protein coatings can be linked to targets for example through amino or sulfhydryl groups.
A variety of other methods of conjugation are available, and a large variety of homobifunctional and heterobifunctional linkers useful in effecting such conjugation are available, for example, from Pierce Chemical Co. Linkers can be of specified lengths also, to effect a specific separation between the particle and target. Techniques for conjugation of specific binding moieties to polysaccharide solid supports are well known in the art and have been applied extensively to specific binding assays, notably immuno-assays. Any suitable derivatization and conjugation method is contemplated by the invention.
An example of suitable particles is provided, for example, in Example 1 of US 5385707 as follows: Supeφaramagnetic particles were prepared by mixing 10 g dextran T40 (Pharmacia, Uppsala), 1.5 g ferric chloride hexahydrate, and 0.64 g fenous chloride tetrahydrate in 20 ml water and heating to 40 degrees C. The solution was stined and 20 ml 4M NaOH was added dropwise with continued stirring. The resulting particle suspension was neutralized with acetic acid, and centrifuged for 10 min at 2,000.times.g followed by filtration through a 0.22 uM pore-size filter (Millex GV) to remove aggregates.
Unbound dextran was removed by washing in a high gradient magnetic field (HGMF). This was performed by washing the magnetic particles in columns of ordinary household steel wool (average diameter about 30 um) which was placed in the chamber of a HGMS device shown in FIG. 1 at a strength of 0.6 Tesla. Ten ml of particle suspension were applied to a 15 times 40 mm column of 2 g of steel wool, and loaded column was washed with 30 ml buffer. About 90%) ofthe particles were washed through the column. The column was then removed from the external field and the remaining 10%> ofthe magnetic particles were eluted.
Molday (U.S. Pat. No. 4,452,773) describes the preparation of further magnetic iron-dextran microparticles and provides a summary describing the various means of preparing particles suitable for attachment to biological materials. A further description of polymeric coatings for magnetic particles used in high gradient magnetic separation (HGMS) methods are found in DE 3720844 (Miltenyi) and U.S. Pat. No. 5,385,707. Methods to prepare supeφaramagnetic particles are described for example in U.S. 4,770,183.
As noted above, direct conjugation of a protein or polypeptide to a particle may be achieved by use of any suitable chemical linking groups. For example, a polysaccharide or other coating of a microparticle may be suitably derivatized to provide functional groups. A wide variety of such modifications is known in the art. For example, amino groups may be introduced before or after forming the particles, or an aldehyde function may be introduced by reacting the polysaccharide with, eg diimidazole or DCCD, and coupling hexane diamine to the sugar molecules. Alternatively, in preparing dextran for coating, aminodextran may be mixed with unsubstituted dextran to provide amino groups. The polysaccharides may be conveniently oxidized using periodate to provide aldehyde functional groups that can be conjugated to amino substituents on a proteinaceous binding moiety, particularly under the conditions of reductive amination. Proteins and polypeptides can then be coupled to the particles through side chain amino or sufhydryl groups and heterofunctional cross-linking reagents.
As described by Miltenyi, a large number of heterofunctional compounds are available for linking to entities. Illustrative entities include but are not limited to: azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3,-[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N-.gamma- maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N- succinimidyl [4-azidophenyl]-l,3'-dithiopropionate, N-succinimidyl [4- iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl 4-[N- maleimidomethyl]cyclohexane-l -carboxylate. A prefened linking group is 3-(2- pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) or 4-(N- maleimidomethyl)-cyclohexane-l -carboxylic acid N-hydroxysuccinimide ester (SMCC) with a reactive sulfhydryl group on the protein or polypeptide and a reactive amino group on the particle.
Carbohydrate/Polysaccharide Particles and Coatings
In one embodiment of the invention the particle may comprise or be coated with a carbohydrate polymer, preferably a polysaccharide polymer. Preferably such a polysaccharide is water-soluble.
As is well-known, polysaccharides are generally made up of a number of monosaccharide units typically joined by glycosidic bonds, such as 1-4 or 1-6 linkages. Suitably the monosaccharide units may be, for example, aldoses (which may for example be trioses, tetroses such as erythrose or threose; pentoses such as ribose, arabinose, xylose or lyxose; hexoses such as allose, altrose, glucose, mannose, gulose, idose, galactose or tulose, or heptoses); or ketoses (which may for example be ketotrioses, ketotetroses such as erythulose; ketopentoses such as ribulose or xylulose; ketohexoses such as fructose, psicose, tagatose or sorbose, or ketoheptoses). Units may be in either D- or L- form, but the D form is generally prefened (eg D-glucose). Likewise, monosaccharide units may be in either alpha or beta forms, for example alpha-D-glucose. The monosaccharides in a polysaccharide may be substantially the same (ie to provide a homopolysaccharide) or combinations of units may be used (ie to provide a heteropolysaccharide). Tens, hunreds or thousands of monosaccharide units may be present in such a polymer, and branching will commonly be present.
Suitable carbohydrate polymers include for example, glucans such as dextrans including aminodextrans and carboxymethyl-dextrans, heparins, celluloses (and derivatives thereof such as methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose and hydroxypropylcellulose), chitosan and hydrolysates of chitosan, starches (and derivatives thereof such as hydroxyethyl-starches and hydroxy propyl- starches), glycogens, heparins, alginates, agaroses and derivatives and activated versions thereof, guar gums, pullulans, inulins, xanthan gums, canageenans, pectins and alginic acid hydrolysates and derivatives and activated versions thereof.
For example, a review of dextran conjugation is provided by Mehvar, Journal of Controlled Release Vol 69 (2000) pages 1 -25.
As noted above, derivatives of such polymers ("derivatised polymers") may also be used in the present invention. Such derivatised polymers may typically for example result from activation processes as described below.
Activation of polymers
If desired, to conjugate modulators of Notch signalling (eg proteins, polypeptides or peptides, or mimetics thereof such as "small molecules") to a polymer support/matrix substrate eg particle (or polymer-coated particle) a number of groups on the polymer may be converted into more reactive functional groups which facilitate conjugation. This process is frequently refened to as "activation" and the product is called an "activated" or "functionalized" polymer.
In particular, if a polymeric molecule to be used as a support is not active (or is not considered sufficiently active) on its own it should preferably be activated by the use of a suitable technique.
Modulators of Notch signalling are preferably covalently attached to a polymer or activated polymer (either directly or via a linker) using chemical techniques. Reaction chemistries resulting in such linkages are well known in the art and may for example involve the use of complementary functional groups (eg on the linker, polymer and/or modulator of Notch signalling) for example as shown below: First Reactive Group Second Reactive Group Linkage carboxyl amine amide sulfonyl halide amine sulfonamide hydroxyl alkyl/aryl halide ether hydroxyl isocyanate urethane amine epoxide beta-hydroxyamine amine alkyl/aryl halide alkylamine hydroxyl carboxyl ester amine aldehyde amide/amine thiol/sulfhydryl maleimide amine succinimide
As described, for example, in US 6303752 (Novozymes), methods and chemistry for activation of polymeric molecules as well as for conjugation of proteins, polypeptides and peptides are well described in the literature. For example, commonly used methods for activation of polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R. F. Taylor, (1991), "Protein immobilisation. Fundamental and applications", Marcel Dekker, N.Y.; S. S. Wong, (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, Boca Raton; G. T. Hermanson et al., (1993), "Immobilized Affinity Ligand Techniques", Academic Press, N.Y. and Hermanson (1995) "Bioconjugate Techniques", Academic Press, N. Y.). Some of these methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc. The functional groups on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which may typically comprise i) activation of polymer, ii) conjugation, and iii) if required, blocking of residual active groups.
For example, coupling polymeric molecules to the free acid groups of polypeptides may be performed for example with the aid of diimide and for example amino-PEG or hydrazino- PEG (Pollak et al., (1976), J. Amr. Chem. Soc, 98, 289-291) or diazoacetate/amide (Wong et al., (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press).
Coupling to free sulfhydryl groups (such as a cysteine residue in a protein or polypeptide) can be achieved for example with groups like maleimido or ortho-pyridyl disulfide. Also vinylsulfone (U.S. Pat. No. 5,414,135, (1995), Snow et al.) has a preference for sulfhydryl groups.
Accessible arginine residues in a polypeptide chain may suitably be targeted by groups comprising two vicinal carbonyl groups.
Techniques involving coupling polymers such as electrophilically activated PEGs to the amino groups of reidues such as lysines may also be useful. Many ofthe usual leaving groups for alcohols give rise to an amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al., (1984), Methods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach, K, Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987), Methods in Enzymology vol. 135, Mosbach, K, Ed., Academic Press: Orlando, 1987; pp 79-84; Crossland et al., (1971), J. Amr. Chem. Soc. 1971, 93, pp. 4217-4219), mesylates (Harris, (1985), supra; Harris et al., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, pp 341-352), aryl sulfonates like tosylates, and para-nitrobenzene sulfonates can be used.
Organic sulfonyl chlorides, e.g. tresyl chloride, effectively convert hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in proteins or polypeptides allow stable linkages to be formed between polymer and polypeptide. In addition to high conjugation yields, the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity). Epoxides may also be used for creating amine bonds.
Converting PEG into a chloroformate with phosgene may facilitate carbamate linkages to lysines. The many variations include substituting the chlorine with N-hydroxy succinimide (U.S. Pat. No. 5,122,614, (1992); Zalipsky et al., (1992), Biotechnol. Appl. Biochem., 15, p. 100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., (1991), Carbohydr. Res., 213, pp 309-319), with para-nitrophenol, DMAP (EP 632 082 Al, (1993), Looze, Y.) etc. The derivatives are typically made for example by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide. Alternatively, isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.
In a further coupling technique, urethane (carbamate) linkages may be formed between an amino acid amino group (eg lysine, histidine, N-terminal residue), and an activated polymer. Suitably, such a urethane linkage is formed using a terminal oxycarbonyl-oxy-N- dicarboximide group such as a succinimidyl carbonate group. Alternative activating groups include N-succinimide, N-phthalimide, N-glutarimide, N-tetrahydrophthalimide and N- norborene-2,3-dicarboxide. These urethane-forming groups are described for example in U.S. Pat. No. 5,122,614, the disclosure of which is hereby incoφorated by reference. This patent also discloses the formation of N-succinimide carbonate derivatives of polyalkylene oxides including polyethylene glycols which are also capable of forming urethane linkages with amino group targets (eg lysine).
Suitable starting materials and reagents for preparing the conjugates ofthe present invention are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis., USA), Bachem (Tonance, Calif, USA), Emka-Chemie, or Sigma (St. Louis, Mo., USA), Pierce Chemical Company (Rockford, IL, US), Molecular Probes Inc (Eugene, OR, US) or Amersham Pharmacia (Little Chalfont, UK and Piscataway, NJ, US); or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).
Additionally, it will be appreciated that conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, N.Y., 1991, and references cited therein.
Preferably a suitable linker reagent for use in the present invention may be a bifunctional reagent with a group for reacting with a modulator of Notch signalling (for example for reacting with a protein or polypeptide modulator of Notch signalling) and a group for reacting with a polymer support structure. After reaction the linker reagent may typically remain in the resulting conjugate as a linker reagent residue (which may also be termed, for example, a "linker").
A wide range of linker reagents are available for example from the Pierce Chemical
Company, Rockford, IL, USA., (see for example Pierce Chemical Company, Cross-linking Technical Section, Pierce Life Science and Analytical Research products Catalog and Handbook, 1994), for example as follows:
p-azidobenzoyl hydrazide (ABH)
3-([2-aminoethyl]dithio)-propionic acid (AEDP)
N-alpha-maleimidoacetoxy)-succinimide ester (AMAS)
N-5-azido-2-nitrobenzyloxysuccinimide ANB-NOS)
N-(4-[p-azidosalicylamido]-butyl)-3'(2'pyridylthio)-propionamide (APDP) p-azidophenyl glyoxal monohydrate (APG)
4-(p-azidosalicylamido)-butylamine (ASBA)
Bis(beta-[4-azidosalicylamido]-ethyl)disulfide (BASED)
1,4-5/s-Maleimidobutane (BMB)
1 ,4-R/5-Maleimidyl-2,3-dihydroxybutane (BMDB) 1 ,6-Rts-maleimidohexane (BMH)
Rw-Maleimidoethane (BMOE)
N-beta-maleimidopropionic acid (BMP A)
1,8-Rts-maleimidotriethylene glycol (BM[PEO]3)
1,1 l-Rts-maleimidotetraethylene glycol BM[PEO]4 N-(beta-maleimidopropionic acid)hydrazide.TFA (BMPH)
N-(beta-maleimidipropyloxy)succinimide ester (BMPS)
Bis(2-[succinimidooxy-carbonyloxy]ethyl)sulfone (BSOCOES) Bis(sulfosuccinimidyl)-suberate (BS3)
1 ,5-difluoro-2,4-dinitrobenzene (DFDNB)
Dimethyladipimidiate (DMA)
Dimethylsuberimidate (DMS) 1 ,4-Di-(3'-[2' pyridylthio]-proρionamido)butane (DPDPB)
Disuccinimidyl glutarate (DSG)
Dithiobis (succinimidylpropionate) (DSP)
Disuccinimidyl suberate (DSS)
Disuccinimidyl tartarate (DST) Dimethyl 3,3'-dithiobis-propionimidate (DTBP)
Dithio-bis-maleimidoethane (DTME)
3 ,3 '-dithiobis(sulfosuccinimidylpropionate) (DTS SP)
Ethylene glycol bis(succinimidylsuccinate) (EGS)
N-epsilon-maleimidocaproic acid (ECMA) N-epsilon-(maleimidocaprolyloxy)succinimide ester (EMCS)
N-gamma-maleimidobutyryloxy-succinimide ester (GMBS)
1 ,6-hexane-bis- vinylsulfone (HBVS)
N-kappa-malaimidoundecanoic acid (KMUA)
Succinimidyl -4-(N-maleimido-methyl)cyclohexane-l-carboxy-(6-amido caproate) (LC- SMCC)
Succinimidyl 6-(3'-[2-pyridyl-dithio]propionamido)hexanoate (LC-SPDP) m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
4-(N-maleimidomethyl)-cyclohexane- 1 -carboxyl-hydrazide (M2C2H)
3-maleimidophenyl boronic acid (MPBA) 4-(4-N-maleimidophenyl)-butyric acid hyrdrazide (MPBH)
Methyl N-succinimidyl adipate (MSA)
N-Hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA)
3-(2-pyridylthio)-propionyl hydrazide (PDPH)
N-(p-maleimidophenyl)isocyanate (PMPI) N-succinimidyl (4'azido-phenyl)l,3'-dithiopropionate (SADP)
Sulfosuccinimidyl-2-[7-azido-4-methylcoumarin-3-acetamido]ethyl-l,3'-dithiopropionate
(SAED) Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido) ethyl l,3'-dithiopropionate (SAND) N-succinimidyl 6-(4'-azido-2'-nitrophenylamino) hexanoate (SANPAH) Sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl 1,3-dithiopropionate (SASD) N-succinimidyl S-acetylthioacetate (SATA) N-succinimidyl S-acetylthiopropionate (SATP)
Succinimidyl 3-(bromoacetamido) propionate (SBAP)
Sulfosuccinimidyl(perfluoroazidobenzamido) ethyl l,3'-dithiopropionate (SFAD) N-succinimidyl iodoacetate (SIA) N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) Succinimidyl 4-(N-maleimido-methyl)cyclohexane-l -carboxylate (SMCC) Succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB) Succinimidyl-6-(beta-maleimido-propionamido) hexanoate (SMPH) 4-succinimidyloxy-carbonyl-methyl-alpha-(2-pyridylthio) toluene (SMPT) Succinimidyl-(4-psoralen-8-yloxy)butyrate (SPB) N-succinimidyl 3-(2-pyridylthio)propionate (SPDP)
Bis(2-[sulfosuccinimidooxycarbonyloxy]ethyl) sulfone (Sulfo-BSOCOES) Sulfodisuccinimidyl tartarate (Sulfo-DST) Ethylene glycol bw(sulfo-succinimidyl) succcinate (Sulfo-EGS) N-(epsilon-maleimidocaproyloxy)sulfosuccinimide ester (Sulfo-EMCS) N-gamma-maleimidobutryloxy-sulfosuccinimide ester (Sulfo-GMBS) N-hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HSAB) N-(kappa-maleimidoundecanoyloxy)-sulfosuccinimide ester (Sulfo-KMUS) Sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate (Sulfo-LC-SPDP) m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS) Sulfosuccinimidyl(4-azido-salicylamido)hexanoate (Sulfo-NHS-LC-ASA) Sulfosuccinimidyl (4-azidophenyldithio)propionate (Sulfo-SADP) Sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)-hexanoate (sulfo-SANPAH) Sulfo-NHS-2-(6-[biotinamido]-2-(p-azidobenzamido)-hexanoamido)ethyl-l,3'- Dithiopropionate (Sulfo-BED; trifunctional) Sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (Sulfo-SIAB)
Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (Sulfo-SMCC) Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (Sulfo-SMPB) Sulfosuccinimidyl 6-(alpha-mathyl-alpha-[2-pyridyldithio]-toluamido)hexanoate (Sulfo-LC- SMPT)
N-succinimidyl-(4-vinylsulfonyl) benzoate (SVSB) Tris-(2-maleimidoethyl) amine (TMEA; tri functional) Tris-(succinimidyl amino-triacetate (TSAT; tri functional).
Suitably a linker used will be a bifunctional reagent, such as a heterobifunctional reagent (although it will be appreciated that homobifunctional reagents may also be used). Trifunctional and higher reagents may also be used if desired.
Suitably the modulators of Notch signalling are presented on the polymer in an orientation suitable for binding to and/or activation of a Notch receptor.
Polymers
As noted above preferably the matrix/substrate eg particle used comprises and/or is coated with, a polymeric structure which is preferably a pharmaceutically acceptable polymer. Prefened polymers are water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. Other suitable polymers include, for example, polyethylene glycol propionaldehyde, monomethoxy-polyethylene glycol, polyvinyl pynolidone (PVP), poly-l,3-dioxolane, poly- 1,3,6-trioxane, ethylene/maleic anhydride copolymer, (either homopolymers or random copolymers), poly(n-vinyl pynolidone)polyethylene glycol, polypropylene glycol homopolymers (PPG) and other polyalkylene oxides, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and other polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, colonic acids or other carbohydrate polymers, Ficoll or dextran and mixtures thereof. It will be appreciated that polymers may also be used in activated, functionalised or derivatised forms
Modulators of Notch signalling may be attached to the support structure at random positions within the molecule, or at predetermined positions within the molecule and may be attached to one, two, three or more chemical moieties. Polymers may be either homopolymers or copolymers, eg random copolymers and may be either straight or branched.
Suitably the polymer may be a polysaccharide polymer, such as a glucan, for example a dextran or a dextran derivative such as amino-dextran.
A polymer where used may, if desired, have a branched structure. For example, branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Moφurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incoφorated herein by reference.
Where the modulator of Notch signalling is a protein, the protein should preferably be attached to the particulate support structure with consideration of effects on functional or antigenic domains ofthe protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incoφorated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polymers such as polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polymer such as polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include, for example, lysine residues and N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydryl groups from cysteine residues may also be used as a reactive group for attaching polymers such as polyethylene glycol molecules. For example, attachment may be at an amino group, such as attachment at the N-terminus or a lysine group, or at a cysteine group, for example a C-terminal cysteine group.
Polymers such as polyethylene glycol may, for example, be attached to proteins and polypeptides via linkage to any of a number of amino acid residues ofthe protein or polypeptide. For example, polymers such as polyethylene glycol can be linked to a protein via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polymers such as polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) ofthe protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) ofthe protein.
In some instances, it may be desirable to have proteins attached to the support structure through their N-termini. For example, using polyethylene glycol as an illustration, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification ofthe N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization ofthe protein at the N-terminus with a carbonyl group containing polymer may be achieved.
Preferably, to provide an optimal orientation, proteins, polypeptides or peptides may be attached to the support structure through a suitably provided terminal residue, for example an C-terminal residue such as a terminal lysine, histidine, aspartic acid, glutamic acid or cysteine residue, which may be readily created or exposed by genetic manipulation techniques if not already present in the protein or peptide to be attached. Attachment at a terminal residue, or at a point close to the protein/peptide terminus (preferably C-terminus), typically provides better presentation of ligands for binding to and/or activation of Notch receptors.
For example, in a prefened form of the invention a multiplicity of protein/peptide modulators of Notch signalling (such as Notch ligand constructs comprising a DSL domain and 1-5, eg 3 EGF domains) are attached to a water-soluble polymeric support such as a polysaccharide, eg a dextran, by C-terminal residues (eg cysteine, lysine, histidine, glutamic or aspartic acid, preferably cysteine) via a linker such as sulfosuccinimidyl 4-[N- maleimidomethyl]-cyclohexane-l -carboxylate (sulfo-SMCC) or the like.
Polypeptide Sequences
As used herein, the term "polypeptide" is synonymous with the term "amino acid sequence" and/or the term "protein". In some instances, the term "polypeptide" is synonymous with the term "peptide".
"Peptide" usually refers to a short amino acid sequence that is 10 to 40 amino acids long, preferably 10 to 35 amino acids.
The polypeptide 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.
Polynucleotide Sequences
As used herein, the term "polynucleotide sequence" is synonymous with the term "polynucleotide" and/or the term "nucleotide sequence".
The polynucleotide sequence may be DNA or RNA of genomic or synthetic or of recombinant origin. They may also be cloned by standard techniques. The polynucleotide sequence may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
"Polynucleotide" refers to a polymeric form of nucleotides of at least 10 bases in length and up to 1,000 to 10,000 bases or more. Longer polynucleotide 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 ofthe 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 ofthe 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 nucleic acid may be RNA or DNA and is preferably DNA. Where it is RNA, manipulations may be performed 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 ofthe 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 ofthe 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.
The polynucleotide sequence may comprise, for example, a protein-encoding domain, an antisense sequence or a functional motif such as a protein-binding domain and includes variants, derivatives, analogues and fragments thereof. The term also refers to polypeptides encoded by the nucleotide sequence. Variants, Derivatives, Analogues, Homologues and Fragments
In addition to the specific polypeptide and polynucleotide sequences mentioned herein, the present invention also encompasses the use of variants, derivatives, analogues, homologues, mimetics 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 polypeptide or polynucleotide which retains at least one of the functions of the endogenous polypeptide or polynucleotide but generally has a different evolutionary origin thereto.
The term "mimetic" as used herein, in relation to polypeptides or polynucleotides, refers to a chemical compound that possesses at least one of the endogenous functions of the polypeptide or polynucleotide which it mimics.
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 transport activity or ability to modulate Notch signalling. 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 Asp,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, CTT ,CTC ,CTA, CTG
M Met Methionine ATG
N Asn Asparagine AAT,AAC
P Pro Proline CCT, CCC, CCA, CCG
Q Gin Glutamine CAA,CAG
R Arg Arginine CGT ,CGC , CGA, CGG,AGA,AGG
S Ser Serine TCT , TCC , TCA, TCG,AGT ,AGC
T Thr Threonine ACT,ACC,ACA,ACG
V Val Valine GTT, GTC, GTA, GTG
Trp Tryptophan TGG
X Xxx Unknown
Y Tyr Tyrosine TAT , TAC z Glu , Gin Glutamic ,
Glutamine GAA,GAG, CAA, CAG
* End Terminator TAA,TAG,TGA Conservative 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 imgf000076_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 ofthe 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 polynucleodtide.
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 conesponding to the naturally-occurring sequence either side ofthe insertion site. The flanking regions will contain convenient restriction sites conesponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative ofthe 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 HIV, for instance, use a large number of rare codons. By changing a nucleotide sequence such that rare codons are replaced by the conesponding 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 ofthe sequence is codon optimised. Even more preferably, the sequence is codon optimised in its entirety.
As used herein, the term "homology" can be equated with "identity". An homologous sequence will be taken to include an amino acid sequence which may be at least 75, 85 or 90%) identical, preferably at least 95 or 98%> identical. In particular, homology should typically be considered with respect to those regions ofthe 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 ofthe present invention it is prefened 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 conesponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion 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 prefened to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
Calculation of maximum % homology therefor firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA (Atschul) and the GENE WORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching. However it is prefened to use the GCG Bestfit program. 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 prefened to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to calculate %> homology, preferably % sequence identity. The software typically does this as part ofthe sequence comparison and generates a numerical result.
Nucleotide sequences which are homologous to or variants of sequences of use in the present invention can be obtained in a number of ways, for example by probing DNA 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 ofthe reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants ofthe 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 software 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.
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 ofthe polynucleotide or encoded polypeptide.
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 ofthe 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 ofthe 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
For recombinant production, host cells can be genetically engineered to incoφorate expression systems or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis et al and Sambrook et al, such as calcium phosphate transfection, DEAE- dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, fransduction, 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 ofthe translated protein into the lumen ofthe endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incoφorated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
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.
In one embodiment ofthe invention, any one or more ofthe above candidate modulators is brought into contact with a cell ofthe immune system. Cells ofthe immune system of use in the present invention are described below.
Cells of the Immune System
Cells of use in the present invention are preferably cells ofthe immune system capable of transducing the Notch signalling pathway.
Most preferably the cells of use in the present invention are T-cells. These include, but are not limited to, CD4+ and CD8+ mature T cells, immature T cells of peripheral or thymic origin and NK-T cells.
Alternatively, the cells may for example be antigen-presenting cells (APCs). APCs include dendritic cells (DCs) such as interdigitating DCs or follicular DCs, Langerhans cells, PBMCs, macrophages, B-lymphocytes, T-lymphocytes, or other cell types such as epithelial cells, fibroblasts or endothelial cells, constitutively expressing or activated to express a MHC Class II molecules 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.
The T cells or APCs may be isolated from a patient, or from a donor individual or another individual. The cells are preferably mammalian cells such as human or mouse cells. Preferably the cells are of human origin. The APC or precursor APC may be provided by a cell proliferating in culture such as an established cell line or a primary cell culture. Examples include hybridoma cell lines, L-cells and human fibroblasts such as MRC-5. Prefened cell lines for use in the present invention include Jurkat, H9, CEM and EL4 T- cells; long-term T-cell clones such as human HA1.7 or mouse D10 cells; T-cell hybridomas such as DO11.10 cells; macrophage-like cells such as U937 or THP1 cells; B-cell lines such as EBV-transformed cells such as Raji, A20 and Ml cells.
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 manow. From peripheral blood, adherent precursors can be treated with a GM-CSF/IL-4 mixture (Inaba et al), or from bone manow, non-adherent CD34+ cells can be treated with GM-CSF and TNF-α (Caux et al). DCs can also be routinely prepared from the peripheral blood of human volunteers, similarly to the method of Sallusto and Lanzavecchia J Exp Med (1994) 179(4) 1109-18 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 et al). Culture conditions may include other cytokines such as GM-CSF or IL-4 for the maintenance and, or activity ofthe dendritic cells or other antigen presenting cells.
T cells and B cells for use in the invention are preferably obtained from cell lines such as lymphoma or leukemia cell lines, T cell hybridomas or B cell hybridomas but may also be isolated from an individual suffering from a disease of the immune system or a recipient for a transplant operation or from a related or unrelated donor individual. T cells and B cells may be obtained from blood or another source (such as lymph nodes, spleen, or bone manow) and may be enriched or purified by standard procedures. 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 prefened to use helper T cells (CD4+). Alternatively other T cells such as CD8+ cells may be used.
Candidate modulators of use in the present invention are brought into contact with a cell of the immune system as described above. In a further step, modulation of Notch signalling by a candidate modulator is detected. Assays for detecting modulation of Notch signalling will be described below. Many of these assays will involve monitoring the expression of a "target gene". Assays
Assays for monitoring expression of the one or more target genes and other methods of detecting modulation of Notch signalling are described below.
For example, an assay may be set up to detect either inhibition or enhancement of Notch signalling in cells ofthe immune system by candidate modulators. The method comprises mixing cells ofthe immune system, where necessary transformed or transfected, etc. with a synthetic reporter gene, in an appropriate buffer, with a sufficient amount of candidate modulator and monitoring Notch signalling. The modulators may be small molecules, proteins, antibodies or other ligands as described above. Amounts or activity ofthe target gene (also described above) will be measured for each compound tested using standard assay techniques and appropriate controls. Preferably the detected signal is compared with a reference signal and any modulation with respect to the reference signal measured.
The assay may also be run in the presence of a known antagonist ofthe Notch signalling pathway in order to identify compounds capable of rescuing the Notch signal.
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 the Notch signalling pathway in cells ofthe immune system 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. Preferably an assay for use with the present invention will be a cell based assay.
Techniques for drug screening may be based 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 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.
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.
It is expected that the assay methods ofthe present invention will be suitable for both small and large-scale screening of test compounds as well as in quantitative assays.
Various nucleic acid assays are also known. Any conventional 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 anays and other hybridization methods.
Target 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 target 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.
Generation of nucleic acids for analysis from samples generally requires nucleic acid amplification. Many amplification methods rely on an enzymatic chain reaction (such as a polymerase chain reaction, a ligase chain reaction, or a self-sustained sequence replication) or from the replication of all or part of the vector into which it has been cloned. Preferably, the amplification according to the invention is an exponential amplification, as exhibited by for example the polymerase chain reaction.
Many target and signal amplification methods have been described in the literature, for example, general reviews of these methods in Landegren, U., et al., Science 242:229-237 (1988) and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990). These amplification methods may be used in the methods of our invention, and include polymerase chain reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase hybridisation, Qbeta bacteriophage replicase, transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based amplification (NASBA) and in situ hybridisation. Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.
PCR is a nucleic acid amplification method described mter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR consists of repeated cycles of DNA polymerase generated primer extension reactions. 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 formation 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. PCR can be used to amplify any known nucleic acid in a diagnostic context (Mok et al., (1994), Gynaecologic
Oncology, 52: 247-252).
Self-sustained sequence replication (3SR) is a variation of TAS, which involves the isothermal amplification of a nucleic acid template via sequential rounds of reverse transcriptase (RT), polymerase and nuclease activities that are mediated by an enzyme cocktail and appropriate oligonucleotide primers (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874). Enzymatic degradation of the RNA of the RNA/DNA heteroduplex is used instead of heat denaturation. RNase H and all other enzymes are added to the reaction and all steps occur at the same temperature and without further reagent additions. Following this process, amplifications of 10 6 to 109 have been achieved in one hour at 42 °C.
Ligation amplification reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two per target strand. This technique is described by Wu, D. Y. and
Wallace, R. B. (1989) Genomics 4:560. The oligonucleotides hybridise to adjacent sequences on the target DNA and are joined by the ligase. The reaction is heat denatured and the cycle repeated.
Alternative amplification technology can be exploited in the present invention. For example, rolling circle amplification (Lizardi et al, (1998) Nat Genet 19:225) is an amplification technology available commercially (RCAT™) which is driven by DNA polymerase and can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions.
In the presence of two suitably designed primers, a geometric amplification occurs via DNA strand displacement and hyperbranching to generate 1012 or more copies of each circle in 1 hour.
If a single primer is used, RCAT generates in a few minutes a linear chain of thousands of tandemly linked DNA copies of a target covalently linked to that target.
A further technique, strand displacement amplification (SDA; Walker et al, (1992) PNAS (USA) 80:392) begins with a specifically defined sequence unique to a specific target. But unlike other techniques which rely on thermal cycling, SDA is an isothermal process that utilises a series of primers, DNA polymerase and a restriction enzyme to exponentially amplify the unique nucleic acid sequence.
SDA comprises both a target generation phase and an exponential amplification phase. In target generation, double-stranded DNA is heat denatured creating two single-stranded copies. A series of specially manufactured primers combine with DNA polymerase (amplification primers for copying the base sequence and bumper primers for displacing the newly created strands) to form altered targets capable of exponential amplification.
The exponential amplification process begins with altered targets (single-stranded partial DNA strands with restricted enzyme recognition sites) from the target generation phase.
An amplification primer is bound to each strand at its complementary DNA sequence. DNA polymerase then uses the primer to identify a location to extend the primer from its 3' end, using the altered target as a template for adding individual nucleotides. The extended primer thus forms a double-stranded DNA segment containing a complete restriction enzyme recognition site at each end.
A restriction enzyme is then bound to the double stranded DNA segment at its recognition site. The restriction enzyme dissociates from the recognition site after having cleaved only one strand ofthe double-sided segment, forming a nick. DNA polymerase recognises the nick and extends the strand from the site, displacing the previously created strand. The recognition site is thus repeatedly nicked and restored by the restriction enzyme and DNA polymerase with continuous displacement of DNA strands containing the target segment.
Each displaced strand is then available to anneal with amplification primers as above. The process continues with repeated nicking, extension and displacement of new DNA strands, resulting in exponential amplification ofthe original DNA target.
PCR technology as described e.g. in section 14 of Sambrook et al., 1989, requires the use of oligonucleotide probes that will hybridise to target nucleic acid sequences. 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.
Prefened 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 ofthe invention are labelled with suitable label means for ready detection upon hybridisation. For example, a suitable label means is a radiolabel. The prefened method of labelling a DNA fragment is by incoφorating 32P 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 P-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.
Prefened are such sequences, probes which hybridise under high-stringency conditions.
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) ofthe 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 refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65-68 °C. High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6x SSC, 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 - O.lx SSC, 0.1 % SDS.
It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g. formamide-based buffers, and temperatures. Denhardt's solution and SSC are well known to those of 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) Cunent Protocols in Molecular Biology, John Wiley & Sons, Inc.). Optimal hybridisation conditions have to be determined empirically, as the length and the GC content ofthe hybridising pair also play a role.
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. ofthe gene being monitored. Fluorescent markers, which can be detected and sorted by FACS, are prefened. Especially prefened are GFP and luciferase. Another type of prefened reporter is cell surface markers, i.e. proteins expressed on the cell surface and therefor easily identifiable. Thus, cell-based screening assays can be designed by constructing cell lines in which the expression of a reporter protein, i.e. an easily assayable protein, such as β- galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase, is dependent on the activation of a Notch. For example, a reporter gene encoding one ofthe above polypeptides may be placed under the control of an response element which is specifically activated by Notch signalling. Alternative assay formats include assays which directly assess responses in a biological system. If a cell-based assay system is employed, the test compound(s) indentified may then be subjected to in vivo testing to determine their effect on Notch signalling pathway.
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 ofthe gene of interest (i.e. of an endogenous target gene), 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 target 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 Laboratory 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 ofthe 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 inteφreted by software 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 target gene expression in cells transfected with recombinant DNA encoding polypeptides. This can be achieved directly, by labelling ofthe 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 therefor 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 therefor 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 either manually, or using FACS sorting. Nucleic acid probes complementary to mRNA may be prepared according to the teaching set forth above, using the general procedures as described by Sambrook et al (1989).
In a prefened embodiment, the invention comprises the use of an antisense nucleic acid molecule, complementary to a target 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). These high density aπays are particularly useful for diagnostic and prognostic puφoses. Use may also be made of In Vivo Expression Technology (IVET) (Camilli). IVET identifies target genes upregulated during say treatment or disease when compared to laboratory culture.
The present invention also provides a method of detection of polypeptides. 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, protein gel assay, Western Blot analysis, antibody sandwich assays, antibody detection, FACS and ELISA assays. For example, polypeptides can be detected by differential mobility on protein gels, or by other size analysis techniques, such as mass spectrometry. The detection means may be sequence-specific. For example, polypeptide or RNA molecules can be developed which specifically recognise polypeptides in vivo or in vitro.
For example, RNA aptamers can be produced by SELEX. SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules. It is described, for example, in U.S. patents 5654151, 5503978, 5567588 and 5270163, as well as PCT publication WO 96/38579
The invention, in certain embodiments, includes antibodies specifically recognising and binding to polypeptides.
Antibodies may be recovered from the serum of immunised animals. Monoclonal antibodies may be prepared from cells from immunised animals in the conventional manner.
The antibodies ofthe invention are useful for identifying cells expressing the genes being monitored.
Antibodies according to the invention may be whole antibodies of natural classes, such as IgE and IgM antibodies, but are preferably IgG antibodies. Moreover, the invention includes antibody fragments, such as Fab, F(ab')2, Fv and ScFv. Small fragments, such Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.
The antibodies may comprise a label. Especially prefened are labels which allow the imaging ofthe antibody in neural cells in vivo. Such labels may be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within tissues. Moreover, they may be fluorescent labels or other labels which are visualisable in tissues and which may be used for cell sorting.
In more detail, antibodies as used herein can be altered antibodies comprising an effector protein such as a label. Especially prefened are labels which allow the imaging ofthe distribution ofthe antibody in vivo. Such labels can be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within the body of a patient. Moreover, they can be fluorescent labels or other labels which are visualisable on tissue Antibodies as described herein can be produced in cell culture. Recombinant DNA technology can be used to produce the antibodies according to established procedure, in bacterial or preferably mammalian cell culture. The selected cell culture system optionally secretes the antibody product, although antibody products can be isolated from non-secreting cells.
Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, e.g. foetal calf serum, or trace elements and growth sustaining supplements, e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone manow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like. Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts ofthe desired antibodies. Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stiner reactor, or immobilised or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges.
Large quantities ofthe desired antibodies can also be obtained by multiplying mammalian cells in vivo. For this puφose, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody-producing tumours. Optionally, the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection. After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.
The foregoing, and other, techniques are discussed in, for example, Kohler and Milstein, (1975) Nature 256:495-497; US 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor, incoφorated herein by reference. Techniques for the preparation of recombinant antibody molecules is described in the above references and also in, for example, EP 0623679; EP 0368684 and EP 0436597, which are incoφorated herein by reference.
The cell culture supematants are screened for the desired antibodies, preferentially by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
For isolation of the antibodies, the immunoglobulins in the culture supematants or in the ascitic fluid can be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like. If necessary and/or desired, the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinity chromatography with the target antigen, or with Protein-A.
The antibody is preferably provided together with means for detecting the antibody, which can be enzymatic, fluorescent, radioisotopic or other means. The antibody and the detection means can be provided for simultaneous, simultaneous separate or sequential use, in a kit.
The antibodies for use with the invention are assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA, sandwich immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays. Such assays are routine in the art (see, for example, Ausubel et al, eds, 1994, Cunent Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incoφorated by reference herein in its entirety). Exemplary immunoassays are described briefly below.
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2,1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e. g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e. g., 1-4 hours) at 4 °C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4 °C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability ofthe antibody of interest to immunoprecipitate a particular antigen can be assessed by, e. g., western blot analysis.
Western blot analysis generally comprises preparing protein samples, electrophoresis ofthe protein samples in a polyacrylamide gel (e. g., 8%-20% SDS-PAGE depending on the molecular weight ofthe antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e. g., PBS with 3%> BSA or non-fat milk), washing the membrane in washing buffer (e. g., PBS-Tween 20), exposing the membrane to a primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, exposing the membrane to a secondary antibody (which recognises the primary antibody, e. g., an antihuman antibody) conjugated to an enzymatic substrate (e. g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e. g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence ofthe antigen.
ELISAs generally comprise preparing antigen, coating the well of a 96 well microtitre plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e. g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence ofthe antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognises the antibody of interest) conjugated to a detectable compound can be added to the well. Further, instead of coating the well with the antigen, the antibody can be coated to the well. In this case, a second antibody conjugated to a detectable 5. compound can be added following the addition ofthe antigen of interest to the coated well.
It is convenient when running assays to immobilise one of more of the reactants, particularly when the reactant is soluble. In the present case it may be convenient to immobilise any one of more of the candidate modulator, Notch ligand, immune cell activator or immune cell 0 costimulus. Immobilisation approaches include covalent immobilisation, such as using amine coupling, surface thiol coupling, ligand thiol coupling and aldehyde coupling, and high affinity capture which relies on high affinity binding of a ligand to an immobilised capturing molecule. Example of capturing molecules include: streptavidin, anti-mouse Ig antibodies, ligand-specific antibodies, protian A, protein G and Tag-specific capture. In one embodiment, immobilisation 5 is achieved through binding to a support, particularly a particulate support which is preferably in the form of a bead.
For assays involving monitoring or detection of tolerised T-cells for use in clinical applications, the assay will generally involve removal of a sample from a patient prior to the step of detecting 0 a signal resulting from cleavage ofthe intracellular domain.
Therapeutic Uses
A. Immunological uses of the present invention 5
In a prefened embodiment, the matrices, substrates, constructs and particles of the present invention may be used to modify immune responses in the immune system of a mammal, such as a human. Preferably such modulation of the immune system is effected by control of immune cell, preferably T-cell, preferably peripheral T-cell, activity. 0 A detailed description ofthe Notch signalling pathway and conditions affected by it may be found for example 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 abenations to the T cell system and infectious diseases such as those caused by Plasmodium species, Microfilariae, Helminths, Mycobacteria, HIV, Cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, Haemophilus influenza type B, measles, Hepatitis C or Toxicara. Thus particular conditions that may be treated or prevented which are mediated by T cells include multiple schlerosis, rheumatoid arthritis and diabetes. The present invention may also be used in organ transplantation or bone manow 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 hypeneactivity, 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 puφura, 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 puφura, 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 anest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases ofthe gastrointestinal tract, hepatic fibrosis, liver cinhosis 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, AJDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders ofthe CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Bane 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 ofthe CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders ofthe 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 carrier, or inflammation associated with AIDS, 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 manow, organs, lenses, pacemakers, natural or artificial skin tissue.
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 manow transplants or blood transfusions).
A brief overview ofthe most common types of organ and tissue transplants is set out below.
i) 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.
ii) 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 often impossible because of the limited supply of hearts and the urgency of the procedure.
iii) 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.
iv) 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 often carried out simultaneously.
v) 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 burn victims.
vi) Liver Transplantation
Liver transplants are used to treat organ damage caused by viral diseases such as hepititis, 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 hemolysis of recipient red blood cells if there is a mismatch of blood groups. In addition, manifestations of GVHD have occuned in liver transplants even when donor and recipient are blood-group compatible.
Vaccines and cancer vaccines
The particles ofthe present invention may also be used in vaccine compositions such as cancer and pathogen vaccines. Vaccine Compositions
Particles 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 ofthe 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.
Prefened spray devices for intranasal use are devices for which the performance ofthe 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 ofthe 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 ofthe above bile acids, or N, N-bis (3DGluconoamidopropyl) deoxycholamide.
Suitably, an adjuvant formulation ofthe 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 ofthe 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 ofthe 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 ofthe 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 Ban 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; HIV-1, (such as tat, nef, gpl20 or gpl60); human heφes viruses, such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSV1 or HSV2; 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); Bonelia 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 ofthe Human Granulocytic Ehrlichiosis; Rickettsia spp, including R.rickettsii; 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), enterohemonagic E. coli, enteropathogenic E. coli (for example shiga toxinlike toxin); Haemophilus spp., including H. influenzae type B (eg PRP), non-typable H. influenzae, for example OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and fimbrin derived peptides (see for example US 5,843,464); Helicobacter spp, including H. pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa;
Legionella spp, including L. pneumophila ; Leptospira spp., including L. intenogans; Listeria spp., including L. monocytogenes; Moraxella spp, including M catanhalis, also known as Branhamella catanhalis (for example high and low molecular weight adhesins and invasins); Morexella Catanhalis (including outer membrane vesicles thereof, and OMP106 (see for example W097/41731)); Mycobacterium spp., including M. tuberculosis (for example ESAT6, Antigen 85 A, -B or -C), M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Neisseria spp, including N. gononhea and N. meningitidis (for example capsular polysaccharides and conjugates thereof, transferrin-binding proteins, lactoferrin 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. enteritidis; Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Staphylococcus spp., including S. aureus, S. epidermidis; Streptococcus spp, including S. pneumonie (eg capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline-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. pallidum (eg the outer membrane proteins), T. denticola, T. hyodysenteriae; 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. lamblia; Leshmania spp., including L. major;
Plasmodium. faciparum (MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin,
PfEMPl, PD32, 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 Coφ); tuberculosis (BCG) vaccines such as TICE BCG (Organon Teknika Coφ) 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-Imune (Lederle Lab Div, American Cyanamid Co); Pneumococcal 7-valent conjugate vaccines (eg diphtheria CRM197 Protein conjugates such as Prevnar from Lederle Lab Div, American Cyanamid Co); poliovirus vaccines such as Poliovax (Aventis Pasteur, Ltd); poliovirus vaccines such as IPOL (Aventis Pasteur, SA); rabies vaccines such as Imovax (Aventis Pasteur, SA) and RabAvert (Chiron Behring GmbH & Co); rubella virus vaccines such as Meruvax II (Merck & Co, Inc); Typhoid Vi polysaccharide vaccines such as TYPHIM Vi (Aventis Pasteur, SA); Varicella virus vaccines such as Varivax (Merck & Co, Inc) 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 (hCG beta) antigen, carcinoembryonic antigen, EGFRvIII antigen, Globo H antigen, GM2 antigen, GP100 antigen, HER2/neu antigen, KSA 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 ofthe 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).
B. Non-immunological uses of the present invention
Cell fate/cancer indications
It will be appreciated however that the particles of the present invention, as modulators of Notch sigalling, may also be used for altering the fate of a cell, tissue or organ type by altering Notch pathway function in a cell by a partially or fully non-immunological mode of action (eg by modifying general cell fate, differentiation or proliferation), as described, for example in WO 92/07474, WO 96/27610, WO 97/01571, US 5648464, US 5849869 and US 6004924 (Yale University/Imperial Cancer Technology), the texts of which are herein incoφorated by reference.
Thus, the particles ofthe present invention are also useful in methods for altering the fate of any cell, tissue or organ type by altering Notch pathway function in the cell. Thus, for example, the present constructs also have application in the treatment of malignant and pre- neoplastic disorders for example by an antiproliferative, rather than immunological mechanism. For example, in the cancer field the conjugates ofthe present invention are especially useful in relation to adenocarcinomas such as: small cell lung cancer, and cancer ofthe 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, breasy 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 neuoma, medulloblastoma, craniopharyngioma, oligodendroglioma, menangioma, melanoma, neutroblastoma 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, heφes zoster or heφes simplex virus, Lyme disease, tuberculosis or syphilis; degenerative lesions and diseases and demyelinated lesions. The present invention may be used to treat, for example, diabetes (including diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, sarcoidosis, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, central pontine myelinolysis, Parkinson's disease, Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis, cerebral infarction or ischemia, spinal cord infarction or ischemia, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and 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).
The present invention may further be useful in the promotion of tissue regeneration and repair, for example by modification of differentiation processes. The present invention, therefore, may also be used to treat diseases associated with defective tissue repair and regeneration such as, for example, cinhosis ofthe liver, hypertrophic scar formation and psoriasis. The invention may also be useful in the treatment of neutropenia or anemia and in techniques of organ regeneration and tissue engineering and stem cell treatments.
Pharmaceutical Compositions
Preferably the active agents of the present invention are administered in the form of pharmaceutical compositions. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and in addition to one or more active agents 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 carrier, 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 also be provided in such a pharmaceutical composition. Examples of -I l l-
preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
Administration
Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response ofthe particular patient. The dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
In one particularly prefened 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 or stem or tissue cells) in an ex vivo manner. For example, leukocytes such as T cells or APCs may be obtained from a patient or donor in known manner, treated incubated ex vivo in the manner of the present invention, and then administered to a patient.
In general, a therapeutically effective daily dose may for example range from 0.01 to 500 mg/kg, for example 0.01 to 50 mg/kg body weight ofthe subject to be treated, for example 0.1 to 20 mg/kg. The particles ofthe present invention may also be administered by intravenous infusion, at a dose which is likely to range from for example 0.001-10 mg/kg/hr.
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. Preferably the pharmaceutical compositions are in unit dosage form.
The agents ofthe present invention can be administered by any suitable means including, but not limited to, for example, oral, rectal, nasal, topical (including intradermal, transdermal, aerosol, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal) routes of administration. Subcutaneous, intramuscular or intradermal administration is generally prefened. Suitably the active agents are administered in combination with a pharmaceutically acceptable carrier or diluent as described under the heading "Pharmaceutical compositions" above. The pharmaceutically acceptable carrier or diluent may be, for example, sterile isotonic saline solutions, or other isotonic solutions such as phosphate-buffered saline (PBS). Active agents ofthe present invention may suitably be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
In one embodiment, it may be desired to formulate an active agent in an orally active form. Thus, 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. Doses such as tablets or capsules comprising the conjugates may be administered singly or two or more at a time, as appropriate. It is also possible to administer the agents in sustained release formulations.
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 incoφorated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. They can also be incoφorated, for example at a concentration of between 1 and 10%) by weight, into an ointment consisting of a white wax or white soft paraffin base together with such 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, heφes 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 also be adminstered by needleless systems, such as ballistic delivery on particles for delivery to the epidermis or dermis or other sites such as mucosal surfaces.
For parenteral administration, active agents may for example be used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
For buccal or sublingual administration, agents may for example 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 ofthe particular patient. It is to be noted that whilst the above- mentioned dosages are exemplary ofthe 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.
As noted above, 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 ofthe patient.
The term treatment or therapy as used herein should be taken to encompass diagnostic and prophylactic applications , and treatment includes both human and veterinary applications.
The active agents ofthe present invention may also be administered with other active agents such as, for example, immunosuppressants, steroids or anticancer agents. 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 10 to 10 treated cells, preferably from 105 to IO7 cells, more preferably about IO6 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 known in the art. For example, an enriched population of T-cells can be obtained using immunoaffmity 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 manow 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.
Antigens
In one embodiment, the constructs/particles 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 (THl) is characterised by high IL-2 and IFNγ but low IL-4 production, whereas in humoral immunity (TH2) the pattern is low IL-2 and IFNγ but high IL-4, IL-5 and IL-13. Since the secretory pattern is modulated at the level of the secondary lymphoid organ or cells, then pharmacological manipulation of the specific TH cytokine pattern can influence the type and extent of the immune response generated.
The TH1-TH2 balance refers to the relative representation of the two different forms of helper T cells. The two forms have large scale and opposing effects on the immune system. If an immune response favours THl 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, it is prefened to use antigens known to be associated with auto-immune diseases such as myelin basic protein (associated with multiple sclerosis), collagen (associated with rheumatoid arthritis), and insulin (diabetes), or antigens associated with rejection of non-self tissue such as MHC antigens or antigenic determinants thereof. Where primed the APCs and/or T cells of the present invention are to be used in tissue transplantation procedures, antigens may be obtained from the tissue donor. Polynucleotides coding for antigens or antigenic determinants which may be expessed in a subject may also be used.
In a further embodiment, such antigens or antigenic determinants or polynucleotides coding for them may be included in or on the matrix/substrate eg particle.
Autoantigens and Bystander antigens
The term "autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in an autoimmune disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction, such as heatshock proteins (HSP), which although not necessarily specific to a particular tissue are normally shielded from the immune system.
"Bystander suppression" is suppression at the locus of autoimmune attack of cells that contribute to autoimmune destruction; this suppression is mediated by the release of one or more immunosuppressive factors (including Th2-enhancing cytokines and Thl -inhibiting cytokines) from suppressor/regulatory T-cells elicited by a bystander antigen and recruited to the site where cells contributing to autoimmune destruction are found. The result may be antigen-nonspecific but locally restricted downregulation ofthe autoimmune responses responsible for tissue destruction.
"Autoimmune disease" includes spontaneous or induced malfunction ofthe immune system of mammals, including humans, in which the immune system fails to distinguish between foreign immunogenic substances within the mammal and/or autologous substances and, as a result, treats autologous tissues and substances as if they were foreign and mounts an immune response against them.
Autoimmune diseases are characterized by immune responses that are directed against self antigens. These responses are maintained by the persistent activation of self-reactive T lymphocytes. T lymphocytes are specifically activated upon recognition of foreign and/or self antigens as a complex with self Major Histocompatibility Complex (MHC) gene products on the surface of antigen-presenting cells (APC). A detailed discussion of autoimmune diseases, autoantigens and bystander antigens is included in the textbook "The Autoimmune Diseases" Third Edition, 1998, edited by Rose and Mackay, Academic Press, San Diego, California, US (Library of Congress Card Catalog No 98-84368, ISBN 0-12-596923-6), the text of which is hereby incoφorated herein by reference.
A non-limiting list of autoimmune diseases and tissue- or organ-specific confirmed or potential bystander antigens and autoantigens of use in the products ofthe present invention is provided below.
Autoimmune disorders
Autoimmune disorders include organ specific diseases and systemic illnesses.
In more detail, organ-specific autoimmune diseases include, for example, several forms of anemia (aplastic, hemolytic), autoimmune hepatitis, iridocyclitis, scleritis, uveitis, orchitis and idiopathic thrombocytopenic puφura.
Systemic autoimmune diseases include, for example: 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 puφura, Behcet's Syndrome, Takayasu arteritis, Giant cell arteritis, Thrombangiitis obliterans, polymyalgia rheumatica, essential (mixed) cryoglobulinemia, psoriasis vulgaris and psoriatic arthritis, diffuse fasciitis with or without eosinophilia, relapsing panniculitis, relapsing polychondritis, lymphomatoid granulomatosis, erythema nodosum, ankylosing spondylitis, Reiter's syndrome and different forms of inflammatory dermatitis.
Autoantigens Autoantigens may be derived from tissues, proteins etc associated with the disease which give rise to the relevant autoimmune response. For example:
Autoimmune condition Source of autoantigens
Addison's disease adrenal cell antigens; 21-hydroxylase, 17-hydroxylase
Alopecia hair follicle antigens
Autoimmune hepatitis liver cell antigens Autoimmune parotitis parotid gland antigens Autoimmune haemolytic anemia red cell membrane proteins; 95-110 kDa membrane protein
Chronic active hepatitis liver cell antigens
Goodpasture's syndrome renal and lung basement membrane antigens; collagens
Guillain-Bane syndrome nerve cell antigens Hypophysial insufficiency Hypophyseal antigens Biermer's gastritis Parietal cell ofthe stomach; intrinsic Factor
Idiopathic leukopenia granulocyte antigens Idiopathic thrombocytopenia platelet membrane proteins; Glycoprotein Ila/IIIb
Isaac's syndrome voltage-gated potassium channels Lambert-Eaton synaptogamin in voltage-gated myasthenic syndrome (LEMS) calcium channels
Myocardial infraction heart cell antigens Paraneoplastic encephalitis RNA-binding protein (HuD) Pemphigus vulgaris "PeV antigen complex"; desmoglein (DG) (see eg Eur. J. Cell Biol. 55:200 (91))
Primary biliary cinhosis mitochondrial antigens; dihydrolipoamide acetyltransferase; pyruvate dehydrogenase complex 2 (PDC-E2)
Progressive systemic sclerosis DNA topoisomerase; RNA polymerase
Spontaneous infertility Sperm antigens (eg post-acrosomal sperm protein (PASP)) (see eg Biol. Reprod. 43:559 (90))
Uveitis Ocular antigen, S-antigen, inteφhotoreceptor retinoid binding protein
(see eg Exp. Eye Res. 56:463 (93))
Vitiligo melanocyte antigens
It will be appreciated that combinations of such autoimmune antigens and autoimmune antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
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 (T-cell) receptor. Preferably the antigen used in the present invention is an immunogen.
The antigen 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.
The antigen moiety may be, for example, a synthetic MHC-peptide complex i.e. a fragment of the MHC molecule bearing the antigen groove bearing an element of the antigen. Such complexes have been described in Altman et al. (1996) Science 274: 94-96. Some prefened autoantigens for use in the products, methods, uses and constructs etc ofthe present invention include the following:
Goodpasture's autoantigens and bystander antigens
In one embodiment ofthe present invention the autoantigen or bystander antigen may be a Goodpasture's autoantigen or bystander antigen for use to treat Goodpasture's disease/syndrome.
The term "Goodpasture's autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in Goodpasture's disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of Goodpasture's disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "Goodpasture's bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in Goodpasture's disease. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Examples of Goodpasture's autoantigens and Goodpasture's bystander antigens include, but are not limited to collagens in particular, type IV, alpha 3 collagens. An amino acid sequence for a human collagen, type IV, alpha 3 (Goodpasture antigen) is reported as follows (GenBank Accession No NM_001723):
MSARTAPRPQVLLLP LLVLLAAAPAASKGCVCKDKGQCFCDGAKGEKGEKGFPGPPGSPGQKGFTGPEGLPGP QGPKGFPGLPGLTGSKGVRGISGLPGFSGSPGLPGTPGNTGPYGLVGVPGCSGSKGEQGFPGLPGTPGYPGIPG AAGLKGQKGAPAKGEDIELDAKGDPGLPGAPGPQGLPGPPGFPGPVGPPGPPGFFGFPGAMGPRGPKGHMGERV IGHKGERGVKGLTGPPGPPGTVIVTLTGPDNRTDLKGEKGDKGAMGEPGPPGPSGLPGESYGSEKGAPGDPGLQ GKPGKDGVPGFPGSEGVKGNRGFPGLMGEDGIKGQKGDIGPPGFRGPTEYYDTYQEKGDEGTPGPPGPRGARGP QGPSGPPGVPGSPGSSRPGLRGAPGWPGLKGSKGERGRPGKDAMGTPGSPGCAGSPGLPGSPGPPGPPGDIVFR KGPPGDHGLPGYLGSPGIPGVDGPKGEPGLLCTQCPYIPGPPGLPGLPGLHGVKGIPGRQGAAG KGSPGSPGN TGLPGFPGFPGAQGDPGLKGEKGETLQPEGQVGVPGDPGLRGQPGRKGLDGIPGTLGVKGLPGPKGELALSGEK GDQGPPGDPGSPGSPGPAGPAGPPGYGPQGEPGLQGTQGVPGAPGPPGEAGPRGELSVSTPVPGPPGPPGPPGH PGPQGPPGIPGSLGKCGDPGLPGPDGEPGIPGIGFPGPPGPKGDQGFPGTKGSLGCPGKMGEPGLPGKPGLPGA KGEPAVAMPGGPGTPGFPGERGNSGEHGEIGLPGLPGLPGTPGNEGLDGPRGDPGQPGPPGEQGPPGRCIEGPR GAQGLPGLNGLKGQQGRRGKTGPKGDPGIPGLDRSGFPGETGSPGIPGHQGEMGPLGQRGYPGNPGILGPPGED GVIGMMGFPGAIGPPGPPGNPGTPGQRGSPGIPGVKGQRGTPGAKGEQGDKGNPGPSEISHVIGDKGEPGLKGF AGNPGEKGNRGVPGMPGLKGLKGLPGPAGPPGPRGDLGSTGNPGEPGLRGIPGSMGNMGMPGSKGKRGTLGFPG RAGRPGLPGIHGLQGDKGEPGYSEGTRPGPPGPTGDPGLPGDMGKKGEMGQPGPPGHLGPAGPEGAPGSPGSPG LPGKPGPHGDLGFKGIKGLLGPPGIRGPPGLPGFPGSPGPMGIRGDQGRDGIPGPAGEKGETGLLRAPPGPRGN PGAQGAKGDRGAPGFPGLPGRKGAMGDAGPRGPTGIEGFPGPPGLPGAIIPGQTGNRGPPGSRGSPGAPGPPGP PGSHVIGIKGDKGSMGHPGPKGPPGTAGDMGPPGRLGAPGTPGLPGPRGDPGFQGFPGVKGEKGNPGFLGSIGP PGPIGPKGPPGVRGDPGTLKIISLPGSPGPPGTPGEPGMQGEPGPPGPPGNLGPCGPRGKPGKDGKPGTPGPAG EKGNKGSKGEPESLFHQL
(see also Turner et al, Molecular cloning ofthe human Goodpasture antigen demonstrates it to be the alpha 3 chain of type IV collagen, J. Clin. Invest. 89 (2), 592-601 (1992))
Further sequences are provided, for example, under GenBank Accession Nos NM 031366.1, NM_031364.1, NM_031363.1, NM_031362.1 and NM_000091.2 (collagen, type IV, alpha 3 (Goodpasture antigen) (COL4A3)) and NM_130778.1 and NM_000494.2 (collagen, type XVII, alpha 1 (COL17A1)).
Renal autoantigens and bystander antigens
In another embodiment the autoantigen or bystander antigen may be a renal autoantigen or renal bystander antigen, for use to treat autoimmune disease ofthe kidney.
The term "renal autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in autoimmune disease ofthe kidney, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease ofthe kidney when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "renal bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe kidney under autoimmune attack in an autoimmune disease ofthe kidney. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Examples of renal autoantigens and renal bystander antigens include, but are not limited to glomerular basement membrane (GBM) antigens (Goodpasture's antigens as described further above) and tubular basement membrane (TBM) antigens associated with tubulointerstitial nephritis (TIN).
Pemphigus autoantigens and bystander antigens
In an alternative embodiment ofthe present invention the autoantigen or bystander antigen may be a Pemphigus autoantigen or bystander antigen for use to treat Pemphigus.
The term "Pemphigus autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in Pemphigus, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of Pemphigus when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "Pemphigus bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in Pemphigus. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Pemphigus includes, for example, pemphigus vulgaris, pemphigus foliaceus and bullous pemphigoid.
Examples of Pemphigus autoantigens and Pemphigus bystander antigens include, but are not limited to desmoglein 1 and desmoglein 3.
An amino acid sequence for a human desmoglein 1 (DSG1) autoantigen protein is reported as follows (GenBank Accession No AF097935):
MD SFFRVVAVLFIFLVVVEVNSEFRIQVRDYNTKNGTIK HSIRRQKRE IKFAAACREGEDNSKRNPIAKIH SDCAANQQVTYRISGVGIDQPPYGIFVINQKTGEINITSIVDREVTPFFIIYCRALNSMGQDLERPLELRVRVL DINDNPPVFSMATFAGQIEENSNANTLVMILNATDADEPNNLNSKIAFKIIRQEPSDSPMFIINRNTGEIRTMN NFLDREQYGQYALAVRGSDRDGGADGMSAECECNIKILDVNDNIPYMEQSSYTIEIQENTLNSNLLEIRVIDLD EEFSANWMAVIFFISGNEGN FEIEMNERTNVGILKVVKPLDYEAMQSLQLSIGVRNKAEFHHSIMSQYKLBCAS AISVTVLNVIEGPVFRPGSKTYVVTGNMGSNDKVGDFVATDLDTGRPSTTVRYVMGNNPADLLAVDSRTGKLTL KNKVTKEQYNMLGGKYQGTILSIDDNLQRTCTGTININIQSFGNDDRTNTEPNTKITTNTGRQESTSSTNYDTS TTSTDSSQVYSSEPGNGAKDLLSDNVHFGPAGIGLLIMGFLVLGLVPFLMICCDCGGAPRSAAGFEPVPECSDG AIHSWAVEGPQPEPRDITTVIPQIPPDNANIIECIDNSGVYTNEYGGREMQDLGGGERMTGFELTEGVKTSGMP EICQEYSGT RRNSMRECREGGLNMNFMESYFCQKAYAYADEDEGRPSNDCLLIYDIEGVGSPAGSVGCCSFIG ED DDSFLDTLGPKFKKLADISLGKESYPDLDPSWPPQSTEPVCLPQETEPVVSGHPPISPHFGTTTVISESTY PSGPGVLHPKPILDPLGYGNVTVTESYTTSDTLKPSVHVHDNRPASNVVVTERVVGPISGADLHGMLEMPDLRD GSNVIVTERVIAPSSSLPTSLTIHHPRESSNVVVTERVIQPTSGMIGSLSMHPELANAHNVIVTERVVSGAGVT GISGTTGISGGIGSSGLVGTSMGAGSGALSGAGISGGGIGLSSLGGTASIGHMRSSSDHHFNQTIGSASPSTAR SRITKYSTVQYSK
(see also Nilles et al, Structural analysis and expression of human desmoglein: a cadherin- like component of the desmosome, J. Cell. Sci. 99 (Pt 4), 809-821 (1991))
An amino acid sequence for a human bullous pemphigoid antigen 1, 230/240kDa (BPAG1) is reported as follows (GenBank Accession No NM_001723):
MHSSSYSYRSSDSVFSNTTSTRTSLDSNENLLLVHCGPTLINSCISFGSESFDGHR EMLQQIANRVQRDSVIC EDKLILAGNALQSDSKRLESGVQFQNEAEIAGYILECENLLRQHVIDVQILIDGKYYQADQLVQRVAKLRDEIM ALRNECSSVYSKGRILTTEQTK MISGITQSLNSGFAQTLHPSLTSG TQSLTPSLTSSSMTSGLSSGMTSRLT PSVTPAYTPGFPSG VPNFSSGVEPNSLQTLKLMQIRKPLLKSS LDQNLTEEEINMKFVQDLLN VDEMQVQL DRTE GSDLPSVESHLENHKNVHRAIEEFESS KEAKISEIQMTAPLKLTYAEKLHRLESQYAKLLNTSRNQER HLDTLHNFVSRATNELI LNEKEEEEVAYDWSERNTNIARKKDYHAELMRELDQKEENIKSVQEIAEQLLLENH PARLTIEAYRAAMQTQ SWILQLCQCVEQHIKENTAYFEFFNDAKEATDYLRNLKDAIQRKYSCDRSSSIHKLE DLVQESMEEKEELLQYKSTIANLMGKAKTIIQLKPRNSDCPLKTSIPIKAICDYRQIEITIYKDDECVLANNSH RAK KVISPTGNEAMVPSVCFTVPPPNKEAVDLANRIEQQYQNVLTL HESHINMKSVVS HYLINEIDRIRAS NVASIKTMLPGEHQQVLSNLQSRFEDFLEDSQESQVFSGSDITQLEKEVNVCKQYYQELLKSAEREEQEESVYN LYISEVRNIRLRLENCEDRLIRQIRTPLERDDLHESVFRITEQEKLKKELERLKDDLGTITNKCEEFFSQAAAS SSVPTLRSELNVVLQNMNQVYSMSSTYIDK KTVNLVLKNTQAAEALVK YETKLCEEEAVIADKNNIENLIST LKQ RSEVDEKRQVFHALEDELQKAKAISDEMFKTYKERDLDFDWHKEKADQLVER QNVHVQIDNRLRD EGI GKSLKYYRDTYHPLDD IQQVETTQRKIQENQPENSKTLATQLNQQKMLVSEIEMKQSKMDECQKYAEQYSATV KDYELQTMTYRAMVDSQQKSPVKRRRMQSSADLIIQEFMDLRTRYTALVTLMTQYIKFAGDSLKRLEEEEIKRC KETSEHGAYSD LQRQKATVLENSKLTGKISELERMVAELKKQKSRVEEELPKVREAAENELRKQQRNVEDISL QKIRAESEAKQYRRELETIVREKEAAERELERVRQLTIEAEAKRAAVEENLLNFRNQLEENTFTRRTLEDHLKR KDLSLNDLEQQKNKLMEELRRKRDNEEELLKLIKQME DLAFQKQVAEKQLKEKQKIELEARRKITEIQYTCRE NALPVCPITQATSCRAVTGLQQEHDKQKAEE KQQVDELTAANRKAEQDMRELTYELNALQLEKTSSEEKARLL KDKLDETNNTLRCLKLELERKDQAEKGYSQQLRELGRQLNQTTGKAEEAMQEASDLKKIKRNYQLELESLNHEK GKLQREVDRITRAHAVAEKNIQHLNSQIHSFRDEKE ERLQICQRKSDHLKEQFEKSHEQ LQNIKAEKENNDK IQRLNEELEKSNECAEMLKQKVEELTRQNNETKLMMQRIQAESENIVLEKQTIQQRCEALKIQADGFKDQLRST NEHLHKQTKTEQDFQRKIKCLEEDLAKSQNLVSEFKQKCDQQNIIIQNTKKEVRNLNAELNASKEEKRRGEQKV QLQQAQVQELNNRLKKVQDELHLKTIEEQMTHRKMVLFQEESGKFKQSAEEFRKKMEKLMESKVITENDISGIR LDFVSLQQENSRAQENAKLCETNIKELERQLQQYREQMQQGQHMEANHYQKCQKLEDELIAQKREVENLKQKMD QQIKEHEHQLVLLQCEIQKKSTAKDCTFKPDFEMTVKECQHSGE SSRNTGHLHPTPRSPLLR TQEPQPLEEK QHRVVEQIPKEVQFQPPGAPLEKEKSQQCYSEYFSQTSTELQITFDETNPITRLSEIEKIRDQALNNSRPPVR YQDNACEMELVKVLTPLEIAKNKQYDMHTEVTTLKQEKNPVPSAEEWMLEGCRASGGLKKGDFLKKG EPETFQ NFDGDHACSVRDDEFKFQGLRHTVTARQLVEAKLLDMRTIEQLRLGLKTVEEVQKTLNKFLTKATSIAGLYLES TKEKISFASAAERIIIDKMVALAFLEAQAATGFIIDPISGQTYSVEDAVLKGVVDPEFRIRLLEAEKAAVGYSY SSKTLSVFQAMENRMLDRQKGKHILEAQIASGGVIDPVRGIRVPPEIALQQG LNNAILQFLHEPSSNTRVFPN PNNKQALYYSELLRMCVFDVESQCFLFPFGERNISNLNVKKTHRISVVDTKTGSELTVYEAFQRN IEKSIYLE LSGQQYQWKEAMFFESYGHSSHM TDTKTGLHFNINEAIEQGTIDKALVKKYQEGLITLTELADSLLSRLVPKK DLHSPVAGY LTASGERISVLKASRRNLVDRITALRCLEAQVSTGGIIDPLTGKKYRVAEALHRGLVDEGFAQQ LRQCELVITGIGHPITNKMMSVVEAVNANIINKEMGIRCLEFQYLTGGLIEPQVHSRLSIEEALQVGIIDVLIA TKLKDQKSYVRNIICPQTKRKLTYKEALEKADFDFHTGLKLLEVSEPLMTGISS YYSS
(see also, for example Sawamura et al, Bullous pemphigoid antigen (BPAG1): cDNA cloning and mapping ofthe gene to the short arm of human chromosome 6, Genomics 8 (4), 722-726 (1990)) Further sequences are provided, for example, under GenBank Accession Nos NM H5548.1, NM_020388.2 and NM_001723.2 (Bullous pemphigoid antigen 1 (230/240kD) (BPAG1)), M91669.1 (Bullous pemphigoid autoantigen BP180), NM_001942.1 (desmoglein 1 (DSG1)) and NM_001944.1 (desmoglein 3 (pemphigus vulgaris antigen; DSG3))
In one embodiment one or more antigenic determinants may be used in place of a full antigen. For example, some specific class II MHC-associated autoantigen peptide sequences are as follows (see US 5783567):
Peptide Sequence Source
LNSKIAFKIVSQEPA desmoglein 3 (aa 190-204) TPMFLLSRNTGEVRT desmoglein 3 (aa 206-220)
Thyroid Autoantigens and Bystander antigens
In an alternative embodiment ofthe present invention the autoantigen or bystander antigen may be a thyroid autoantigen or bystander antigen for use to treat thyroid autoimmune disease.
The term "thyroid autoimmune disease" as used herein includes any condition in which there is an autoimmune reaction to the thyroid or a component thereof. The best known autoimmune diseases ofthe thyroid include Graves' disease (also known as thyrotoxicosis), Hashimoto's thyroiditis and primary hypothyroidism. Further examples include atrophic autoimmune thyroiditis, primary myxoedema, asymptomatic thyroiditis, postpartal thyroiditis and neonatal hypothyroidism.
Diagnosis is typically based on the detection of autoantibodies in the patient. The three main thyroid autoantigens are the TSH receptor, thyroperoxidase (TPO, also known as microsomal antigen) and thyroglobulin (Tg) (Dawe, K., Hutchings, P., Champion, B., Cooke, A., Roitt, I., "Autoantigens in Thyroid diseases", Springer Semin. Immunopathol. 14, 285-307, 1993). The term "thyroid autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in a thyroid autoimmune disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of a thyroid autoimmune disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ (usually the thyroid gland) under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "thyroid bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe thyroid gland under autoimmune attack. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
It will be appreciated that combinations of thyroid autoimmune/bystander antigens and thyroid autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
Examples of thyroid autoantigens and thyroid bystander antigens include, but are not limited to, the thyroid stimulatory hormone (TSH) receptor (associated in particular with Grave's disease), thyroperoxidase (TPO; associated with Hashimoto's thyroiditis) and thyroglobulin (Tg). For example, an amino acid sequence for a human thyroid stimulatory hormone receptor (TSHR) is reported as follows (GenBank Accession No M32215):
MRPADLLQLVLLLDLPRDLGGMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSN LPNISRIYVSIDVTLQQLESHSFYN SKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVY STDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFG GVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGIL ESLMCNESSMQSLRQRKSVNALNSPLHQEYEENLGDSIVGYKEKSKFQDTHNNAHYYVFFEEQEDEIIGFGQEL KNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMGYKFLRIVV FVSLLALLGNVFVL ILLTSHY KLNVPRF MCNLAFADFCMGMYLLLIASVDLYTHSEYYNHAIDWQTGPGCNTAGFFTVFASE SVYTLTVITLE RWYAITFAMRLDRKIRLRHACAIMVGG VCCFLLALLPLVGISSYAKVSICLPMDTETPLALAYIVFVLTLNIV AFVIVCCCYVKIYITVRNPQYNPGDKDTKIAKRMAVLIFTDFICMAPISFYALSAILNKPLITVSNSKI LVLF YPLNSCANPFLYAIFTKAFQRDVFILLSKFGICKRQAQAYRGQRVPPKNSTDIQVQKVTHEMRQGLHNMEDVYE LIEKSHLTPKKQGQISEEYMQTVL
An amino acid sequence for a human thyroperoxidase (described as the primary autoantigen in human autoimmune thyroiditis (Hashimoto's thyroiditis) is reported as follows (GenBank Accession No M17755 ):
MRALAVLSVTLVMACTEAFFPFISRGKELL GKPEESRVSSVLEESKR VDTAMYATMQRNLKKRGILSGAQLL SFSKLPEPTSGVIARAAEIMETSIQAMKRKVN KTQQSQHPTDALSEDLLSIIANMSGCLPYMLPPKCPNTCLA NKYRPITGACNNRDHPRWGASNTALARWLPPVYEDGFSQPRGWNPGFLYNGFPLPPVREVTRHVIQVSNEVVTD DDRYSDLLMAWGQYIDHDIAFTPQSTSKAAFGGGSDCQMTCENQNPCFPIQLPEEARPAAGTACLPFYRSSAAC GTGDQGALFGNLSTANPRQQMNGLTSFLDASTVYGSSPALERQLRN TSAEGLLRVHGRLRDSGRAYLPFVPPR APAACAPEPGNPGETRGPCFLAGDGRASEVPSLTALHTLW REHNRLAAALKALNAH SADAVYQEARKVVGAL HQIITLRDYIPRILGPEAFQQYVGPYEGYDSTANPTVSNVFSTAAFRFGHATIHPLVRRLDASFQEHPDLPGLW LHQAFFSP TLLRGGGLDPLIRGLLARPAKLQVQDQ MNEELTERLFVLSNSSTLDLASINLQRGRDHG PGYN E REFCGLPRLETPADLSTAIASRSVADKILDLYKHPDNIDVWLGGLAENFLPRARTGPLFACLIGKQMKA RD GDWFWWENSHVFTDAQRRELEKHSLSRVICDNTGLTRVPMDAFQVGKFPEDFESCDSITGMNLEAWRETFPQDD KCGFPESVENGDFVHCEESGRRVLVYSCRHGYELQGREQLTCTQEG DFQPPLCKDVNECADGAHPPCHASARC RNTKGGFQCLCADPYELGDDGRTCVDSGRLPRVTWISMS AALLIGGFAGLTSTVICRWTRTGTKSTLPISETG GGTPELRCGKHQAVGTSPQRAAAQDSEQESAGMEGRDTHRLPRAL
Wegener's autoantigens and bystander antigens
In an alternative embodiment ofthe present invention the autoantigen or bystander antigen may be a Wegener's autoantigen or bystander antigen for use to treat Wegener's disease. The term "Wegener's autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in Wegener's disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of Wegener's disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "Wegener's bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in Wegener's disease. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Examples of Wegener's autoantigens and Wegener's bystander antigens include, but are not limited to myeloblastin/proteinase 3.
An amino acid sequence for a Wegener's autoantigen/myeloblastin proteinase 3 autoantigen is reported as follows (GenBank Accession No M75154):
MAHRPPSPALASVLLALLLSGAARAAEIVGGHEAQPHSRPYMASLQMRGNPGSHFCGGTLIHPSFVLTAPHCLR DIPQRLVNVVLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNDILLIQLSSPANLSASVTSVQLPQQDQPVPH GTQCLAMG GRVGAHDPPAQVLQELNVTVVTFFCRPHNICTFVPRRKAGICFGDSGGPLICDGIIQGIDSFVI GCATRLFPDFFTRVALYVDWIRSTLRRVEAKGRP (see also Labbaye et al, Wegener autoantigen and myeloblastin are encoded by a single mRNA, Proc. Natl. Acad. Sci. U.S.A. 88 (20), 9253-9256 (1991))
Autoimmune anemia autoantigens and bystander antigens
In a further alternative embodiment ofthe present invention the autoantigen or bystander antigen may be an autoimmune anemia autoantigen or bystander antigen fo use to treat autoimmune anemia.
The term "autoimmune anemia" as used herein includes any disease in which red blood cells (RBCs) or a component thereof come under autoimmune attack. The term includes, for example, autoimmune haemolytic anemia, including both "warm autoantibody type" and "cold autoantibody type".
The term "autoimmune anemia autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in autoimmune anemia, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of autoimmune anemia when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "autoimmune anemia bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe red blood cells (RBCs) under autoimmune attack in autoimmune anemia. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Autoimmune anemia includes, in particular, autoimmune hemolytic anemia. Examples of autoimmune hemolytic anemia autoantigens and bystander antigens include, but are not limited to Rhesus (Rh) antigens such as E, e or C, red cell proteins and glycoproteins such as red cell protein band 4.1 and red cell membrane band 3 glycoprotein. Further examples include Wrb, Ena, Ge, A, B and antigens within the Kidd and Kell blood group systems.
Autoimmune thrombocytopenia autoantigens and bystander antigens
In a further alternative embodiment ofthe present invention the autoantigen or bystander antigen may be an autoimmune thrombocytopenia autoantigen or bystander antigen for use to treat autoimmune thrombocytopenia.
The term " autoimmune thrombocytopenia autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in autoimmune thrombocytopenia, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of autoimmune thrombocytopenia when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells. The term "autoimmune thrombocytopenia bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe platelets under autoimmune attack in autoimmune thrombocytopenia. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Autoimmune thrombocytopenia includes, in particular, autoimmune thrombocytopenia puφura. Examples of autoimmune thrombocytopenia puφura autoantigens and bystander antigens include, but are not limited to platelet glycoproteins such as GPIIb/IIIa and/or GPIb/IX.
For example, an amino acid sequence for a human platelet glycoprotein lib (GPIIb) is reported as follows (GenBank Accession No M34480)
MARALCPLQALWLLE VLLLLGACAAPPAWALNLDPVQLTFYAGPNGSQFGFSLDFHKDSHGRVAIVVGAPRTL GPSQEETGGVFLCPWRAEGGQCPSLLFDLRDETRNVGSQTLQTFKARQGLGASVVS SDVIVACAP QH NVLE KTEEAEKTPVGSCFLAQPESGRRAEYSPCRGNTLSRIYVENDFS DKRYCEAGFSSVVTQAGELVLGAPGGYYF LGLLAQAPVADIFSSYRPGILL HVSSQSLSFDSSNPEYFDGY GYSVAVGEFDGDLNTTEYVVGAPT SWTLG AVEILDSYYQRLHRLRAEQMASYFGHSVAVTDVNGDGRHDLLVGAPLYMDSRADRKLAEVGRVYLFLQPRGPHA LGAPSLLLTGTQLYGRFGSAIAPLGDLDRDGYNDIAVAAPYGGPSGRGQVLVFLGQSEGLRSRPSQVLDSPFPT GSAFGFSLRGAVDIDDNGYPDLIVGAYGANQVAVYRAQPVVKASVQLLVQDSLNPAVKSCVLPQTKTPVSCFNI QMCVGATGHNIPQKLS NAELQLDRQKPRQGRRVLLLGSQQAGTTLDLDLGGKHSPICHTTMAFLRDEADFRDK LSPIVLSLNVSLPPTEAGMAPAVVLHGDTHVQEQTRIVLDCGEDDVCVPQLQ TASVTGSPLLVGADNVLELQM DAANEGEGAYEAELAVHLPQGAHYMRALSNVEGFERLICNQKKENETRVVLCELGNPMKKNAQIGIAMLVSVGN LEEAGESVSFQLQIRSKNSQNPNSKIVLLDVPVRAEAQVELRGNSFPASLVVAAEEGEREQNSLDS GPKVEHT YELHNNGPGTVNGLHLSIHLPGQSQPSDLLYILDIQPQGGLQCFPQPPVNPLKVDWGLPIPSPSPIHPAHHKRD RRQIFLPEPEQPSRLQDPVLVSCDSAPCTVVQCDLQEMARGQRAMVTVLAFL PSLYQRPLDQFVLQSHA FN VSSLPYAVPPLSLPRGEAQV TQLLRALEERAIPI WV VGVLGGLLLLTILVLAMWKVGFFKRNRHTLEEDDE EGE
An amino acid sequence for a human platelet glycoprotein Ilia (GPIIIa) is reported as follows (GenBank Accession No M35999)
MRARPRPRPLWVTVLALGALAGVGVGGPNICTTRGVSSCQQCLAVSPMCAWCSDEALPLGSPRCDLKENLLKDN CAPESIEFPVSEARVLEDRPLSDKGSGDSSQVTQVSPQRIA RLRPDDSKNFSIQVRQVEDYPVDIYYLMDLSY SMKDDLWSIQNLGTKLATQMRKLTSNLRIGFGAFVDKPVSPYMYISPPEALENPCYDMKTTCLPMFGYKHVLTL TDQVTRFNEEVKKQSVSRNRDAPEGGFDAIMQATVCDEKIG RNDASHLLVFTTDAKTHIALDGRLAGIVQPND GQCHVGSDNHYSASTTMDYPSLGLMTEKLSQKNINLIFAVTENVVNLYQNYSELIPGTTVGVLSMDSSNVLQLI VDAYGKIRSKVELEVRDLPEELSLSFNATCLNNEVIPGLKSCMGLKIGDTVSFSIEAKVRGCPQEKEKSFTIKP VGFKDSLIVQVTFDCDCACQAQAEPNSHRCNNGNGTFECGVCRCGPGWLGSQCECSEEDYRPSQQDECSPREGQ PVCSQRGECLCGQCVCHSSDFGKITGKYCECDDFSCVRYKGEMCSGHGQCSCGDC CDSD TGYYCNCTTRTDT CMSSNGLLCSGRGKCECGSCVCIQPGSYGDTCEKCPTCPDACTFKKECVECKKFDRGALHDENTCNRYCRDEIE SVKELKDTGKDAVNCTYKNEDDCVVRFQYYEDSSGKSILYVVEEPECPKGPDILVVLLSVMGAILLIGLAAL I KLLITIHDRKEFAKFEEERARAK DTANNPLYKEATSTFTNITYRGT
Autoimmune gastritis autoantigens and bystander antigens
In a further alternative embodiment ofthe present invention the autoantigen or bystander antigen may be an autoimmune gastritis autoantigen or bystander antigen for use to treat autoimmune gastritis.
The term "autoimmune gastritis" as used herein includes any disease in which gastric tissue or a component thereof comes under autoimmune attack.
The term includes, for example, pernicious anemia.
The term "autoimmune gastritis autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in autoimmune gastritis, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of autoimmune gastritis when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells. The term "autoimmune gastritis bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe gastric tissue under autoimmune attack in autoimmune gastritis. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Autoimmune gastritis includes, in particular, pernicious anemia. Examples of autoimmune gastritis autoantigens and bystander antigens include, but are not limited to parietal cell antigens such as gastric H+/K+ ATPase, (lOOkDa alpha subunit and 60-90kDa beta subunit; especially the beta subunit) and intrinsic factor.
For example an amino acid sequence for a human H,K- ATPase beta subunit is reported as follows (GenBank Accession No M75110):
MAALQEKKTCGQRMEEFQRYC NPDTGQMLGRTLSR V ISLYYVAFYVVMTG FALCLYVLMQTVDPYTPDYQ DQLRSPGVTLRPDVYGEKGLEIVYNVSDNRTWADLTQTLHAFLAGYSPAAQEDSINCTSEQYFFQESFRAPNHT KFSCKFTADMLQNCSGLADPNFGFEEGKPCFIIKMNRIVKFLPSNGSAPRVDCAFLDQPRELGQPLQVKYYPPN GTFSLHYFPYYGKKAQPHYSNPLVAAKLLNIPRNAEVAIVCKVMAEHVTFNNPHDPYEGKVEFKLKIEK
(see also GenBank Accession No J05451; human gastric (H+/K+)-ATPase gene and GenBank Accession No M63962; human gastric H,K-ATPase catalytic subunit gene).
Autoimmune hepatitis autoantigens and bystander antigens
In an alternative embodiment ofthe present invention the autoantigen or bystander antigen may be an autoimmune hepatitis autoantigen or bystander antigen for use to treat autoimmune hepatitis.
The term "autoimmune hepatitis" as used herein includes any disease in which the liver or a component ofthe liver comes under autoimmune attack. The term thus includes, for example, primary biliary cinhosis (PBC) and primary sclerosing cholangitis.
The term " autoimmune hepatitis autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in autoimmune hepatitis, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of autoimmune hepatitis when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term " autoimmune hepatitis bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in autoimmune gastritis. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Examples of autoimmune hepatitis autoantigens and bystander antigens include, but are not limited to cytochrome P450s such as cytochrome P450 2D6, cytochrome P450 2C9 and cytochrome P450 1 A2, the asialoglycoprotein receptor (ASGP R) and UDP- glucuronosyltransferases (UGTs).
For example, cDNA encoding human cytochrome P450-2d6 (coding for antigen for AIH Type2a LKM1 antibody) is reported as follows (GenBank Accession No E15820): 1 atggggctag aagcactggt gcccctggcc atgatagtgg ccatcttcct gctcctggtg
61 gacctgatgc accggcgcca acgctgggct gcacgctacc caccaggccc cctgccactg
121 cccgggctgg gcaacctgct gcatgtggac ttccagaaca caccatactg cttcgaccag
181 ttgcggcgcc gacttcggga cgtgttcagc ctgcanctgg cctggacgcc ggtggtcgtg 241 ctcaatgggc tggcggccgt gcgcgaggcg ctggtgaccc acggcgagga caccgccgac
301 cgcccgcctg tgcccatcac ccagatcctg ggcttcgggc cgcgttccca aggggtgttc
361 ctggcgcgct atgggcccgc gtggcgcgag cagaggcgct tctccgtctc caccttgcgc
421 aacttgggcc tgggcaagaa gtcgctggag cagtgggtga ccgaggaggc ngcctgcctt
481 tgtgccgcct tcgccaacca ctccggacgc ccctttcgcc ccaacggtct cttggacaaa 541 gccgtgagca acgtgatcgc ctccctcacc tgcgggcgcc gcttcgagta cgacgaccct
601 cgcttcctca ggctgctgga cctagctcag gagggactga aggaggagtc gggctttctg
661 cgcgaggtgc tgaatgctgt ccccgtcctc ctgcatatcc cngcgctggc tggcaaggtc
721 ctacgcttcc aaaaggcttt cctgacccag ctggatgagc tgctaactga gcacaggatg
781 acctgggacc cagcccagcc cccccgagac ctgactgagg ccttcctggc agagatggag 841 aaggccaagg ggaaccctgc gagcagcttc aatgatgaga acctgcgcat agtggtggct
901 gacctgttct ctgccgggat ggtgaccacc tcgaccacgc tggcctgggg cctcctgctc
961 atgatcctac atccggatgt gcagcgccgt gtccaacagg agatcgacga cgtgataggg
1021 caggtgcggc gaccagagat gggtgaccag gctcacatgc cctacaccac tgccgtgatt
1081 catgaggtgc agcgctttgg ggacatcgtc cccctgggtg tgacccatat gacatcccgt 1141 gacatcgagg tacagggctt cngcatccct aagggaacga cactcatcac caacctgtca
1201 tcggtnctga aggatgaggc cgtctgggag aagcccttcc gcttccaccc cgaacacttc
1261 ctggatgccc agggccactt tgtgaagccg gaggccttcc tgcctttctc agcaggccgc
1321 cgtgcatgcc tcggggagcc cctggcccgc atggagctct tcctcttctt cacctccctg
1381 ctgcagcact tcagcttctc ggtgcccact ggacagcccc ggcccagcca ccatggtgtc 1441 tttgctttcc tggtgagccc atccccctat gagctttgtg ctgtgccccg ctagaatggg
1501 gtacctagtc cccagcctgc tcctagccca gaggctctaa tgtac
An amino acid sequence for a human cytochrome P450-1A2 (CYP1A2) is reported as follows (GenBank Accession No AF182274): MALSQSVPFSATELLLASAIFCLVFWVLKGLRPRVPKGLKSPPEP G PLLGHVLTLGKNPHLALSRMSQRYGD VLQIRIGSTPVLVLSRLDTIRQALVRQGDDFKGRPDLYTSTLITDGQSLTFSTDSGPVWAARRRLAQNALNTFS IASDPASSSSCYLEEHVSKEAMALISRLQELMAGPGHFDPYNQVVVSVANVIGAMCFGQHFPESSDEMLSLVKN THEFVETASSGNPLDFFPILRYLPNPALQRFKAFNQRFL FLQKTVQEHYQDFDKNSVRDITGALFKHSKKGPR ASGNLIPQEKIVNLVNDVFGAGFDTVTTAIS SLMYLVTKPEIQRKIQKELDTVIGRERRPRLSDRPQLPYLEA FILETFRHSSFLPFTIPHSTTRDTTLNGFYIPKKCCVFVNQ QVNHDPEL EDPSEFRPERFLTADGTAINKPL SEKMMLFGMGKRRCIGEVLAK EIFLFLAILLQQLEFSVPPGVKVDLIPIYGLTMKHARCEHVQARLRFSIN
Examples of primary biliary cinhosis (PBC) autoantigens and bystander antigens include, but are not limited to mitochondrial antigens such as pyruvate dehydrogenase (El -alpha decarboxylase, El -beta decarboxylase and E2 acetyltransferase), branched-chain 2-oxo-acid dehydrogenases and 2-oxoglutarate dehydrogenases.
Autoimmune vasculitis autoantigens and bystander antigens In a further alternative embodiment ofthe present invention the autoantigen or bystander antigen may be an autoimmune vasculitis autoantigen or bystander antigen for use to treat autoimmune vasculitis.
The term "autoimmune vasculitis" as used herein includes any disease in which blood vessels or a component thereof come under autoimmune attack and includes, for example, large vessel vasculitis such as giant cell arteritis and Takayasu's disease, medium-sized vessel vasculitis such as polyarteritis nodosa and Kawasaki disease and small vessel vasculitis such as Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis, Henoch Schonlein puφura, essential cryoglobulinaemic vasculitis and cutaneous leukocytoclastic angiitis.
The term "autoimmune vasculitis autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in autoimmune vasculitis, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of autoimmune vasculitis when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "autoimmune vasculitis bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe blood vessel tissue under autoimmune attack in autoimmune vasculitis. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction. Examples of vasculitis autoantigens and bystander antigens include, but are not limited to basement membrane antigens (especially the noncollagenous domain ofthe alpha 3 chain of type IV collagen) and endothelial cell antigens.
Ocular autoantigens and bystander antigens
In a further alternative embodiment ofthe present invention the autoantigen or bystander antigen may be an ocular autoantigen or bystander antigen for use to treat an autoimmune disease of the eye.
The term "autoimmune disease ofthe eye" includes any disease in which the eye or a component thereof comes under autoimmune attack. The term thus includes, for example, cicatricial pemphigoid, uveitis, Mooren's ulcer, Reiter's syndrome, Behcet's syndrome, Vogt- Koyanagi-Harada Syndrome, scleritis, lens-induced uveitis, optic neuritis and giant-cell arteritis.
The term "ocular autoantigen" as used herein includes any substance or a component thereof normally found within the eye of a mammal that, in an autoimmune disease ofthe eye, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of autoimmune disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "ocular bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe eye under autoimmune attack. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Examples of ocular autoantigens and bystander antigens include, but are not limited to retinal antigens such as ocular antigen, S-antigen, inteφhotoreceptor retinoid binding protein (see eg Exp. Eye Res. 56:463 (93)) in uveitis and alpha crystallin in lens-induced uveitis.
An amino acid sequence for a human retinal S-antigen (48 KDa protein) is reported as follows (GenBank Accession No X12453):
MAASGKTSKSEPNHVIFKKISRDKSVTIYLGNRDYIDHVSQVQPVDGVVLVDPDLVKGKKVYVTLTCAFRYGQE DVDVIGLTFRRDLYFSRVQVYPPVGAASTPTKLQESLLKKLGSNTYPFLLTFPDYLPCSVMLQPAPQDSGKSCG VDFEVKAFATDSTDAEEDKIPKKSSVRYLIRSVQHAPLEMGPQPRAEATWQFFMSDKP HLAVSLNREIYFHGE PIPVTVTVTNNTEKTVKKIKACVEQVANVVLYSSDYYVKPVAMEEAQEKVPPNSTLTKTLTLLPLLANNRERRG IALDGKIKHEDTNLASSTIIKEGIDRTVLGILVSYQIKVKLTVSGFLGELTSSEVATEVPFRLMHPQPEDPAKE SIQDANLVFEEFARHNLKDAGEAEEGKRDKNDADE
An amino acid sequence for a human alpha crystallin is reported as follows (GenBank Accession No U05569):
MDVTIQHP FKRTLGPFYPSRLFDQFFGEGLFEYDLLPFLSSTISPYYRQSLFRTVLDSGISEVRSDRDKFVIF LDVKHFSPEDLTVKVQDDFVEIHGKHNERQDDHGYISREFHRRYRLPSNVDQSALSCSLSADGMLTFCGPKIQT GLDATHAERAIPVSREEKPTSAPSS
Adrenal autoantigens and bystander antigens
In a further alternative embodiment ofthe present invention the autoantigen or bystander antigen may be an adrenal autoantigen or bystander antigen for use to treat adrenal autoimmune disease.
The term "adrenal autoimmune disease" as used herein includes any disease in which the adrenal gland or a component thereof comes under autoimmune attack. The term includes, for example, Addison's disease. The term "adrenal autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in adrenal autoimmune disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of adrenal autoimmune disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "adrenal bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe adrenal gland under autoimmune attack in adrenal autoimmune disease. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Examples of adrenal autoantigens and bystander antigens include, but are not limited to adrenal cell antigens such as the adrenocorticotropic hormone receptor (ACTH receptor) and enzymes such as 21-hydroxylase and 17-hydroxylase.
For example, an amino acid sequence for a human steroid 17-alpha-hydroxylase is reported as follows (GenBank Accession No NM_000102):
M ELVALLLLTLAYLF PKRRCPGAKYPKSLLSLPLVGSLPFLPRHGHMHNNFFKLQKKYGPIYSVRMGTKTTV IVGHHQLAKEVLIKKGKDFSGRPQMATLDIASNNRKGIAFADSGAH QLHRRLAMATFALFKDGDQKLEKIICQ EISTLCDMLATHNGQSIDISFPVFVAVTNVISLICFNTSYKNGDPELNVIQNYNEGIIDNLSKDSLVD VPWLK IFPNKTLEKLKSHVKIRNDLLNKILENYKEKFRSDSITNMLDTLMQAKMNSDNGNAGPDQDSELLSDNHILTTI GDIFGAGVETTTSVVK TLAFLLHNPQVKKKLYEEIDQNVGFSRTPTISDRNRLLLLEATIREVLR RPVAPML IPHKANVDSSIGEFAVDKGTEVIINL ALHHNEKE HQPDQFMPERFLNPAGTQLISPSVSYLPFGAGPRSCIG EILARQELFLIMAWLLQRFD EVPDDGQLPSLEGIPKVVFLIDSFKVKIKVRQAWREAQAEGST (see also Krohn et al: Identification by molecular cloning of an autoantigen associated with Addison's disease as steroid 17 alpha-hydroxylase, Lancet 339 (8796), 770-773 (1992))
Cardiovascular autoantigens and bystander antigens
In a further alternative embodiment ofthe present invention the autoantigen or bystander antigen may be a cardiac autoantigen or bystander antigen for use to treat cardiac autoimmune disease.
The term "cardiac autoimmune disease" as used herein includes any disease in which the heart or a component thereof comes under autoimmune attack. The term includes, for example, autoimmune myocarditis, dilated cardiomyopathy, autoimmune rheumatic fever and Chagas' disease.
The term "cardiac autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in cardiac autoimmune disease, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of cardiac autoimmune disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "cardiac bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe heart tissue under autoimmune attack in cardiac autoimmune disease. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Examples of cardiac autoantigens and bystander antigens include, but are not limited to heart muscle cell antigens such as mysosin, laminin, beta-1 adrenergic receptors, adenine nucleotide translocator (ANT) protein and branched-chain ketodehydrogenase (BCKD).
Scleroderma/Polymyositis Autoantigens and Bystander antigens
In an alternative embodiment ofthe present invention the autoantigen or bystander antigen may be a scleroderma or myositis autoantigen or bystander antigen for use to treat scleroderma or myositis.
The term "myositis/scleroderma autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in myositis (particularly in dermatomyositis or polymyositis) or scleroderma, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of myositis (particularly in dermatomyositis or polymyositis) or scleroderma when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "myositis/scleroderma bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in myositis (particularly in dermatomyositis or polymyositis) or scleroderma. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
As described, for example, in US 5862360, scleroderma, or systemic sclerosis, is characterized by deposition of fibrous connective tissue in the skin, and often in many other organ systems. It may be accompanied by vascular lesions, especially in the skin, lungs, and kidneys. The course of this disease is variable, but it is usually slowly progressive. Scleroderma may be limited in scope and compatible with a normal life span. Systemic involvement, however, can be fatal.
Scleroderma may be classified as either diffuse or limited, on the basis ofthe extent of skin and internal organ involvement. The diffuse form is characterized by thickening and fibrosis of skin over the proximal extremities and trunk. The heart, lungs, kidneys, and gastrointestinal tract below the esophagus are often involved. Limited scleroderma is characterized by cutaneous involvement ofthe hands and face. Visceral involvement occurs less commonly. The limited form has a better prognosis than the diffuse form, except when pulmonary hypertension is present.
Antinuclear antibodies are found in over 95 percent of patients with scleroderma. Specific antinuclear antibodies have been shown to be directed to topoisomerase I, centromere proteins, RNA polymerases, or nucleolar components. Different antibodies are associated with particular clinical patterns of scleroderma. For example, antibodies to topoisomerase I (Scl-70) and to RNA polymerases (usually RNA polymerase III) are seen in patients with diffuse scleroderma. Antibodies to nuclear ribonucleoprotein (nRNP) are associated with diffuse and limited scleroderma.
Patients with scleroderma typically show autoreactivity against centrosomes (Tuffanelli, et al., Arch. Dermatol., 119:560-566, 1983). Centrosomes are essential structures that are highly conserved, from plants to mammals, and are important for various cellular processes. Centrosomes play a crucial role in cell division and its regulation. Centrosomes organize the mitotic spindle for separating chromosomes during cell division, thus ensuring genetic fidelity. In most cells, the centrosome includes a pair of centrioles that lie at the center of a dense, partially filamentous matrix, the pericentriolar material (PCM). The microtubule cytoskeleton is anchored to the centrosome or some other form of microtubule organizing center (MTOC), which is thought to serve as a site of microtubule nucleation.
As discussed in US 6160107 the idiopathic inflammatory myopathies polymyositis, dermatomyositis and the related overlap syndromes disorder, such as polymyositis- sclerodeπna overlap, are inflammatory myopathies that are characterized by chronic muscle inflammation and proximal muscle weakness. The muscle inflammation causes muscle tenderness, muscle weakness, and ultimately muscle atrophy and fibrosis (see, for example, Plotz, et al. Annals of Internal Med. I l l : 143-157(1989)). Also associated with the muscle inflammation are elevated serum levels of aldolase, creatine kinase, transaminases, such as alanine aminotransferase and aspartate aminotransferase, and lactic dehydrogenase. Other systems besides muscle can be affected by these conditions, resulting in arthritis, Raynaud's phenomenon, and interstitial lung disease. Clinically, polymyositis and dermatomyositis are distinguished by the presence of a characteristic rash in patients with dermatomyositis. Differences in the myositis of these conditions can be distinguished in some studies of muscle pathology.
Autoantibodies can be detected in about 90%> of patients with polymyositis and dermatomyositis (Reichlin and Arnett, Arthritis and Rheum. 27: 1150-1156 (1984)). Sera from about 60%> of these patients form precipitates with bovine thymus extracts on Ouchterlony immunodiffusion (ID), while sera from other patients stain tissue culture substrates, such as HEp-2 cells, by indirect immunofluorescence (IIF) (see, e.g., Targoff and Reichlin Arthritis and Rheum. 28: 796-803 (1985); Nishikai and Reichlin Arthritis and Rheum. 23: 881-888 (1980); Reichlin, et al., J. Clin. Immunol. 4:40-44 (1984)). There are numerous precipitating autoantibody specificities in myositis patients, but each individual antibody specificity occurs in only a fraction ofthe patients.
Many autoantibodies associated with myositis or myositis-overlap syndromes have been defined, and, in some cases, the antibodies have been identified. These include antibodies that are present in other disorders and also disease-specific antibodies (see, e.g., (Targoff and Reichlin Mt. Sinai J. of Med. 55: 487-493 (1988)). For example, a group of myositis- associated autoantibodies have been identified which are directed at cytoplasmic proteins that are related to tRNA and protein synthesis, particularly aminoacyl-tRNA synthetases. These include anti-Jo-1, which is the most common autoantibody associated with myositis autoimmune disorders (about 20%> of such patients (Nishikai, et al. Arthritis Rheum. 23: 881-888 (1980)) and which is directed against histidyl-tRNA synthetase; anti-PL-7, which is directed against threonyl-tRNA synthetase; and anti-PL12, which is directed against alanyl- tRNA synthetase. Anti-Ul RNP, which is frequently found in patients with SLE, may also be found in mixed connective tissue disease, overlap syndromes involving myositis, or in some cases of myositis alone. This antibody reacts with proteins that are uniquely present on the UI small nuclear ribonucleoprotein, which is one ofthe nuclear RNPs that are involved in splicing mRNA. Autoantibodies such as anti-Sm, anti-Ro/SSA, and anti-La/SSB, that are usually associated with other conditions, are sometimes found in patients with overlap syndromes. Anti-Ku has been found in myositis-scleroderma overlap syndrome and in SLE. The Ku antigen is a DNA binding protein complex with two polypeptide components, both of which have been cloned.
Anti Jo-1 and other anti-synthetases are disease specific. Other myositis-associated antibodies are anti-PM-Scl, which is present in about 5-10% of myositis patients, many of whom have polymyositis-scleroderma overlap, and anti-Mi-2, which is present in about 8%> of myositis patients, almost exclusively in dermatomyositis. Mi-2 is found in high titer in about 20%) of all dermatomyositis patients and in low titer in less than 5%> of polymyositis patients (see, e.g., Targoff and Reichlin, Mt. Sinai J. of Med. 55: 487-493 (1988)).
Anti -Mi was first described by Reichlin and Mattioli, Clin. Immunol, and Immunopathol. 5: 12-20 (1976)). A complement-fixation reaction was used to detect it and, in that study, patients with dermatomyositis, polymyositis and polymyositis overlap syndromes had positive reactions. The prototype or reference serum, from patient Mi, forms two precipitin lines on immunodiffusion (ID) with calf thymus antigens, Mi-1 and Mi-2. Mi-1, which has been purified from bovine thymus nuclear extracts (Nishikai, et al. Mol. Immunol. 17: 1129- 141 (1980)) is rarely found in other sera and is not myositis specific (Targoff, et al., Clin. Exp. Immunol. 53: 76-82 (1983)).
Anti-Mi-2 was found to be a myositis-specific autoantibody by Targoff, et al. Arthritis and Rheum. 28: 796-803 (1985). Furthermore, all patients with the antibody have the dermatomyositis rash.
Bovine thymus Mi-2 antigen was originally found to be a nuclear protein that separates in SDS polyacrylamide (SDS-PAGE) gels into two bands with apparent molecular weights of 53 kilodaltons (hereinafter kDa) and 61 KDa, respectively. Recently, additional higher molecular weight bands have been found. The bovine thymus antigenic activity is destroyed by SDS-PAGE and is trypsin sensitive, but not RNAse sensitive (Targroff et al. Arthritis and Rheum. 28: 796-803 (1985)).
Anti-PM-1 was first identified as an antibody found in 61%> of dermatomyositis/polymyositis patients, including patients; with polymyositis-scleroderma overlap (Wolfe, et al. J. Clin. Invest. 59: 176-178 (1977)). PM-1 was subsequently shown to be more than one antibody. The unique specificity component of PM-1 was later named PM-Scl (Reichlin, et al. J. Clin. Immunol. 4: 40-44 (1984)). Anti-PM-Scl is found in the sera of about 5-10% of myositis patients, but is most commonly associated with polymyositis-scleroderma overlap syndrome. It also occurs in patients with polymyositis or dermatomyositis alone or in patients with scleroderma without myositis.
Anti-PM-Scl antibody immunoprecipitates a complex from HeLa cell extracts of at least eleven polypeptides that have molecular weights ranging from about 20 to 110 kDa (see, Reimer, et al., J. Immunol. 137:3802-3808 (1986). The antigen is trypsin-sensitive, occurs in nucleoli (see, e.g., Targoff and Reichlin Arthritis Rheum. 28: 226-230 (1985)) and is believed to be a preribosomal particle.
In an abstract, Bluthner, et al., First Int. Workshop on the Mol. and Cell Biology of Autoantibodies and Autoimmunity in Heidelberg (Springer-Veriag July 27-29, 1989) report that sera from patients suffering from polymyositis/scleroderma-overlap syndrome (PM/Scl) recognize two major nucleolar proteins of 95 and 75 kDa molecular weight in Western blots of a Hela cell extract. They also report that cDNA that encodes a 20 kDa protein reactive with autoantibodies eluting from the 95 kDa PM-Scl HeLa antigen subunit has been cloned from a HeLa cDNA library. The sequence ofthe cloned DNA has not as yet been reported.
It will be appreciated that combinations of myositis/scleroderma autoimmune/bystander antigens and myositis/scleroderma autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
Examples of myositis/scleroderma autoantigens and myositis/scleroderma bystander antigens include, but are not limited to, Jo-1 (his-tRNA synthetase), PM-Scl, Mi-2, Ku, PL-7 (thr- tRNA synthetase), PL- 12 (ala-tRNA-synthetase), SRP (signal recognition particle), Anti- nRNP (UI small nuclear RNP), Ro/SS-A, and La SS-B.
For example, an amino acid sequence for a human 100 kD Pm-Scl autoantigen protein (PM/Scl-lOOa) is reported as follows (GenBank Accession No L01457):
MAPPSTREPRVLSATSATKSDGEMVLPGFPDADSFVKFALGSVVAVTKASGGLPQFGDEYDFYRSFPGFQAFCE TQGDRLLQCMSRVMQYHGCRSNIKDRSKVTELEDKFDLLVDANDVILERVGILLDEASGVNKNQQPVLPAGLQV PKTVVSS NRKAAEYGKKAKSETFRLLHAKNIIRPQLKFREKIDNSNTPFLPKIFIKPNAQKPLPQALSKERRE RPQDRPEDLDVPPA ADFIHQQRTQQVEQDMFAHPYQYELNHFTPADAVLQKPQPQLYRPIEETPCHFISSLDE LVELNEKL NCQEFAVDLEHHSYRSFLGLTCLMQISTRTEDFIIDTLELRSDMYILNESLTDPAIVKVFHGADS DIE LQKDFGLYVVNMFDTHQAARLLNLGRHSLDHLLKLYCNVDSNKQYQLAD RIRPLPEEMLSYARDDTHYL LYIYDKMRLEMWERGNGQPVQLQVVWQRSRDICLKKFIKPIFTDESYLELYRKQKKHLNTQQLTAFQLLFA RD KTARREDESYGYVLPNHMMLKIAEELPKEPQGIIACCNPVPPLVRQQINEMHLLIQQAREMPLLKSEVAAGVKK SGPLPSAERLENVLFGPHDCSHAPPDGYPIIPTSGSVPVQKQASLFPDEKEDNLLGTTCLIATAVITLFNEPSA EDSKKGPLTVAQKKAQNIMESFENPFRMISNRWKLAQVQVQKDSKEAVKKKAAEQTAAREQAKEACKAAAEQAI SVRQQVVLENAAKKRERATSDPRTTEQKQEKKRLKISKKPKDPEPPEKEFTPYDYSQSDFKAFAGNSKSKVSSQ FDPNKQTPSGKKCIAAKKIKQSVGNKSMSFPTGKSDRGFRYN PQR
(see also Gee et al, Cloning of a complementary DNA coding for the 100-kD antigenic protein ofthe PM-Scl autoantigen, J. Clin. Invest. 90 (2), 559-570 (1992))
An amino acid sequence for a human 100 kD Pm-Scl autoantigen protein (PM/Scl-lOOb) is reported as follows (GenBank Accession No X66113):
MAPPSTREPRVLSATSATKSDGEMVLPGFPDADSFVKFALGSVVAVTKASGGLPQFGDEYDFYRSFPGFQAFCE TQGDRLLQCMSRVMQYHGCRSNIKDRSKVTELEDKFDLLVDANDVILERVGILLDEASGVNKNQQPVLPAGLQV PKTVVSS NRKAAEYGKKAKSETFR LHAKNIIRPQLKFREKIDNSNTPFLPKIFIKPNAQKPLPQALSKERRE RPQDRPEDLDVPPALADFIHQQRTQQVEQDMFAHPYQYELNHFTPADAVLQKPQPQLYRPIEETPCHFISSLDE LVELNEKLLNCQEFAVDLEHHSYRSFLGLTCLMQISTRTEDFIIDTLELRSDMYILNESLTDPAIVKVFHGADS DIE LQKDFGLYVVNMFDTHQAARLLNLGRHSLDHLLKLYCNVDSNKQYQLAD RIRPLPEEMLSYARDDTHYL LYIYDKMRLEMWERGNGQPVQLQVVWQRSRDICLKKFIKPIFTDESYLELYRKQKKHLNTQQLTAFQ LFAWRD KTARREDESYGYVLPNHMMLKIAEELPKEPQGIIACCNPVPPLVRQQINEMHLLIQQAREMPLLKSEVAAGVKK SGPLPSAERLENVLFGPHDCSHAPPDGYPIIPTSGSVPVQKQASLFPDEKEDNLLGTTCLIATAVITLFNEPSA EDSKKGPLTVAQKKAQNIMESFENPFRMFLPSLGHRAPVSQAAKFDPSTKIYEISNR KLAQVQVQKDSKEAVK KKAAEQTAAREQAKEACKAAAEQAISVRQQVVLENAAKKRERATSDPRTTEQKQEKKRLKISKKPKDPEPPEKE FTPYDYSQSDFKAFAGNSKSKVSSQFDPNKQTPSGKKCIAAKKIKQSVGNKSMSFPTGKSDRGFRYNWPQR
(see also Bluthner and Bautz, Cloning and characterization ofthe cDNA coding for a polymyositis-scleroderma overlap syndrome-related nucleolar 100-kD protein, J. Exp. Med. 176 (4), 973-980 (1992))
An amino acid sequence for a human75 kD Pm-Scl autoantigen protein (PM/Scl-75a) is reported as follows (GenBank Accession No M58460):
MAAPAFEPGRQSDLLVKLNRLMERCLRNSKCIDTESLCVVAGEKV QIRVDLHLLNHDGNIIDAASIAAIVALC HFRRPDVSVQGDEVTLYTPEERDPVPLSIHHMPICVSFAFFQQGTYLLVDPNEREERVMDGLLVIAMNKHREIC TIQSSGGIMLLKDQVLRCSKIAGVKVAEITELILKALENDQKVRKEGGKFGFAESIANQRITAFKMEKAPIDTS DVEEKAEEIIAEAEPPSEVVSTPVLWTPGTAQIGEGVENS GDLEDSEKEDDEGGGDQAII DGIKMDTGVEVS DIGSQDAPIILSDSEEEEMIILEPDKNPKKIRTQTTSAKQEKAPSKKPVKRRKKKRAAN
(see also Alderuccio et al, Molecular characterization of an autoantigen of PM-Scl in the polymyositis/scleroderma overlap syndrome: a unique and complete human cDNA encoding an apparent 75-kD acidic protein ofthe nucleolar complex, J. Exp. Med. 173 (4), 941-952 (1991))
An amino acid sequence for a human 75 kD Pm-Scl autoantigen protein (PM/Scl-75b) is reported as follows (GenBank Accession No U09215):
MAAPAFEPGRQSDLLVKLNRLMERCLRNSKCIDTESLCVVAGEKV QIRVDLHLLNHDGNIIDAASIAAIVALC HFRRPDVSVQGDEVTLYTPEERDPVPLSIHHMPICVSFAFFQQGTYLLVDPNEREERVMDGLLVIAMNKHREIC TIQSSGGIML KDQVLRCSKIAGVKVAEITELILKALENDQKVRKEGGKFGFAESIANQRITAFKMEKAPIDTS DVEEKAEEIIAEAEPPSEVVSTPVLWTPGTAQIGEGVENS GDLEDSEKEDDEGGGDQAIILDGIKMDTGVEVS DIGSQELGFHHVGQTGLEFLTSDAPIILSDSEEEEMIILEPDKNPKKIRTQTTSAKQEKAPSKKPVKRRKKKRA AN
An amino acid sequence for a Jo-1 (histidyl-tRNA synthetase) autoantigen protein is reported as follows (GenBank Accession No Zl 1518): MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKLKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVRE KVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIK RYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFA ICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQAL EGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMF DPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKK LEERLKLVSEL DAGIKAE LYKK NPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPLCIC
(see also Raben et al, Human histidyl-tRNA synthetase: recognition of amino acid signature regions in class 2a aminoacyl-tRNA synthetases, Nucleic Acids Res. 20 (5), 1075-1081 (1992))
An amino acid sequence for a PL-7 (threonyl-tRNA synthetase) autoantigen protein is reported as follows (GenBank Accession No M63180):
MGGEEKPIGAGEEKQKEGGKKKNKEGSGDGGRAELNPWPEYIYTRLEMYNILKAEHDSILAEKAEKDSKPIKVT LPDGKQVDAES KTTPYQIACGISQGLADNTVIAKVNNVV DLDRPLEEDCTLE LKFEDEEAQAVY HSSAHI MGEGMERVYGGCLCYGPPIENGFYYDMYLEEGGVSSNDFSSLEALCKKIIKEKQAFERLEVKKETLLAMFKYNK FKCRILNEKVNTPTTTVYRCGPLIDLCRGPHVRHTGKIKALKIHKNSSTY EGKADMETLQRIYGISFPDPKML KE EKFQEEAKNRDHRKIGRDQELYFFHELSPGSCFFLPKGVYIYNALIEFIRSEYRKRGFQEVVTPNIFNSRL MTSGH QHYSENMFSFEVEKELFALKPMNCPGHSLMFDHRPRS RELPLRLADFGGLHRNELSGALTGLTRVR RFQQDDAHIFCAMEQIEDEIKGCLDFLRTVYSVFGFSFKLNLSTRPEKFLGDIEV DQAEKQLENSLNEFGEK ELNSGDGAFYGPKIDIQIKDAIGRYHQCATIQLDFQLPIRFNLTYVSHDGEDKKRPVIVHRAILGSVERMIAIL TENYGGKLAPFWLSPRQVMVVPVGPTCDEYAQNVRQQFHDAKFMADIDLDPGCTLNKKIRNAQLAQYNFILVVG EKEKITGTVNIRTRDNKVHGERTISETIERLQQLKEFRSKQAEEEF
(See also Cruzen et al, Nucleotide and deduced amino acid sequence of human threonyl- tRNA synthetase reveals extensive homology to the Escherichia coli and yeast enzymes, J. Biol. Chem. 266 (15), 9919-9923 (1991))
An amino acid sequence for a PL- 12 (alanyl-tRNA synthetase) autoantigen protein is reported as follows (GenBank Accession No D32050):
MDSTLTASEIRQRFIDFFKRNEHTYVHSSATIPLDDPTL FANAGMNQFKPIFLNTIDPSHPMAK SRAANTQK CIRAGGKQNDLDDVGKDVYHHTFFEMLGS SFGDYFKE ACKMALELLTQEFGIPIERLYVTYFGGDEAAG EA DLECKQIWQNLGLDDTKILPGNMKDNFWEMGDTGPCGPCSEIHYDRIGGRDAAHLVNQDDPNVLEIWN VFIQY NREADGILKPLPKKSIDTGMGLERLVSVLQNKMSNYDTDLFVPYFEAIQKGTGARPYTGKVGAEDADGIDMAYR VLADHARTITVALADGGRPDNTGRGYVLRRILRRAVRYAHEKLNASRGFFATLVDVVVQSLGDAFPELKKDPDM VKDIINEEEVQFLKTLSRGRRILDRKIQSLGDSKTIPGDTA LLYDTYGFPVD TGLIAEEKGLVVDMDGFEEE RKLAQLKSQGKGAGGEDLIMLDIYAIEELRARGLEVTDDSPKYNYHLDSSGSYVFENTVATVMALRREKMFVEE VSTGQECGVVLDKTCFYAEQGGQIYDEGYLVKVDDSSEDKTEFTVKNAQVRGGYVLHIGTIYGDLKVGDQV LF IDEPRRRPIMSNHTATHILNFALRSVLGEADQKGSLVAPDRLRFDFTAKGAMSTQQIKKAEEIANEMIEAAKAV YTQDCPLAAAKAIQGLRAVFDETYPDPVRVVSIGVPVSELLDDPSGPAGSLTSVEFCGGTHLRNSSHAGAFVIV TEEAIAKGIRRIVAVTGAEAQKALRKAESLKKCLSVMEAKVKAQTAPNKDVQREIADLGEALATAVIPQ QKDE LRETLKSLKKVMDDLDRASKADVQKRVLEKTKQFIDSNPNQP VI EMESGASAKALNEALKLFKMHSPQTSAM LFTVDNEAGKITCLCQVPQNAANRGLKASEWVQQVSGLMDGKGGGKDVSAQATGKNVGCLQEALQLATSFAQLR LGDVKN
An amino acid sequence for an EJ (glycyl-tRNA synthetase) autoantigen protein is reported as follows (GenBank Accession No U09587):
MDGAGAEEVLAPLRLAVRQQGDLVRKLKEDKAPQVDVDKAVAELKARKRVLEAKELALQPKDDIVDRAKMEDTL KRRFFYDQAFAIYGGVSGLYDFGPVGCALKNNIIQT RQHFIQEEQILEIDCTMLTPEPVLKTSGHVDKFADFM VKDVKNGECFRADHLLKAHLQKLMSDKKCSVEKKSEMESVLAQLDNYGQQELADLFVNYNVKSPITGNDLSPPV SFNLMFKTFIGPGGNMPGYLRPETAQGIFLNFKRLLEFNQGKLPFAAAQIGNSFRNEISPRSGLIRVREFTMAE IEHFVDPSEKDHPKFQNVADLHLYLYSAKAQVSGQSARKMRLGDAVEQGVINNTVLGYFIGRIYLYLTKVGISP DKLRFRQHMENEMAHYACDCWDAESKTSYGWIEIVGCADRSCYDLSCHARATKVP VAEKPLKEPKTVNVVQFE PSKGAIGKAYKKDAKLVMEYLAICDECYITEMEMLLNEKGEFTIETEGKTFQLTKDMINVKRFQKTLYVEEVVP NVIEPSFGLGRIMYTVFEHTFHVREGDEQRTFFSFPAVVAPFKCSVLP SQNQEFMPFVKELSEALTRHGVSHK VDDSSGSIGRRYARTDEIGVAFGVTIDFDTVNKTPHTATLRDRDSMRQIRAEISELPSIVQDLANGNITWADVE ARYPLFEGQETGKKETIEE
Further sequences are provided, for example, under GenBank Accession Nos AF241268.1, AF353396.1 (scleroderma-associated autoantigen); NM_005033.1 (polymyositis/scleroderma autoantigen 1 (75kDa) (PMSCL1)); XM_001527.4, NM_002685.1 (polymyositis/scleroderma autoantigen 2 (lOOkDa) (PMSCL2)).
Nervous system Autoantigens and Bystander antigens
In an alternative embodiment ofthe present invention the autoantigen or bystander antigen may be a nervous system autoantigen or bystander antigen for use to treat an autoimmune disease ofthe nervous system.
The term "autoimmune disease ofthe nervous system" includes any disease in which nervous tissue or a component thereof comes under autoimmune attack. The term includes, for example central nervous system diseases having an autoimmune etiology such as multiple sclerosis (MS), perivenous encephalomyelitis, autoimmune myelopathies, paraneoplastic cerebellar degeneration, paraneoplastic limbic (cortical) degeneration, stiff man syndrome, choreas (such as Sydenham's chorea), stroke, focal epilepsy and migraine; and peripheral nervous system diseases having an autoimmune etiology such as Guillain-Bane syndrome, Miller Fisher syndrome, chronic inflammatory demyelinating neuropathy, multifocal motor neuropathy with conduction block, demyelinating neuropathy associated with anti-myelin-associated glycoprotein antibodies, paraneoplastyic sensory neuropathy, POEMS, dorsal root ganglion neuronitis, acute panautonomic neuropathy and brachial neutritis.
The term "nervous system autoantigen" as used herein includes any nervous system substance or a component thereof normally found within a mammal that, in an autoimmune disease ofthe nervous system, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease ofthe nervous system when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "nervous system bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in an autoimmune disease ofthe nervous system. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
Preferably the nervous system autoantigen or nervous system bystander antigen is an MS autoantigen or MS bystander antigen. The term "MS autoantigen" as used herein includes any nervous system substance or a component thereof normally found within a mammal that, in multiple sclerosis (MS), becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of MS when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "MS bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component of nervous tissue under autoimmune attack in MS. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
It will be appreciated that combinations of nervous system autoimmune/bystander antigens and nervous system autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
Examples of nervous system autoantigens and nervous system bystander antigens include, but are not limited to, myelin basic protein (MBP), DM20,central nervous system white matter; proteolipid protein (PLP); myelin oligodendrocyte- associated protein (MOG), myelin associated glycoprotein (MAG), alpha B-crystallin (see eg J. Chromatog. Biomed. Appl. 526:535 (90)) The protein components of myelin proteins, including myelin basic protein (MBP) I proteolipid protein (PLP), myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG), are of particular interest. The suppression of T cell responsiveness to these antigens may be used to prevent or treat demyelinating diseases.
Proteolipid is a major constituent of myelin, and is known to be involved in demyelinating diseases (see, for example Greer et al. (1992) J. Immunol. 149: 783-788 and Nicholson (1997) Proc. Natl. Acad. Sci. USA 94: 9279-9284).
The integral membrane protein PLP is a dominant autoantigen of myelin.
Determinants of PLP antigenicity have been identified in several mouse strains, and includes residues 139-151 (Tuohy et al. (1989) J. Immunol. 142: 1523-1527), residues 103-116 (Tuohy et al. (1988) J. Immunol. 141: 1126-1130), residues 215-232 (Endoh et al. (1990) Int. Arch. Allerqv Appl. Immunol. 92: 433-438), residues 43-64 (Whitham et al (1991) J. Immunol. 147: 3803-3808) and residues 178-191 (Greer, et al. (1992) J. Immunol. 149: 783- 788). Immunization with native PLP or with synthetic peptides conesponding to PLP epitopes induces experimental allergic encephalomyelitis (EAE). Analogues of PLP peptides generated by amino acid substitution can prevent EAE induction and progression (Kuchroo et al. (1994) J. Immunol. 153: 3326-3336, Nicholson et al. (1997) Proc. Natal. Acad. Sci. USA 94:9279-9284).
An amino acid sequence for a human proteolipid protein is reported as follows (GenBank Accession No M27110): MGLLECCARCLVGAPFASLVATGLCFFGVALFCGCGHEALTGTEKLIETYFSKNYQDYEYLINVIHAFQYVIYG TASFFFLYGALLLAEGFYTTGAVRQIFGDYKTTICGKGLSATVTGGQKGRGSRGQHQAHSLERVCTCLGK LGH PDKFVGITYALTVVWLLVFACSAVPVYIYFNTWTTCQSIAFPSKTSASIGSLCADARMYGVLP NAFPGKVCGS NLLSICKTAEFQMTFHLFIAAFVGAAATLVSLLTFMIAATYNFAVLKLMGRGTKF
MBP is an extrinsic myelin protein that has been studied extensively. At least 26 MBP epitopes have been reported (Meinl et al (1993) J. Clin. Invest. 92: 2633-2643). Of particular interest are residues 1-11, 59-76 and 87-99. Analogues of MBP peptides generated by truncation have been shown to reverse EAE (Karin et al (1998) J. Immunol. 160: 5188- 5194). DNA encoding polypeptide fragments may comprise coding sequences for immunogenic epitopes, e. g. myelin basic protein p84-102, more particularly myelin basic protein p87-99, VHFFKNIVTPRTP (ρ87-99), or the truncated 7-mer peptide FKNIVTP. The sequences of myelin basic protein exon 2, including the immunodominant epitope bordered by amino acids 59-85, are also of interest. For examples, see Sakai et al. (1988) J Neuroimmunol 19: 21-32; Baxevanis et al (1989) J Neuroimmunol 22: 23-30; Ota et al (1990) Nature 346: 183-187; Martin et al (1992) J Immunol. 148: 1350-1366, Valli et al (1993) J Clin In 91 : 616. The immunodominant MBP (84102) peptide has been found to bind with high affinity to DRB1*1501 and DRB5*0101 molecules ofthe disease-associated DR2 haplotype. Overlapping but distinct peptide segments were important for binding to these molecules; hydrophobic residues (Vall89 and Phe92) in the MBP (88-95) segment for peptide binding to DRB1*1501 molecules; hydrophobic and charged residues (Phe92, Lys93) in the MBP (89-101/102) sequence contributed to DRB5*0101 binding.
An amino acid sequence for a human myelin basic protein (MBP) is reported as follows (GenBank Accession No Ml 3577):
MASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKDSHHPARTAHYGSLP QKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFS GAEGQRPGFGYGGRASDYKSAHKGFKGVDAQ GTLSKIFKLGGRDSRSGSPMARR
The transmembrane glycoprotein MOG is a minor component of myelin that has been shown to induce EAE. Immunodominant MOG epitopes that have been identified in several mouse strains include residues 1-22,35-55,64-96 (deRosbo et al. (1998) J. Autoimmunity 11 : 287- 299, deRosbo ef al. (1995) Eur J Immunol. 25: 985-993) and 41-60 (Leadbetter et al (1998) J Immunol 161 : 504-512).
An amino acid sequence for a human myelin/oligodendrocyte glycoprotein (MOG) protein (25.1kD) is reported as follows (GenBank Accession No U64564):
MASLSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVG YRPPFS RVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEGGFTCFFRDHSYQEEAAMELKVEDPF YWVSPGVLVLLAVLPVLLLQITVGLVFLCLQYR RGKLRAEIENLHRTFDPHFLRVPCWKITLFVIVPVLGPLV ALIICYNWLHRRLAGQFLEELRNPF
An amino acid sequence for a human myelin-associated glycoprotein (MAG) is reported as follows (GenBank Accession No M29273): MIF TALPLFWIMISASRGGH GA MPSSISAFEGTCVSIPCRFDFPDELRPAVVHGVWYFNSPYPKNYPPVVF KSRTQVVHESFQGRSRLLGDLGLRNCTLLLSNVSPELGGKYYFRGDLGGYNQYTFSEHSVLDIVNTPNIVVPPE VVAGTEVEVSCMVPDNCPELRPELS LGHEGLGEPAVLGRLREDEGT VQVSL HFVPTREANGHRLGCQASFP NTTLQFEGYASMDVKYPPVIVEMNSSVEAIEGSHVSLLCGADSNPPPLLTWMRDGTVLREAVAESLL ELEEVT PAEDGVYACLAENAYGQDNRTVGLSVMYAPWKPTVNGTMVAVEGETVSILCSTQSNPDPILTIFKEKQILSTVI YESELQLELPAVSPEDDGEY CVAENQYGQRATAFNLSVEFAPVLLLESHCAAARDTVQCLCVVKSNPEPSVAF ELPSRNVTVNESEREFVYSERSGLVLTSILTLRGQAQAPPRVICTARNLYGAKSLELPFQGAHRLM AKIGPVG AVVAFAILIAIVCYITQTRRKKNVTESPSFSAGDNPPVLFSSDFRISGAPEKYESERRLGSERRLLGLRGEPPE LDLSYSHSDLGKRPTKDSYTLTEE AEYAEIRVK
In one embodiment one or more antigenic determinants may be used in place of a full antigen. For example, some specific class II MHC-associated autoantigen peptide sequences are as follows (see US 5783567):
Peptide Sequence Source
GRTQDENPVVHFFKNIVTPRTPP MBP (aa 80-102)
AVYVYIYFNTWTTCQFIAFPFK PLP (aa 170-191)
SQRHGSKYLATASTMDHARHG MBP (aa 7-27) RDTGILDSIGRFFGGDRGAP MBP (aa 33-52)
QKSHGRTQDENPVVHFFKNI MBP (aa 74-93)
DENPVVHFFKNIVT MBP (aa 84-97)
ENPVVHFFKNIVTPR MBP (aa 85-99)
HFFKNIVTPRTPP MBP (aa 90-102) KGFKGVDAQGTLSK MBP (aa 139-152)
VDAQGTLSKIFKLGGRDSRS MBP (aa 144-163)
Autoimmune Arthritis Autoantigens and Bystander antigens
In an alternative embodiment ofthe present invention the autoantigen or bystander antigen may be autoimmune arthritis autoantigen or bystander antigen for use to treat autoimmune arthritis.
The term "autoimmune arthritis autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in autoimmune arthritis
(especially rheumatoid arthritis (RA)), becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of autoimmune arthritis when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "autoimmune arthritis bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in autoimmune arthritis, especially rheumatoid arthritis (RA). The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
The term "autoimmune arthritis" includes rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, spondylo arthritis, relapsing polychondritis and other connective tissue diseases having an autoimmune disease component.
It will be appreciated that combinations of RA autoimmune/bystander antigens and RA autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
Some examples of RA autoantigens and RA bystander antigens include, but are not limited to, antigens from connective tissue, collagen (especially types I, II, III, IX, and XI), heat shock proteins and immunoglobulin Fc domains (see, eg J. Immunol. Methods 121:21 9 (89) and 151 : 177 (92)). Collagen is a family of fibrous proteins that have been classified into a number of structurally and genetically distinct types (Stryer, L. Biochemistry, 2nd Edition, W. H. Freeman & Co., 1981, pp. 184-199). Type I collagen is the most prevalent form and is found inter alia, in skin, tendons, cornea and bones and consists of two subunits of alphal(I) collagen and one subunit of a different sequence termed alpha2. Other types of collagen, including type II collagen, have three identical subunits or chains, each consisting of about 1,000 amino acids. Type II collagen ("CH") is the type of collagen found wter alia, in cartilage, the interverbebral disc and the vitreous body. Type II collagen contains three alphal(II) chains (alphal(II) ). Type III collagen is found inter alia, in blood vessels, the cardiovascular system and fetal skin and contains three alphal(III) chains (alphal (111)3). Type IV collagen is localized, inter alia, in basement membranes and contains three alpha 1 (IV) chains (alphal (IV)3).
Diabetes Autoantigens and Bystander antigens
In an alternative embodiment of the present invention the autoantigen or bystander antigen may be a diabetes autoantigen or bystander antigen for use to treat autoimmune diabetes.
The term "autoimmune diabetes" as used herein includes all forms of diabetes having an autoimmune component, and, in particular, Type I diabetes (also known as juvenile diabetes or insulin-dependent diabetes mellitus; IDDM). Type I diabetes is a disease that affects mainly children and young adults. The clinical features ofthe disease are caused by an insufficiency in the body's own insulin production due to a significant or even total reduction in of insulin production. It has been found that this type of diabetes is an autoimmune disease (cf. Castano, L. and G. S. Eisenbirth (1990) Type I diabetes: A chronic autoimmune disease of human, mouse and rat. Annu. Rev. Immunol. 8:647-679).
All cells ofthe immune system play a more or less important role. The B lymphocytes produce autoantibodies, whereas the monocytes/macrophages are probably involved in the induction of autoimmunity as antigen presenting cells. It is understood that T lymphocytes play a major role as effector cells in the destruction reaction. Like most autoimmune diseases type I diabetes arises because the tolerance ofthe T cells towards the body's own tissue ("self ') is lost. In particular, loss of tolerance towards pancreatic beta cells will result in the destruction thereof and diabetes will arise.
It is reported that about 30%> to 40% of diabetic children will eventually develop nephropathy requiring dialysis and transplantation (see US 5624895) Other significant complications include cardiovascular disease, stroke, blindness and gangrene. Moreover, diabetes mellitus accounts for a significant proportion of morbidity and mortality among dialysis and transplant patients.
Onset of Type I diabetes mellitus normally results from a well-characterized insulitis. During this condition, the inflammatory cells are typically directed against the beta cells ofthe pancreatic islets. It has been demonstrated that a large proportion ofthe infiltrating T lymphocytes produced during Type I diabetes mellitus are CD8-positive cytotoxic cells, which confirms the cytotoxic activity ofthe cellular infiltrate. CD4-positive lymphocytes are also present, the majority of which are helper T cells (Bottazzo et at., 1985, New England Journal of Medicine, 313, 353-359). The infiltrating cells also include lymphocytes or B cells that produce immunoglobulin-G (IgG) which suggest that these antibody-producing cells infiltrate the pancreatic islets (Glerchmann et at., 1987, Immunology Today, 8, 167- 170).
The term "diabetes autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in autoimmune diabetes, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of autoimmune diabetes when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells. The term "diabetes bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue (usually the pancreas) under autoimmune attack. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
It will be appreciated that combinations of diabetes autoimmune/bystander antigens and diabetes autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
Examples of diabetes autoantigens and bystander antigens include, but are not limited to, pancreatic beta cell (Type I) antigens, insulins, insulin receptors, insulin associated antigens (IA-w), glucagons, amylins, gamma amino decarboxylases (GADs) and heat shock proteins (HSPs), carboxypeptidases, peripherins and gangliosides. Some of these are discussed in more detail below.
a) Preproinsulin
Human insulin mRNA is translated as a 110 amino acid single chain precursor called preproinsulin, and removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin. Proinsulin consists of three domains: an amino-terminal B chain, a carboxy-terminal A chain and a connecting peptide in the middle known as the C peptide. Within the endoplasmic reticulum, proinsulin is exposed to several specific endopeptidases which excise the C peptide, thereby generating the mature form of insulin which consists ofthe A and B chain. Insulin and free C peptide are packaged in the Golgi into secretory granules which accumulate in the cytoplasm. The preproinsulin peptide sequence is reported as follows: MAL MRLLPL LALLAL GPD PAAAFVNQHL CGSHLVEALY LVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPL ALEGSLQKRG IVEQCCTSIC SLYQLENYCN
The insulin A chain includes amino acids 90-110 of this sequence. The B chain includes amino acids 25-54. The connecting sequence (amino acids 55-89) includes a pair of basic amino acids at either end. Proteolytic cleavage of proinsulin at these dibasic sequences liberates the insulin molecule and free C peptide, which includes amino acids 57-87. The human preproinsulin or an immunologically active fragment thereof, e. g., B chain or an immunogenic fragment thereof, e. g., amino acids 33-47 (conesponding to residues 9-23 of the B-chain), are useful as autoantigens in the methods and compositions described herein.
b) GAD65
Gad65 is a primary beta-cell antigen involved in the autoimmune response leading to insulin dependent diabetes mellitus (Christgau et al. (1991) J Biol Chem. 266 (31): 21257-64). The presence of autoantibodies to GAD65 is used as a method of diagnosis of type 1 diabetes. Gad65 is a 585 amino acid protein with a sequence reported as follows:
MASPGSGF S FGSEDGSGDS ENPGTARA C QVAQKFTGGI GNKLCALLYG DAEKPAESGG
SQPPRAAARK AACACDQKPC SCSKVDVNYA FLHATDLLPA CDGERPTLAF LQDVMNILLQ
YVVKSFDRST KVIDFHYPNE LLQEYNWELA DQPQNLEEIL MHCQTTLKYA IKTGHPRYFN
QLSTGLDMVG LAAD LTSTA NTNMFTYEIA PVFVLLEYVT LKKMREIIG PGGSGDGIFS
PGGAISNMYA MMIARFKMFP EVKEKGMAAL PRLIAFTSEH SHFSLKKGAA ALGIGTDSVI LIKCDERGKM IPSDLERRIL EAKQKGFVPF LVSATAGTTV YGAFDPLLAV ADICKKYKIW
MHVDAA GGG LLMSRKHK K LSGVERANSV T NPHKMMGV PLQCSALLVR EEGLMQNCNQ
MHASYLFQQD KHYDLSYDTG DKALQCGRHV DVFKL LM R AKGTTGFEAH VDKCLELAEY
LYNIIKNREG YEMVFDGKPQ HTNVCF YIP PSLRTLEDNE ERMSRLSKVA PVIKARMMEY
GTTMVSYQPL GDKVNFFRMV ISNPAATHQD IDFLIEEIER LGQDL
c) Islet tyrosine phosphatase IA-2
IA-2/ICA512, a member ofthe protein tyrosine phosphatase family, is another major autoantigen in type 1 diabetes (Lan et al. DNA Cell Biol 13 : 505-514, 1994). It is reported that 70%> of diabetic patients have autoantibodies to IA-2, which may appear years before the development of clinical disease. The IA-2 molecule is 979 amino acids in length and consists of an intracellular, transmembrane, and extracellular domain (Rabin et al. (1994) J. Immunol. 152 (6), 3183-3188). Autoantibodies are typically directed to the intracellular domain, e. g., amino acids 600-979 and fragments thereof (Zhang et al. (1997) Diabetes 46: 40-43 ; Xie et al. (1997) J Immunol 159: 3662-3667). The amino acid sequence of IA-2 is reported as follows:
MRRPRRPGGL GGSGGLRLLL CLLLLSSRPG GCSAVSAHGC LFDRRLCSHL EVCIQDGLFG QCQVGVGQAR PLLQVTSPVL QRLQGVLRQL MSQGLS HDD LTQYVISQEM ERIPRLRPPE PRPRDRSGLA PKRPGPAGEL LLQDIPTGSA PAAQHRLPQP PVGKGGAGAS SSLSPLQAEL LPPLLEHLLL PPQPPHPSLS YEPALLQPYL FHQFGSRDGS RVSEGSPGMV SVGPLPKAEA PALFSRTASK GIFGDHPGHS YGDLPGPSPA QLFQDSGLLY LAQELPAPSR ARVPRLPEQG SSSRAEDSPE GYEKEGLGDR GEKPASPAVQ PDAALQRLAA VLAGYGVELR QLTPEQLSTL LTLLQLLPKG AGRNPGGVVN VGADIKKTME GPVEGRDTAE LPARTSPMPG HPTASPTSSE VQQVPSPVSS EPPKAARPPV TPVLLEKKSP LGQSQPTVAG QPSARPAAEE YGYIVTDQKP LSLAAGVKLL EILAEHVHMS SGSFINISVV GPALTFRIRH NEQNLSLADV TQQAGLVKSE LEAQTGLQIL QTGVGQREEA AAVLPQTAHS TSPMRSVLLT LVALAGVAGL LVALAVALCV RQHARQQDKE RLAALGPEGA HGDTTFEYQD LCRQHMATKS LFNRAEGPPE PSRVSSVSSQ FSDAAQASPS SHSSTPS CE EPAQANMDIS TGHMILAYME DHLRNRDRLA KE QALCAYQ AEPNTCATAQ GEGNIKKNRH PDFLPYDHAR IKLKVESSPS RSDYINASPI IEHDPRMPAY IATQGPLSHT IADF QMV E SGCTVIVMLT PLVEDGVKQC DRY PDEGAS LYHVYEVNLV SEHI CEDFL VRSFYLKNVQ TQETRTLTQF HFLS PAEGT PASTRPLLDF RRKVNKCYRG RSCPIIVHCS DGAGRTGTYI LIDMVLNRMA KGVKEIDIAA TLEHVRDQRP GLVRSKDQFE FALTAVAEEV NAILKALPQ
d) ICA12
ICA12 (Kasimiotis et al. (2000) Diabetes 49 (4): 555-61; GenBank Accession
No. AAD16237) is one of a number of islet cell autoantigens associated with diabetes. The amino acid sequence of ICA12 is reported as follows:
MSMRSPISAQ LALDGVGTMV NCTIKSEEKK EPCHEAPQGS ATAAEPQPGD PARASQDSAD PQAPAQGNFR GS DCSSPEG NGSPEPKRPG ASEAASGSQE KLDFNRNLKE VVPAIEKLLS SD KERFLGR NSMEAKDVKG TQESLAEKEL QLLVMIHQLS TLRDQLLTAH SEQKNMAAML FEKQQQQMEL ARQQQEQIAK QQQQLIQQQH KINLLQQQIQ QVNMPYVMIP AFPPSHQPLP VTPDSQLALP IQPIPCKPVE YPLQLLHSPP APVVKRPGAM ATHHPLQEPS QPLNLTAKPK APELPNTSSS PSLKMSSCVP RPPSHGGPTR DLQSSPPSLP LGFLGEGDAV TKAIQDARQL LHSHSGALDG SPNTPFRKDL ISLDSSPAKE RLEDGCVHPL EEAMLSCDMD GSRHFPESRN SSHIKRPMNA FMV AKDERR KILQAFPDMH NSSISKILGS RWKSMTNQEK QPYYEEQARL SRQHLEKYPD YKYKPRPKRT CIVEGKRLRV GEYKALMRTR RQDARQSYVI PPQAGQVQMS SSDVLYPRAA GMPLAQPLVE HYVPRSLDPN MPVIVNTCSL REEGEGTDDR HSVADGEMYR YSEDEDSEGE EKSDGELVVL TD
e) ICA69
ICA69 is another autoantigen associated with type 1 diabetes (Pietropaolo et al.
J Clin Invest 1993; 92: 359-371). An amino acid sequence of ICA69 is reported as follows: MSGHKCSYP DLQDRYAQDK SVVNKMQQRY WETKQAFIKA TGKKEDEHVV ASDADLDAKL ELFHSIQRTC LDLSKAIVLY QKRICFLSQE ENELGKFLRS QGFQDKTRAG KMMQATGKAL CFSSQQRLAL RNPLCRFHQE VETFRHRAIS DT LTVNRME QCRTEYRGAL LWMKDVSQEL DPDLYKQMEK FRKVQTQVRL AKKNFDKLKM DVCQKVDLLG ASRCNLLSHM LATYQTTLLH F EKTSHTMA AIHESFKGYQ PYEFTTLKSL QDPMKKLVEK EEKKKINQQE STDAAVQEPS QLISLEEENQ RKESSSFKTE DGKSILSALD KGSTHTACSG PIDELLDMKS EEGACLGPVA GTPEPEGADK DDLLLLSEIF NASSLEEGEF SKE AAVFGD GQVKEPVPTM ALGEPDPKAQ TGSGFLPSQL LDQNMKDLQA SLQEPAKAAS DLTA FSLFA DLDPLSNPDA VGKTDKEHEL LNA
f) Glima 38
Glima 38 is a 38 kDa islet cell membrane autoantigen which is specifically immunoprecipitated with sera from a subset of prediabetic individuals and newly diagnosed type 1 diabetic patients. Glima 38 is an amphiphilic membrane glycoprotein, specifically expressed in islet and neuronal cell lines, and thus shares the neuroendocrine expression patterns of GAD65 and IA2 (Aanstoot et al. J Clin Invest. 1996 Jun 15; 97 (12): 2772-2783).
g) Heat shock protein 60 (HSP60)
HSP60, e. g., an immunologically active fragment of HSP60, e. g., p277 (see Elias et al., Eur JImmunol 1995 25 (10): 2851-7), can also be used as an autoantigen in the methods and compositions described herein. Other useful epitopes of HSP 60 are described, for example, in US 6110746.
h) Carboxypeptidase H
Carboxypeptidase H has been identified as an autoantigen, e. g., in pre-type 1 diabetes patients (Castano et al. (1991) J Clin Endocrinol Metab 73 (6): 1197-201 ; Alcalde et al. J Autoimmun. 1996 Aug; 9 (4): 525-8.). Therefore, carboxypeptidase H or immunologically reactive fragments thereof (e. g., the 136-amino acid fragment of carboxypeptidase-H described in Castano, supra) can be used in the methods and compositions described herein,
i) Peripherin
Peripherin is a 58 KDa diabetes autoantigen identified in nod mice (Boitard et al. (1992) Proc Natl Acad Sci U S A 89 (1): 172-6). A human peripherin sequence is reported as follows:
MSHHPSGLRA GFSSTSYRRT FGPPPSLSPG AFSYSSSSRF SSSRLLGSAS PSSSVRLGSF RSPRAGAGAL LRLPSERLDF SMAEALNQEF LATRSNEKQE LQELNDRFAN FIEKVRFLEQ
QNAALRGELS QARGQEPARA DQLCQQELRE LRRELELLGR ERDRVQVERD GLAEDLAALK
QRLEEETRKR EDAEHNLVLF RKDVDDATLS RLELERKIES LMDEIEFLKK LHEEELRDLQ
VSVESQQVQQ VEVEATVKPE LTAALRDIRA QYESIAAKNL QEAEEWYKSK YADLSDAANR
NHEALRQAKQ EMNESRRQIQ SLTCEVDGLR GTNEALLRQL RELEEQFALE AGGYQAGAAR LEEELRQLKE EMARHLREYQ ELLNVKMALD IEIATYRKLL EGEESRISVP VHSFASLNIK
TTVPEVEPPQ DSHSRKTVLI KTIETRNGEQ VTESQKEQR SELDKSSAHS Y j) Ganghosides
Ganghosides can also be useful autoantigens in the methods and compositions described herein. Ganghosides are sialic acid-containing glycolipids which are formed by a hydrophobic portion, the ceramide, and a hydrophilic part, i. e. the oligosaccharide chain. Ganghosides are expressed, inter alia, in cytosol membranes of secretory granules of pancreatic islets. Auto-antibodies to ganghosides have been described in type 1 diabetes, e. g., GMl-2 ganglioside is an islet autoantigen in diabetes autoimmunity and is expressed by human native (3 cells (Dotta et al. Diabetes. 1996 Sep; 45 (9): 1193-6). Ganghosides GT3, GD3 and GM-1 are also the target of autoantibodies associated with autoimmune diabetes (reviewed in Dionisi et al. Aim 1st Super Sanita 1997; 33 (3): 433-5). Ganglioside GM3 participates in the pathological conditions of insulin resistance (Tagami et al. J Biol Chem 2001 Nov 13; online publication ahead of print).
Further sequences are provided, for example, under GenBank Accession Nos U26593.1, BC008640.1, NM_022308.1, NM_022307.1, NM_004968.1, AF146363.1, AF147807.1, AH008870.1, U37183.1, U38260.1, AH005787.1, U71264.1, U71263.1, U71262.1,
U71261.1, U71260.1, U71259.1, U71258.1, U71257.1, U71256.1, U71255.1, U71254.1, U71253.1, U71252.1, U01882.1, U17989.1 (diabetes mellitus type I autoantigen (ICAp69)), X62899.2 (islet cell antigen 512), A28076.1 (islet GAD sequence (HIGAD-FL)) and AF098915.1 (type 1 diabetes autoantigen ICA12).
Myasthenia Gravis Autoantigens and Bystander antigens
In an alternative embodiment ofthe present invention the autoantigen or bystander antigen may be a Myasthenia Gravis autoantigen or bystander antigen for use to treat Myasthenia Gravis.
The term "Myasthenia Gravis autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in Myasthenia Gravis, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of Myasthenia Gravis when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "Myasthenia Gravis bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in Myasthenia Gravis. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction. It will be appreciated that combinations of Myasthenia Gravis autoimmune/bystander antigens and Myasthenia Gravis autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
Some examples of Myasthenia Gravis autoantigens and Myasthenia Gravis bystander antigens include, but are not limited to, acetyl choline receptors and components thereof, preferably human acetyl choline receptors and components thereof (see eg Eur. J. Pharm. 172:231(89)).
An amino acid sequence for a human gravin (A kinase (PRKA) anchor protein) autoantigen is reported as follows (GenBank Accession No M96322):
DCQAKSTPVIVSATTKKGLSSDLEGEKTTSLK KSDEVDEQVACQEVKVSVAIEEDLEPENGILELETKSSKLV QNIIQTAVDQFVRTEETATEMLTSELQTQAHMIKADSQDAGQETEKEGEEPQASAQDETPITSAKEESESTAVG QAHSDISKDMSEASEKTMTVEVEGSTVNDQQLEEVVLPSEEEGGGAGTKSVPEDDGHALLAERIEKSLVEPKED EKGDDVDDPENQNSALADTDASGGLTKESPDTNGPKQKEKEDAQEVELQEGKVHSESDKAITPQAQEELQKQER ESAKSELTES
An amino acid sequence for a human cholinergic receptor (gamma subunit) autoantigen is reported as follows (GenBank Accession No NM_005199): MHGGQGPLLLLLLLAVCLGGTQRNLRNQEERLLADLMQNYDPNLRPAERDSDVVNVSLKLTLTNLISLNEREEA LTTNV IEMQ CDYRLR DPRDYEGL VLRVPSTMVWRPDIVLENNVDGVFEVALYCNVLVSPDGCIY LPPAI FRSACSISVTYFPFDWQNCSLIFQSQTYSTNEIDLQLSQEDGQTIE IFIDPEAFTENGE AIQHRPAKMLLDP AAPAQEAGHQKVVFYLLIQRKPLFYVINIIAPCVLISSVAILIHFLPAKAGGQKCTVAINVLLAQTVFLFLVAK KVPETSQAVPLISKYLTFLLVVTILIVVNAVVVLNVSLRSPHTHSMARGVRKVFLRLLPQLLRMHVRPLAPAAV QDTQSRLQNGSSG SITTGEEVALCLPRSELLFQQ QRQGLVAAALEKLEKGPELGLSQFCGSLKQAAPAIQAC VEACNLIACARHQQSHFDNGNEE FLVGRVLDRVCFLAMLSLFICGTAGIFLMAHYNRVPALPFPGDPRPYLPS PD
An amino acid sequence for a human cholinergic receptor (alpha subunit) autoantigen is reported as follows (GenBank Accession No S77094):
MEP PLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNV RLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYNNADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYC EIIVTHFPFDEQNCSMKLGT TYDGSVVAINPESDQPDLSNFMESGEWVIKESRG KHSVTYSCCPDTPYLDIT YHFVMQRLPLYFIVNVIIPCLLFSFLTGLVFYLPTDSGEKMTLSISVLLSLTVFLLVIVELIPSTSSAVPLIGK YMLFTMVFVIASIIITVIVINTHHRSPSTHVMPN VRKVFIDTIPNIMFFSTMKRPSREKQDKKIFTEDIDISD ISGKPGPPPMGFHSPLIKHFEVKSAIEGIKYIAETMKSDQESNNAAAE KYVAMVMDHILLGVFMLVCIIGTLA VFAGRLIELNQQG
(see also Gattenlohner et al, Cloning of a cDNA coding for the acetylcholine receptor alpha- subunit from a thymoma associated with myasthenia gravis, Thymus 23 (2), 103-113 (1994)) Purified acetylcholine receptor can be isolated, for example, by the method of Mcintosh et al. J Neuroimmunol. 25: 75, 1989.
In an alternative embodiment one or more antigenic determinants may be used in place of a full antigen. For example, some specific class II MHC-associated autoantigen peptide sequences are as follows (see US 5783567):
Peptide Sequence Source
TVGLQLIQLINVDEVNQIVTTNVRLK AChR alpha (aa 32-67)
QQ VDYNLK
QIVTTNVRLKQQ VDYNLK AChR alpha aa 48-67) Q VDYNL AChR alpha aa 59-65)
GGVKKIHIPSEKI RPDL AChR alpha aa 73-90)
AIVKFTKVLLQY AChR alpha aa 101-112) TPPAIFKSYCEIIVTHFPF AChR alpha aa 118-137)
MKLGT TYDGSVV AChR alpha aa 144-156) MKLGIWTYDGSVV AChR alpha aa 144-157) analog(I-148) TYDGSVVA AChR alpha aa 149-157)
SCCPDTPYLDITYHFVM AChR alpha aa 191-207)
DTPYLDITYHFVMQRLPL AChR alpha aa 195-212)
FIVNVIIPCLLFSFLTGLVFY AChR alpha aa 214-234) LLVIVELIPSTSS AChR alpha aa 257-269)
STHVMPNWVRKVFIDTIPN AChR alpha aa 304-322)
N VRKVFIDTIPNIMFFS AChR alpha aa 310-327)
IPNIMFFSTMKRPSREKQ AChR alpha aa 320-337)
AAAE KYVAMVMDHIL AChR alpha aa 395-410) IIGTLAVFAGRLIELNQQG AChR alpha aa 419-437)
GQTIE IFIDPEAFTENGE AChR gamma aa 165-184)
MAHYNRVPALPFPGDPRPYL AChR gamma aa 476-495)
SLE Autoantigens and SLE Bystander antigens In an alternative embodiment ofthe present invention the autoantigen or bystander antigen may be a Systemic Lupus Erythematosus (SLE) autoantigen or bystander antigen for use to treat SLE.
The term "SLE autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in Systemic Lupus Erythematosus (SLE), becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "SLE bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe organ or tissue under autoimmune attack in SLE. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction, such as heatshock proteins (HSP), which although not necessarily specific to a particular tissue are normally shielded from the immune system.
It will be appreciated that combinations of SLE autoimmune/bystander antigens and SLE autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate. Some examples of SLE autoantigens and SLE bystander antigens include, but are not limited to, ds-DNA, chromatin, histones, nucleolar antigens, soluble RNA protein particles (such as U1RNP, Sm, Ro/SSA and La/SSB) erythrocyte antigens and platelet antigens. Examples of proteins include, for example, the human Ku and La antigens.
For example, an amino acid sequence for a human lupus p70 (Ku) autoantigen protein is reported as follows (GenBank Accession No J04611):
MSGWESYYKTEGDEEAEEEQEENLEASGDYKYSGRDSLIFLVDASKAMFESQSEDELTPFDMSIQCIQSVYISK IISSDRDLLAVVFYGTEKDKNSVNFKNIYVLQELDNPGAKRILELDQFKGQQGQKRFQDMMGHGSDYSLSEVL VCANLFSDVQFKMSHKRIMLFTNEDNPHGNDSAKASRARTKAGDLRDTGIFLDLMHLKKPGGFDISLFYRDIIS IAEDEDLRVHFEESSKLEDLLRKVRAKETRKRALSRLKLKLNKDIVISVGIYNLVQKALKPPPIKLYRETNEPV KTKTRTFNTSTGGLLLPSDTKRSQIYGSRQIILEKEETEELKRFDDPGLMLMGFKPLVLLKKHHYLRPSLFVYP EESLVIGSSTLFSALLIKCLEKEVAALCRYTPRRNIPPYFVALVPQEEELDDQKIQVTPPGFQLVFLPFADDKR KMPFTEKIMATPEQVGKMKAIVEKLRFTYRSDSFENPVLQQHFRNLEALALDLMEPEQAVDLTLPKVEAMNKRL GSLVDEFKELVYPPDYNPEGKVTKRKHDNEGSGSKRPKVEYSEEELKTHISKGTLGKFTVPMLKEACRAYGLKS GLKKQELLEALTKHFQD
(see also Reeves,W.H. and Sthoeger,Z.M., Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen, J. Biol. Chem. 264 (9), 5047-5052 (1989))
An amino acid sequence for a human lupus p80 (Ku) autoantigen protein is reported as follows (GenBank Accession No J04977 ):
MVRSGNKAAVVLCMDVGFTMSNSIPGIESPFEQAKKVITMFVQRQVFAENKDEIALVLFGTDGTDNPLSGGDQY QNITVHRHLMLPDFDLLEDIESKIQPGSQQADFLDALIVSMDVIQHETIGKKFEKRHIEIFTDLSSRFSKSQLD IIIHSLKKCDISLQFFLPFSLGKEDGSGDRGDGPFRLGGHGPSFPLKGITEQQKEGLEIVKMVMISLEGEDGLD EIYSFSESLRKLCVFKKIERHSIH PCRLTIGSNLSIRIAAYKSILQERVKKT TVVDAKTLKKEDIQKETVYC LNDDDETEVLKEDIIQGFRYGSDIVPFSKVDEEQMKYKSEGKCFSVLGFCKSSQVQRRFFMGNQVLKVFAARDD EAAAVALSSLIHALDDLDMVAIVRYAYDKRANPQVGVAFPHIKHNYECLVYVQLPFMEDLRQYMFSSLKNSKKY APTEAQLNAVDALIDSMSLAKKDEKTDTLEDLFPTTKIPNPRFQRLFQCLLHRALHPREPLPPIQQHI NMLNP PAEVTTKSQIPLSKIKTLFPLIEAKKKDQVTAQEIFQDNHEDGPTAKKLKTEQGGAHFSVSSLAEGSVTSVGSV NPAENFRVLVKQKKASFEEASNQLINHIEQFLDTNETPYFMKSIDCIRAFREEAIKFSEEQRFNNFLKALQEKV EIKQLNHF EIVVQDGITLITKEEASGSSVTAEEAKKFLAPKDKPSGDTAAVFEEGGDVDDLLDMI
(see also Yaneva,M., Wen,J., Ayala,A. and Cook,R., cDNA-derived amino acid sequence of the 86-kDa subunit ofthe Ku antigen, J. Biol. Chem. 264 (23), 13407-13411 (1989))
An amino acid sequence for a human La protein/SS-B antigen is reported as follows (GenBank Accession No J04205 Ml 1108): MAENGDNEKMAALEAKICHQIEYYFGDFNLPRDKFLKEQIKLDEGWVPLEIMIKFNRLNRLTTDFNVIVEALSK SKAELMEISEDKTKIRRSPSKPLPEVTDEYKNDVKNRSVYIKGFPTDATLDDIKE LEDKGQVLNIQMRRTLHK AFKGSIFVVFDSIESAKKFVETPGQKYKETDLLILFKDDYFAKKNEERKQNKVEAKLRAKQEQEAKQKLEEDAE MKSLEEKIGCLLKFSGDLDDQTCREDLHILFSNHGEIKWIDFVRGAKEGIILFKEKAKEALGKAKDANNGNLQL RNKEVT EVLEGEVEKEALKKIIEDQQESLNKWKSKGRRFKGKGKGNKAAQPGSGKGKVQFQGKKTKFASDDEH DEHDENGATGPVKRAREETDKEEPASKQQKTENGAGDQ
(see also Chambers et al, Genomic structure and amino acid sequence domains ofthe human La autoantigen, J. Biol. Chem. 263 (34), 18043-18051 (1988))
Bowel Autoantigens and Bystander Antigens
In an alternative embodiment ofthe present invention the autoantigen or bystander antigen may be a bowel autoantigen or bystander antigen for use to treat an autoimmune disease of the bowel.
The term "autoimmune disease ofthe bowel" as used herein includes any disease in which the bowel or a component of the bowel comes under autoimmune attack. The main autoimmune diseases ofthe bowel are inflammatory bowel disease (IBD) and celiac (also known as coeliac) disease.
Inflammatory bowel disease (IBD) is the term generally applied to four diseases ofthe bowel, namely Crohn's disease, ulcerative colitis, indeterminate colitis, and infectious colitis.
Ulcerative colitis is a chronic inflammatory disease mainly affecting the large intestine. The course ofthe disease may be continuous or relapsing, mild or severe. The earliest lesion is typically an inflammatory infiltration with abscess formation at the base ofthe crypts of Lieberkuhn. Coalescence of these distended and ruptured crypts tends to separate the overlying mucosa from its blood supply, leading to ulceration. Signs and symptoms ofthe disease include cramping, lower abdominal pain, rectal bleeding, and frequent, loose discharges consisting mainly of blood, pus, and mucus with scanty fecal particles. A total colectomy may be required for acute severe or chronic, unremitting ulcerative colitis.
Crohn's disease (also known as regional enteritis or ulcerative ileitis) is also a chronic inflammatory disease of unknown etiology but, unlike ulcerative colitis, it can affect any part of the bowel. The most prominent feature ofthe disease is the granular, reddish-puφle edematous thickening ofthe bowel wall. With the development of inflammation, these granulomas often lose their circumscribed borders and integrate with the sunounding tissue. Dianhea and obstruction ofthe bowel are the predominant clinical features. As with ulcerative colitis, the course ofthe disease may be continuous or relapsing, mild or severe but, unlike ulcerative colitis, it is not curable by resection ofthe involved segment of bowel. Many patients with Crohn's disease require surgery at some point, but subsequent relapse is common and continuous medical treatment is usual.
Celiac disease (CD) is a disease ofthe intestinal mucosa and is usually identified in infants and children. Celiac disease is associated with an inflammation ofthe mucosa, which causes malabsoφtion. Individuals with celiac disease are intolerant to the protein gluten, which is present in foods such as wheat, rye and barley. When exposed to gluten, the immune system of an individual with celiac disease responds by attacking the lining ofthe small intestine.
The term "bowel autoantigen" as used herein includes any substance or a component thereof normally found within a mammal that, in an autoimmune disease ofthe bowel, becomes a target of attack by the immune system, preferably the primary (or a primary) target of attack. The term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease ofthe gut when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of autoantigens. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens from (and preferably specific to) the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
The term "bowel bystander antigen" as used herein includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, polysaccharides or any other immunogenic substance that is, or is derived from, a component ofthe bowel under autoimmune attack in an autoimmune disease ofthe bowel. The term includes but is not limited to autoantigens and fragments thereof such as antigenic determinants (epitopes) involved in autoimmune attack. In addition, the term includes antigens normally not exposed to the immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
It will be appreciated that combinations of bowel autoimmune/bystander antigens and bowel autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
Examples of bowel autoantigens and bystander antigens include, but are not limited to, gliadins and tissue transglutaminase (tTG) (associated with celiac disease; see Marsh, Nature Medicine 1997;7:725-6) and tropomyosins, in particular tropomyosin isoform 5, (associated with ulcerative colitis).
Allergens and antigenic determinants thereof
In an alternative embodiment ofthe present invention the antigen or bystander antigen may be a an allergen or bystander antigen for use to treat an allergic condition.
The term "allergen" as used herein means any substance which can induce an allergic response, especially a type I hypersensitive response. Typical allergens include, but are not limited to, pollens, molds, foods, animal danders or their excretions, smuts and insects, their venoms or their excretions. Allergens may, for example, be natural or synthetic organic molecules such as peptides/proteins, polysaccharides or lipids. They may be administered singly or as a mixture. Allergens may be chemically or physically modified. Such modified allergens, or allergen derivatives, are known in the art. Examples include, but are not limited to, peptide fragments, conjugates or polymerized allergen derivatives. Thus, the term "allergen" as used herein includes naturally occurring (native) allergens as well as any biologically active fragment, derivative, homologue or variant thereof or any antigenic determinant or epitope (especially immunodominant epitope) thereof or any polynucleotide coding for an allergen (including any biologically active fragment, derivative, homologue or variant) or antigenic determinant or epitope (especially immunodominant epitope) thereof. The amount of allergen to be administered can be determined empirically and depends on the sensitivity ofthe individual as well as the desired clinical result. Generally, a regimen of desensitization initially involves the periodic administration of smaller amounts of allergen, which level is increased over the course ofthe regimen until a predetermined (planned) upper limit is reached or the individual can tolerate exposure to such allergen without a significant adverse allergic response. The particular regimen often is tailored to individual patient needs. The embodiment and potential advantage ofthe present invention is that it may be possible to meaningfully decrease the level of allergens administered and/or the number of injections and, thereby, the length ofthe desensitization regimen. Further, with a meaningful decrease ofthe level (dose) of allergen administered to particularly sensitive individuals, there is a possible diminished risk of severe allergic reaction to the administration ofthe allergen.
The progress of immunotherapy can be monitored by any clinically acceptable diagnostic tests. Such tests are well known in the art and include symptom levels and requirement levels for ancillary therapy recorded in a daily diary, as well as skin testing and in vitro serological tests for specific IgE antibody and/or specific IgG antibody.
The present invention may be used for preventing and treating all forms of allergy and allergic disorder, including without limitation: ophthalmic allergic disorders, including allergic conjunctivitis, vernal conjunctivitis, vernal keratoconjunctivitis, and giant papillary conjunctivitis; nasal allergic disorders, including allergic rhinitis and sinusitis; otic allergic disorders, including eustachian tube itching; allergic disorders ofthe upper and lower airways, including intrinsic and extrinsic asthma; allergic disorders ofthe skin, including dermatitis, eczema and urticaria; and allergic disorders ofthe gastrointestinal tract.
Any form of allergen (including any biologically active fragment, derivative, homologue or variant) or antigenic determinant or epitope (especially immunodominant epitope) thereof or any polynucleotide coding for an allergen (including any biologically active fragment, derivative, homologue or variant) or antigenic determinant or epitope (especially immunodominant epitope) thereof may be used, including but not limited to pollen allergens, mite allergens, animal dander allergens, latexes, food allergens, insect allergens (eg mite or cockroach allergens), fungal allergens, drug allergens and venom allergens and antigenic determinants or epitopes (especially immunodominant epitopes) thereof, for example:
Pollen allergens and antigenic determinants thereof
1. Grass Pollen Antigens and Antigenic Determinants
a. Ryegrass pollen antigens and antigenic determinants
For example, Lol p 1 (eg GenBank Accession No M57474), Lol p lb (eg GenBank Accession No M59163), Lol p 2 (eg GenBank Accession No X73363), Lol p 2a (eg SwissProt Accession No PI 4947), Lol p 2b (eg PIR Accession No A48595), Lol p 3 (eg SwissProt Accession No PI 4948), Lol p 4 (eg PIR Accession No A60737), Lol p 5 (eg PIR Accession No S38288), Lol p 9 (eg GenBank Accession No L13083) or Lol p 11 (eg PIR Accession No A54002); and antigenic determinants thereof.
For example, an amino acid sequence for Lol p 1 is reported as follows (GenBank Accession No M57474):
MASSSSVLLVVALFAVFLGSAHGIAKVPPGPNITAEYGDK LDAKST YGKPTGAGPKDNGGACGYKNVDKAPF NGMTGCGNTPIFKDGRGCGSCFEIKCTKPESCSGEAVTVTITDDNEEPIAPYHFDLSGHAFGSMAKKGEEQNVR SAGELELQFRRVKCKYPDDTKPTFHVEKASNPNYLAILVKYVDGDGDVVAVDIKEKGKDKWTELKES GAVWRI DTPDKLTGPFTVRYTTEGGTKSEFEDVIPEG KADTSYSAK
b. Timothy Grass pollen antigens and antigenic determinants
For example, Phi p 1 (eg GenBank Accession No X78813), Phi p 2 (eg GenBank Accession No X75925), Phi p 5 (eg GenBank Accession No Z27083), Phi p 5a (eg GenBank Accession No X70942), Phi p 5b (eg GenBank Accession No Z27083), Phi p 6 (eg GenBank Accession No Z27082), Phi p 11 (eg GenBank Accession No X77583), Phi p 32K (eg PIR Accession No S38294) and Phi p 38K (eg PIR Accession No S38293); and antigenic determinants thereof. For example, an amino acid sequence for Phi p 1 is reported as follows (GenBank Accession No X78813):
MASSSSVLLVVVLFAVFLGSAYGIPKVPPGPNITATYGDK LDAKST YGKPTGAGPKDNGGACGYKDVDKPPF SGMTGCGNTPIFKSGRGCGSCFEIKCTKPEACSGEPVVVHITDDNEEPIAPYHFDLSGHAFGAMAKKGDEQKLR SAGELELQFRRVKCKYPEGTKVTFHVEKGSNPNYLALLVKYVNGDGDVVAVDIKEKGKDK IELKES GAIWRI DTPDKLTGPFTVRYTTEGGTKTEAEDVIPEGWKADTSYESK
c. Bent Grass pollen antigens and antigenic determinants
For example, Agr a 1 (eg PIR Accession No E37396); and antigenic determinants thereof.
d. Bermuda Grass pollen antigens and antigenic determinants
For example, Cyn d 1 (eg PIR Accession No A61226) and Cyn d 2 (eg GenBank Accession No AJ131335); and antigenic determinants thereof.
e. Blue Grass pollen antigens and antigenic determinants
For example, Poa p 1 (eg PIR Accession No F37396), Poa p 2 (eg GenBank Accession No AJ131337) and Poa p 9 (eg GenBank Accession No M38342); and antigenic determinants thereof.
f. Canary Grass pollen antigens and antigenic determinants
For example, Pha a 1 (eg SwissProt Accession No Q41260) Pha a 5.1 (eg SwissProt Accession No P56154) Pha a 5.2 (eg SwissProt Accession No P56165) Pha a 5.3 (eg SwissProt Accession No P56166) and Pha a 5.4 (eg SwissProt Accession No P56167); and antigenic determinants thereof.
g. Orchard Grass pollen antigens and antigenic determinants For example, Dae g l(eg PIR Accession No D58493) Dae g 2 (eg GenBank Accession No S45354) Dae g 3 (eg PIR Accession No A60359); and antigenic determinants thereof.
2. Tree pollen antigens and antigenic determinants
a. Birch pollen antigens and antigenic determinants
For example, Bet v la (eg GenBank Accession No X15877), Bet v lb (eg GenBank Accession No X77200), Bet v lc (eg GenBank Accession No X77265), Bet v Id (eg GenBank Accession No X77266), Bet v le (eg GenBank Accession No X77267), Bet v If (eg GenBank Accession No X77268), Bet v lg (eg GenBank Accession No X77269), Bet v lj (eg GenBank Accession No X77271), Bet v Ik (eg GenBank Accession No X77272), Bet v IL (eg GenBank Accession No X77273), Bet v lm (eg GenBank Accession No X81972), Bet v 2 (eg GenBank Accession No M65179), Bet v 3 (eg GenBank Accession No X79267), or Bet v 4 (eg GenBank Accession No Y12560); and antigenic determinants thereof.
For example, an amino acid sequence for Bet v la is reported as follows (GenBank
Accession No XI 5877): MGVFNYETETTSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKYVKDRVDE VDHTNFKYNYSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAV ESYLLAHSDAYN
b. Chestnut tree pollen antigens and antigenic determinants
For example, Cas s 1 (eg PIR Accession No PC2001); and antigenic determinants thereof.
c. Hornbeam tree pollen antigens and antigenic determinants
For example, Car b 1 (eg GenBank Accession No X66932); and antigenic determinants thereof.
d. Oak tree pollen antigens and antigenic determinants For example, Que a 1 (eg PIR Accession No D53288); and antigenic determinants thereof.
e. Olive Tree pollen antigens and antigenic determinants
For example, Ole e 1 (eg GenBank Accession No S75766), Ole e 3 (eg GenBank Accession No AF015810), Ole e 4 (eg SwissProt Accession No P80741) Ole e 5 (eg SwissProt Accession No P80740) Ole e 6 (eg GenBank Accession No U86342); and antigenic determinants thereof.
3. Weed pollen antigens and antigenic determinants
a. Ragweed (A. artemisiifloria)
For example, Amb a 1.1 (eg GenBank Accession No M80558), Amb a 1.2 (eg GenBank Accession No M80559), Amb a 1.3 (eg GenBank Accession No M62961), Amb a 1.4 (eg GenBank Accession No M80562), Amb a 2 (eg GenBank Accession No M80561) Amb a 3 (eg GenBank Accession No P00304) and Amb a 5 (eg SwissProt Accession No P02878); and antigenic determinants thereof. For example, an amino acid sequence for Amb a 1.1 is reported as follows (GenBank Accession No M80558):
MGIKHCCYILYFTLALVTLLQPVRSAEDLQEILPVNETRRLTTSGAYNIIDGC RGKADWAENRKALADCAQGF GKGTVGGKDGDIYTVTSELDDDVANPKEGTLRFGAAQNRPL IIFERDMVIRLDKEM VNSDKTIDGRGAKVEI INAGFTLNGVKNVIIHNINMHDVKVNPGGLIKSNDGPAAPRAGSDGDAISISGSSQIWIDHCSLSKSVDGLVDA KLGTTRLTVSNSLFTQHQFVLLFGAGDENIEDRGMLATVAFNTFTDNVDQRMPRCRHGFFQVVNNNYDK GSYA IGGSASPTILSQGNRFCAPDERSKKNVLGRHGEAAAESMKWN RTNKDVLENGAIFVASGVDPVLTPEQSAGMI PAEPGESALSLTSSAGVLSCQPGAPC
b. Ragweed (A. psilostachya) For example, Amb p 5 (eg GenBank Accession No L24465); and antigenic determinants thereof.
c. Ragweed (A. trifida)
For example, Amb t 5 (eg GenBank Accession No M38782); and antigenic determinants thereof.
4. Crop pollen antigens and antigenic determinants
a. Brassica (Rape)
For example, Bra n 1 (eg GenBank Accession No D63151) and Bra n 2 (eg GenBank Accession No D63152); and antigenic determinants thereof.
For example, an amino acid sequence for Bra n 1 is reported as follows (GenBank Accession No D63151):
MADAEHERI FKKFDTDGDGKI SAAELEEALKKLGSVTPDDVTRMMAKI DTDGDGNI SFQEFTEFASANPGLMKD AKVF
b. Maize pollens
For example, Zea m 1 (eg GenBank Accession No L14271); and antigenic determinants thereof.
c. Rice pollens
For example, Ory s l(eg GenBank Accession No U31771); and antigenic determinants thereof.
Mite allergens Mite allergens include all types of allergens found in mites. Common types of mite allergen include, for example, enzymes such as proteases (eg trypsin, chymotrypsin) amylase, and glutathione transferase or structural proteins such as tropomyosin. Suitably the mite allergen is a dust mite allergen.
Any form of mite antigen or antigenic determinant or any polynucleotide coding for a mite antigen or antigenic determinant (including any biologically active fragment, derivative, homologue or variant) may be used, including but not limited to epitopic polypeptide or polynucleotide sequences ofthe following:
antigens or antigenic determinants from dust mites such as Dermatophagoides farinae such as Der f 1 (eg SwissProt Accession Nos PI 6311, Q9GYY0), Der f 2 (eg SwissProt Accession Nos Q00855, Q9BIX2), Der f 3 (eg SwissProt Accession Nos P49275+, Q94508, Q9TWV8), Der f 6 (eg SwissProt Accession No P49276), Der f 7 (eg SwissProt Accession No Q26456), Der f mag (eg SwissProt Accession No P39673), Der f mag29 (eg SwissProt Accession No P39674), Der f mag3 (eg SwissProt Accession No Q94507), Der f 15 (eg SwissProt Accession No Q9U6R7);
antigens or antigenic determinants from mites such as Glycyphagus domesticus, such as Gly d 2.02 (eg SwissProt Accession No Q9NFQ4);
antigens or antigenic determinants from dust mites such as Dermatophagoides pteronyssinus such as Der p 1 (eg SwissProt Accession No P08176), Der p 2 (eg SwissProt Accession No P49278), Der p 3 (eg SwissProt Accession No P39675),
Der p 4 (eg SwissProt Accession Nos P49274, Q9Y197), Der p 5 (eg SwissProt Accession No PI 4004) Der p 6 (eg SwissProt Accession No P49277), Der p 7 (eg SwissProt Accession No P49273), Der p 10 (eg SwissProt Accession No 018416), Der p 8 (eg SwissProt Accession No P46419);
antigens or antigenic determinants from dust mites such as Dermatophagoides microceras, such as Der m 1 (eg SwissProt Accession No PI 6312); antigens or antigenic determinants from mites such as Euroglyphus, such as Eur m 1 (eg SwissProt Accession No P25780), Eur m 2.0101 (eg SwissProt Accession No Q9TZZ2) or Eur m 3.0101 (eg SwissProt Accession No O97370);
antigens or antigenic determinants from mites such as Lepidoglyphus, such as Lep d 1 (eg SwissProt Accession No P80384+), Lep d 5 (eg SwissProt Accession No Q9U5P2), Lep d 7 (eg SwissProt Accession No Q9U1G2), Lep d 10 (eg SwissProt Accession No Q9NFZ4), Lep d 13 (eg SwissProt Accession No Q9U5P1);
For example, an amino acid sequence for Der p I is reported as follows (SwissProt Accession No P08176):
1 m ivlaiasl lalsavyarp ssiktfeeyk kafnksyatf edeeaarknf lesvkyvqsn
61 ggainhlsdl sldefknrfl msaeafehlk tqfdlnaetn acsingnapa eidlrqmrtv 121 tpirmqggcg scwafsgvaa tesaylayrn qsldlaeqel vdcasqhgch gdtiprgiey
181 iqhngvvqes yyryvareqs crrpnaqrfg isnycqiypp nvnkireala qthsaiavii
241 gikdldafrh ydgrtiiqrd ngyqpnyhav nivgysnaqg vdy ivrns dtn gdngyg
301 yfaanidlmm ieeypyvvil
For example, an amino acid sequence for Der f I is reported as follows (eg SwissProt Accession No P16311):
1 mkfvlaiasl lvlstvyarp asiktfeefk kafnknyatv eeeevarknf leslkyvean
61 kgainhlsdl sldefknryl msaeafeqlk tqfdlnaets acrinsvnvp seldlrslrt 121 vtpirmqggc gscwafsgva atesaylayr ntsldlseqe lvdcasqhgc hgdtiprgie
181 yiqqngvvee rsypyvareq rcrrpnsqhy gisnycqiyp pdvkqireal tqthtaiavi
241 igikdlrafq hydgrtiiqh dngyqpnyha vnivgygstq gddy ivrns wdtt gdsgy
301 gyfqagnnlm mieqypyvvi m
Animal Food Allergens
Animal food allergens include all types of allergens found in foods originating with animals, such as milk, eggs and fish seafoods. Common types of animal food allergen include, for example antigens or antigenic determinants from tropomyosins, parvalbumins, ovomucoids, ovalbumins, ovotransferrins, lysozymes, vitellogenins, apovitellenins, serum albumins (such as Bovine Serum Albumin; BSA), beta-lactoglobulins, alpha-lactalbumins and caseins (such as Casein, alpha-Si Casein and Alpha-S2 Casein). Any form of animal food antigen or antigenic determinant or any polynucleotide coding for an animal food antigen or antigenic determinant (including any biologically active fragment, derivative, homologue or variant) may be used, including but not limited to polypeptide or polynucleotide sequences ofthe following:
Fish allergens such as those from Caφ (eg Cyc p 1.02 and Cyc p 1.01)
Cod (eg Gad cl; eg SwissProt Accession No P02622), Mackerel (eg Traj 1; eg SwissProt
Accession No Q91482) and Salmon (eg Sal s 1; eg SwissProt Accession No Q91482);
Marine mollusc allergens such as those from Crab (eg Cha f 1 ; eg SwissProt Accession No Q9N2R3), Lobster (eg Horn a 1 ; eg SwissProt Accession No 044119), Shrimp (eg Met el ; eg SwissProt Accession No Q25456) and Spiny Lobster (eg Pan s 1 ; eg SwissProt Accession No 061379);
Egg allergens such as ovomucoids (eg Gal dl ; eg SwissProt Accession No P01005), ovalbumins (eg Gal d2; eg SwissProt Accession No P01014), ovotransfemns (eg Gal d3; eg SwissProt Accession No P02789), lysozymes (eg Gal d4; eg SwissProt Accession No P00698), vitellogenins, apovitellenins and tropomyosins (eg Horn a 1);
Milk allergens such as those from cow milk, such as BSA (eg Bos d 6; eg SwissProt Accession No P02769), beta-lactoglobulins, alpha-lactalbumins, alpha-Si caseins and alpha-S2 caseins.
Plant Food Allergens
Plant food allergens include all types of allergens found in plant matter used as food. Common examples include, for example plant enzymes such as papains, pectate lyases, superoxide dismutases, glyoxalases, beta-fructofuranosidases and phosphate isomerases; plant enzyme inhibitors such as amylase inhibitors and trypsin inhibitors; plant profilins, patatins, actinidins, glycinins, beta-conglycinins, agglutinins and gliadins Any form of plant food allergen or antigenic determinant or any polynucleotide coding for a plant food allergen or antigenic determinant (including any biologically active fragment, derivative, homologue or variant) may be used in the present invention, including but not limited to polypeptide or polynucleotide sequences ofthe following:
Avocado allergens and antigenic determinants such as those from Prs a 1 (eg SwissProt
Accession No P93680);
Apple allergens and antigenic determinants such as those from Mai d 1 (eg SwissProt Accession No P43211 ), Mai d 4 (eg SwissProt Accession No Q9XF42), Mai d 3 (eg SwissProt Accession No Q9M5X7);
Apricot allergens and antigenic determinants such as those from Pru ar 3 (eg SwissProt Accession No P81651), Pru ar 1 (eg SwissProt Accession No O50001);
Barley allergens and antigenic determinants such as those from Hor v 1 (eg SwissProt Accession No P16968) and Hor v 9 (eg SwissProt Accession No Q9S8H1);
Buckwheat allergens and antigenic determinants such as those from Fag ag 1 (eg SwissProt Accession No Q9XFM4);
Canot allergens and antigenic determinants such as those from Dau c 1 (eg SwissProt Accession No O04298);
Castor Bean allergens and antigenic determinants such as those from Ric c 1 (eg SwissProt Accession No P01089);
Celery allergens and antigenic determinants such as those from Api g 1 (eg SwissProt Accession No P49372) Api g 5 (eg SwissProt Accession No P81943) and Api g 1.0201 (eg SwissProt Accession No P92918), Api g 3 (eg SwissProt Accession No P92919) Api g 4 (eg SwissProt Accession No Q9XF37); Cherry allergens and antigenic determinants such as those from Pru a 1 (eg SwissProt Accession No 024248), Pru a 2 (eg SwissProt Accession No P50694), Pru av 3 (eg SwissProt Accession No Q9M5X8), Pru av 4 (eg SwissProt Accession No Q9XF39);
Kidney Bean allergens and antigenic determinants such as those from PR Protein (eg SwissProt Accession Nos P25985+ and P25986);
Kiwi allergens and antigenic determinants such as those from Act c 1 (eg SwissProt
Accession No P00785);
Maize allergens and antigenic determinants such as those from Zea m 14 (eg SwissProt
Accession No PI 9656), Profilin (eg SwissProt Accession No 022655), Zea m 1 (eg
SwissProt Accession No Q07154);
Mustard leaf allergens and antigenic determinants such as those from Braj 1 L (eg SwissProt Accession No P80215);
Mustard white allergens and antigenic determinants such as those from Sin a 1 (eg SwissProt Accession No Q41196);
Olive allergens and antigenic determinants such as those from Ole e 1 (eg SwissProt Accession No PI 9963), Ole e 3 (eg SwissProt Accession No 081092), Ole e 4 (eg SwissProt Accession No P80741), Ole e 5 (eg SwissProt Accession No P80740), Ole e 6 (eg SwissProt Accession No 024172), Ole e 7 (eg SwissProt Accession No P81430), Ole e 8 (eg SwissProt Accession No Q9M7R0), Ole e 9 (eg Entez Accession No AAK58515), Ole e 2 (eg SwissProt Accession No 024169);
Papaya allergens and antigenic determinants such as those from papain (eg SwissProt Accession No P00784);
Peach allergens and antigenic determinants such as those from Pru p 1 (eg SwissProt Accession No P81402); Pear allergens and antigenic determinants such as those from Pyr c 1 (eg SwissProt Accession No O65200), Pyr c 3 (eg SwissProt Accession No Q9M5X6), Pyr c 4 (eg SwissProt Accession No Q9XF38);
Pineapple allergens and antigenic determinants such as those from pineapple profilin (eg Entrez Accession No AAK54835);
Plantain allergens and antigenic determinants such as those from Pla 1 1 (eg SwissProt Accession No P82242), Pla 1 1.0101 (eg SwissProt Accession No CAC41633), Pla 1 1.0102 (eg SwissProt Accession No CAC41634), Pla 1 1.0103 (eg SwissProt Accession No CAC41635);
Plum allergens and antigenic determinants such as those from Pru d 3 (eg SwissProt Accession No P82534);
Potato allergens and antigenic determinants such as those from patatins (eg SwissProt
Accession Nos P07745, P15476, P15477, PI 1768 and P15478) Sol a t 2 (eg SwissProt
Accession No P16348) and Sol a 1 4 (eg SwissProt Accession No P30941);
Rice allergens and antigenic determinants such as those from RA 1 (eg SwissProt Accession
No Q01884), RA 2 (eg SwissProt Accession No Q01885), RA 5 (eg SwissProt Accession
No Q01881), RA 14 (eg SwissProt Accession No Q01882), RA 17 (eg SwissProt Accession
No Q01883) Glyoxalase (eg SwissProt Accession No Q9ZWJ2), Ory s 1 (eg SwissProt Accession No Q40638);
Soybean allergens and antigenic determinants such as those from AlaBx (eg SwissProt Accession No P04776), A2Bla (eg SwissProt Accession No P04405), A3B4 (eg SwissProt Accession No P04347) A5A4B3 (eg SwissProt Accession No P02858), Gy3 (eg SwissProt Accession No PI 1828), Gy4 (eg SwissProt Accession No Q43452), A5 A4B3 (eg SwissProt Accession No Q39921), Beta -Conglycinin (eg SwissProt Accession No P13916), Lectin (eg SwissProt Accession No P05046), Trypsin Inhibitor (eg SwissProt Accession Nos Q39869, Q39898 and Q39899), Gly m 1 (eg SwissProt Accession No P22895), Gly m 1 A (eg
SwissProt Accession No Q9S8F3), Gly m 2 (eg SwissProt Accession No Q39802), Gly m 3
(eg SwissProt Accession No O65809), Gly m 3 (eg SwissProt Accession No O65810), Gly m bd (eg SwissProt Accession No Q9AVK8);
Tomato allergens and antigenic determinants such as those from Lye e 1 (eg SwissProt
Accession No PI 3447);
Turnip allergens and antigenic determinants such as those from Bra r 2 (eg SwissProt Accession No P81729);
Wheat allergens and antigenic determinants such as those from agglutinins (eg SwissProt Accession Nos PI 0968, P02876 and PI 0969), alpha amylase and trypsin inhibitors (eg SwissProt Accession Nos P01084, P10846, P01083, P01085, P16852, P16159, P17314, P16851, P16850 and Q41540), gliadins (eg SwissProt Accession Nos P04728, P04726, P02863, Q41546, P04727, P04721, P04722, P04723, P04724, P04725, P18573, P02863, P21292, P08453, P06659, P04729 , P04730, P08079, P02865, Q41548 and Q41543), phosphate isomerases (eg SwissProt Accession No Q9FS79), glutenins (eg SwissProt Accession Nos P08488, P10386, P10387, P02861, P02862, P08489, P10388, P10385, P16315, Q03872, Q41603, Q03871, Q41549, Q41550, Q41551, Q41552 and Q9S8D7), Tri a 2 (CAA10349), Tri a 3 (eg SwissProt Accession No Q41576) and profilins (eg SwissProt Accession Nos P49232, P49233 and P49234).
Nut allergens
Nut allergens include all types of allergens found in nuts. Common examples include, for example albumins, profilins, vicilins, agglutinins, arachins, glycinins and profilins.
Any form of nut allergen or antigenic determinant or any polynucleotide coding for a nut allergen or antigenic determinant (including any biologically active fragment, derivative, homologue or variant) may be used in the present invention, including but not limited to polypeptide or polynucleotide sequences ofthe following: allergens or antigenic determinants from peanut such as Ara h 1 (eg SwissProt Accession Nos P43238, P43237), Ara h 2 (eg ENTREZ Accession No AAK96887), Ara h 3 (eg SwissProt Accession No O82580), Ara h 4 (eg SwissProt Accession No Q9SQH7), Ara h 5 (eg SwissProt Accession No Q9SQI9), Ara h 6 (eg SwissProt Accession No Q9SQG5), Ara h 7 (eg SwissProt Accession No Q9SQH1);
allergens or antigenic determinants from brazil nut such as Ber e 1 (eg SwissProt Accession No P04403);
allergens or antigenic determinants from chestnut (eg Castanea sativa) such as Cas s 1 (eg SwissProt Accession No Q9S8Q4); and
allergens or antigenic determinants from hazel nut such as Cor a 1-5 (eg SwissProt Accession No P43216), Cor a 1 (eg SwissProt Accession Nos Q08407, Q39454, Q39453), Cor a 1.0401 (eg SwissProt Accession No Q9SWR4), Cor a 1.0402 (eg SwissProt Accession No Q9FPK4), Cor a 1.0403 (eg SwissProt Accession No Q9FPK3), Cor a 1.0404 (eg SwissProt Accession No Q9FPK2), Cor a 9 (eg ENTREZ Accession No AAL73404).
Animal allergens
Animal allergens include all types of allergens generated by animals. Common examples include, for example lipocalins, serum albumins and protease inhibitors, which are commonly present, for example, in animal danders.
Any form of animal antigen or antigenic determinant or any polynucleotide coding for an animal antigen or antigenic determinant (including any biologically active fragment, derivative, homologue or variant) may be used, including but not limited to polypeptide or polynucleotide sequences ofthe following:
antigens or antigenic determinants from cat danders such as Fel d 1 (eg SwissProt Accession No P30440), Fel d 2 (eg SwissProt Accession No P49064) and Fel d 3 (eg Entrez Accession No AAL49391); antigens or antigenic determinants from cow danders such as Bos d 2 (eg PIR Accession No B59225);
antigens or antigenic determinants from dog danders such as Can f 1 (eg SwissProt Accession No 018873), Can f 2 (eg SwissProt Accession No 018874) or Can f 3 (eg SwissProt Accession No P49822); and
antigens or antigenic determinants from horse danders such as Equ cl (eg SwissProt Accession No Q95182), Equ c 2.0101 (eg SwissProt Accession No P81216) and Equ c 2.0102 (eg SwissProt Accession No P81217).
Cockroach allergens
Any form of cockroach antigen or antigenic determinant or any polynucleotide coding for a cockroach antigen or antigenic determinant (including any biologically active fragment, derivative, homologue or variant) may be used, such as allergens from Blatella and Periplanta, including but not limited to polypeptide or polynucleotide sequences of: Blag 1.0101 (eg SwissProt Accession No Q9UAM5), Bla g 1.02 (eg SwissProt Accession No 096522), Bla g 2 (eg SwissProt Accession No P54958), Bla g 4 (eg SwissProt Accession No P54962), Bla g 5 (eg SwissProt Accession No 018598) Per a 1.0104 (eg SwissProt Accession No 018528), Per a 1.02 (eg SwissProt Accession No 018527), Per a 1.0101 (eg SwissProt Accession No Q9TZR6), Per a 3 (eg SwissProt Accession No Q25641), Per a 1.0102 (eg SwissProt Accession No 018535), Per a 1 (eg SwissProt Accession No O18530) and Per a 7 (eg SwissProt Accession No Q9UB83).
Venom allergens
Venom allergens include all types of allergens found in venoms, especially insect venoms. Common types of venom allergen include, for example enzyme inhibitors such as melittin and venom enzymes such as phospholipases, hyaluronidases, and diphosphatases. Any form of venom antigen or antigenic determinant or any polynucleotide coding for a venom antigen or antigenic determinant (including any biologically active fragment, derivative, homologue or variant) may be used, including but not limited to polypeptide or polynucleotide sequences ofthe following:
Bee venom allergens such as allergens from the honey bee and bumble bee, for example Api m 1 (eg SwissProt Accession No P00630), Api m 2 (eg SwissProt Accession No Q08169), Api m 3 (eg SwissProt Accession No P01501) and Bom 1 1 (eg SwissProt Accession No P82971);
Hornet venom allergens from hornets such as, for example, the European Hornet, D. arenaria, D. maculata, Vespa crabro and Vespa mandarinia, for example Ves c 5.01 (eg SwissProt Accession No P35781), Ves c 5.02 (eg SwissProt Accession No P35782), Dol a 5 (eg SwissProt Accession No Q05108), Dol m 1.01 (eg SwissProt Accession No Q06478), Dol m 1.02 (eg SwissProt Accession No P53357), Dol m 2 (eg SwissProt
Accession No P49371), Dol m 5.01 (eg SwissProt Accession No PI 0736), Dol m 5.02 (eg SwissProt Accession No P10737), Vesp c 5.01 (eg SwissProt Accession No P35781), Vesp c 5.02 (eg SwissProt Accession No P35782)Nesp m 5 (eg SwissProt Accession No P81657);
Ant venom allergens from ants such as, for example, common ants and the fire ants S. invicta, S. richteri and S. geminata , for example Myr p 1 (eg SwissProt Accession No Q07932), Myr p 2 (eg SwissProt Accession No Q26464), Sol i 2 (eg SwissProt Accession No P35775), Sol i 3 (eg SwissProt Accession No P35778), Sol i 4 (eg SwissProt Accession No P35777), Sol j 4 (eg Entrez Accession No AAC97369), Sol r 2 (eg SwissProt Accession No P35776), Sol r 3 (eg SwissProt Accession No P35779), Sol g 4 (eg SwissProt Accession No Q9NH75).
Mosquito venom allergens from mosquitos such as Aedes aegypti, for example Aed a l(eg SwissProt Accession No P50635), Aed a 2 (eg SwissProt Accession No P18153) and Aed a 3 (eg SwissProt Accession No O01949). Wasp venom allergens from wasps such as P. annularis, P. dominulus, P. exclamans and P. fascatus, such as Pol a 5 (eg SwissProt Accession No Q05109), Pol a 1 (eg SwissProt Accession No Q9U6W0) Pol a 2 (eg SwissProt Accession No Q9U6V9), Pol d 5 (eg SwissProt Accession No P81656), Pol e 5 (eg SwissProt Accession No P35759) and Pol f 5 (eg SwissProt Accession No P35780);
Yellow jacket venom allergens from yellow jackets such as V. flavopilosa, V. germanica, V. maculifrons, V. pensylvanica, V. squamosa, V. vidua and V. vulgaris, such as Ves f 5 (eg SwissProt Accession No P35783), Ves g 5 (eg SwissProt Accession No P35784), Ves ml (eg SwissProt Accession No P51528), Ves m 5 (eg SwissProt Accession No P35760), Ves p 5 (eg SwissProt Accession No P35785), Ves s 5 (eg SwissProt Accession No P35786), Ves vi 5 (eg SwissProt Accession No P35787), Ves v 1 (eg SwissProt Accession No P49369), Ves v 2 (eg SwissProt Accession No P49370) and Ves v 5 (eg SwissProt Accession No Q05110).
Fungal allergens
Fungal allergens include all types of allergens originating with fungi. Common examples include, for example, ribosomal proteins, heat shock proteins and enzymes (such as proteases, enolases, alcohol dehydrogenases and superoxide dismutases (SODs)). Fungi may include, for example, strains of Alternaria, Aspergillus, Candida, Cladosporium, Fusarium, Penicillium and Trichophyton.
Any form of fungal antigen or antigenic determinant or any polynucleotide coding for a fungal antigen or antigenic determinant (including any biologically active fragment, derivative, homologue or variant) may be used in the present invention, including but not limited to polypeptide or polynucleotide sequences ofthe following:
antigens or antigenic determinants from Alternaria alternata such as Alt a 1 (eg SwissProt Accession Nos P79085, Q00021), Alt a 2 (eg SwissProt Accession No O94095), Alt a 3 (eg SwissProt Accession No P78983), Alt a 6 (eg SwissProt Accession No P42037) Alt a 7 (eg SwissProt Accession No P42058), Alt a 10 (eg SwissProt Accession No P42041), Alt a 11 (eg SwissProt Accession No Q9HDT3), Alt a 12 (eg SwissProt Accession No P49148);
antigens or antigenic determinants from Aspergillus mitogillin such as Asp f 1 (eg SwissProt Accession Nos P04389, P82261, O60023, Q9P4F0), Asp f 2 (eg SwissProt Accession Nos P79017, P82262), Asp f 3 (eg SwissProt Accession Nos O43099, P82263, O43099), Asp f 4 (eg SwissProt Accession No O60024), Asp f 6 (eg SwissProt Accession No Q92450), Asp f 7 (eg SwissProt Accession No 042799), Asp f 8 (eg SwissProt Accession No Q9UUZ6), Asp f 9 (eg SwissProt Accession No O42800), Asp f 13 (eg SwissProt Accession No O60022), Asp 1 1 (eg SwissProt Accession No P82257), Asp f 11 (eg SwissProt Accession No Q9Y7F6), Asp 1 2 (eg SwissProt Accession No P82258), Asp 1 3 (eg SwissProt Accession No P82259) or Asp fl 1 (eg SwissProt Accession No Q9UVU3);
antigens or antigenic determinants from Candida such as Can a 1 (eg SwissProt Accession No P43067); antigens or antigenic determinants from Cladosporium such as Cla h 6 (eg SwissProt Accession No P42040), Cla h 3 (eg SwissProt Accession No P40108), Cla h 4 (eg SwissProt Accession No P42039), Cla h 5 (eg SwissProt Accession No P42059) or Cla h 12 (eg SwissProt Accession No P50344);
antigens or antigenic determinants from Penicillium citrinum such as Pen c 19 (eg SwissProt Accession No Q92260) or Pen c 2 (eg SwissProt Accession No Q9Y755);
antigens or antigenic determinants from Penicillium notatum such as Pen n 13 (eg PIR Accession No JC7208 );
antigens or antigenic determinants from Penicillium oxalicum such as Pen o 18 (eg ENTREZ Accession No AAG44478);
antigens or antigenic determinants from Trichophyton such as Tri r 4 (eg SwissProt Accession No Q9UW98) or Tri r 2 (eg SwissProt Accession No Q9LTW97). Drug allergens
Drugs or drug-like agents capable of causing allergic reactions (drug allergens) include for example:
Antibiotics such as penicillins, sulphonamides, chloramphenicol, cephalosporins, neomycin, streptomycin, bacitracin, clindamycin, dapsone, cephalosporins and vancomycin; cardiovascular agents such as ACE inhibitors, quinidine, amiodarone and methyldopa; anaesthetic drugs and muscle relaxants such as thiopentone and halothane; analgesic agents, for example moφhine derivatives such as moφhine, pethidine and codeine; anti- inflammatory drugs such as diclofenac, ibuprofen and indomethacin; cancer chemotherapy drugs such as cisplatin, cyclophosphamide, methotrexate, bleomycin and cytarabine; antiseptics such as chlorhexidine, iodine and mercurochrome; solvents such as cremophor; vaccines such as tetanus toxoid and diphtheria vaccine; preservatives such as parabens, sulphites and benzalkonium chlorides; biological therapeutics such as erythropoietins (EPO), insulins, blood factors such as Factor VIII, therapeutic antibodies (eg anti-TNF antibodies) and therapeutic enzymes (eg chymopapain and strep tokinase); dyes such as erythrosine and tartrazine; diagnostic agents such as fluoroscein and iodine contrast reagents; hormones such as ACTH, calcitonin, glucocorticoids, and insulins; antivenoms; serum albumins such as human serum albumin; and allergy immunotherapy vaccines.
It will be appreciated that combinations of such allergens and antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
In addition, it will be appreciated that modulation of an immune response to one particular antigen or antigenic determinant may also modulate responses to other similar antigens and antigenic determinants by operation of a "bystander effect" and/or by so-called epitope spreading or linked suppression. 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 (T-cell) receptor. Preferably the antigen used in the present invention is an immunogen.
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 (THl) is characterised by high IL-2 and IFNγ but low IL-4 production, whereas in humoral immunity (TH2) the pattern is low IL-2 and IFNγ but high IL-4, IL-5 and IL-13. Since the secretory pattern is modulated at the level of the secondary lymphoid organ or cells, then pharmacological manipulation of the specific TH cytokine pattern can influence the type and extent of the immune response generated.
The TH1-TH2 balance refers to the relative representation of the two different forms of helper T cells. The two forms have large scale and opposing effects on the immune system. If an immune response favours THl 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 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.
The antigen moiety may be, for example, a synthetic MHC-peptide complex i.e. a fragment of the MHC molecule bearing the antigen groove bearing an element of the antigen. Such complexes have been described in Altman et al. (1996) Science 274: 94-96.
Graft (transplant) antigens The term "graft antigen" as used herein means an antigen or antigenic determinant from a graft which is at least partly responsible for immune response against the graft, and in extreme cases contributes to graft rejection. Typically, a graft antigen will be a Type I or Type II MHC antigen (especially HLA antigen) present on cells ofthe graft.
Combination treatments
Combination treatments wherein active agents ofthe present invention are administered in combination with other active agents, antigens or antigenic determinants are also within the scope ofthe present invention.
By "simultaneously" is meant that the active agents are administered at substantially the same time, and preferably together in the same formulation.
By "contemporaneously" it is meant that the active agents are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the agents 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 agents are preferably 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.
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 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 known manner, treated/incubated ex vivo in the manner of the present invention, and then administered to a patient. In addition, it will be appreciated that a combination of routes of administration may be employed if desired. For example, where appropriate one component (such as the modulator of Notch signalling) may be administered ex-vivo and the other may be administered in vivo, or vice versa.
Tolerisation assays
Any of the assays described above (see "Assays") can be adapted to monitor or to detect reduced reactivity (eg reduation of immune responses) and increased tolerisation in immune cells for use in clinical applications. Such assays will involve, for example, detecting increased Notch-ligand expression or activity in host cells or monitoring Notch cleavage in donor cells. Further methods of monitoring immune cell activity are set out below.
Immune cell activity may be monitored by any suitable method known to those skilled in the art. For example, cytotoxic activity may be monitored. Natural killer (NK) 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: III, 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 incoφorated 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 conelates with the development of cytotoxicity and because the appearance of Leu 23 on certain T-cells conelates 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 ppl87-376; 'Cytokines of the Immune Response' Chapter 7 in "Handbook of Immune Response Genes". Mak T.W. and J.J.L. Simard 1998, which are incoφorated herein by reference.
The present invention will now further be described with reference to the following non- limiting Examples: Example 1
CD4+ cell purification
Spleens were removed from mice (variously Balb/c females, 8-10 weeks, C57B/6 females, 8- 10 weeks, CARDl females, 8-10 weeks (D011.10 transgenic, CAR transgenic)) and passed through a 0.2μM cell strainer into 20ml R10F medium (RIOF-RPMI 1640 media (Gibco Cat No 22409) plus 2mM L-glutamine, 50μg/ml Penicillin, 50μg/ml Streptomycin, 5 x IO"5 M β-mercapto-ethanol in 10% fetal calf serum). The cell suspension was spun (1150φm 5min) and the media removed.
The cells were incubated for 4 minutes with 5ml ACK lysis buffer (0.15M NH4C1, 1.0M KHC03, O.lmM Na EDTA 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.
Example 2
Antibody Coating
The following protocols were used for coating 96 well flat-bottomed plates with antibodies.
A) The plates were coated with Dulbecco's Phosphate Buffered Saline (DPBS) plus lμg/ml anti-CD3 antibody (Pharmingen, San Diego, US: Cat No 553058, Clone No 145-2C11) plus lμg/ml anti-IgG4 antibody (Pharmingen Cat No 555878). lOOμl of coating mixture was used per well. Plates were incubated overnight at 4°C then washed with DPBS. Each well then received either lOOμl DPBS or lOOμl DPBS plus lOμg/ml Notch ligand (mouse Delta 1 extracellular domain/Ig4Fc fusion protein; Fc-delta). The plates were incubated for 2-3 hours at 37°C then washed again with DPBS before cells (prepared as in Example 1) were added.
B) Alternatively, the plates were coated with DPBS plus lμg/ml anti-hamsterlgG antibody (Pharmingen Cat No 554007) plus lμg/ml anti-IgG4 antibody. lOOμl of coating mixture was added per well. Plates were incubated overnight at 4°C then washed with DPBS. Each well then received either lOOμl DPBS plus anti-CD3 antibody (lμg/ml) or, lOOμl DPBS plus anti-CD3 antibody (lμg/ml) plus Fc-delta (lOμg/ml). The plates were incubated for 2-3 hours at 37°C then washed again with DPBS before cells (prepared as in Example 1) were added.
Example 3
Primary Polyclonal Stimulation
CD4+ cells were cultured in 96 well, flat-bottomed plates pre-coated according to Example 2 (A) or 2 (B). Cells were re-suspended, following counting, at 2 x 10° /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-10, IFNg and IL-13using antibody pairs from R & D Systems (Abingdon, UK). The cells were then split 1 in 3 into new wells (not coated) and fed with R10F medium plus recombinant human IL-2 (2.5ng/ml, PeproTech Inc, London, UK: Cat No 200-02).
Results are shown in Figure 9. -196A-
Reducing immune pathology in infectious disease
Immune pathology may result from an inappropriate or excessive host response. Nonspecific immune responses such as inflamation provide an essential first line of host defense against many pathogens, and are subsequently driven and refined by the activities of antigen- specific T-cell responses, which serve to recruit the most effective inflammatory cells to deal with the pathogen. Normally this is a transient phase, which terminates once the host has acquired specific effector mechanisms such as antibodies or cytotoxic T lymphocytes and eliminated the organism. In some cases however, especially with chronic pathogenic infections, these non-specific responses may be excessive or persist to an inappropriate degree, perhaps without significantly reducing the infection; in these situations the vigorous immune response may become counteφroductive. In these scenarios, it may be appropriate to reduce the inappropriate immune response to relieve symptoms in the patient. The infection may then clear over time or where necessary may be further treated for example with anti-infective drugs.
Thus, in a further aspect of the invention there is provided a product comprising i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling (preferably being Notch receptor activators); and ii) a pathogen antigen or antigenic determinant, or a polynucleotide coding for a pathogen antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for reduction of an immune response to said pathogen antigen or antigenic determinant.
In a further aspect there is provided a method for reducing an immune response to a pathogen antigen or antigenic determinant in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering in vivo, in either order: i) an effective amount of a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling (preferably being Notch receptor activators); and ii) an effective amount of a pathogen antigen or antigenic determinant, or a polynucleotide coding for a pathogen antigen or antigenic determinant. -197-
Example 4
Real Time PCR analysis of primary stimulated CD4+ cells
Murine (Balb/c) stimulated CD4+ T-cells from Example 3 were harvested at 4, 16 and 24 hours. Total cellular RNA was isolated using the RNeasy™ RNA isolation kit (Qiagen, Crawley, UK) according to the manufacturer's guidelines.
In each case 1 μg of total RNA was reverse transcribed using Superscript™ II Reverse Transcriptase (Invitrogen, Paisley, UK) using Oligo dT(]2-ι8) or a random decamer mix according to the manufacturer's guidelines. After synthesis, Oligo dT(] -ι8)- 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 18s rRNA: Forward GTAACCCGTTGAACCCCATT
Reverse CCATCCAATCGGTAGTAGCG
mouse Hes-1: Forward GGTGCTGATAACAGCGGAAT
Reverse ATTTTTGGAATCCTTCACGC
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. Hes-1 was then analysed by the same method using serially-diluted Hes-1 -specific -198-
standards, and the Hes-1 value divided by the 18S rRNA value to generate a value, which represents the relative expression of Hes-1 in each cDNA sample. All Cp analysis was performed using the Second Derivative Maximum algorithm within the Lightcycler system software.
Results (HES-1 expression relative to 18S rRNA expression with and without Fc-delta) are shown in Figure 10.
Example 5
Screening under polarising conditions
Plates were coated and CD4+ cells added as in Example 2 (A).
The procedure of Example 3 was then followed, except that instead of adding lOOμl R10F medium per well as in Example 3, lOOμl of polarising cocktail was added per well as follows:
Un-polarised cells: R10F medium.
Thl polarised cells: R10F medium plus anti-IL-4 antibody (lOμg/ml, Pharmingen Cat No 554432) plus IL-12 (lOng/ml, Peprotech 210-12). Th2 polarised cells: RlOFmedium plus anti-IL-12 antibody (lOμg/ml, Pharmingen Cat No 554475) plus anti-IFNg antibody (lμg/ml, Pharmingen Cat No 554408) plus IL-4 (lOng/ml, Peprotech Cat No 214-14).
Cells were then stimulated and cytokines (IL-10, IFNγ and IL-13) measured by ELISA as described in Example 3. Results are shown in Figure 11.
Example 6 -199-
Soluble Ligand
The procedure of Example 2(A) (with the modification that ligand was not added to the plate) and Example 3 (with the modification that soluble Fc-delta was added with the R10F medium) was used to compare soluble Fc-delta with plate-bound Fc-delta against controls. Results are shown in Figure 12.
Example 7
Secondary stimulation
7 days after primary stimulation all cells were harvested and counted then stimulated in one of three ways as follows:
Re-stimulation Cells were re-stimulated exactly as for primary stimulation (Example 3).
Re-challenge on anti-CD3/CD28
96-well flat-bottomed plates were coated with PBS plus lμg/ml anti-CD3 antibody. The plates were incubated overnight at 4°C then washed with DPBS.
The cells were re-suspended at 2 x 10° /ml in R10F medium plus anti-CD28 antibody
(4μg/ml). lOOμl cell suspension was added per well. lOOμl of R10F medium was then added per 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. After 72 hours supematants were removed for ELISA as described in Example 3 (primary stimulation).
Re-stimulation with APC plus anti-CD3
Primary stimulated cells from Example 3 were harvested after 7 days and restimulated with APCs ofthe same strain (2 x IO4 per well) plus anti-CD3 antibody. -200-
Mouse spleen cells were isolated as described in Example 1 up to the counting step. Thy- 1.2 antibody-binding cells were then removed on a MACS column and the flowthrough was recovered and treated with mitomycin-C for 45 minutes then added to a 96 well plate in lOOμl R10F medium with equal numbers of cells from Example 3 and 0.5 μg/ml anti-CD3 antibody.
Cell proliferation was measured using a kit from Roche Molecular Biochemicals, Cell Proliferation ELISA, BrdU (chemiluminescent) 1 669 915, according to the manufacturer's instructions. Plates were pulsed at 72 hours and read on a luminometer.
Cytokines (IL-10 and IFN-γ) were measured as described in Example 3. Results are shown in Figure 13.
Example 8
CHO-N2 (N27) Luciferase Reporter Assay
A) Construction of Luciferase Reporter Plasmid lOxCBFl-Luc (pLOR91)
An adenovirus major late promoter TATA-box motif with Bglll and Hindlll cohesive ends was generated as follows:
Bglll Hindlll
GATCTGGGGGGCTATAAAAGGGGGTA ACCCCCCGATATTTTCCCCCATTCGA
This was cloned into plasmid pGL3 -Basic (Promega) between the Bgill and Hindlll sites to generate plasmid pGL3-AdTATA.
A TPl promoter sequence (TPl; equivalent to 2 CBFl repeats) with BamHl and Bglll cohesive ends was generated as follows: -201-
BamHl Bgl l l
5' GATCCCGACTCGTGGGAAAATGGGCGGAAGGGCACCGTGGGAAAATAGTA 3'
3' GGCTGAGCACCCTTTTACCCGCCTTCCCGTGGCACCCTTTTATCATCTAG 5'
This sequence was pentamerised by repeated insertion into a Bglll site and the resulting TPl pentamer (equivalent to 10 CBFl repeats) was inserted into pGL3-AdTATA at the Bglll site to generate plasmid pLOR91.
B) Generation of a stable CHO cell reporter cell line expressing full length Notch2 and the lOxCBFl-Luc reporter cassette
A cDNA clone spanning the complete coding sequence of the human Notch2 gene (see, eg GenBank Accession No AF315356) was constructed as follows. A 3' cDNA fragment encoding the entire intracellular domain and a portion of the extracellular domain was isolated from a human placental cDNA library (OriGene Technologies Ltd., USA) using a PCR-based screening strategy. The remaining 5* coding sequence was isolated using a RACE (Rapid Amplification of cDNA Ends) strategy and ligated onto the existing 3' fragment using a unique restriction site common to both fragments (Cla I). The resulting full-length cDNA was then cloned into the mammalian expression vector pcDNA3.1-V5-HisA (Invitrogen) without a stop codon to generate plasmid pLOR92. When expressed in mammalian cells, pLOR92 thus expresses the full-length human Notch2 protein with V5 and His tags at the 3' end ofthe intracellular domain.
Wild-type CHO-Kl cells (eg see ATCC No CCL 61) were transfected with pLOR92 (pcDNA3.1-FLNotch2-V5-His) using Lipfectamine 2000™ (Invitrogen) to generate a stable CHO cell clone expressing full length human Notch2 (N2). Transfectant clones were selected in Dulbecco's Modified Eagle Medium (DMEM) plus 10%> heat inactivated fetal calf serum ((HI)FCS) plus glutamine plus Penicillin-Streptomycin (P/S) plus 1 mg/ml G418 (Geneticin™ - Invitrogen) in 96-well plates using limiting dilution. Individual colonies were expanded in DMEM plus 10%(HI)FCS plus glutamine plus P/S plus 0.5 mg/ml G418. Clones were tested for expression of N2 by Western blots of cell lysates using an anti-V5 -202-
monoclonal antibody (Invitrogen). Positive clones were then tested by transient transfection with the reporter vector pLOR91 (lOxCBFl-Luc) and co-culture with a stable CHO cell clone (CHO-Delta) expressing full length human delta-like ligand 1 (DLLl; eg see GenBank Accession No AF196571). (CHO-Delta was prepared in the same way as the CHO Notch 2 clone, but with human DLLl used in place of Notch 2. A strongly positive clone was selected by Western blots of cell lysates with anti-V5 mAb.)
One CHO-N2 stable clone, N27, was found to give high levels of induction when transiently transfected with pLOR91 (lOxCBFl-Luc) and co-cultured with the stable CHO cell clone expressing full length human DLLl (CHO-Deltal). A hygromycin gene cassette (obtainable from pcDNA3.1/hygro, Invitrogen) was inserted into pLOR91 (lOxCBFl-Luc) using BamHl and Sail and this vector (lOxCBFl-Luc-hygro) was transfected into the CHO-N2 stable clone (N27) using Lipfectamine 2000 (Invitrogen). Transfectant clones were selected in DMEM plus 10%>(HI)FCS plus glutamine plus P/S plus 0.4 mg/ml hygromycin B (Invitrogen) plus 0.5 mg/ml G418 (Invitrogen) in 96-well plates using limiting dilution. Individual colonies were expanded in DMEM plus 10%>(HI)FCS plus glutamine plus P/S + 0.2 mg/ml hygromycin B plus 0.5 mg/ml G418 (Invitrogen).
Clones were tested by co-culture with a stable CHO cell clone expressing FL human DLLl. Three stable reporter cell lines were produced N27#l l, N27#17 and N27#36. N27#l l was selected for further use because of its low background signal in the absence of Notch signalling, and hence high fold induction when signalling is initiated. Assays were set up in 96-well plates with 2 x IO4 N27#l l cells per well in 100 μl per well of DMEM plus 10%(HI)FCS plus glutamine plus P/S.
C) Transient Transfection of CHO-N2 Cells with lOxCBFl-Luc
Alternatively, for transient transfection, CHO-N2 (Clone N27) cells were maintained in DMEM plus 10%(HI)FCS plus glutamine plus P/S plus 0.5 mg/ml G418 and a T80 flask of the CHO-N2 cells was transfected as follows. The medium on the cells was replaced with 8 -203-
ml of fresh in DMEM plus 10%(HI)FCS plus glutamine plus P/S. In a sterile bijou 10 μg of pLOR91 (lOxCBFl-Luc) was added to OptiMem (Invitrogen) to give a final volume of 1 ml and mixed. In a second sterile bijou 20 μl of Lipofectamine 2000 reagent was added to 980 μl of OptiMem and mixed.
The contents of each bijou were mixed and left at room temperature for 20 minutes. The 2 ml of transfection mixture was added to the flask of cells containing 8 ml of medium and the resulting mixture was left in a CO2 incubator overnight before removing the transfected cells and adding to the 96-well plate containing the immobilised Notch ligand protein.
The following day the transfected CHO-N2 cells were removed using 0.02%> EDTA solution (Sigma), spun down and resuspended in 10 ml DMEM plus 10%(HI)FCS plus glutamine plus P/S. 10 μl of cells were counted and the cell density was adjusted to 2.0 x 105 cells/ml with fresh DMEM plus 10%(HI)FCS plus glutamine plus P/S. 100 μl per well was added to a 96-well tissue culture plate (flat bottom), i.e. 2.0 x IO4 transfected cells per well, using a multi-channel pipette and the plate was then incubated overnight.
D) Immobilisation of Notch Ligand protein directly onto a 96-well Tissue Culture Plate
10 μg of purified Notch ligand protein was added to sterile PBS in a sterile Eppendorf tube to give a final volume of 1 ml. Serial 1 :2 dilutions were made by adding 500 μl into sterile Eppendorf tubes containing 500 μl of sterile PBS to generate dilutions of 10 μg/ml, 5 μg/ml, 2.5 μg/ml, 1.25 μg/ml, 0.625 μg/ml and 0 μg/ml.
The lid of the plate was sealed with parafilm and the plate was left at 4 °C overnight or at 37 °C for 2 hours. The protein was then removed and the plate was washed with 200 μl of PBS.
E) A20-Delta cells
The IVS, IRES, Neo and pA elements were removed from plasmid pIRESneo2 (Clontech, USA) and inserted into a pUC cloning vector downstream of a chicken beta-actin promoter -204-
(eg see GenBank Accession No E02199). Mouse Delta-1 (eg see GenBank Accession No NM_007865) was inserted between the actin promoter and IVS elements and a sequence with multiple stop codons in all three reading frames was inserted between the Delta and IVS elements.
The resulting construct was transfected into A20 cells using electroporation and G418 to provide A20 cells expressing mouse Deltal on their surfaces (A20-Delta).
F) CHO and CHO-hDeltal-V5-His Assay Control
CHO cells were maintained in DMEM plus 10%>(HI)FCS plus glutamine plus P/S and CHO-hDeltal-V5-His (clone#10) cells were maintained in DMEM plus 10%(HI)FCS plus glutamine plus P/S plus 0.5mg/ml G418.
Cells were removed using 0.02%> EDTA solution (Sigma), spun down and resuspended in 10 ml DMEM plus 10%(HI)FCS plus glutamine plus P/S. 10 μl of cells were counted and the cell density was adjusted to 5.0 x 105 cells/ml with fresh DMEM plus 10%(HI)FCS plus glutamine plus P/S. 300 μl of each cell line at 5.0 x 105 cells/ml was added into duplicate wells of a 96-well tissue culture plate. 150 μl of DMEM plus 10%(HI)FCS plus glutamine plus P/S was added in to the next 5 wells below each well. 150 μl of cells were serially diluted into the next 4 wells giving cell density dilution of 5.0 x 105 cells/ml, 2.5 x 105 cells/ml, 1.25 x IO5 cells/ml, 0.625 x IO5 cells/ml, 0.3125 x 105 cells/ml and 0 cells/ml.
100 μl from each well was added into the 96-well plate containing 100 μl of CHO-N2 cells transfected with lOxCBFl-Luc (2.0 xlO4 transfected CHO-N2 cells/well) and the plate was left in an incubator overnight.
G) Cell Co-Culture -205-
5 x IO4 CHO-N2 cells were plated on a 96 well plate. CHO-Delta or A20-Delta cells were titrated in as required (max ratio CHO-N2: CHO-Delta was 1 :1, max ratio CHO-N2: A20- Delta was 1 :2). The mixture was incubated overnight before conducting a luciferase assay.
H) Luciferase Assay
Supernatant was removed from all wells. 100 μl of PBS and 100 μl of SteadyGlo™ luciferase assay reagent (Promega) was added and the cells were left at room temperature for 5 minutes. The mixture was pipetted up and down 2 times to ensure cell lysis and contents from each well were transfened into a white 96-well OptiPlate™ (Packard). Luminescence was measured in a TopCount™ counter (Packard).
Results of sample assays (using the stable CHO-Notch2-10xCBFl-Luc reporter cell line described above with (A) plate-immobilised human Delta- l/Ig4Fc fusion protein, (B) plate- immobilised mouse Delta-l/Ig4Fc fusion protein, (C) CHO / CHO-human Deltal co- cultured cells and (D) A20/ A20-mouse Deltal co-cultured cells as actives against conesponding controls) are shown in Figures 14 A to D.
Example 9
Dynabeads Luciferase Assay Method For Detecting Notch Ligand Activity
Fc-tagged Notch ligands were 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:
2.5 x IO7 beads (62.5 μl of beads at 4.0 x 108 beads/ml) and 5 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 φm 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 shaker at room -206-
temperature for 30 minutes. PBS to was added to 1 ml and the mixture was spun down at 13,000 φm for 1 minute and then washed twice more with 1 ml of PBS.
The mixture was then spun down at 13,000 φm for 1 minute and the beads were resupsended in a 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 φm for 5 minutes at room temperature.
The beads then were washed with 10 ml of PBS, spun down, resupended in 1 ml of PBS, transfened to a sterile Eppendorf tube, washed with a further 2 x 1 ml of PBS, spun down and resuspended beads in a final volume of 250 μl of DMEM plus 10%(HI)FCS plus glutamine plus P/S, i.e. at 1.0 x 105 beads/μl.
Stable N27#l l cells from Example 8 (T80 flask) were removed using 0.02% EDTA solution (Sigma), spun down and resuspended in 10 ml DMEM plus 10%(HI)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 IO5 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.
100 μl of beads were then added in duplicate to the first pair of wells to give 1.0 x IO7 beads / well (100 beads / cell); 20 μl of beads added in duplicate to the second pair of wells to give 2.0 x 106 beads / well (20 beads / cell); 4 μl of beads added in duplicate to the third pair of wells to give 4.0 x IO5 beads / well (4 beads / cell) and 0 μl of beads added to the fourth pair of wells. The plate was left in a CO2 incubator overnight.
Luciferase assay
Supernatant was then removed from all the wells, 150 μl of PBS and 150 μl of SteadyGlo luciferase assay reagent (Promega) were added and the resulting mixture left at room temperature for 5 minutes. -207-
The mixture was then pipetted up and down 2 times to ensure cell lysis and the contents from each well were transfened into an Eppendorf tube, spun at 13,000 φm for 1 minute and the cleared supernatant was transfened to a white 96-well OptiPlate™ (Packard), leaving the bead pellet behind. Luminescence was then read in a TopCount™ (Packard) counter.
Example 10
Dynabeads ELISA Assay Method For Detecting Notch Ligand Activity
M450 Streptavidin Dynabeads were coated with anti-hamster-IgGl biotinylated monoclonal antibody, anti-human-IgG4 biotinylated monoclonal antibody or both antibodies and rotated for 2 hours at room temperature.
Beads were washed three times with PBS (1ml). The anti-hamster-IgGl beads were then further incubated with anti-CD3ε chain monoclonal antibody, the anti-human-IgG4 beads were further incubated with Fc-Delta, and the double coated beads incubated with both anti- CD3ε chain monoclonal antibody and Fc-Delta. Beads were rotated overnight at 4°C, washed three times with PBS (1ml) and resuspended.
T-cell assays were carried out with CD4+ T-cells and the beads. Supematants were removed after 72 hours and cytokines measured by ELISA as described in Example 3. Results are shown in Figure 15.
Example 11
Modulation of cytokine production by human CD4+ T cells in the presence of Deltal- h!gG4 immobilised on Dynal microbeads.
Human peripheral blood mononuclear cells (PBMC) were purified from blood using Ficoll- Paque separation medium (Pharmacia). Briefly, 28 ml of blood were overlaid on 21 ml of -208-
Ficoll-Paque separation medium and centrifuged at 18-20°C for 40 minutes at 400g. PBMC were recovered from the interface and washed 3 times before use for CD4+ T cell purification.
Human CD4+ T cells were isolated by positive selection using anti-CD4 microbeads from Miltenyi Biotech according to the manufacturer's instructions.
The CD4+ T cells were incubated in triplicates in a 96-well-plate (flat bottom) at 105 CD4/well/200μl in RPMI medium containing 10%> FCS, glutamine, penicillin, streptomycin and β2-mercaptoethanol.
Cytokine production was induced by stimulating the cells with anti-CD3/CD28 T cell expander beads from Dynal at a 1 :1 ratio (bead/cell) or plate bound anti-CD3 (clone UCHT1, BD Biosciences, 5μg/ml) and soluble anti-CD28 (clone CD28.2, BD Biosciences, 2μg/ml). Beads coated with mouse DeltalEC domain-hIgG4 fusion protein (prepared as described above with the modifications that incubation with human IgG4 was for 30-40 minutes at room temperature and incubation with Delta-Fc was for two hours at room temperature) or control beads were added in some of the wells at a 10:1 ratio (beads/cell). The supematants were removed after 3 or 4 days of incubation at 37°C/ 5%>CO2/humidified atmosphere and cytokine production was evaluated by ELISA using Pharmingen kits OptEIA Set human IL10 (catalog No. 555157), OptEIA Set human IL-5 (catalog No. 555202) and OptEIA Set human IFNg (catalog No 555142) for IL-10, IL-5 and IFNg respectively and a human TNFa DuoSet from R&D Systems (catalog. No. DY210) for TNFa according to the manufacturer's instructions.
Results are shown in Figures 16 to 18.
Example 12
Variation of bead: cell ratios -209-
The procedure of Example 11 was repeated except that the ratio of control beads to cells and mouse Deltal-hIgG4 fusion protein coated beads to cells was varied between 16:1 and 0.25:1 (variously 16:1, 8:1, 4:1, 2:1, 1 :1, 0.5:1, 0.25:1) and human Deltal-hIgG4 fusion protein coated beads were also used at the same ratios for comparison.
Results are shown in Figure 19.
Example 13
Comparison of CD45RO+ (memory cells) and CD45RO- (naive cells)
The procedure of Example 11 was repeated except that prior to the stimulation the human CD4+ were separated into CD45RO+ (memory cells) and CD45RO- (naive cells, data not shown on the slide). The magnetic separation was done using anti-CD4 Multisort microbeads (cat.No. 551-01) and then anti-CD45RO microbeads (cat.No.460-01) supplied by Miltenyi Biotech and following Miltenyi's protocol.
Results are shown in Figure 20.
Example 14
Measurement of cytokine production in stimulated mouse CD4+ cells under polarising conditions
(i) CD4+ cell purification
Spleens were removed from mice (variously Balb/c females, 8-10 weeks, C57B/6 females, 8-
10 weeks, CARDl females, 8-10 weeks (DO 11.10 transgenic, CAR transgenic)) 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 -210-
β-mercapto-ethanol in 10%> fetal calf serum). The cell suspension was spun (1150φm 5min) and the media removed.
The cells were incubated for 4 minutes with 5ml ACK lysis buffer (0.15M NH4C1, 1.0M KHC0 , 0. ImM 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 Dulbecco's Phosphate Buffered Saline (DPBS) plus lμg/ml anti-CD3 antibody (Pharmingen, San Diego, US: Cat No 553058, Clone No 145-2C11) plus lμg/ml anti-IgG4 antibody (Pharmingen Cat No 555878). lOOμl of coating mixture was used per well. Plates were incubated overnight at 4°C then washed with DPBS. Each well then received either lOOμl DPBS or lOOμl DPBS plus lOμg/ml Notch ligand (mouse Delta 1 extracellular domain/Ig4Fc fusion protein; Fc-delta). The plates were incubated for 2-3 hours at 37°C then washed again with DPBS before cells (prepared as in (i)) were added.
(iii) Primary Polyclonal Stimulation
CD4+ cells were cultured in 96 well, flat-bottomed plates pre-coated as in (ii) above. Cells were re-suspended, following counting, at 2 x IO6 /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 polarising or control medium was then added to each well to give a final volume of 200μl (2 x IO5 cells/well, anti-CD28 final concentration 2μg/ml) as follows:
Un-polarised cells: R10F medium.
Thl polarised cells: R10F medium plus anti-IL-4 antibody (lOμg/ml, Pharmingen Cat No 554432) plus IL-12 (lOng/ml, Peprotech 210-12). -211-
Th2 polarised cells: RlOFmedium plus anti-IL-12 antibody (lOμg/ml, Pharmingen Cat No 554475) plus anti-IFNg antibody (lμg/ml, Pharmingen Cat No 554408) plus IL-4 (lOng/ml, Peprotech Cat No 214-14).
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-10 and TNFa using antibody pairs from R & D Systems (Abingdon, UK). The cells were then split 1 in 3 into new wells (not coated) and fed with R10F medium plus recombinant human IL-2 (2.5ng/ml, PeproTech Inc, London, UK: Cat No 200-02).
Results are shown in Figure 21.
Example 15
i) Preparation of human Deltal-IgG4Fc Fusion Protein
A fusion protein comprising the extracellular domain of human Deltal fused to the Fc domain of human IgG4 (refened to herein as "hDeltal-IgG4Fc" and also refened to in the accompanying Figures as "D1E8G4" and "DlE8Fc4") 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.
i) Cloning
A 1622bp extracellular (EC) fragment of human Delta-like ligand 1 (hECDLL-1; see GenBank Accession No AF003522) was gel purified using a Qiagen QIAquick™ Gel Extraction Kit (cat 28706) according to the manufacturer's instructions (Qiagen, Valencia, CA, US). The fragment was then ligated into a pCR Blunt cloning vector (Invitrogen, UK) cut Hindlll - BsiWI, thus eliminating a Hindlll, BsiWI and Apal site. -212-
The ligation was transformed into DH5α cells, streaked onto LB + Kanamycin (30ug/ml) plates and incubated at 37°C overnight. Colonies were picked from the plates into 3ml LB + Kanamycin (30ugmr') and grown up overnight at 37°C. Plasmid DNA was purified from the cultures using a Qiagen Qiaquick Spin Miniprep kit (cat 27106) according to the manufacturer's instructions, then diagnostically digested with Hindlll. A clone was chosen and streaked onto an LB + Kanamycin (30ug/ml) plate with the glycerol stock of modified pCRBlunt-hECDLL-1 and incubated at 37°C overnight. A colony was picked off this plate into 60ml LB + Kanamycin (30ug/ml) and incubated at 37°C overnight. The culture was maxiprepped using a Clontech Nucleobond Maxi Kit (cat K3003-2) according to the manufacturer's instructions (Clontech, PaloAlto, CA, US), and the final DNA pellet was resuspended in 300ul dH2O and stored at -20°C.
5ug of modified pCR Blunt-hECDLL-1 vector was linearised with Hindlll and partially digested with Apal. The 1622bp hECDLL-1 fragment was then gel purified using a Clontech
Nucleospin® Extraction Kit (K3051-1) according to the manufacturer's instructions. The
DNA was then passed through another Clontech Nucleospin® column and followed the isolation from PCR protocol, concentration of sample was then checked by agarose gel analysis ready for ligation.
Plasmid pconγ (Lonza Biologies, UK) was cut with Hindlll - Apal and the following oligos were ligated in (SEQ ID NO: 1):
agcttgcggc cgcgggccca gcggtggtgg acctcactga gaagctagag gcttccacca aaggcc acgccg gcgcccgggt cgccaccacc tggagtgact cttcgatctc cgaaggtggt tt
The ligation was transformed into DH5α cells and LB + Amp (lOOug/ml) plates were streaked with 200ul ofthe transformation and incubated at 37°C overnight. The following day 12 clones were picked into 2 x YT + Ampicillin (lOOugmf1) and grown up at 37°C throughout the day. Plasmid DNA was purified from the cultures using a Qiagen Qiaquick Spin Miniprep kit (cat 27106) and diagnostically digested with Notl. A clone -213-
(designated "pDev41") was chosen and an LB + Amp (lOOug/ml) plate was streaked with the glycerol stock of pDev41 and incubated at 37°C overnight. The following day a clone was picked from this plate into 60ml LB + Amp (lOOug/ml) and incubated with shaking at 37°C overnight. The clone was maxiprepped using a Clontech Nucleobond Maxi Kit (cat K3003- 2) according to the manufacturer's instructions and stored at -20°C.
The pDev41 clone 5 maxiprep was then digested with Apal - EcoRI to generate the IgG4Fc fragment (1624bp). The digest was purified on a 1% agarose gel and the main band was cut out and purified using a Clontech Nucleospin Extraction Kit (K3051-1).
The polynucleotide was then cloned into the polylinker region of pEE14.4 (Lonza Biologies, UK) downstream ofthe strong hCMV promoter enhancer region (hCMV-MIE) and upstream of S V40 polyadenylation signal (encodes the GS gene required for selection in glutamine free media; contains the GS minigene - GS cDNA which includes the last intron and polylinker adenylation signals ofthe wild type hamster GS gene) which is under the control ofthe late SV40 promoter, has the hCMV promoter to drive transcription ofthe desired gene. 5ug ofthe maxiprep of pEE14.4 was digested with Hindlll - EcoRI, and the product was gel extracted and treated with alkaline phosphatase.
ii) Generation of Expression Constructs
A 3 fragment ligation was set up with pEE14.4 cut Hindlll - EcoRI, ECDLL-1 from modified pCR Blunt (Hindlll - Apal) and the IgG4Fc fragment cut from pDev41 (Apal - EcoRI). This was transformed into DH5 cells and LB + Amp (lOOug/ml) plates were streaked with 200ul ofthe transformation and incubated at 37C overnight. The following day 12 clones were picked into 2 x YT + Amp (lOOug/ml) and minipreps were grown up at 37°C throughout the day. Plasmid DNA was purified from the preps using a Qiagen Qiaquick spin miniprep kit (Cat No 27106), diagnostically digested (with EcoRI and Hindlll) and a clone (clone 8; designated "pDev44") was chosen for maxiprepping. The glycerol stock of pDev44 clone 8 was streaked onto an LB + Amp (lOOugmf1) plate and incubated at 37°C overnight. The following day a colony was picked into 60ml LB + Amp (lOOugmf1) broth and -214-
incubated at 37°C overnight. The plasmid DNA was isolated using a Clontech Nucleobond Maxiprep Kit (Cat K3003-2).
iii) Addition of optimal KOZAK Sequence
A Kozak sequence was inserted into the expression construct as follows. Oligonucleotides were kinase treated and annealed to generate the following sequences:
AGCTTGCCGCCACCATGGGCAGTCGGTGCGCGCTGGCCCTGGCGGTGCTC ACGGCGGTGGTACCCGTCAGCCACGCGCGACCGGGACCGC (SEQ ID NO: 2)
TCGGCCTTGCTGTGTCAGGTCTGGAGCTCTGGGGTGTT CACGAGAGCCGGAACGACACAGTCCAGACCTCGAGACCCCACAAGC (SEQ ID NO: 3)
pDev44 was digested with Hindlll - BstBI, gel purified and treated with alkaline phosphatase. The digest was ligated with the oligos, transformed into DH5α cells by heat shock . 200ul of each transformation were streaked onto LB + Amp plates (lOOug/ml) and incubated at 37°C overnight. Minipreps were grown up in 3 ml 2 x YT + Ampicillin (lOOugmf1). Plasmid DNA was purified from the minipreps using a Qiagen Qiaquick spin miniprep kit (Cat No 27106) and diagnostically digested with Ncol. A clone
(pDev46) was selected and the sequence was confirmed. The glycerol stock was streaked, broth grown up and the plasmid maxiprepped.
iv) Transfection
Approx lOOug pDev46 Clone 1 DNA was linearised with restriction enzyme Pvu I. The resulting DNA preparation was cleaned up using phenol/chloroform/IAA extraction followed by ethanol wash and precipitation. The pellets were resuspended in sterile water and linearisation and quantification was checked by agarose gel electrophoresis and UV spectrophotometry. -215-
40ug linearised DNA (pDev46 Clone 1) and 1 x IO7 CHO-Kl cells were mixed in serum free DMEM in a 4mm cuvette, at room temp. The cells were then electroporated at 975uF 280 volts, washed out into non-selective DMEM, diluted into 96 well plates and incubated. After 24 hours media were removed and replaced with selective media (25uM L-MSX). After 6 weeks media were removed and analysed by IgG4 sandwich ELISA. Selective media were replaced. Positive clones were identified and passaged in selective media 25um L-MSX.
v) Expression
Cells were grown in selective DMEM (25um L-MSX) until semi-confluent. The media was then replaced with serum free media (UltraCHO) for 3-5 days. Protein (hDeltal-IgG4Fc fusion protein) was purified from the resulting media by FPLC.
The amino acid sequence ofthe resulting expressed fusion protein was as follows (SEQ ID NO: 4):
MGSRCALALAVLSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCA CRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFG FTWPGTFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDL KYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICL PGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWG GLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDECDPSPC KNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCP VGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQAGFSGRHCDDNVDDCASS PCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCEHAPCHNGATCHERGHGYVC ECARGYGGPNCQFLLPELPPGPAVVDLTEKLEASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NNDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK Wherein the first underlined sequence is the predicted signal peptide (cleaved from the mature protein) and the second underlined sequence is the IgG4 Fc sequence. The protein -216-
normally exists as a dimer linked by cysteine disulphide bonds (see eg schematic representation in Figure 6).
Example 16
i) Preparation of Notch ligand extracellular domain fragment with free Cysteine tail for particle coupling
A protein fragment comprising amino acids 1 to 332 of human Delta 1 (DLL-1; for sequence see GenBank Accession No AF003522) and ending with a free cysteine residue ("DlE3cys") was prepared as follows:
A template containing the entire coding sequence for the extracellular (EC) domain of human DLL-1 (with two silent mutations) was prepared by a PCR cloning strategy from a placental cDNA library made from placental polyA+ RNA (Clontech; cat no 6518-1) and combined with a C-terminal V5HIS tag in a pCDNA3.1 plasmid (Invitrogen, UK) The template was cut Hindlll to Pmel to provide a fragment coding for the EC domain and this was used as a template for PCR using primers as follows:
5'-primer: CAC CAT GGG CAG TCG GTG CGC GCT GG (SEQ ID NO: 5)
3'-primer: GTC TAC GTT TAAACT TAA CAC TCG TCAATC CCC AGC TCG CAG GTG (SEQ IDNO: 6)
PCR was carried out using Pfu turbo polymerase (Stratagene, La Jolla, CA, US) with cycling conditions as follows: 95C 5min, 95C lmin, 45-69C lmin, 72C lmin for 25 cycles, 72C lOmin.
The products at 58C, 62C & 67C were purified from 1%> agarose gel in 1 x TAE using a Qiagen gel extraction kit according to the manufacturer's instructions, ligated into pCRIIblunt vector (InVitrogen TOPO-blunt kit) and then transformed into TOP 10 cells -217-
(InVitrogen). The resulting clone sequence was verified, and only the original two silent mutations were found to be present in the parental clone.
The resulting sequence coding for "DlE3Cys" was excised using Pmel and Hindlll, purified on 1%) agarose gel, lx TAE using a Qiagen gel extraction kit and ligated into pCDNA3.1V5HIS (Invitrogen) between the Pmel and Hindlll sites, thereby eliminating the V5HIS sequence. The resulting DNA was transformed into TOP 10 cells. The resulting clone sequence was verified at the 3 '-ligation site.
The DlE3Cys-coding fragment was excised from the pCDNA3.1 plasmid using Pmel and Hindlll. A pEE14.4 vector plasmid (Lonza Biologies, UK) was then restricted using EcoRI, and the 5 '-overhangs were filled in using Klenow fragment polymerase. The vector DNA was cleaned on a Qiagen PCR purification column, restricted using Hindlll, then treated with Shrimp Alkaline Phosphatase (Roche). The pEE14.4 vector and DlE3cys fragments were purified on 1%» agarose gel in 1 x TAE using a Qiagen gel extraction kit prior to ligation (T4 ligase) to give plasmid pEE14.4 DLLΔ4-8cys. The resulting clone sequence was verified.
The DlE3Cys coding sequence is as follows (SEQ ID NO: 7):
1 atgggcagtc ggtgcgcgct ggccctggcg gtgctctcgg ccttgctgtg
51 tcaggtctgg agctctgggg tgttcgaact gaagctgcag gagttcgtca
101 acaagaaggg gctgctgggg aaccgcaact gctgccgcgg gggcgcgggg
151 ccaccgccgt gcgcctgccg gaccttcttc cgcgtgtgcc tcaagcacta
201 ccaggccagc gtgtcccccg agccgccctg cacctacggc agcgccgtca 251 cccccgtgct gggcgtcgac tccttcagtc tgcccgacgg cgggggcgcc
301 gactccgcgt tcagcaaccc catccgcttc cccttcggct tcacctggcc
351 gggcaccttc tctctgatta ttgaagctct ccacacagat tctcctgatg
401 acctcgcaac agaaaaccca gaaagactca tcagccgcct ggccacccag
451 aggcacctga cggtgggcga ggagtggtcc caggacctgc acagcagcgg 501 ccgcacggac ctcaagtact cctaccgctt cgtgtgtgac gaacactact
551 acggagaggg ctgctccgtt ttctgccgtc cccgggacga tgccttcggc
601 cacttcacct gtggggagcg tggggagaaa gtgtgcaacc ctggctggaa
651 agggccctac tgcacagagc cgatctgcct gcctggatgt gatgagcagc
701 atggattttg tgacaaacca ggggaatgca agtgcagagt gggctggcag 751 ggccggtact gtgacgagtg tatccgctat ccaggctgtc tccatggcac
801 ctgccagcag ccctggcagt gcaactgcca ggaaggctgg gggggccttt
851 tctgcaacca ggacctgaac tactgcacac accataagcc ctgcaagaat -218-
901 ggagccacct gcaccaacac gggccagggg agctacactt gctcttgccg 951 gcctgggtac acaggtgcca cctgcgagct ggggattgac gagtgttaa
The DNA was prepared for stable cell line transfection selection in a Lonza GS system using a Qiagen endofree maxi-prep kit.
ii) Expression of DlE3Cys
Linearisation of DNA
The pEE14.4 DLLΔ4-8cys plasmid DNA from (i) above was linearised by restriction enzyme digestion with Pvul, and then cleaned up using phenol chloroform isoamyl alcohol (IAA), followed by ethanol precipitation. Plasmid DNA was checked on an agarose gel for linearisation, and spec'd at 260/280nm for quantity and quality of prep.
Transfection
CHO-Kl cells were seeded into 6 wells at 7.5 x 105 cells per well in 3ml media (DMEM 10%) FCS) 24hrs prior to transfection, giving 95% confluency on the day of transfection. Lipofectamine 2000 was used to transfect the cells using 5ug of linearised DNA. The transfection mix was left on the cell sheet for 5 Vi hours before replacing with 3ml semi- selective media (DMEM, 10% dFCS, GS) for overnight incubation.
At 24 hours post-transfection the media was changed to full selective media (DMEM (Dulbecco's Modified Eagle Medium), 10%odFCS (fetal calf serum), GS (glutamine synthase), 25uM L-MSX (methionine sulphoximine)) and incubated further.
Cells were plated into 96 wells at 10 5 cells per well on days 4 and 15 after transfection. -219-
96 well plates were screened under a microscope for growth 2 weeks post clonal plating. Single colonies were identified and scored for %> confluency. When colony size was >30%> media was removed and screened for expression by dot blot against anti -human-Delta- 1 antisera. High positives were confirmed by the presence of a 36kDa band reactive to anti- human-Delta- 1 antisera in PAGE Western blot of media.
Cells were expanded by passaging from 96 well to 6 well to T25 flask before freezing. The fastest growing positive clone (LC09 0001) was expanded for protein expression.
DlE3Cys expression and purification
T500 flasks were seeded with lx IO7 cells in 80ml of selective media. After 4 days incubation the media was removed, cell sheet rinsed with DPBS and 150ml of 325 media with GS supplement added to each flask. Flasks were incubated for 7 further days before harvesting. Harvest media was filtered through a 0.65- 0.45um filter to clarify prior to freezing. Frozen harvests were purified by FPLC as follows:
Frozen harvest was thawed and filtered. A 17ml Q Sepharose column was equilibrated in 0.1M Tris pH8 buffer, for 10 column volumes. The harvest was loaded onto the column using a PI pump set at 3ml/min, the flowthrough was collected into a separate container (this is a reverse purification - a lot ofthe BSA contaminant binds to the Q Sepharose FF and our target protein does not and hence remains in the flowthrough). The flowthrough was concentrated in a TFF rig using a lOkDa cut off filter cartridge, during concentration it was washed 3 x with 0.1M Sodium phosphate pH 7 buffer. The 500ml was concentrated down to 35ml, to a final concentration of 3mg/ml.
Samples were run on SDS PAGE reduced and non-reduced (gels are shown in Figure 22)
The amino acid sequence ofthe resulting expressed DlE3Cys protein was as follows (SEQ ID NO: 8):
MGSRCALALAVLSAL CQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTF -220-
FRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPG TFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRF VCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGF CDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQP QCNCQEGWGG FCNQDLNYCTHH KPCKNGATCTNTGQGSYTCSCRPGYTGATCELGIDEC
(wherein the sequence in italics is the leader peptide, the underlined sequence is the DSL domain, the bold sequences are the three EGF repeats, and the terminal Cys residue is shown bold underlined).
iii) Reduction of DlE3cys Protein
40μg DlE3Cys protein from (ii) above was made up to lOOμl to include lOOmM sodium phosphate pH 7.0 and 5mM EDTA. 2 volumes of immobilised TCEP (tris[2- carboxyethyljphosphine hydrochloride; Pierce, Rockford, IL, US, Cat No: 77712; previously washed 3 times 1ml lOOmM sodium phosphate pH 7.0) were added and the mixture was incubated for 30 minutes at room temperature, with rotating.
The resin was pelleted at room temperature in a micro uge (13,000 revs/min, 5 minutes) and the supernatant was transfened to a clean Eppendorf tube and stored on ice. Protein concentration was measured by Warburg-Christian method.
Example 17
Coating M-450 Epoxy Dynabeads with Notch Ligand proteins
Dynabeads M-450 Epoxy (Dynal, Cat. no. 140.01; 4.5 μm average diameter) are supplied by Dynal (Dynal Biotech, Oslo, Norway) as ethanol-washed beads in distilled water at 4 x 108 -221-
beads/ml. These beads are magnetic polystyrene beads that have a surface epoxy (glycidyl ether) reactive group which does not require further activation.
Proteins are adsorbed hydrophobically on initial coupling with covalent coupling of primary amine groups occurring after 24 h. Coupling reactions occur at neutral pH over a 24 h incubation time at a temperature between 4 °C and 37 °C.
The appropriate quantity of epoxy beads were washed in PBS using a magnet (3 x 1 ml).
Purified hDeltal-IgG4Fc fusion protein from Example 15 or DlE3Cys protein from Example 16 above was added (~ 5 μg per 107 beads typical starting concentration) to beads at a final concentration of 4 - 8 x 108 beads/ml.
In some cases a blocking protein was added to assist binding orientation as follows: The beads were incubated for 15-30 min at 4 - 37 °C, with shaking. 0.1 %> BSA was added as blocking protein (Dynal typically suggest 0.1-0.5%) BSA or HAS for antibody binding) The beads were then left for 16 - 20 h at 4 - 37 °C, depending on stability of protein, with shaking to ensure covalent coupling. Coated beads were washed x 3 with PBS/0.1 % BSA using a magnet. The addition of 0.1 % BSA in the wash buffer here ensures complete blocking ofthe beads after coating. Coated beads were stored at 4 °C.
The activity of the various beads was tested in a CHO-N2 stable reporter assay as described above (with parallel experiments using hIgG4-coated beads under the same conditions as control). CHO/CHO-hDl co-culture assays (using CHO cells expressing full length Delta 1 or native CHO cells in place of beads) were run at the same time as further controls. Results are shown in Figure 23.
Example 18
Coating M-270 Amine Dynabeads with Notch Ligand protein -222-
To activate, 1.5 x IO8 M-270 Amine Dynabeads (2.7 μm average diameter) were washed 3 times with 0.5ml lOOmM sodium phosphate pH 7.0, then made to 2.5 x 108/ml in lOOmM sodium phosphate pH 7.
Sulfo-SMCC (sulphosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1 -carboxylate; Pierce, Cat No: 22322) was added to 0.2 mg/ml and the mixture was incubated for 30 minutes at room temperature, with rotating. The beads were washed 3 times with 0.5ml lOOmM sodium phosphate pH 7.0, then made up to 1 x 109/ml in lOOmM sodium phosphate pH 7.
5 x 107 'activated' amine beads in lOOmM sodium phosphate pH7 were combined with 20ug TCEP reduced DlE3cys protein from Example 16 above and made up to 250 μl in sodium phosphate pH 7. The resulting mixture was incubated for 18 hours at 4C with rotation.
The resulting protein coupled beads were washed 3 times with 0.5ml DPBS, then made up to
2 x 108/ml in DPBS. Activity was measured using a CHO-Notch 2 reporter assay as described above (with parallel experiments using non-reduced DlE3Cys protein-coated beads and uncoated beads under the same conditions as controls). Results are shown in
Figure 24.
Example 19
Coating Sulphate-Polystyrene/Latex Beads with Notch ligand protein Surfactant-free sulphate white polystyrene latex beads (product number 1-5000) were supplied by Interfacial Dynamics Coφoration (Portland, Oregon, US). The polystyrene microspheres have a mean diameter of 4.9 μm and were supplied dispersed in distilled de- ionised water at 6.3 x 108 beads/ml. The beads are negatively charged and have sulphate groups on the surface - the surface is hydrophobic in nature.
To coat the beads with D1E8G4 (prepared as above) or human IgG4 (hIgG4, Sigma; as a control) an aliquot ofthe supplied beads was removed and placed into 1 ml of sterile PBS, spun at 13K for 10 min and re- washed with a further 1 ml of PBS. The beads were -223-
resuspended in PBS at 50 μl per IO7 beads. Protein used to coat the beads was added at a concentration of 10 μg per 10 beads in a final concentration of 200 μg/ml in PBS in a 500 μl Eppendorf tube and placed on a rotating wheel overnight at 4 °C. The following day the beads were washed by pelleting in a micro fuge at 13 K for 10 min and washing with 3 x 1 ml of PBS. After the final wash the beads were resuspended in complete medium (DMEM + 10%) HI FCS + glutamine + P/S) at 2 x IO7 beads/ml and assayed in the CHO-N2 signalling assay starting at 2 x IO6 beads per well with serial 1:2 dilutions ofthe beads made in complete medium. Results are shown in Figure 25.
Example 20
CD4+ cell purification
Spleens were removed from mice (Balb/c females, 8-10 weeks) and treated with 1 mg/ml
Collagenase D (Boehringer Mannheim) in RPMI medium with no supplements for 40 min.
Tissue was passed through a 0.2μ cell strainer (Falcon) 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 (1150φm 5min) and the media removed.
The cells were incubated for 4 minutes with 5ml ACK lysis buffer (0.15M NH4C1, 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.
Example 21 -224-
Antibody Coating
The following protocol was used for coating 96 well flat-bottomed plates with antibodies.
The plates were coated with Dulbecco's Phosphate Buffered Saline (DPBS) plus 1 μg/mL anti-hamster IgG antibody (Pharmingen, San Diego, US: Cat No 554007). lOOμL of coating mixture was used per well. Plates were incubated overnight at 4°C then washed with DPBS. Each well then received lOOμL DPBS plus 0.1-1 μg/mL anti-CD3 (Pharmingen Cat No 553058, Clone No 145-2C11).
The plates were incubated for 2-3 hours at 37°C then washed again with DPBS before cells (prepared as in Example 20) were added.
Example 22
Primary Polyclonal Stimulation
CD4+ cells were cultured in 96 well, flat-bottomed plates pre-coated according to Example 21. Cells were resuspended following counting at 4xl06/mL in R10F medium and 50μL suspension added per well. R10F medium plus 8μg/mL CD28 antibody (Pharmingen, Cat No 553294, Clone No 37.51) was added at 50μL per well. Beads coated with Notch ligand were added in appropriate volumes to give final ratios of 0.1-20:1 beadsxell. R10F medium was added to give a final volume of 200μL per well (2xl05 cells/well, anti-CD28 final concentration 2μg/mL). The plates were then incubated at 37°C for 72 hours.
170μL supernatant was then removed from each well and stored at -20°C until tested by ELISA for IL-10, IFNγ, IL-2 and IL-13 using antibody pairs from R&D Systems (Abingdon, UK).
Results for various beads prepared as described above (alongside conesponding uncoated beads as controls) are shown in Figures 26 to 33.
Example 23 -225-
lμm, Dynal Beads
MyOne Streptavidin beads (lμm, Dynal 650.01) and CELLection Biotin Binder (4.5 μm, Dynal 115.21) were coated with anti-hIgG4-Biotin antibodies based on the binding capacity recommended by the supplier.
Briefly, 20 μg of anti-hIgG4-biotin (BD Biosciences) were incubated 30 minutes at room temperature with either 1 mg of MyOne beads (equivalent to 7-12x10 beads) or 10 CELLection beads. The beads were then washed and further incubated with either 100 μg of human Deltal EC domain-hIgG4 fusion protein or 100 μg of MgG4 (Sigma) for 2 hours at room temperature. After washing, the MyOne beads and the CELLection beads were resuspended in 500 μl of RPMI/BSA 0.1% and stored at 4°C.
Beads were tested (alongside uncoated beads as controls) in a CHO-N2 reporter assay as described above. Results are shown in Figure 34.
Example 24
Modulation of cytokine production by human CD4+ T cells in the presence of Deltal- h!gG4 immobilised on MyOne or CELLection Dynal microbeads.
Human peripheral blood mononuclear cells (PBMC) were purified from blood using Ficoll- Paque separation medium (Pharmacia). Briefly, 28 ml of blood were overlaid on 21 ml of Ficoll-Paque separation medium and centrifuged at 18-20°C for 40 minutes at 400g. PBMC were recovered from the interface and washed 3 times before use for CD4+ T cell purification.
Human CD4+ T cells were isolated by positive selection using anti-CD4 microbeads from Miltenyi Biotech according to the manufacturer's instructions. -226-
The CD4+ T cells were incubated in triplicates in a 96-well-plate (flat bottom) at IO5 CD4/well/200μl in RPMI medium containing 10%> FCS, glutamine, penicillin, streptomycin and β2-mercaptoethanol.
Cytokine production was induced by stimulating the cells with anti-CD3/CD28 T cell expander beads from Dynal at a 1:1 ratio (bead/cell). 10, 5, 2.5, 1.25, 0.62 μl of beads coated with human Deltal EC domain-hIgG4 fusion protein (prepared as described above) or control beads were added in some of the wells. The supematants were removed after 3 days of incubation at 37°C/ 5%CO2/humidified atmosphere and cytokine production was evaluated by ELISA using Pharmingen kits OptEIA Set human ILl 0 (catalog No. 555157) and OptEIA Set human IL-5 (catalog No. 555202) for IL-10, IL-5 respectively and a human IL-2 DuoSet from R&D Systems (catalog. No DY202) for IL-2 according to the manufacturer's instructions.
Results are shown in Figure 35.
Example 25
Modulation of cytokine production by Deltal -h!gG4 immobilised on MyOne or CELLection Dynal microbeads during a Mixed Lymphocyte Reaction.
Human peripheral blood mononuclear cells (PBMC) were purified from blood of 2 donors (donor A and donor B) as indicated above.
Human CD 14+ monocytes and CD4+ T cells were isolated from PBMC from donor A and B respectively by positive selection using anti-CD 14 and anti-CD4 microbeads from Miltenyi Biotech according to the manufacturer's instructions.
The CD 14+ cells (donor A) were differentiated into dendritic cells (DC) by incubation for 6 days in medium [RPMI / 10%FCS / glutamine / B2-mercaptoethanol / antibiotics] in the presence of hGM-CSF 50 ng/ml and hIL-4 50 ng/ml (both from Peprotech). Dendritic cell -227-
maturation was induced by addition into the culture of LPS lμg/ml (Sigma L-2654) for the last 24 hours.
Matured-DC were treated for 1 hour with 50 μg/ml Mitomycin C (Sigma) in RPMI and washed 4 times. These cells were then plated at 4xl04, lxlO4, 2.5xl03, 6.25xl02 cells/well in triplicates in a 96-well-plate in RPMI medium containing 10%> FCS, glutamine, penicillin, streptomycin and β2-mercaptoethanol. 2x105 AUogenic CD4+ T cells (donor B) were added into each well given a final volume of 200 μl/well.
10 μl of beads coated with human Deltal EC domain-hIgG4 fusion protein (prepared as described above) or control beads were added in some ofthe wells.
The supematants were removed after 5 days of incubation at 37°C/ 5%CO2/humidified atmosphere and cytokine production was evaluated by ELISA using Pharmingen kits OptEIA Set human IL10 (catalog No. 555157) and OptEIA Set human IFNg (catalog No 555142) for IL-10 and IFNg respectively and a human TNFa DuoSet from R&D Systems (catalog. No. DY210) for TNFa according to the manufacturer's instructions.
Results are shown in Figure 36.
Example 26
Coupling of DlE3cys to iron/dextran microbeads
i) Purification of expressed DlE3Cys by HIC
DlE3Cys Harvests from Example 16 above were purified using Hydrophobic Interaction Chromatography (HIC), the eluate was then concentrated and buffer exchanged using centrifugal concentrators according to the manufacturers' instructions. The purity ofthe product was determined by SDS PAGE (see Figure 37).
ii) Partial reduction of DlE3cys -228-
DlE3cys protein (purified as in (i) above) at 1 mg/ml in lOOmM sodium phosphate pH7.0 was reduced using TCEP.HC1 (Tris(2-carboxyethyl)phosphine hydrochloride; Pierce, 20490) at a 10-fold molar excess of reducing agent for lh at 22°C. The protein was purified by buffer exchange using Sephadex G-25, PD-10 columns (Amersham Biosciences, 17-0851- 01) into lOOmM sodium phosphate pH7.0 followed by concentration in Vivaspin 6ml concentrators. Protein concentration was estimated using the Warburg-Christian A280/A260 method.
The efficiency of reduction can be estimated using the Ellman's assay. The supplied DlE3cys protein has no free thiol groups, whereas partially reduced DlE3cys is predicted to have a single free thiol group per mole of protein. Using a 96-well microtitre plate, aliqouts of DlE3cys protein or L-cysteine hydrochloride (Sigma, C-1276) were made to 196 ul in lOOmM sodium phosphate pH7.0 and 4ul 4 mg/ml Ellman's reagent (in lOOmM sodium phosphate pH 7.0) was added. Reactions were incubated for 15 min at 22°C and absorbance was recorded at 405nm.
iii) Coupling of Reduced DlE3cys to Beads.
DlE3Cys was coupled to beads from Miltenyi Biotec (Bisley, Suney, UK and Auburn, CA, US; eg product reference 130-048-001) by reductive coupling. The beads are super- paramagnetic iron-dextran particles with a mean particle diameter of approximately 50 nm.
Example 27
CHO-N2 (N27) Luciferase Reporter Assay
DlE3Cys coupled microbeads from Example 26 above were added to individual wells of a 96-well plate at concentrations of up to 150 ug/ml in PBS (pre-addition) and the mixture was incubated overnight. Wells were washed with 1 x PBS (phosphate buffered saline). -229-
N27#l 1 cells (CHO cells expressing full length human Notch2 and a CBFl -luciferase reporter construct; T80 flask; as described in Example 8 above) were removed using 0.02%ι EDTA (with trypsin) solution (Sigma), spun down and resuspended in 10 ml DMEM plus 10%(HI)FCS plus glutamine plus P/S. 10 μl of cells were counted and the cell density was adjusted to 2.0 x 105 cells/ml with fresh DMEM plus 10%(HI)FCS plus glutamine plus P/S. The reporter cells were plated out at 100 μl per well of a 96-well plate (i.e. 2 x IO4 cells per well) and were placed in an incubator to settle down for at least 30 minutes.
The following day 100 μl of supernatant was 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 are transfened to a white 96-well plate (Nunc). Luminescence is then read in a TopCount™ (Packard) counter. An increase in luminescence compared to control indicates activation of Notch signalling.
CHO cells expressing full length human Deltal (CHO-Delta cells; prepared as described in WO 03/0102441 in the name of Lorantis Ltd; eg see Example 8 therein) and native CHO cells were used as controls at a cell ratio of 1 :1 to the reporter cells.
Results are shown in Figure 38 alongside the conesponding CHO/CHO-Delta and other controls.
Example 28
Co-administration of KLH beads and DlE3Cys-coupled microbeads in vivo
i) Coating of beads with KLH
Imject® Mariculture Keyhole Limpet Hemocyanin (mcKLH) in PBS Buffer (lyophilized from PBS) 20mg (Pierce product number 77600) was reconstituted with 2.0ml dH2O to make a lOmg/ml solution containing PBS, pH 7.2 with proprietary stabilizer. -230-
Surfactant-free White Aldehyde/Sulfate Latex Beads (Interfacial Dynamics coφ Portland or USA batch number 1813) concentration 5.8xl08 beads/ml were washed in PBS x3 (spun for lOmins at 13k RT). The beads were then resuspended at 2xl08 beads/ml in 500μg/ml mcKLH in PBS and horizontally rotated at 37°C overnight. Beads were then washed again in PBS x3 (spun for lOmins at 13k RT) and resuspended in PBS at the required concentration. Successful coating of the beads was checked by their ability to neutralize an anti-KLH antiserum in an ELISA system.
ii) in vivo administration with DlE3Cys-coupled beads
6-8 weeks old female Balb/c mice were injected s.c. at the base of the tail with 2 x 10° KLH coated beads (prepared as described in Example 26 above) per mouse. Particles bearing modulators of Notch signalling (DlE3cys-coupled beads from Example 26 above; 0.6 or 7 μg protein per mouse); DlE3Cys protein alone (7 μg per mouse; control); Protein G-coupled beads (Miltenyi Cat No 130-071-101; control); or LPS 0.76 ng/mouse in Na2PO4 buffer (100 ul) were injected s.c. in a close separate site of the tail base (all agents were administered as aqueous solutions; 100 mM sodium phosphate at pH 7). In each case 8 mice were used in each group and one group was left untreated.
Groups were thus as follows:
A: n=8, 2xl06 KLH beads/mouse, lOOul s.c.,(l site 100 ul each) tail base, + 7 ug
DlE3cys-coated Miltenyi beads/mouse, 100 ul (1 site 100 ul each) s.c. tail base B: n=8, 2x10 KLH beads/mouse, lOOul s.c. .,(1 site 100 ul each) tail base - DlE3cys-coated Miltenyi beads/mouse, 100 ul (1 site 100 ul each) s.c. tail base C: n=8, 2xl06 KLH beads/mouse, lOOul s.c.,(l site 100 ul each) tail base, coated Miltenyi beads ,100 ul/mouse (1 site 100 ul each) s.c.tail base D D:: nn==88,, 22xxl1006 KKLLHH bbeeaaddss//mmoouussee,, llOOOOuull ss..cc,, ((11 site 100 ul each) tail base + 7 ug soluble DlE3cys 100 ul (1 site 100 ul each) tail base -231-
E: n=8, 2xl06KLH beads/mouse, lOOul s.c, (1 site 100 ul each) tail base + LPS 0.76 ng/mouse in Na2PO4 buffer 100 ul (1 site 100 ul each)s.c tail base F: n=8, 2xl06 KLH beads/mouse, lOOul s.c, (1 site 100 ul each) tail base + Saline 100 ul (1 site 100 ul each)s.c. tail base G: Untreated
Mice were challenged after 7 days in the right ear with 20 μg of KLH. The increase in ear swelling (right ear - left ear) was measured for the following four days using a digital calliper.
Results are shown in Figure 39.
References
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Inaba et al. (1992) J. Exp. Med. 175:1157-1167. Caux et al. (1992) Nature 360:258-261. Coffin et al. (1998) Gene Therapy 5:718-722. Kroll et al. (1993) DNA Cell Biol. 12:441-453. Osborne and Miele (1999) Immunity 11:653-663. Matsuno et al. (1995) Development 121(8):2633-44. Matsuno et al. (1998) Nat. Genet. 19:74-78.
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Claims

-233-CLAIMS
1. A product comprising i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation ofthe immune system.
2. A product comprising i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of an immune response to said antigen or antigenic determinant.
3. A product as claimed in claim 1 or claim 2 wherein the antigen or antigenic determinant is an allergen or antigenic determinant thereof.
4. A product as claimed in claim 1 or claim 2 wherein the antigen or antigenic determinant is an autoantigen or antigenic determinant thereof.
5. A product as claimed in claim 1 or claim 2 wherein the antigen or antigenic determinant is a graft antigen or antigenic determinant thereof.
6. A product as claimed in any ofthe preceding claims wherein the pharmaceutically acceptable support matrix bears at least 5 modulators of Notch signalling.
7. A product as claimed in any ofthe preceding claims wherein the pharmaceutically acceptable support matrix bears at least 10 modulators of Notch signalling.
8. A product as claimed in any ofthe preceding claims wherein the pharmaceutically acceptable support matrix bears at least 20 modulators of Notch signalling. -234-
9. A product as claimed in any ofthe preceding claims wherein the pharmaceutically acceptable support matrix bears at least 50 modulators of Notch signalling.
10. A product as claimed in any ofthe preceding claims wherein the pharmaceutically acceptable support matrix bears at least 100 modulators of Notch signalling.
11. A product as claimed in any ofthe preceding claims wherein the pharmaceutically acceptable support matrix is a particulate matrix.
12. A product as claimed in claim 11 wherein the pharmaceutically acceptable support matrix is a bead.
13. A product as claimed in claim 12 wherein the pharmaceutically acceptable support matrix is a microbead or microsphere.
14. A product as claimed in any of claims 11 to 13 wherein the pharmaceutically acceptable support matrix has a maximum linear dimension of from about 1 nanometres to about 1000 micrometres
15. A product as claimed in claim 14 wherein the pharmaceutically acceptable support matrix has a maximum linear dimension of from about 10 nanometres to about 100 micrometres.
16. A product as claimed in claim 15 wherein the pharmaceutically acceptable support matrix has a maximum linear dimension of from about 10 nanometres to about 10 micrometres.
17. A product as claimed in claim 16 wherein the pharmaceutically acceptable support matrix has a maximum linear dimension of from about 40 nanometres to about 10 micrometres. -235-
18. A product as claimed in claim 17 wherein the pharmaceutically acceptable support matrix has a maximum linear dimension of from about 40 nanometres to about 1000 nanometres
19. A product as claimed in any ofthe preceding claims for modulation of peripheral T- cell activation.
20. A product as claimed in any ofthe preceding claims for generation of regulatory T-
Cells (Tregs).
21. A product as claimed in any ofthe preceding claims for use in reducing an immune response to an antigen or antigenic determinant.
22. A product as claimed in any ofthe preceding claims for use in promoting immune tolerance to an antigen or antigenic determinant.
23. A product as claimed in any ofthe preceding claims for use in the treatment of autoimmune disease, allergy or transplant rejection.
24. A product as claimed in any ofthe preceding claims wherein the modulator of Notch signalling comprises a Notch ligand protein or a fragment, derivative, homologue, analogue or allelic variant thereof.
25. A product as claimed in claim 24 wherein the modulator of Notch signalling comprises a Delta or Senate/Jagged protein or a fragment, derivative, homologue, analogue or allelic variant thereof. -236-
26. A product as claimed in claim 24 or claim 25 wherein the modulator of Notch signalling comprises a protein comprising at least an active portion of a Notch ligand extracellular domain
27. A product as claimed in any ofthe preceding claims wherein the modulator of Notch signalling comprises a protein or polypeptide comprising a DSL or EGF-like domain or a polynucleotide sequence coding for such a protein or polypeptide.
28. A product as claimed in claim 27 wherein the modulator of Notch signalling comprises or codes for a Notch ligand DSL domain and at least 2 to 8 or more EGF-like domains.
29. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide comprising at least one Notch ligand DSL domain and at least 2 Notch ligand EGF domains.
30. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide comprising at least one Notch ligand DSL domain and at least 3 Notch ligand EGF domains.
31. A product as claimed in any of the preceding claims wherein at least one of the modulators of Notch signalling comprises a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 1-5 or more 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.
32. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 2-4 or more Notch ligand EGF domains; iii) optionally all or part of a Notch ligand N-terminal domain; and -237-
iv) optionally one or more heterologous amino acid sequences.
33. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 2-3 or more 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.
34. A product as claimed in any ofthe preceding claims wherein at least one of the modulators of Notch signalling comprises a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 3 or more 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.
35. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide consisting essentially of the following components: i) a Notch ligand DSL domain; ii) 1-5 and no 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.
36. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide consisting essentially of the following components: i) a Notch ligand DSL domain; ii) 2-4 and no 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. -238-
37. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide consisting essentially of the following components: i) a Notch ligand DSL domain; ii) 2-3 and no 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.
38. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide consisting essentially of the following components: i) a Notch ligand DSL domain; ii) 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.
39. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide which comprises Delta DSL or EGF domains.
40. A product as claimed in any ofthe claims 1 to 38 wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide which comprises Jagged DSL or EGF domains.
41. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide which has at least 50% amino acid sequence similarity to the following sequence along the entire length ofthe latter:
MGSRCA-I---AIAVLSALLCQVWSSGVFELK EFVNKKGLLGNR CCRGGAGPPPCACRTF FRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPG TFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRF -239-
VCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGF CDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHH KPCK GATCTNTGQGSYTCSCRPGYTGATCELGIDEC
42. A product as claimed in claim 41 wherein the protein or polypeptide has at least 70%> amino acid sequence similarity to the sequence of claim 41 along the entire length ofthe latter.
43. A product as claimed in claim 42 wherein the protein or polypeptide has at least 90%> amino acid sequence similarity to the sequence of claim 41 along the entire length ofthe latter.
44. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises an antibody.
45 . A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide which is bound to the particle through a cysteine residue.
46. A product as claimed in any ofthe preceding claims wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide which is bound to the particle through a C-terminal cysteine residue.
47. A product as claimed in any ofthe preceding claims in the form of a pharmaceutical composition.
48. A combination of i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) an autoantigen or antigenic determinant thereof, or a polynucleotide coding for an autoantigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use for the treatment of autoimmune disease. -240-
49. A combination of i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) an allergen or antigenic determinant thereof, or a polynucleotide coding for an allergen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use for the treatment of allergy.
50. A combination of i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) a graft antigen or antigenic determinant thereof, or a polynucleotide coding for a graft antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use for the treatment of graft rejection.
51. A pharmaceutical composition comprising i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of modulators of Notch signalling; ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant; and iii) a pharmaceutically acceptable carrier.
52. A method for treating an immune disorder in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering in vivo, in either order: i) an effective amount of a pharmaceutically acceptable support matrix bearing a multiplicity of modulators of Notch signalling; and ii) an effective amount of an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant.
53. A method for reducing an immune response to an antigen or antigenic determinant in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering in vivo, in either order: i) an effective amount of a pharmaceutically acceptable support matrix suitable bearing a multiplicity of modulators of Notch signalling; and ii) an effective amount of an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant. -241-
54. A method for promoting immune tolerance to an antigen or antigenic determinant in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering in vivo, in either order: i) an effective amount of a pharmaceutically acceptable support matrix bearing a multiplicity of modulators of Notch signalling; and ii) an effective amount of an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant.
55. A pharmaceutically acceptable support matrix for in vivo administration bearing a multiplicity of modulators of Notch signalling for use to treat autoimmune disease in simultaneous, contemporaneous, separate or sequential combination with an autoantigen or antigenic deteriminant thereof or a polynucleotide coding for an autoantigen or antigenic determinant thereof.
56. The use of a combination of i) a pharmaceutically acceptable support matrix for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) an autoantigen or antigenic determinant thereof or a polynucleotide coding for an autoantigen or antigenic determinant thereof, in the manufacture of a medicament for the treatment of autoimmune disease.
57. The use of a combination of i) a pharmaceutically acceptable support matrix for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) an allergen or antigenic determinant thereof or a polynucleotide coding for an allergen or antigenic determinant thereof, in the manufacture of a medicament for the treatment of allergy.
58. The use of a combination of i) a pharmaceutically acceptable support matrix for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) a graft antigen or antigenic determinant thereof or a polynucleotide coding for a graft antigen or antigenic determinant thereof, in the manufacture of a medicament for the treatment of graft rejection. -242-
59. The use of a pharmaceutically acceptable support matrix for in vivo administration bearing a multiplicity of modulators of Notch signalling in the manufacture of a medicament for treatment of autoimmune disease in simultaneous, contemporaneous, separate or sequential combination with an autoantigen or antigenic determinant thereof or a polynucleotide coding for an autoantigen or antigenic determinant thereof.
60. The use of a pharmaceutically acceptable support matrix for in vivo administration bearing a multiplicity of modulators of Notch signalling in the manufacture of a medicament for treatment of allergy in simultaneous, contemporaneous, separate or sequential combination with an allergen or antigenic determinant thereof or a polynucleotide coding for an allergen or antigenic determinant thereof.
61. The use of a pharmaceutically acceptable support matrix for in vivo administration bearing a multiplicity of modulators of Notch signalling in the manufacture of a medicament for treatment of transplant rejection in simultaneous, contemporaneous, separate or sequential combination with a graft antigen or antigenic determinant thereof or a polynucleotide coding for a graft antigen or antigenic determinant thereof.
62. A pharmaceutical or veterinary kit comprising i) a pharmaceutically acceptable support matrix for in vivo administration bearing a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant
63. A construct comprising i) a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant.
64. A construct as claimed in claim 63 in the form of a particle bearing both i) a multiplicity of modulators of Notch signalling; and ii) an antigen or antigenic determinant, or a polynucleotide coding for an antigen or antigenic determinant. -243-
65. A construct as claimed in claim 64 in the form of a bead.
66. A construct as claimed in claim 64 or claim 65 in the form of a microparticle or microbead.
67. A particle having a multiplicity of modulators of Notch signalling bound thereto, wherein the particle has a maximum linear dimension of less than 500 nm.
68. A particle having a multiplicity of modulators of Notch signalling bound thereto, wherein the particle has a maximum linear dimension of less than 300 nm
69. A particle having a multiplicity of modulators of Notch signalling bound thereto, wherein the particle has a maximum linear dimension of less than 200 nm
70. A particle having a multiplicity of modulators of Notch signalling bound thereto, wherein the particle has a maximum linear dimension of less than 150 nm
71. A particle having a multiplicity of modulators of Notch signalling bound thereto, wherein the particle has a maximum linear dimension of less than 100 nm
72. A particle having a multiplicity of modulators of Notch signalling bound thereto, wherein the particle has a maximum linear dimension of from 1 to 100 nm
73. A particle having a multiplicity of modulators of Notch signalling bound thereto, wherein the particle has a maximum linear dimension of from 10 to 80 nm
74. A particle having a multiplicity of modulators of Notch signalling bound thereto, wherein the particle has a maximum linear dimension of from 30 to 70 nm
75. A particle as claimed in any of claims 67 to 74 which is biocompatible.
76. A particle as claimed in claim 75 which is substantially biodegradable. -244-
77. A particle as claimed in any of claims 67 to 76 in the form of a microbead.
78. A particle as claimed in any of claims 67 to 77 which comprises any one or more of polystyrene, polyacrylamide, latex, chitosan, cellulose, silica, dextran, agarose, cellulose, polylactide, or poly(methylmethacrylate) (PMMA) optionally in modified, crosslinked or derivatized form, or an inorganic salt or oxide.
79. A particle as claimed in any of claims 67 to 78 wherein at least one ofthe modulators of Notch signalling is an activator of a Notch receptor.
80. A particle as claimed in any of claims 67 to 79 wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide.
81. A particle as claimed in any of claims 67 to 80 wherein at least one ofthe modulators of Notch signalling comprises a Notch ligand or a fragment, derivative, homologue, analogue or allelic variant thereof.
82. A particle as claimed in in any of claims 67 to 81 wherein at least one ofthe modulators of Notch signalling comprises Delta or Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof.
83. A particle as claimed in any of claims 67 to 82 wherein at least one ofthe modulators of Notch signalling comprises a protein or polypeptide comprising a Notch ligand DSL domain.
84. A particle as claimed in any of claims 67 to 83 wherein at least one ofthe modulators of Notch signalling comprises a Notch ligand DSL domain and at least one Notch ligand EGF-like domain. -245-
85. A particle as claimed in any of claims 67 to 85 wherein at least one ofthe modulators of the Notch signalling pathway comprises a protein or polypeptide comprising at least one Notch ligand DSL domain and at least 2 Notch ligand EGF domains.
86. A particle as claimed in any of claims 67 to 85 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide comprising at least one Notch ligand DSL domain and at least 3 Notch ligand EGF domains.
87. A particle as claimed in any of claims 67 to 86 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 1-5 or more 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.
88. A particle as claimed in any of claims 67 to 87 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 2-4 or more 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.
89. A particle as claimed in any of claims 67 to 88 wherein at least one of the modulators ofthe Notch signalling pathway comprises a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 2-3 or more 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.
90. A particle as claimed in any of claims 67 to 89 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide comprising: -246-
i) a Notch ligand DSL domain; ii) 3 or more 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.
91. A particle as claimed in any of claims 67 to 90 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide consisting essentially of the following components: i) a Notch ligand DSL domain; ii) 1-5 and no 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.
92. A particle as claimed in any of claims 67 to 91 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide consisting essentially of the following components: i) a Notch ligand DSL domain; ii) 2-4 and no 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.
93. A particle as claimed in any of claims 67 to 92 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide consisting essentially of the following components: i) a Notch ligand DSL domain; ii) 2-3 and no 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.
94. A particle as claimed in any of claims 67 to 93 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide consisting essentially of the following components: i) a Notch ligand DSL domain; -247-
ii) 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.
95. A particle as claimed in any of claims 67 to 94 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide which comprises Delta DSL or EGF domains.
96. A particle as claimed in any of claims 67 to 95 wherein wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide which comprises Jagged DSL or EGF domains.
97. A particle as claimed in any of claims 67 to 96 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide which comprises human Delta DSL or EGF domains.
98. A particle as claimed in any of claims 67 to 97 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide which comprises human Jagged DSL or EGF domains.
99. A particle as claimed in any of claims 67 to 98 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide which has at least 50%> amino acid sequence similarity to the following sequence along the entire length ofthe latter:
MGSRCΛIALAVLSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTF FRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPG TFSLIIEALHTDSPDDLATENPERLISRLATQRHLTVGEE SQDLHSSGRTDLKYSYRF VCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGF CDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQP QCNCQEGWGGLFCNQDLNYCTHH KPCKNGATC TGQGSYTCSCRPGYTGATCELGIDEC -248-
100. A particle as claimed in claim 99 wherein the protein or polypeptide has at least 70%> amino acid sequence similarity to the sequence of claim 99 along the entire length ofthe latter:
101. A particle as claimed in claim 100 wherein the protein or polypeptide has at least 90%) amino acid sequence similarity to the sequence of claim 99 along the entire length of the latter:
102. A particle as claimed in any of claims 67 to 101 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises an antibody.
103. A particle as claimed in any of claims 67 to 102 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide which is bound to the particle through a cysteine residue.
104. A particle as claimed in claim 103 wherein at least one ofthe modulators ofthe Notch signalling pathway comprises a protein or polypeptide which is bound to the particle through a C-terminal cysteine residue.
105. A particle as claimed in any of claims 67 to 104 which further comprises an antigen or antigenic determinant or a polynucleotide coding for an antigen or antigenic determinant.
106. A particle as claimed in claim 105 wherein the antigen or antigenic determinant is an allergen or autoantigen or an antigenic determinant thereof or a polynucleotide coding for an allergen or autoantigen or antigenic determinant thereof.
107. A particle as claimed in any of claims 67 to 106 having at least 10 modulators of Notch signalling bound thereto.
108. A particle as claimed in any of claims 67 to 107 having at least 100 modulators of Notch signalling bound thereto. -249-
109. A particle as claimed in any of claims 67 to 108 having at least 1000 modulators of Notch signalling bound thereto.
110. A particle as claimed in any of claims 67 to 109 wherein at least one ofthe modulators of Notch signalling is chemically bound to the particle.
111. A pharmaceutical composition comprising a particle as claimed in any of claims 67 to 110.
112. A pharmaceutical composition for in vivo administration comprising a particle as claimed in any of claims 67 to 111.
113. A method for modulating Notch signalling in a subject by administering a particle as claimed in any of claims 67 to 111.
114. A method for modulating an immune response in a subject by administering a particle as claimed in any of claims 67 to 111.
115. A method for reducing the immune response to an antigen or antigenic determinant by administering a pharmaceutically acceptable support matrix suitable for in vivo administration bearing said antigen or antigenic determinant.
116. A method for promoting immune tolerance to an antigen or antigenic determinant by administering a pharmaceutically acceptable support matrix suitable for in vivo administration bearing said antigen or antigenic determinant.
117. A method as claimed in claim 115 or claim 116 wherein the antigen or antigenic determinant is an autoantigen or bystander antigen or antigenic determinant thereof for the treatment of autoimmune disease. -250-
118. A method as claimed in claim 115 or claim 116 wherein the antigen or antigenic determinant is an allergen or antigenic determinant thereof for the treatment of allergy.
119. A method as claimed in claim 115 or claim 116 wherein the antigen or antigenic determinant is a graft antigen or antigenic determinant thereof for the treatment of graft rejection.
120. The use for immunotherapy of a pharmaceutically acceptable support matrix suitable for in vivo administration bearing an antigen or antigenic determinant thereof.
121. The use of a pharmaceutically acceptable support matrix suitable for in vivo administration bearing an antigen or antigenic determinant thereof to reduce an immune response to said antigen or antigenic determinant.
122. The use of a pharmaceutically acceptable support matrix suitable for in vivo administration bearing an antigen or antigenic determinant thereof to promote immune tolerance to said antigen or antigenic determinant.
123. A construct comprising a pharmaceutically acceptable support matrix suitable for in vivo administration bearing an allergen or antigenic determinant thereof.
124. A construct comprising a pharmaceutically acceptable support matrix suitable for in vivo administration bearing an autoantigen or bystander antigen or antigenic determinant thereof.
125. A construct comprising a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a graft antigen or antigenic determinant thereof.
126. A construct as claimed in any of claims 123 to 125 wherein the pharmaceutically acceptable support matrix is a particulate matrix.
127. A construct as claimed in claim 126 wherein the particulate matrix is a bead. -251-
128. A construct as claimed in claim 127 wherein the particulate matrix is a microbead or microsphere.
129. A construct as claimed in any of claims 123 to 128 wherein the pharmaceutically acceptable support matrix has a maximum linear dimension of from about 1 nanometres to about 1000 micrometres
130. A construct as claimed in claim 129 wherein the pharmaceutically acceptable support matrix has a maximum linear dimension of from about 10 nanometres to about 100 micrometres.
131. A construct as claimed in claim 130 wherein the pharmaceutically acceptable support matrix has a maximum linear dimension of from about 10 nanometres to about 10 micrometres.
132. A construct as claimed in claim 131 wherein the pharmaceutically acceptable support matrix has a maximum linear dimension of from about 40 nanometres to about 10 micrometres.
133. A construct as claimed in claim 132 wherein the pharmaceutically acceptable support matrix has a maximum linear dimension of from about 40 nanometres to about 1000 nanometres.
134. A method for increasing an immune response to a tumour or pathogen antigen or antigenic determinant in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering in vivo, in either order: i) an effective amount of a pharmaceutically acceptable support matrix bearing a multiplicity of inhibitors of Notch signalling; and ii) an effective amount of a tumour or pathogen antigen or antigenic determinant, or a polynucleotide coding for a tumour or pathogen antigen or antigenic determinant. -252-
135. A vaccine composition comprising i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of inhibitors of Notch signalling; and ii) a vaccine antigen or antigenic determinant thereof, or a polynucleotide coding for a vaccine antigen or antigenic determinant thereof; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation ofthe immune system.
136. A vaccine composition comprising i) a pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of inhibitors of Notch signalling; and ii) a vaccine antigen or antigenic determinant, or a polynucleotide coding for a vaccine antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for enhancing an immune response to said vaccine antigen or antigenic determinant.
137. A vaccine composition as claimed in claim 135 or claim 136 in the form of a tumour vaccine wherein the vaccine antigen or antigenic determinant is a tumour antigen or antigenic determinant thereof.
138. A vaccine composition as claimed in claim 135 or claim 136 wherein the vaccine antigen or antigenic determinant is a pathogen antigen or antigenic determinant thereof.
139. A vaccine composition as claimed in any of claims 135 to 138 for use as a prophylactic vaccine.
140. A vaccine composition as claimed in any of claims 135 to 138 for use as a therapeutic vaccine.
141. A pharmaceutically acceptable support matrix suitable for in vivo administration bearing a multiplicity of inhibitors of Notch signalling
142. A construct comprising a pharmaceutically acceptable support matrix suitable for in vivo administration bearing both i) a multiplicity of inhibitors of Notch signalling; and ii) a -253-
vaccine antigen or antigenic determinant, or a polynucleotide coding for a vaccine antigen or antigenic determinant.
143. A construct as claimed in claim 142 wherein the vaccine antigen or antigenic determinant is a tumour antigen or antigenic determinant.
144. A construct as claimed in claim 142 wherein the vaccine antigen or antigenic determinant is a pathogen antigen or antigenic determinant.
PCT/GB2004/000046 2003-01-07 2004-01-07 Modulators of notch signalling for use in immunotherpapy WO2004060262A2 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
JP2006500232A JP2006517533A (en) 2003-01-23 2004-01-23 Treatment of autoimmune diseases using activators of the Notch signaling pathway
PCT/GB2004/000263 WO2004064863A1 (en) 2003-01-23 2004-01-23 Treatment of autoimmune diseases using an activator for the notch signaling pathway
AT04722319T ATE474593T1 (en) 2003-03-21 2004-03-22 TREATMENT OF ALLERGIC DISEASES USING A MODULATOR OF THE NOTCH SIGNALING PATHWAY
PCT/GB2004/001252 WO2004082710A1 (en) 2003-03-21 2004-03-22 Treatment of allergic diseases using a modulator of the notch signaling pathway
EP04722319A EP1646400B1 (en) 2003-03-21 2004-03-22 Treatment of allergic diseases using a modulator of the notch signaling pathway
US11/188,417 US20060204508A1 (en) 2003-01-23 2005-07-25 Treatment of autoimmune diseases using an activator for the notch signalling pathway
US11/231,494 US20060205823A1 (en) 2003-03-21 2005-09-21 Treatment of allergic diseases using a modulator of the Notch signaling pathway
US12/766,738 US20100303867A1 (en) 2003-03-21 2010-04-23 a composition comprising a notch ligand and an allergenb or allergen bystander antigen

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GB0300234A GB0300234D0 (en) 2003-01-07 2003-01-07 Medical treatment
GB0300234.2 2003-01-07
GB0301521A GB0301521D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301515.3 2003-01-23
GB0301513.8 2003-01-23
GB0301519.5 2003-01-23
GB0301515A GB0301515D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301524.5 2003-01-23
GB0301510.4 2003-01-23
GB0301522A GB0301522D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301513A GB0301513D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301512A GB0301512D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301526.0 2003-01-23
GB0301518.7 2003-01-23
GB0301529A GB0301529D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301521.1 2003-01-23
GB0301519A GB0301519D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301526A GB0301526D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301529.4 2003-01-23
GB0301522.9 2003-01-23
GB0301518A GB0301518D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301510A GB0301510D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301527A GB0301527D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301524A GB0301524D0 (en) 2003-01-23 2003-01-23 Medical treatment
GB0301512.0 2003-01-23
GB0301527.8 2003-01-23
GB0306621A GB0306621D0 (en) 2003-03-22 2003-03-22 Medical treatment
GB0306621.4 2003-03-22
GBPCT/GB03/001525 2003-04-04
PCT/GB2003/001525 WO2003087159A2 (en) 2002-04-05 2003-04-04 Modulators of the notch signalling pathway and uses thereof in medical treatment
GB0312062.3 2003-05-24
GB0312062A GB0312062D0 (en) 2003-05-24 2003-05-24 Medical treatment
PCT/GB2003/003285 WO2004013179A1 (en) 2002-08-03 2003-08-01 Conjugate of notch signalling pathway modulators and their use in medical treatment
GBPCT/GB03/003285 2003-08-01
GB0323130.5 2003-10-03
GB0323130A GB0323130D0 (en) 2003-10-03 2003-10-03 Medical treatment

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