EP1585543A1 - Traitement de maladies autoimmunes au moyen d'un activateur de la voie de signalisation notch - Google Patents

Traitement de maladies autoimmunes au moyen d'un activateur de la voie de signalisation notch

Info

Publication number
EP1585543A1
EP1585543A1 EP04704657A EP04704657A EP1585543A1 EP 1585543 A1 EP1585543 A1 EP 1585543A1 EP 04704657 A EP04704657 A EP 04704657A EP 04704657 A EP04704657 A EP 04704657A EP 1585543 A1 EP1585543 A1 EP 1585543A1
Authority
EP
European Patent Office
Prior art keywords
autoantigen
bystander antigen
antigenic determinant
antigen
bystander
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP04704657A
Other languages
German (de)
English (en)
Inventor
Brian Robert Champion
Silvia Ragno
Lesley Lynn Young
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Celldex Therapeutics Ltd
Original Assignee
Lorantis Ltd
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 GB0301526A external-priority patent/GB0301526D0/en
Priority claimed from GB0301524A external-priority patent/GB0301524D0/en
Priority claimed from GB0301512A external-priority patent/GB0301512D0/en
Priority claimed from GB0301515A external-priority patent/GB0301515D0/en
Priority claimed from GB0301519A external-priority patent/GB0301519D0/en
Priority claimed from GB0301522A external-priority patent/GB0301522D0/en
Priority claimed from GB0301529A external-priority patent/GB0301529D0/en
Priority claimed from GB0301521A external-priority patent/GB0301521D0/en
Priority claimed from GB0301518A external-priority patent/GB0301518D0/en
Priority claimed from GB0301527A external-priority patent/GB0301527D0/en
Priority claimed from GB0301513A external-priority patent/GB0301513D0/en
Priority claimed from GB0301510A external-priority patent/GB0301510D0/en
Priority claimed from PCT/GB2003/001525 external-priority patent/WO2003087159A2/fr
Priority claimed from GB0312062A external-priority patent/GB0312062D0/en
Priority claimed from PCT/GB2003/003285 external-priority patent/WO2004013179A1/fr
Priority claimed from GB0323130A external-priority patent/GB0323130D0/en
Priority claimed from PCT/GB2004/000263 external-priority patent/WO2004064863A1/fr
Application filed by Lorantis Ltd filed Critical Lorantis Ltd
Publication of EP1585543A1 publication Critical patent/EP1585543A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • Y02B60/45—

Definitions

  • the present invention relates to the modulation of immune function.
  • WO 98/20142 describes how manipulation of the Notch signalling pathway can be used in immunotherapy and in the prevention and/or treatment of T-cell mediated diseases.
  • regulatory T cells which are able to transmit antigen-specific tolerance to other T cells, a process termed infectious tolerance (WO98/20142).
  • infectious tolerance WO98/20142
  • the functional activity of these cells can be mimicked by over- expression of a Notch ligand protein on their cell surfaces or on the surface of antigen presenting cells.
  • regulatory T cells can be generated by over-expression of a member of the Delta or Serrate family of Notch ligand proteins.
  • 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/GB02/04390 (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
  • 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/GB2003/001525 (filed on 4 April 2003), published as WO 03/087159; and PCT/GB2003/003285 filed on 1 August 2003 (claiming priority from GB 0312062.3 and others).
  • PCT/GB97/03058 (WO 98/20142), PCT/GB99/04233 (WO 00/36089), PCT/GBOO/04391 (WO 0135990), PCT/GBOl/03503 (WO 02/12890), PCT/GB 02/02438 (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) and PCT/GB2003/001525 (WO 03/087159) and PCT/GB2003/003285, is hereby incorporated herein by reference
  • the present invention seeks to provide further methods of modulating the immune system particularly, but without limitation, in the prevention and/or treatment of autoimmune disease.
  • Notch signalling provides a "bystander effect” or “bystander suppression effect” which may be used in a wide variety of ways to suppress unwanted immune responses in immune diseases and disorders.
  • this "Notch bystander effect” may, for example, be used to provide targeted immune suppression at a disease locus with less of an undesirable general immunosuppressant effect on the whole body as compared, for example, to immunoppressant drugs or steroids, which are relatively indiscriminate in action.
  • the "Notch bystander effect” identified in one aspect of the present invention is particularly suited to treatment of autoimmune disease, but is not limited to such treatment, and may also be used to treat other immune related disorders. Use of the effect makes it possible to provide localised immune suppression in an autoimmune disease even where the primary autoantigen or autoantigens are uncertain or not fully characterised, so long as a relevant "bystander antigen" can be identified. Thus, when using this "bystander effect” it is not always necessary to identify key pathogenic autoantigens as targets for immune suppression (although this will be possible in some cases) . Chosen antigens or antigenic determinants may simply need to be expressed in or delivered to the diseased tissue (or lymphatic tissue draining these sites).
  • a product comprising a modulator of the Notch signalling pathway and an autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoantigen or bystander antigen or antigenic determinant thereof as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of an autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and an autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with an autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response 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 pharmaceutical kit comprising a modulator of the Notch signalling pathway and an autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoantigen or bystander antigen or antigenic determinant thereof.
  • the modulator of the Notch signalling pathway will be an activator of the Notch signalling pathway, and preferably a direct activator of a Notch receptor ("Notch receptor agonist”), such as a Notch ligand or fragment, derivative or variant thereof.
  • Notch receptor agonist a Notch receptor
  • the methods, uses, products and compositions etc of the present invention are for in vivo (rather than ex-vivo or in vitro) administration.
  • the autoantigen or bystander antigen or antigenic determinant for use with the invention in any of its aspects will be a human autoantigen, bystander antigen or antigenic determinant thereof, or a polynucleotide coding for any of the foregoing.
  • the modulation of immune response comprises immunotherapy.
  • the modulation of immune response comprises reducing the immune response to the autoantigen or bystander antigen.
  • the modulation of immune response comprises modulation of T-cell activity, preferably peripheral T-cell activity.
  • the modulation of immune response comprises treatment of an organ-specific autoimmune disease.
  • the modulation of immune response comprises treatment of a systemic autoimmune disease.
  • the autoantigen or bystander antigen may be a Goodpasture's autoantigen or bystander antigen for treatment of Goodpasture's disease.
  • a product comprising a modulator of the Notch signalling pathway and a Goodpasture's autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a Goodpasture's autoantigen or bystander antigen or antigenic determinant thereof as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a Goodpasture's autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a Goodpasture's autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a Goodpasture's autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a Goodpasture's autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a Goodpasture's autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a Goodpasture's autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a Goodpasture's autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a Goodpasture's autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a renal autoantigen or renal bystander antigen for treatment of an autoimmune disease of the kidney.
  • a product comprising a modulator of the Notch signalling pathway and a renal autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a renal autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a renal autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a renal autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a renal autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a renal autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a renal autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a renal autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a renal autoantigen or bystander antigen or antigenic determinant thereof ,or a polynucleotide coding for a renal autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a Pemphigus autoantigen or bystander antigen for treatment of Pemphigus.
  • a product comprising a modulator of the Notch signalling pathway and a Pemphigus autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Pemphigus autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a Pemphigus autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Pemphigus autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and a Pemphigus autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a Pemphigus autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a Pemphigus autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Pemphigus autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a Pemphigus autoantigen or bystander antigen or antigemc determinant thereof, or a polynucleotide coding for a Pemphigus autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a Pemphigus autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Pemphigus autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a Wegener's autoantigen or bystander antigen or antigenic determinant thereof for treatment of Wegener's disease.
  • a product comprising a modulator of the Notch signalling pathway and a Wegener's autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Wegener's autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a Wegener's autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Wegener's autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and a Wegener's autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a Wegener's autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a Wegener's autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Wegener's autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a Wegener's autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Wegener's autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a Wegener's autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Wegener's autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be an autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof, for treatment of autoimmune anemia.
  • a product comprising a modulator of the Notch signalling pathway and a autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and a effective amount of an autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and an autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with an autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with an autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and an autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune anemia autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof, for treatment of autoimmune thrombocytopenia.
  • a product comprising a modulator of the Notch signalling pathway and an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune thrombocytopenia autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof, for treatment of autoimmune gastritis.
  • a product comprising a modulator of the Notch signalling pathway and an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune gastritis autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof, for treatment of autoimmune hepatitis.
  • a product comprising a modulator of the Notch signalling pathway and an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune hepatitis autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof, for treatment of autoimmune vasculitis.
  • a product comprising a modulator of the Notch signalling pathway and an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune vasculitis autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be an ocular autoantigen or bystander antigen or antigenic determinant thereof, for treatment of an autoimmune disease of the eye.
  • a product comprising a modulator of the Notch signalling pathway and an ocular autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an ocular autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of an ocular autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an ocular autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and an ocular autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an ocular autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with an ocular autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an ocular autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with an ocular autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an ocular autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and an ocular autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an ocular autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be an adrenal autoantigen or bystander antigen or antigenic determinant thereof, for treatment of an adrenal autoimmune disease.
  • a product comprising a modulator of the Notch signalling pathway and an adrenal autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an adrenal autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of an adrenal autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an adrenal autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with an adrenal autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an adrenal autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with an adrenal autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an adrenal autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and an adrenal autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an adrenal autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a cardiac autoantigen or bystander antigen or antigenic determinant thereof, for treatment of cardiac autoimmune disease.
  • a product comprising a modulator of the Notch signalling pathway and a cardiac autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a cardiac autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a cardiac autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a cardiac autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a cardiac autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a cardiac autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a cardiac autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a cardiac autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a cardiac autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a cardiac autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof, for treatment of scleroderma or myositis.
  • a product comprising a modulator of the Notch signalling pathway and a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a scleroderma or myositis autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a nervous system autoantigen or bystander antigen or antigenic determinant thereof, for use to treat an autoimmune disease of the nervous system.
  • a product comprising a modulator of the Notch signalling pathway and a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a nervous system especially MS
  • a polynucleotide coding for a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof.
  • a nervous system especially MS
  • a nervous system especially MS
  • a modulator of the Notch signalling pathway and a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof; in the manufacture of a medicament for modulation of immune response.
  • a nervous system especially MS
  • a polynucleotide coding for a nervous system especially MS
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a nervous system (especially MS) autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a nervous system
  • the autoantigen or bystander antigen may be an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof, for use to treat autoimmune arthritis.
  • a product comprising a modulator of the Notch signalling pathway and an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune arthritis autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof, for use to treat autoimmune diabetes.
  • a product comprising a modulator of the Notch signalling pathway and an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an autoimmune diabetes autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a Myasthenia Gravis autoantigen or bystander antigen or antigenic determinant thereof, for use to treat Myasthenia Gravis.
  • a product comprising a modulator of the Notch signalling pathway and a Myasthenia Gravis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Myasthenia Gravis autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a Myasthenia Gravis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Myasthenia Gravis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a Myasthenia Gravis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Myasthenia Gravis autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a Myasthenia Gravis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Myasthenia Gravis autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a Myasthenia Gravis autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Myasthenia Gravis autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a Systemic Lupus Erythematosus (SLE) autoantigen or bystander antigen or antigenic determinant thereof, for use to treat SLE.
  • SLE Systemic Lupus Erythematosus
  • a product comprising a modulator of the Notch signalling pathway and a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof; in the manufacture of a medicament for modulation of immune response.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Systemic Lupus Erythematosus autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a bowel autoantigen or bystander antigen or antigenic determinant thereof, for use to treat an autoimmune disease of the bowel.
  • a product comprising a modulator of the Notch signalling pathway and a bowel autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a bowel autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a bowel autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a bowel autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and a bowel autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a bowel autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a bowel autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a bowel autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a bowel autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a bowel autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a bowel autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a bowel autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a thyroid autoantigen or bystander antigen or antigenic determinant thereof, for use to treat an autoimmune disease of the thyroid.
  • a product comprising a modulator of the Notch signalling pathway and a thyroid autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a thyroid autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a thyroid autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a thyroid autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a thyroid autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a thyroid autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a thyroid autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a thyroid autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a thyroid autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a thyroid autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof for use to treat Sjogren's syndrome.
  • a product comprising a modulator of the Notch signalling pathway and a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a Sjogren's autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be an endocrine autoantigen or bystander antigen or antigenic determinant thereof, for use to treat an autoimmune disease of an endocrine gland.
  • a product comprising a modulator of the Notch signalling pathway and an endocrine autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an endocrine autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of an endocrine autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an endocrine autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and an endocrine autoantigen or bystander antigen or antigenic determinant thereof or a polynucleotide coding for an endocrine autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with an endocrine autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an endocrine autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with an endocrine autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an endocrine autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and an endocrine autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for an endocrine autoantigen or bystander antigen or antigenic determinant thereof.
  • the autoantigen or bystander antigen may be a skin autoantigen or bystander antigen or antigenic determinant thereof, for use to treat an autoimmune disease of the skin.
  • a product comprising a modulator of the Notch signalling pathway and a skin autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a skin autoantigen or bystander antigen or antigenic determinant thereof, as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of immune response.
  • a method of modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentially administering to a mammal in need thereof an effective amount of a modulator of the Notch signalling pathway and an effective amount of a skin autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a skin autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway and a skin autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a skin autoantigen or bystander antigen or antigenic determinant thereof; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • a modulator of the Notch signalling pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a skin autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a skin autoantigen or bystander antigen or antigenic determinant thereof.
  • a modulator of the Notch signalling pathway in the manufacture of a medicament for modulation of immune response in simultaneous, contemporaneous, separate or sequential combination with a skin autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a skin autoantigen or bystander antigen or antigenic determinant thereof.
  • a pharmaceutical kit comprising a modulator of the Notch signalling pathway and a skin autoantigen or bystander antigen or antigenic determinant thereof, or a polynucleotide coding for a skin autoantigen or bystander antigen or antigenic determinant thereof.
  • the modulator of the Notch signalling pathway is an agent which activates the Notch signalling pathway, or a polynucleotide which codes for such an agent.
  • the modulator of the Notch signalling pathway is an agent which activates, preferably directly activates, the Notch receptor (eg human Notchl, human Notch2, human Notch3 or human Notch4), or a polynucleotide which codes for such an agent.
  • the Notch receptor eg human Notchl, human Notch2, human Notch3 or human Notch4
  • the Notch receptor is activated in immune cells, preferably T-cells.
  • the modulator of Notch signalling is preferably not an agent which acts initially by upregulating expression of a Notch ligand (although this may be an indirect effect of action).
  • the modulator of the Notch signalling pathway is not a Notch IC protease, and in particular is preferably not a modulator of presenilin-dependent gamma secretase activity.
  • the modulator of the Notch signalling pathway is not a cytokine.
  • the modulator of the Notch signalling pathway may comprise a fusion protein or a polynucleotide which codes for a fusion protein.
  • the modulator may be a fusion protein comprising a segment of a Notch ligand extracellular domain and an immunoglobulin F c segment or a polynucleotide encoding such a fusion protein.
  • the modulator of the Notch signalling pathway comprises a protein or polypeptide comprising a Notch ligand DSL or EGF-like domain or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide sequence coding for such a protein, polypeptide, fragment, derivative, homologue, analogue or allelic variant.
  • the modulator of the Notch signalling pathway comprises a Notch ligand DSL domain and at least 1 to 20, suitably at least 2 to 15, suitably at least 2 to 10, for example at least 3 to 8 EGF-like domains.
  • the DSL and EGF-like domain sequences are or correspond to mammalian sequences.
  • Preferred sequences include human sequences such as human Deltal, Delta3, Delta4, Jaggedl or Jagged2 sequences.
  • the modulator of the Notch signalling pathway may comprise, for example, Notch intracellular domain (Notch IC) or a fragment, derivative, homologue, analogue or allelic variant thereof, or a polynucleotide sequence which codes for Notch intracellular domain or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • Notch intracellular domain may, for example, be an active part of the intracellular domain of human Notchl, human Notch2, human Notch3 or human Notch4.
  • the modulator of the Notch signalling pathway comprises a Notch ligand or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide encoding a Notch ligand or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • the modulator of the Notch signalling pathway comprises Delta or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide encoding Delta or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • the modulator of the Notch signalling pathway may comprise Serrate/Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide encoding Serrate/Jagged or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • the modulator of the Notch signalling pathway may comprise Notch (eg human Notchl , Notch2, Notch3 or Notch4) or a fragment, derivative, homologue, analogue or allelic variant thereof or a polynucleotide encoding Notch or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • Notch eg human Notchl , Notch2, Notch3 or Notch4
  • the modulator of the Notch signalling pathway may comprise a dominant negative version of a Notch signalling repressor, or a polynucleotide which codes for a dominant negative version of a Notch signalling repressor.
  • a modulator of Notch signalling for use in any aspect of the present invention may comprise a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 1-5 (and in one embodiment not more than 5) Notch ligand EGF domains; iii) optionally all or part of a Notch ligand N-terminal domain; and iv) optionally one or more heterologous amino acid sequences; or a polynucleotide coding therefor.
  • a modulator of Notch signalling for use in any aspect of the present invention may comprise a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 2-4 (and in one embodiment not more than 4) Notch ligand EGF domains; iii) optionally all or part of a Notch ligand N-terminal domain; and iv) optionally one or more heterologous amino acid sequences; or a polynucleotide coding therefor.
  • a modulator of Notch signalling for use in any aspect of the present invention may comprise a protein or polypeptide comprising: i) a Notch ligand DSL domain; ii) 2-3 (and in one embodiment not more than 3) Notch ligand EGF domains; iii) optionally all or part of a Notch ligand N-terminal domain; and iv) optionally one or more heterologous amino acid sequences; or a polynucleotide coding therefor.
  • such a protein or polypeptide may have at least 50%, preferably at least 70%, preferably at least 90%, for example at least 95% amino acid sequence similarity (or preferably sequence identity) to the following sequence along the entire length of the latter (SEQ ID NO: 1):
  • a modulator of Notch signalling will be in a multimerised form, and may preferably comprise a construct comprising at least 3, preferably at least 5, preferably at least 10, at least 30, or at least 50 or 100 or more modulators of Notch signalling.
  • modulators of Notch signalling in the form of Notch ligand proteins/polypeptides coupled to particulate supports such as beads are described in WO 03/011317 (Lorantis) and in Lorantis' co-pending PCT application PCT/GB2003/001525 (filed on 4 April 2003), published as WO 03087159, the texts of which are hereby incorporated by reference (eg see in particular Examples 17, 18, 19 of PCT/GB2003/001525); and Lorantis Ltd's co-pending PCT application filed on 7 January 2004 claiming priority from GB 0300234.2, the text of which is also hereby incorporated by reference.
  • the modulator of the Notch signalling pathway may comprise an antibody, antibody fragment or antibody derivative or a polynucleotide which codes for an antibody, antibody fragment or antibody derivative.
  • 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. HB12655.
  • An anti-human- Jagged 1 antibody is available from R & D Systems, Inc, reference MAB12771 (Clone 188323).
  • a conjugate comprising first and second sequences, wherein the first sequence comprises an autoantigen or bystander antigen or a polynucleotide sequence coding for such an antigen or antigenic determinant and the second sequence comprises a polypeptide or polynucleotide for Notch signalling modulation.
  • the conjugate may be in the form of a nucleotide vector, preferably an expression vector, comprising a first polynucleotide sequence coding for an activator of the Notch signalling pathway (such as a Notch ligand or active fragment thereof) and a second polynucleotide sequence coding for an autoantigen or bystander antigen or antigenic dete ⁇ ninat thereof.
  • a nucleotide vector preferably an expression vector, comprising a first polynucleotide sequence coding for an activator of the Notch signalling pathway (such as a Notch ligand or active fragment thereof) and a second polynucleotide sequence coding for an autoantigen or bystander antigen or antigenic dete ⁇ ninat thereof.
  • Suitable vectors include 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 SN40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retro viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • two or more separate vectors may be used, such that a first vector comprises a polynucleotide sequence coding for a modulator of the Notch signalling pathway and a second vector comprises a polynucleotide sequence coding for an autoantigen or bystander antigen antigenic determinant thereof.
  • a first vector comprises a polynucleotide sequence coding for a modulator of the Notch signalling pathway and a second vector comprises a polynucleotide sequence coding for an autoantigen or bystander antigen antigenic determinant thereof.
  • such vectors may be co-coated onto particles for delivery as described infra under the heading "Particles and Particle Delivery".
  • a method for producing a lymphocyte or antigen presenting cell capable of promoting tolerance to an autoantigen or bystander antigen which method comprises incubating a lymphocyte or APC obtained from a human or animal patient with (i) a modulator of the Notch signalling pathway 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.
  • the method comprises incubating a lymphocyte or APC obtained from a human or animal patient with an APC in the presence of (i) a modulator of the Notch signalling pathway 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.
  • a method for producing an APC capable of inducing tolerance in a T cell to an autoantigen or bystander antigen comprises contacting an APC with (i) a modulator of the Notch signalling pathway and (ii) an autoantigen or bystander antigen or antigenic detenninant thereof or a polynucleotide coding for an autoantigen or bystander antigen or antigenic determinant thereof.
  • a method for producing a T cell capable of promoting tolerance to an autoantigen or bystander antigen comprises incubating an antigen presenting cell (APC) simultaneously or sequentially, in any order, with:
  • a method for producing a lymphocyte or APC capable of promoting tolerance to an autoantigen or bystander antigen or antigenic determinant thereof, which method comprises incubating a lymphocyte or APC obtained from a human or animal patient with a lymphocyte or APC produced as described above.
  • lymphocyte or APC is incubated ex-vivo.
  • a method of promoting tolerance to an autoantigen or bystander antigen comprises administering to the patient a lymphocyte or APC produced by a method as described above.
  • APC includes any vehicle capable of presenting the desired NotcMigand to the T cell population.
  • suitable APCs include dendritic cells, L cells, hybridomas, lymphomas, macrophages, B cells or synthetic APCs such as lipid membranes.
  • the transfection may be brought about by a virus such as a retrovirus or adenovirus, or by any other vehicle or method capable of delivering a gene to the cells.
  • viruses such as a retrovirus or adenovirus
  • vehicle or method capable of delivering a gene to the cells.
  • retroviruses include any vehicles or methods shown to be effective in gene therapy and include retroviruses, liposomes, electroporation, other viruses such as adenovirus, adeno-associated virus, herpes virus, vaccinia, calcium phosphate precipitated D ⁇ A, DEAE dextran assisted transfection, microinjection, nucleofection, polyethylene glycol, protein-D ⁇ A complexes.
  • a method for reducing an immune response to a target disease antigen or antigenic determinant thereof by administering a bystander antigen or antigenic determinant thereof (or a polynucleotide coding for such an antigen or antigenic determinant) and simultaneously, separately or sequentially administering an activator of Notch signalling.
  • a method for reducing an immune response to a target disease autoantigen or antigenic determinant thereof by administering a bystander antigen or antigenic determinant thereof (or a polynucleotide coding for such an antigen or antigenic determinant) and simultaneously, separately or sequentially administering an activator of Notch signalling.
  • a product for reducing an immune response to a target disease antigen or antigenic determinant thereof comprising i) a bystander antigen or antigenic determinant thereof (or a polynucleotide coding for such an antigen or antigenic determinant) and ii) an activator of Notch signalling, for simultaneous, separate or sequential administration for reducing an immune response to a target disease antigen.
  • an activator of Notch signaling in simultaneous, separate or sequential combination with a bystander antigen or antigenic determinant thereof (or a polynucleotide coding for such an antigen or antigenic determinant) for reducing an immune response to a target antigen.
  • target disease antigen herein preferably means an antigen, which may or may not be explicitly identified, which is presented as part of an immune disease process, preferably being presented in an affected locus (eg organ or tissue) or lymphatic tissues draining this locus, together with one or more bystander antigens, wherein an unwanted or overly severe immune response against such target disease antigen contributes significantly to an immune disease or disorder.
  • the antigen may for example be an autoantigen, allergen or graft antigen.
  • bystander antigen herein preferably means an antigen presented as part of an immune disease process, preferably being presented in an affected locus (eg organ or tissue) or lymphatic tissues draining this locus, together with a target antigen, whether or not the bystander antigen contributes significantly to an unwanted or overly severe immune response.
  • the "bystander antigen” is not the or a primary causative antigen of the relevant disease state and may not itself contribute significantly to unwanted or overly severe immune response, but is frequently present at the site of that response (disease locus) as a "bystander”.
  • the bystander antigen may be an exogenous (foreign) antigen or antigenic determinant (eg KLH or any other suitable exogenous antigen) that is delivered to the affected target tisse (eg by direct physical introduction, such as by injection or other such means, or targeted with an agent which concentrates it at the requires site, such as an antibody specific for an antigen present at the target site) to trigger suppressive immune cells (preferably T-cells, preferably regulatory T-cells) in the target tissue or lymphatic tissues draining this tissue.
  • an exogenous (foreign) antigen or antigenic determinant eg KLH or any other suitable exogenous antigen
  • suppressive immune cells preferably T-cells, preferably regulatory T-cells
  • a method for generating immune suppression at a disease locus by: i) administering an exogenous antigen or antigenic determinant thereof (or a polynucleotide coding for such an exogenous antigen or antigenic determinant) and simultaneously, separately or sequentially administering an activator of Notch signalling and ii) administering or targeting said exogenous antigen or antigenic determinant (or a polynucleotide coding for such an antigen or antigenic determinant) to the disease locus to generate bystander immune suppression in said locus.
  • Figure 1 shows a schematic representation of the Notch signalling pathway
  • Figure 2 shows schematic representations of the Notch ligands Jagged and Delta
  • Figure 3 shows an example of a nucleotide vector according to one embodiment of the present invention
  • Figure 4 shows aligned amino acid sequences of DSL domains from various Drosophila and mammalian Notch ligands
  • Figure 5 shows amino acid sequences of human Delta- 1, Delta-2 and Delta-3
  • Figure 6 shows amino acid sequences of human Jagged-1 and Jagged-2
  • Figure 7 shows schematic representations of fusion proteins which may be used in the present invention
  • FIGS 8 and 9 show results from Example 5; and Figure 10 shows results from Example 7.
  • Drosophila and vertebrate names for genes and proteins are used interchangeably and all homologues are included within the scope of the invention.
  • 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 of the 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; without wishing to be bound by any theory of mode of action, it is believed that this suppression may be mediated at least in part 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 for example be antigen-nonspecific but locally restricted downregulation of the autoimmune responses responsible for tissue destruction.
  • Autoimmune disease includes spontaneous or induced malfunction of the 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 Histocompatibihty Complex (MHC) gene products on the surface of antigen-presenting cells (APC).
  • MHC Histocompatibihty Complex
  • autoimmune diseases and tissue- or organ-specific confirmed or potential bystander antigens and autoantigens effective in the treatment of these diseases is provided below.
  • 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 purpura.
  • 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 purpura, Behcet's Syndrome, Takayasu arteritis, Giant cell arteritis, Thrombangutis 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 derma
  • a more extensive list of disorders includes: unwanted immune reactions and inflammation hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis, 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, 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, complications and/or side effects from treatment of Parkinson's disease, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of strokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan- encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components
  • Autoimmune antigens may be derived from tissues, proteins etc associated with the disease which give rise to the relevant autoimmune response. For example:
  • 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
  • Idiopathic leukopenia granulocyte antigens Idiopathic thrombocytopenia platelet membrane proteins; Glycoprotein Ila/IIIb
  • RNA polymerase Primary biliary cirrhosis 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)
  • PASP post-acrosomal sperm protein
  • 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 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 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.
  • the autoantigen or bystander antigen may be a Goodpasture's autoantigen or bystander antigen for treatment of Goodpasture's disease.
  • 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 of the 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 IN, alpha 3 collagens.
  • the autoantigen or bystander antigen may be a renal autoantigen or renal bystander antigen for treatment of an autoimmune disease of the kidney.
  • autoimmune disease of the kidney includes any disease in which the kidney or renal system or a component thereof comes under autoimmune attack.
  • renal autoantigen includes any substance or a component thereof normally found within a mammal that, in autoimmune disease of the 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 of the 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 of the kidney under autoimmune attack in an autoimmune disease of the 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 treatment of 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 tenn 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 of the 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 desmogleins such as desmoglein 1 and desmoglein 3.
  • DSG1 desmoglein 1
  • NM_015548.1, NM_020388.2 and NM_001723.2 Bullous pemphigoid antigen 1
  • NM_001942.1 desmoglein 1 (DSG1)
  • NM_001944.1 desmoglein 3 (pemphigus vulgaris antigen; DSG3)
  • 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 Wegener's autoantigen or bystander antigen for treatment of Wegener's disease.
  • Wegener's autoantigen 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.
  • 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 of the 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.
  • autoantigen or bystander antigen may be an autoimmune anemia autoantigen or bystander antigen for treatment of 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 of the 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. Further examples include Wr , En a , Ge, A, B and antigens within the Kidd and Kell blood group systems.
  • the autoantigen or bystander antigen may be an autoimmune thrombocytopenia autoantigen or bystander antigen for treatment of 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 immuno genie substance that is, or is derived from, a component of the 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 purpura.
  • autoimmune thrombocytopenia purpura 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
  • MRARPRPRPL VTVLALGALAGVGVGGPNICTTRGVSSCQQCLAVSPMCAWCSDEALPLGSPRCDLKENLL KDNCAPESIEFPVSEARVLEDRPLSDKGSGDSSQVTQVSPQRIALRLRPDDSKNFSIQVRQVEDYPVDIYY LMDLSYSMKDDL SIQNLGTKATQMRKLTSNLRIGFGAFVDKPVSPYMYISPPEALENPCYDMKTTCLPM FGYKHVLT TDQVTRFNEEVKKQSVSRNRDAPEGGFDAIMQATVCDEKIG RNDASHLLVFTTDAKTHIAL DGRLAGIVQPNDGQCHVGSDNHYSASTT DYPSLGLMTEKLSQKNINLIFAVTENVVNLYQNYSELIPGTT VGVLSMDSSNVLQLIVDAYGKIRSKVELEVRDLPEELSLSFNATCLNNEVIPGLKSC GLKIGDTVSFSIE AKVRGCPQEKEKSFTIK
  • the autoantigen or bystander antigen may be an autoimmune gastritis autoantigen or bystander antigen for treatment of 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 of the 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, (eg lOOkDa alpha subunit and 60- 90kDa beta subunit; especially the beta subunit) and intrinsic factor.
  • MAALQEKKTCGQRMEEFQRYC NPDTGQMLGRTLSRWV ISLYYVAFYVVMTGLFALCLYVLMQTVDPYTP DYQDQLRSPGVTLRPDVYGEKGLEIVYNVSDNRTWADLTQTLHAFLAGYSPAAQEDSINCTSEQYFFQESF RAPNHTKFSCKFTADMLQNCSGLADPNFGFEEGKPCFIIK NRIVKFLPSNGSAPRVDCAFLDQPRELGQP LQVKYYPPNGTFSLHYFPYYGKKAQPHYSNPLVAAKLLNIPRNAEVAIVCKVMAEHVTFNNPHDPYEGKVE FKLKIEK
  • GenBank Accession No J05451 human gastric (H+/K+)-ATPase gene and GenBank Accession No M63962; human gastric H,K- ATPase catalytic subunit gene).
  • the autoantigen or bystander antigen may be an autoimmune hepatitis autoantigen or bystander antigen for treatment of autoimmune hepatitis.
  • autoimmune hepatitis includes any disease in which the liver or a component of the liver comes under autoimmune attack.
  • the term thus includes, for example, primary biliary cirrhosis (PBC) and primary sclerosing cholangitis.
  • PBC primary biliary cirrhosis
  • 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.
  • 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 of the 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 cytochromes, especially 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): i atggggctag aagcactggt gcccctggcc atgatagtgg ccatcttcct gctcctggtg
  • CYPl A2 An amino acid sequence for a human cytochrome P450-1 A2 (CYPl A2) is reported as follows (GenBank Accession No AF 182274): MALSQSVPFSATELLLASAIFCLVF VLKGLRPRVPKGLKSPPEP GWPLLGHVLTLGKNPHLALSRMSQR YGDVLQIRIGSTPVLVLSRLDTIRQALVRQGDDFKGRPDLYTSTLITDGQSLTFSTDSGPVWAARRRLAQN ALNTFSIASDPASSSSCYLEEHVSKEAMALISRLQELMAGPGHFDPYNQVVVSVSVANVIGAMCFGQHFPESS DEMLSLVKNTHEFVETASSGNPLDFFPILRYLPNPALQRFPAFNQRFL FLQKTVQEHYQDFDKNSVRDIT GALFKHSKKGPRASGNLIPQEKIVNLVNDVFGAGFDTVTTAISWSL YLVTKPEIQRKIQKELDTVIGRER RPRLSDRP
  • PBC primary biliary cirrhosis
  • bystander antigens include, but are not limited to mitochondrial antigens such as pyruvate dehydrogenases (eg El- alpha decarboxylase, El -beta decarboxylase and E2 acetyltransferase), branched-chain 2- oxo-acid dehydrogenases and 2-oxoglutarate dehydrogenases.
  • mitochondrial antigens such as pyruvate dehydrogenases (eg 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 treatment of 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 purpura, 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 purpura,
  • 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 immuno genie substance that is, or is derived from, a component of the 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.
  • vasculitis autoantigens and bystander antigens include, but are not limited to basement membrane antigens (especially the noncollagenous domain of the alpha 3 chain of type IN collagen) and endothelial cell antigens.
  • the autoantigen or bystander antigen may be an ocular autoantigen or bystander antigen for treatment of an autoimmune disease of the eye.
  • autoimmune disease of the 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, Nogt-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 of the 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 of the 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, interphotoreceptor retinoid binding protein (see eg Exp. Eye Res. 56:463 (93)) in uveitis and alpha crystallin in lens-induced uveitis.
  • retinal antigens such as ocular antigen, S-antigen, interphotoreceptor 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 treatment of an 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 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 of the 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. 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.
  • ACTH receptor adrenocorticotropic hormone receptor
  • MWELVALLLLTLAYLFWPKRRCPGAKYP SLLSLPLVGSLPFLPRHGHMHNNFFKLQKKYGPIYSVRMGTK TTVIVGHHQLAKEVLIKKGKDFSGRPQMATLDIASNNRKGIAFADSGAH QLHRRLAMATFALFKDGDQKL EKIICQEISTLCDMLATHNGQSIDISFPVFVAVTNVIS ICFNTSYKNGDPELNVIQNYNEGIIDNLSKDS LVDLVPWLKIFPNKTLEKLKSHVKIRNDLLNKILENYKEKFRSDSITNMLDTLMQAKMNSDNGNAGPDQDS ELLSDNHILTTIGDIFGAGVETTTSVVK TLAFLLHNPQVKKKLYEEIDQNVGFSRTPTISDRNRLLLLEA TIREVLRLRPVAPMLIPHKANVDSSIGEFAVDKGTEVIIN WALHHNEKEWHQPDQFMPERFLNPAGTQLI SPSVSYLPFGAGPRSCIGEI
  • the autoantigen or bystander antigen may be a cardiac autoantigen or bystander antigen for treatment of 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 of the 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 mysosins, laminins, beta-1 adrenergic receptors, adenine nucleotide translocator (ANT) protein and branched-chain ketodehydrogenase (BCKD).
  • heart muscle cell antigens such as mysosins, laminins, 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 treatment of scleroderma or myositis.
  • 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.
  • 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 of the 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 of the 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 of the 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.
  • the centrosome includes a pair of centrioles that lie at the center of a dense, partially filamentous matrix, the pericentriolar material (PCM).
  • PCM pericentriolar material
  • 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.
  • MTOC microtubule organizing center
  • idiopathic inflammatory myopathies polymyositis, dermatomyositis and the related overlap syndromes disorder, such as polymyositis- scleroderma 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. Ill: 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. 23: 881-888 (1980); Reichlin, et al., J. Clin. Immunol. 4:40-44 (1984)).
  • 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 Ul small nuclear ribonucleoprotein, which is one of the 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 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 (Ul 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
  • MAAPAFEPGRQSDLLV LNRMERC RNSKCIDTESLCVVAGEKVWQIRVDLHLLNHDGNIIDAASIAAIV ALCHFRRPDVSVQGDEVTLYTPEERDPVPLSIHHMPICVSFAFFQQGTYLLVDPNEREERVMDGLLVIA N KHREICTIQSSGGIMLLKDQVLRCSKIAGVKVAEITELILKALENDQKVRKEGGKFGFAESIANQRITAFK MEKAPIDTSDVEEKAEEIIAEAEPPSEWSTPVL TPGTAQIGEGVENS GDLEDSEKEDDEGGGDQAIIL DGIKMDTGVEVSDIGSQELGFHHVGQTGLEFLTSDAPIILSDSEEEEMIILEPDKNPKKIRTQTTSAKQEK APSKKPVKRRKKKRAAN
  • 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/sclerodenna 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 of the nervous system.
  • autoimmune disease of the 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-Barre 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 dors
  • neural system autoantigen includes any nervous system substance or a component thereof normally found within a mammal that, in an autoimmune disease of the 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 of the 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 of the organ or tissue under autoimmune attack in an autoimmune disease of the 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.
  • 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.
  • nervous system autoantigens and nervous system bystander antigens include, but are not limited to, myelin basic proteins (MBPs), DM20,central nervous system white matter; proteolipid proteins (PLPs); myelin oligodendrocyte-associated proteins (MOGs), myelin associated glycoproteins (MAGs), alpha B-crystallins (see eg J. Chromatog. Biomed. Appl. 526:535 (90))
  • MBPs myelin basic proteins
  • PBPs proteolipid proteins
  • MOGs myelin oligodendrocyte-associated proteins
  • MAGs myelin associated glycoproteins
  • alpha B-crystallins see eg J. Chromatog. Biomed. Appl. 526:535 (90)
  • myelin proteins including myelin basic protein (MBP) I proteolipid protein (PLP), myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG), are of particular interest.
  • MBP myelin basic protein
  • PBP I proteolipid protein
  • MAG myelin-associated glycoprotein
  • MOG myelin oligodendrocyte glycoprotein
  • 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.
  • 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, NHFFKNIVTPRTP (p87-99), 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.
  • the immunodominant MBP (84102) peptide has been found to bind with high affinity to DRB1*1501 and DRB5*0101 molecules of the disease-associated DR2 haplotype. Overlapping but distinct peptide segments were important for binding to these molecules; hydrophobic residues (Vail 89 and Phe92) in the MBP (88-95) segment for peptide binding to DRB 1*1501 molecules; hydrophobic and charged residues (Phe92, Lys93) in the MBP (89-101/102) sequence contributed to DRB5*0101 binding.
  • MBP myelin basic protein
  • 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).
  • MAG myelin-associated glycoprotein
  • 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):
  • HFFKNIVTPRTPP MBP (aa 90-102)
  • VDAQGTLSKIFKLGGRDSRS MBP (aa 144-163)
  • the autoantigen or bystander antigen may be autoimmune arthritis autoantigen or bystander antigen for use to treat autoimmune arthritis.
  • autoimmune arthritis autoantigen 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.
  • 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 arthritis 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 the organ or tissue under autoimmune attack in autoimmune arthritis, especially rheumatoid arthritis (RA).
  • RA rheumatoid arthritis
  • 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 arthritis includes rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, spondylo arthritis, relapsing polychondritis and other connective tissue diseases having an autoimmune disease component.
  • 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 inter alia, in cartilage, the interverbebral disc and the vitreous body.
  • Type II collagen contains three alphal(II) chains (alphal(II) 3 ).
  • Type III collagen is found inter alia, in blood vessels, the cardiovascular system and fetal skin and contains three alphal(III) chains (alphal(III) 3 ).
  • Type IN collagen is localized, inter alia, in basement membranes and contains three alpha 1 (IN) chains (alphal(IN) ).
  • the autoantigen or bystander antigen may be a diabetes autoantigen or bystander antigen for use to treat autoimmune diabetes.
  • autoimmune diabetes includes all fonns 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 of the 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).
  • T lymphocytes play a major role as effector cells in the destruction reaction.
  • autoimmune diseases type I diabetes arises because the tolerance of the 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.
  • Type I diabetes mellitus normally results from a well-characterized insulitis. During this condition, the inflammatory cells are typically directed against the beta cells of the pancreatic islets. It has been demonstrated that a large proportion of the infiltrating T lymphocytes produced during Type I diabetes mellitus are CD8-positive cytotoxic cells, which confirms the cytotoxic activity of the 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).
  • IgG immunoglobulin-G
  • diabetes autoantigen 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.
  • 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.
  • diabetes 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 the 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.
  • 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.
  • diabetes autoimmune/bystander antigens and diabetes autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
  • 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, peripherals and gangliosides. Some of these are discussed in more detail below.
  • Type I pancreatic beta cell
  • IA-W insulin associated antigens
  • GADs gamma amino decarboxylases
  • HSPs heat shock proteins
  • 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.
  • proinsulin is exposed to several specific endopeptidases which excise the C peptide, thereby generating the mature form of insulin which consists of the 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:
  • 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 (corresponding to residues 9-23 of the B-chain), are useful as autoantigens in the methods and compositions described herein.
  • 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:
  • IA-2/ICA512 a member of the 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:
  • 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:
  • 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:
  • 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).
  • HSP60 Heat shock protein 60
  • HSP60 e. g., an immunologically active fragment of HSP60, e. g., p277 (see Elias et al., Eur Jlmmunol 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.
  • 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 ;
  • carboxypeptidase H or immunologically reactive fragments thereof e. g., the 136-amino acid fragment of carboxypeptidase-H described in Castano, supra
  • carboxypeptidase H or immunologically reactive fragments thereof can be used in the methods and compositions described herein.
  • 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:
  • Gangliosides are sialic acid-containing glycolipids which are formed by a hydrophobic portion, the ceramide, and a hydrophilic part, i. e. the oligosaccharide chain. Gangliosides are expressed, inter alia, in cytosol membranes of secretory granules of pancreatic islets. Auto-antibodies to gangliosides have been described in type 1 diabetes, e. g., GM1-2 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). Gangliosides GT3, GD3 and GM-1 are also the target of autoantibodies associated with autoimmune diabetes (reviewed in Dionisi et al. Aim 1st
  • Ganglioside GM3 participates in the pathological conditions of insulin resistance (Tagami et al. J Biol Chem 2001 Nov 13; online publication ahead of print).
  • ICAp69 islet cell antigen 512
  • A28076.1 islet GAD sequence (HIGAD- FL)
  • AF098915.1 type 1 diabetes autoantigen ICA12
  • the autoantigen or bystander antigen may be a Myasthenia Gravis autoantigen or bystander antigen for use to treat Myasthenia Gravis.
  • Myasthenia Gravis autoantigen 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.
  • 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.
  • Myasthenia Gravis 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 the 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.
  • 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.
  • 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):
  • acetylcholine receptor can be isolated, for example, by the method of Mcintosh et al. J Neuroimmunol. 25: 75, 1989.
  • 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): Peptide Sequence Source
  • MAHYNRVPALPFPGDPRPYL AChR gamma (aa 476-495)
  • the autoantigen or bystander antigen may be a Systemic Lupus Erythematosus (SLE) autoantigen or bystander antigen for use to treat SLE.
  • SLE Systemic Lupus Erythematosus
  • SLE autoantigen 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.
  • SLE Systemic Lupus Erythematosus
  • 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.
  • SLE 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 the 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.
  • 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
  • SLE autoimmune/bystander antigens and SLE autoimmune/bystander antigenic determinants and/or polynucleotide sequences coding for them may also be used as appropriate.
  • SLE autoantigens and SLE bystander antigens include, but are not limited to, ds-DNA, chromatins, histones, nucleolar antigens, soluble RNA protein particles (such as U1RNP, Sm, Ro/SSA and La/SSB) erythrocyte antigens and platelet antigens.
  • proteins include, for example, the human Ku and La antigens.
  • an amino acid sequence for a human lupus p70 (Ku) autoantigen protein is reported as follows (GenBank Accession No J04611): MSG ESYYKTEGDEEAEEEQEENLEASGDYKYSGRDSLIFLVDASKAMFESQSEDELTPFDMSIQCIQSVY ISKIISSDRDLLAVVFYGTEKDKNSVNFKNIYVLQELDNPGAKRILELDQFKGQQGQKRFQDMMGHGSDYS LSEVL VCANLFSDVQFKMSHKRIMLFTNEDNPHGNDSAKASRARTKAGDLRDTGIFLDLMHLKKPGGFDI SLFYRDIISIAEDEDLRVHFEESSKLEDLLRKVRAKETRKRALSRLKLKLNKDIVISVGIYNLVQKAL PP PIKLYRETNEPVKTKTRTFNTSTGGLLLPSDTKRSQIYGSRQIILEKEETEELKRFDDPGLMLMGFKPLVL LKKH
  • the autoantigen or bystander antigen may be a bowel autoantigen or bystander antigen for use to treat an autoimmune disease of the bowel.
  • autoimmune disease of the bowel includes any disease in which the bowel or a component of the bowel comes under autoimmune attack.
  • the main autoimmune diseases of the bowel are inflammatory bowel disease (IBD) and celiac (also known as coeliac) disease.
  • IBD Inflammatory bowel disease
  • Ulcerative colitis is a chronic inflammatory disease mainly affecting the large intestine.
  • the course of the disease may be continuous or relapsing, mild or severe.
  • the earliest lesion is typically an inflammatory infiltration with abscess formation at the base of the 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 of the 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
  • ulcerative colitis 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 of the disease is the granular, reddish- purple edematous thickening of the bowel wall. With the development of inflammation, these granulomas often lose their circumscribed borders and integrate with the surrounding tissue. Diarrhea and obstruction of the bowel are the predominant clinical features.
  • ulcerative colitis the course of the disease may be continuous or relapsing, mild or severe but, unlike ulcerative colitis, it is not curable by resection of the 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 is a disease of the intestinal mucosa and is usually identified in infants and children. Celiac disease is associated with an inflammation of the mucosa, which causes malabsorption. 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 of the small intestine.
  • bowel autoantigen includes any substance or a component thereof normally found within a mammal that, in an autoimmune disease of the 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 of the 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.
  • 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.
  • bowel bystander antigen includes any substance capable of eliciting an immune response, including proteins, protein fragments, polypeptides, peptides, glycoproteins, nucleic acids, pofysaccliarides or any other immunogenic substance that is, or is derived from, a component of the bowel under autoimmune attack in an autoimmune disease of the bowel.
  • 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 - Ill - immune system which become exposed in the locus of autoimmune attack as a result of autoimmune tissue destruction.
  • Examples of bowel autoantigens and bystander antigens include, but are not limited to, gliadins and tissue transglutaminases (tTG) (associated with celiac disease; see Marsh, Nature Medicine 1997;7:725-6) and tropomyosins, in particular tropomyosin isoform 5, (associated with ulcerative colitis).
  • tTG tissue transglutaminases
  • the autoantigen or bystander antigen may be a Sjogren's syndrome autoantigen or bystander antigen or antigenic determinant thereof, for use to treat an autoimmune disease of the bowel.
  • Sjogren's syndrome autoantigen includes any substance or a component thereof normally found within a mammal that, in Sjogren's syndrome, 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 Sjogren's syndrome when administered to mammals. Additionally, the term includes fragments comprising antigenic determinants (epitopes; preferably immunodominant epitopes) or epitope regions (preferably immunodominant epitope regions) of Sjogren's syndrome 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.
  • Sjogren's syndrome 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 the organ or tissue under autoimmune attack in Sjogren's syndrome.
  • 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.
  • Sjogren's syndrome autoantigens and Sjogren's syndrome bystander antigens include, but are not limited to, the following:
  • amino acid sequence for a human 52-kD SS-A/Ro autoantigen is reported as follows (GenBank Accession No M62800 M35041):
  • NM_003141.2 Sjogren syndrome antigen Al (52kDa, ribonucleoprotein autoantigen SS- A Ro) (SSAl)); NM_004600.1 (Sjogren syndrome antigen A2 (60kDa, ribonucleoprotein autoantigen SS-A/Ro) (SSA2)); NM_003142.1, BC001289.1, BC020818.1 (Sjogren syndrome antigen B (autoantigen La) (SSB)); NM_003731.1, BC000864.1 (Sjogren's syndrome nuclear autoantigen 1 (SSNA1)); NM_006396.1, BC014791.1
  • the autoantigen or bystander antigen may be a thyroid autoantigen or bystander antigen or antigenic determinant thereof, for use to treat an autoimmune disease of the thyroid.
  • 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 of the 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 of the 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 autoantigens and thyroid bystander antigens include, but are not limited to, the thyroid stimulatory hormone (TSH) receptor (associated in particular with Grave's disease), thyroperoxidases (TPO; associated with Hashimoto's thyroiditis) and thyro globulins (Tg).
  • TSH thyroid stimulatory hormone
  • TPO thyroperoxidases
  • Tg thyro globulins
  • the autoantigen or bystander antigen may be a skin autoantigen or bystander antigen or antigenic determinant thereof, for use to treat an autoimmune disease of the skin.
  • skin autoantigen as used herein includes any substance or a component thereof normally found within a mammal that, in an autoimmune disease of the skin, such as Psoriasis or Nitiligo (or eg Pemphigus as mentioned above), 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 of the skin 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.
  • skin 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 the organ or tissue under autoimmune attack in an autoimmune disease of the skin.
  • 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 enocrine autoantigen or bystander antigen or antigenic determinant thereof, for use to treat an autoimmune disease of an endocrine gland.
  • endocrine autoantigen as used herein includes any substance or a component thereof normally found within a mammal that, in an autoimmune disease of an endocrine gland, such as Autoimmune oophoritis (or eg Grave's disease or diabetes as mentioned above), 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 of the skin 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.
  • endocrine 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 the organ or tissue under autoimmune attack in an autoimmune disease of an endocrine gland.
  • 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.
  • modulate refers to a change or alteration in the biological activity of the 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 of the Notch signalling pathway. Conveniently such compounds may be referred to herein as inhibitors or antagonists.
  • 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 of the Notch signalling pathway. Conveniently such compounds may be referred to as upregulators or agonists.
  • the modulator is an agonist of Notch signalling, and preferably an agonist of the Notch receptor (eg an agonist of the Notchl, Notch2, Notch3 and/or Notch4 receptor).
  • the active agent of the present invention may be an organic compound or other chemical.
  • a 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. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc.
  • substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc.
  • a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other.
  • the carbons may be linked via a suitable element or group.
  • the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
  • the candidate modulator may comprise at least one cyclic group.
  • the cyclic group may be a polycyclic group, such as a non-fused polycyclic group.
  • the agent comprises at least the one of said cyclic groups linked to another hydrocarbyl group.
  • the modulator will be an amino acid sequence or a chemical derivative thereof, or a combination thereof.
  • the modulator will be a nucleotide sequence - which may be a sense sequence or an anti- sense sequence.
  • the modulator may also be an antibody.
  • antibody includes intact molecules as well as fragments 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;
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
  • F(ab') 2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction;
  • F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds;
  • scFv including a genetically engineered fragment containing the variable region of a heavy and a light chain as a fused single chain molecule.
  • Modulators may be synthetic compounds or natural isolated compounds.
  • the modulator of the Notch signalling pathway may be a protein for Notch signalling transduction.
  • a protein which is for Notch signalling transduction is meant a molecule which participates in signalling through Notch receptors including activation of Notch, the downstream events of the Notch signalling pathway, transcriptional regulation of downstream target genes and other non-transcriptional downstream events (e.g. post- translational modification of existing proteins). More particularly, the protein is a domain that allows activation of target genes of the Notch signalling pathway, or a polynucleotide sequence which codes therefor.
  • a very important component of the Notch signalling pathway is Notch receptor/Notch ligand interaction.
  • Notch signalling may involve changes in expression, nature, amount or activity of Notch ligands or receptors or their resulting cleavage products.
  • Notch signalling may involve changes in expression, nature, amount or activity of Notch signalling pathway membrane proteins or G-proteins or Notch signalling pathway enzymes such as proteases, kinases (e.g. serine/threonine kinases), phosphatases, ligases (e.g. ubiquitin ligases) or glycosyltransferases.
  • the signalling may involve changes in expression, nature, amount or activity of DNA binding elements such as transcription factors.
  • the signalling may be specific signalling, meaning that the signal 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.
  • the Notch signalling pathway is described in more detail below.
  • E[spl] Enhancer of split complex
  • these genes have been shown to be direct targets for binding by the Su(H) protein and to be transcriptionally activated in response to Notch signalling.
  • EBNA2 a viral coactivator protein that interacts with a mammalian Su(H) homologue CBF1 to convert it from a transcriptional repressor to a transcriptional activator
  • the Notch intracellular domain may combine with Su(H) to contribute an activation domain that allows Su(H) to activate the transcription of E(spl) as well as other target genes.
  • the active agent may be Notch or a fragment thereof which retains the signalling transduction ability of Notch or an analogue of Notch which has the signalling transduction ability of Notch.
  • analogue of Notch includes variants thereof which retain the signalling transduction ability of Notch.
  • analogue we include a protein which has Notch signalling transduction ability, but generally has a different evolutionary origin to Notch.
  • Analogues of Notch include proteins from the Epstein Barr virus (EBN), such as EB ⁇ A2, BARFO or LMP2A.
  • a protein which is for Notch signalling activation we mean a molecule which is capable of activating Notch, the Notch signalling pathway or any one or more of the components of the Notch signalling pathway.
  • the modulator of Notch signalling may be a Notch ligand, or a polynucleotide encoding a Notch ligand.
  • Notch ligands of use in the present invention include endogenous Notch ligands which are typically capable of binding to a Notch receptor polypeptide present in the membrane of a variety of mammalian cells, for example hemapoietic stem cells.
  • Notch ligand means an agent capable of interacting with a Notch receptor to cause a biological effect.
  • the term as used herein therefore includes naturally occurring protein ligands such as Delta and Serrate/ Jagged and their biologically active fragments as well as antibodies to the Notch receptor, peptidomimetics and small molecules which have corresponding biological effects to the natural ligands.
  • the Notch ligand interacts with the Notch receptor by binding.
  • Delta particularly examples of naturally occurring mammalian Notch ligands identified to date include the Delta family, for example Delta or Delta-like 1 (Genbank Accession No. AF003522 - Homo sapiens), Delta-3 (Genbank Accession No. AF084576 - Rattus norvegicus) and Delta-like 3 (Mus musculus) (Genbank Accession No. NM_016941 - Homo sapiens) and US 6121045 (Millennium), Delta-4 (Genbank Accession Nos.
  • Serrate- 1 and Serrate-2 WO97/01571, WO96/27610 and WO92/19734
  • Jagged-1 Genbank Accession No. U73936 - Homo sapiens
  • Jagged-2 Genbank Accession No. AF029778 - Homo sapiens
  • LAG-2 LAG-2. Homology between family members is extensive.
  • an activator of Notch signalling may be a constitutively active Notch receptor or Notch intracellular domain, or a polynucleotide encoding such a receptor or intracellular domain.
  • an activator of Notch signalling may act downstream of the Notch receptor.
  • the activator of Notch signalling may be a constitutively active Deltex polypeptide or a polynucleotide encoding such a polypeptide.
  • Other downstream components of the Notch signalling pathway of use in the present invention include the polypeptides involved in the Ras/MAPK cascade catalysed by Deltex, polypeptides involved in the proteolytic cleavage of Notch such as Presenilin and polypeptides involved in the transcriptional regulation of Notch target genes, suitably in a constitutively active form.
  • polypeptide for Notch signalling activation is also meant any polypeptides expressed as a result of Notch activation and any polypeptides involved in the expression of such polypeptides, or polynucleotides coding for such polypeptides.
  • a modulator of Notch signalling may be a molecule which is capable of enhancing Notch-Notch ligand interactions.
  • a molecule may be considered to enhance Notch-Notch ligand interactions if it is capable of enhancing the interaction of Notch with its ligands, preferably to an extent sufficient to provide therapeutic efficacy.
  • the inhibitor is a receptor or a nucleic acid sequence encoding a receptor
  • the receptor is activated.
  • the agent is a nucleic acid sequence
  • the receptor is preferably constitutively active when expressed.
  • 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 and/or a targeting molecule in any of a variety of drug screening techniques.
  • the target employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • Techniques for drug screening may be based on the method described in Geysen, European Patent No. 0138855, published on September 13, 1984.
  • large numbers of different small peptide candidate modulators or targeting molecules are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with a suitable target or fragment thereof and washed. Bound entities are then detected - such as by appropriately adapting methods well known in the art.
  • a purified target can also be coated directly onto plates for use in drug screening techniques. Plates of use for high throughput screening (HTS) will be multi-well plates, preferably having 96, 384 or over 384 wells/plate. Cells can also be spread as "lawns".
  • non-neutralising antibodies can be used to capture the peptide and immobilise it on a solid support.
  • High throughput screening as described above for synthetic compounds, can also be used for identifying organic candidate modulators and targeting molecules.
  • This invention also contemplates the use of competitive drug screening assays in which neutralising antibodies capable of binding a target specifically compete with a test compound for binding to a target.
  • amino acid sequence is synonymous with the term amino acid sequence
  • polypeptide and/or the term “protein”.
  • amino acid sequence is synonymous with the term “peptide”.
  • amino acid sequence is synonymous with the term “protein”.
  • Protein usually refers to a short amino acid sequence that is, for example, about 10 to 40 amino acids long, preferably 10 to 35 amino acids.
  • amino acid sequence may be prepared and isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
  • variant protein can be modified by addition, deletion and/or substitution of at least one amino acid present in the naturally-occurring protein.
  • amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence retains the required target activity or ability to modulate Notch signalling.
  • Amino acid substitutions may include the use of non-naturally occurring analogues.
  • a protein used 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 target 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, phenylalanin ⁇ , and tyrosine.
  • 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.
  • a peptide useful in the invention will at least have a target or signalling modulation capability.
  • “Fragments” are also variants and the term typically refers to a selected region of the protein that is of interest in a binding assay and for which a binding partner is known or determinable.
  • “Fragment” thus refers to an amino acid sequence that is a portion of a full-length polypeptide, suitably between about 8 and about 1500 amino acids in length, for example between about 8 and about 745 amino acids in length, preferably about 8 to about 300, more preferably about 8 to about 200 amino acids, for example about 10 to about 50 or 100 amino acids in length.
  • “Peptide” refers to a short amino acid sequence that is 10 to 40 amino acids long, preferably 10 to 35 amino acids.
  • Such variants may be prepared using standard recombinant DNA techniques such as site- directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.
  • Variants of the nucleotide sequence may also be made. Such variants will preferably comprise codon optimised sequences. Codon optimisation is known in the art as a method of enhancing RNA stability and therefore 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. Niruses such as HIN, for instance, use a large number of rare codons. By changing a nucleotide sequence such that rare codons are replaced by the corresponding commonly used mammalian codons, increased expression of the sequences in mammalian target cells can be achieved. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other
  • the modulator of Notch signalling or antigen/antigenic determinant comprises a nucleotide sequence it may suitably be codon optimised for expression in mammalian cells. In a preferred embodiment, such sequences are optimised in their entirety.
  • “Polynucleotide” refers to a polymeric form of nucleotides of at least 10 bases in length and up to 10,000 bases or more, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA and also derivatised versions such as protein nucleic acid (PNA).
  • PNA protein nucleic acid
  • 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 of the appropriate techniques.
  • Nucleic acid encoding the second region may likewise be provided in a similar vector system.
  • Sources of nucleic acid may be ascertained by reference to published literature or databanks such as GenBank.
  • Nucleic acid encoding the desired first or second sequences may be obtained from academic or commercial sources where such sources are willing to provide the material or by synthesising or cloning the appropriate sequence where only the sequence data are available. Generally this may be done by reference to literature sources which describe the cloning of the gene in question.
  • nucleic acids can be characterised as those nucleotide sequences winch hybridise to the nucleic acid sequences known in the art. It will be understood by a skilled person that numerous different nucleotide sequences can encode the same protein used in the present invention as a result of the degeneracy of the genetic code.
  • nucleotide substitutions that do not affect the protein encoded by the nucleotide sequence of the present invention to reflect the codon usage of any particular host organism in which the target protein or protein for Notch signalling modulation of the present invention is to be expressed.
  • variant in relation to the nucleotide sequence used in the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for an active protein, peptide or polypeptide.
  • sequence homology preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology, similarity or identity to the reference sequences, preferably over the entire length of the reference sequences. More preferably there is at least 95%, more preferably at least 98%, homology, similarity or identityover the entire length of the reference sequences.
  • Nucleotide homology comparisons may be conducted as described below.
  • a preferred sequence comparison program is the GCG Wisconsin Bestfit program described above.
  • the default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch.
  • the default gap creation penalty is -50 and the default gap extension penalty is - 3 for each nucleotide.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences. Percent homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • blastp - compares an amino acid query sequence against a protein sequence database.
  • blastx compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.
  • tblastn compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands).
  • tblastx compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • BLAST uses the following search parameters:
  • HISTOGRAM - Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual).
  • DESCRIPTIONS Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page).
  • EXPECT The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).
  • CUTOFF - Cutoff score for reporting high-scoring segment pairs.
  • the default value is calculated from the EXPECT value (see above).
  • HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual). Typically, significance thresholds can be more intuitively managed using EXPECT.
  • ALIGNMENTS Restricts database sequences to the number specified for which high- scoring segment pairs (HSPs) are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual).
  • MATRIX - Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX.
  • the default matrix is BLOSUM62 (Henikoff & Henikoff, 1992).
  • the valid alternative choices include: PAM40, PAM120, PAM250 and IDENTITY.
  • No alternate scoring matrices are available for BLASTN; specifying the MATRIX directive in BLASTN requests returns an error response.
  • FILTER - Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17:149-163, or segments consisting of short-periodicity internal repeats, as determined by the XNU program of Claverie & States (1993) Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov). Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.
  • Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs.
  • NCBI-gi causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.
  • sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.
  • no gap penalties are used when determining sequence identity.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • the present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the reference sequences, or any variant, fragment or derivative thereof, or to the complement of any of the above.
  • Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
  • hybridization includes "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
  • Nucleotide sequences useful in the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement will be generally at least 75%, preferably at least 85 or 90% and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
  • Preferred nucleotide sequences of the invention will comprise regions homologous to the nucleotide sequence, preferably at least 80 or 90% and more preferably at least 95% homologous to the nucleotide sequence.
  • the term "selectively hybridizable" means that the nucleotide sequence used as a probe is used under conditions where a target nucleotide sequence of the invention is found to hybridize to the probe at a level significantly above background.
  • the background hybridization may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened, hi this event, background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA.
  • the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P.
  • Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Nol 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
  • maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
  • a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.
  • the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention under stringent conditions (e.g.
  • lxSSC 0.15 M NaCl, 0.015 M Na 3 Citrate pH 7.0.
  • nucleotide sequence of the invention is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the present invention.
  • nucleotide sequence is single-stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also included within the scope of the present invention.
  • Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skill in the field. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrid which decreases approximately 1 to 1.5°C with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridisation reaction is performed under conditions of higher stringency, followed by washes of varying stringency.
  • high stringency preferably refers to conditions that permit hybridisation of only those nucleic acid sequences that 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.
  • Nucleotide sequences can be obtained in a number of ways. Variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of sources. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of the reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the amino acid and/or nucleotide sequences useful in the present invention.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer 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 of the target protein or protein for T cell signalling modulation encoded by the nucleotide sequences.
  • the nucleotide sequences such as a DNA polynucleotides useful in the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • PCR polymerase chain reaction
  • This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction (PCR) under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • 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 incorporate expression systems or polynucleotides of the invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis el al and Sambrook et al, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • 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 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 such as CHO, COS, NSO, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells.
  • Proteins or polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein or precursor.
  • an additional amino acid sequence which contains secretory or leader sequences or pro-sequences (such as a HIS oligomer, immunoglobulin Fc, glutathione S- transferase, FLAG etc) to aid in purification.
  • secretory or leader sequences or pro-sequences such as a HIS oligomer, immunoglobulin Fc, glutathione S- transferase, FLAG etc
  • additional sequence may sometimes be desirable to provide added stability during recombinant production.
  • the additional sequence may be cleaved (eg chemically or enzymatically) to yield the final product.
  • the additional sequence may also confer a desirable pharmacological profile (as in the case of IgFc fusion proteins) in which case it may be preferred that the additional sequence is not removed so that it is present in
  • vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard.
  • the appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al.
  • secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • 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.
  • Chemically coupled sequences can be prepared from individual proteins sequences and coupled using known chemically coupling techniques.
  • the conjugate can be assembled using conventional solution- or solid-phase peptide synthesis methods, affording a fully protected precursor with only the terminal amino group in deprotected reactive form.
  • This function can then be reacted directly with a protein or polypeptide or a suitable reactive derivative thereof.
  • this amino group may be converted into a different functional group suitable for reaction with a cargo moiety or a linker.
  • reaction of the amino group with succinic anhydride will provide a selectively addressable carboxyl group, while further peptide chain extension with a cysteine derivative will result in a selectively addressable thiol group.
  • a protein or polypeptide or a derivative thereof may be attached through e.g. amide, ester, or disulphide bond formation.
  • Cross-linking reagents which can be utilized are discussed, for example, in Neans, G.E. and Feeney, R.E., Chemical Modification of Proteins, Holden-Day, 1974, pp. 39-43.
  • Notch signalling pathway directs binary cell fate decisions in the embryo. Notch was first described in Drosophila as a transmembrane protein that functions as a receptor for two different ligands, Delta and Serrate. Vertebrates express multiple Notch receptors and ligands (discussed below). At least four Notch receptors (Notch-1, Notch-2, Notch-3 and Notch-4) have been identified to date in human cells (see for example GenBank Accession Nos. AF308602, AF308601 and U95299 - Homo sapiens).
  • Notch proteins are synthesized as single polypeptide precursors that undergo cleavage via a Furin-like convertase that yields two polypeptide chains that are further processed to form the mature receptor.
  • the Notch receptor present in the plasma membrane comprises a heterodimer of two Notch proteolytic cleavage products, one comprising an N-terminal fragment consisting of a portion of the extracellular domain, the transmembrane domain and the intracellular domain, and the other comprising the majority of the extracellular domain.
  • the proteolytic cleavage step of Notch to activate the receptor occurs in the Golgi apparatus and is mediated by a furin-like convertase.
  • 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
  • the Notch ligands also display multiple EGF-like repeats in their extracellular domains together with a cysteine-rich DSL (Delta-Serrate Lag2) domain that is characteristic of all Notch ligands (Artavanis-Tsakonas).
  • the Notch receptor is activated by binding of extracellular ligands, such as Delta, Serrate and Scabrous, to the EGF-like repeats of Notch's extracellular domain. Delta may require cleavage for activation. It is cleaved by the ADAM disintegrin metalloprotease Kuzbanian at the cell surface, the cleavage event releasing a soluble and active form of Delta.
  • Su(H) is the Drosophila homologue of C-promoter binding factor-1 [CBF-1], a mammalian DNA binding protein involved in the Epstein-Barr virus-induced immortalization of B-cells. It has been demonstrated that, at least in cultured cells, Su(H) associates with the cdclO/ankyrin repeats in the cytoplasm and translocates into the nucleus upon the interaction of the Notch receptor with its ligand Delta on adjacent cells. Su(H) includes responsive elements found in the promoters of several genes and has been found to be a critical downstream protein in the Notch signalling pathway. The involvement of Su(H) in transcription is thought to be modulated by Hairless.
  • NotchlC The intracellular domain of Notch (NotchlC) also has a direct nuclear function (Lieber). Recent studies have indeed shown that Notch activation requires that the six cdclO/ankyrin repeats of the Notch intracellular domain reach the nucleus and participate in transcriptional activation.
  • the site of proteolytic cleavage on the intracellular tail of Notch has been identified between glyl743 and vall744 (termed site 3, or S3) (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 CBF1 (suppressor of hairless, Su(H) in Drosophila, Lag-2 in C. elegans) (Schroeter; Struhl).
  • CSL family protein CBF1 suppressor of hairless, Su(H) in Drosophila, Lag-2 in C. elegans
  • the NotchlC- CBF1 complexes then activate target genes, such as the bHLH proteins HES (hairy- enhancer of split like) 1 and 5 (Weinmaster).
  • This nuclear function of Notch has also been shown for the mammalian Notch homologue (Lu).
  • the post-translational modification of the nascent Notch receptor in the Golgi appears, at least in part, to control which of the two types of ligand is expressed on a cell surface.
  • the Notch receptor is modified on its extracellular domain by Fringe, a glycosyl transferase enzyme that binds to the Lin/Notch motif. Fringe modifies Notch by adding O-linked fucose groups to the EGF-like repeats (Moloney; Bruckner). This modification by Fringe does not prevent ligand binding, but may influence ligand induced conformational changes in Notch.
  • Fringe modifies Notch to prevent it from interacting functionally with Serrate/Jagged ligands but allow it to preferentially bind Delta (Panin; Hicks).
  • Drosophila has a single Fringe gene, vertebrates are known to express multiple genes (Radical, Manic and Lunatic Fringes) (Irvine).
  • Notch IC proteolytic cleavage of the intracellular domain of Notch
  • CBF1 transcriptional activator complex
  • NotchlC-CBFl complexes then activate target genes, such as the bHLH proteins HES (hairy-enhancer of split like) 1 and 5.
  • Notch can also signal in a CBF1- independent manner that involves the cytoplasmic zinc finger containing protein Deltex.
  • Target genes of the Notch signalling pathway include Deltex, genes of the Hes family (Hes-1 in particular), Enhancer of Split [E(spl)] complex genes, IL-10, CD-23, CD-4 and DIM.
  • 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-Tsakonas; Osborne). It interacts with the ankyrin repeats of the Notch intracellular domain.
  • Deltex also acts as a docking protein which prevents Su(H) from binding to the intracellular tail of Notch (Matsuno).
  • Su(H) is released into the nucleus where it acts as a transcriptional modulator.
  • Hes-1 (Hairy-enhancer of Split- 1) (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) 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.
  • 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.
  • IL-10 interleukin-10
  • Th2 helper T-cells It is a co-regulator of mast cell growth and shows extensive homology with the Epstein-Barr bcrfi gene. Although it is not known to be a direct downstream target of the Notch signalling pathway, its expression has been found to be strongly upregulated 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. Although it is not thought to be a direct downstream target of the Notch signalling pathway, its expression has been found to be strongly upregulated coincident with Notch activation.
  • FCE2 human leukocyte differentiation antigen CD23
  • Dlx-1 distalless-1 (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.
  • 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 Serrate family, for example Serrate-1 and Serrate-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.
  • homologues of known mammalian Notch ligands may be identified using standard techniques.
  • a “homologue” it is meant a gene product that exhibits sequence homology, either amino acid or nucleic acid sequence homology, to any one of the 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 corresponding 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 of the 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 of the protein and up to 14 or more EGF-like repeats on the extracellular surface. It is therefore preferred that homologues of Notch ligands also comprise a DSL domain and up to 14 or more EGF-like repeats on the extracellular surface.
  • suitable homologues will 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.
  • 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.
  • Notch ligands typically comprise a number of distinctive domains. Some predicted/potential domain locations for various naturally occurring human Notch ligands (based on amino acid numbering in the precursor proteins) are shown below:
  • a typical DSL domain may include most or all of the following consensus amino acid sequence:
  • DSL domain may include most or all of the 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.
  • the DSL domain may include most or all of the following consensus amino acid sequence: Cys Xaa Xaa Xaa Tyr Tyr Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Arg Pro Arg Asx Asp Xaa Phe Gly His Xaa Xaa Cys Xaa Xaa Xaa Gly Xaa Xaa Cys Xaa Xaa Gly Trp Xaa Gly Xaa Xaa Cys
  • Xaa may be any amino acid and Asx is either aspartic acid or asparagine).
  • the DSL domain used may be derived from any suitable species, including for example Drosophila, Xenopus, rat, mouse or human.
  • the DSL domain is derived from a vertebrate, preferably a mammalian, preferably a human Notch ligand sequence.
  • DSL domain includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturally occurring domains.
  • a DSL domain for use in the present invention may have at least 30%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 the DSL domain of human Jagged 1.
  • a DSL domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Jagged 2.
  • a DSL domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%>, preferably at least 70%, preferably at least 80%>, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Delta 1.
  • a DSL domain for use in the present invention may, for example, have at least 30%), preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Delta 3.
  • a DSL domain for use in the present invention may, for example, have at least 30%), preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80% ⁇ , preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Delta 4.
  • the EGF-like motif has been found in a variety of proteins, as well as EGF and Notch and Notch ligands, including those involved in the blood clotting cascade (Furie and Furie, 1988, Cell 53: 505-518).
  • this motif has been found in extracellular proteins such as the blood clotting factors 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 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 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
  • 'C conserved cysteine involved in a disulfide bond.
  • the EGF-like domain used may be derived from any suitable species, including for example Drosophila, Xenopus, rat, mouse or human.
  • the EGF-like domain is derived from a vertebrate, preferably a mammalian, preferably a human Notch ligand sequence.
  • EGF domain includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturally occurring domains.
  • an EGF-like domain for use in the present invention may have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Jagged 1.
  • an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Jagged 2.
  • an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 1.
  • an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 3.
  • an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%>, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 4.
  • any particular amino acid sequence is at least X% identical to another sequence can be determined conventionally using known computer programs.
  • the best overall match between a query sequence and a subject sequence also referred to as a global sequence alignment
  • the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of the global sequence alignment is given as percent identity.
  • Notch ligand N-terminal domain means the part of a Notch ligand sequence from the N-terminus to the start of the DSL domain. It will be appreciated that this term includes sequence variants, fragments, derivatives and mimetics having activity corresponding to naturally occurring domains.
  • a Notch ligand N-terminal domain for use in the present invention may have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%), preferably at least 80%, preferably at least 90%>, preferably at least 95% amino acid sequence identity to a Notch ligand N-terminal domain of human Jagged 1.
  • a Notch ligand N-terminal domain for use in the present invention may, for example, have at least 30%>, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to a Notch ligand N-terminal domain of human Jagged 2.
  • a Notch ligand N-terminal domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to a Notch ligand N-terminal domain of human Delta 1.
  • a Notch ligand N-terminal domain for use in the present invention may, for example, have at least 30%, preferably at least 50%>, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to a Notch ligand N-terminal domain of human Delta 3.
  • a Notch ligand N-terminal domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to a Notch ligand N-terminal domain of human Delta 4.
  • heterologous amino acid sequence or “heterologous nucleotide sequence” as used herein means a sequence which is not found in the native sequence (eg in the case of a Notch ligand sequence is not found in the native Notch ligand sequence) or its coding sequence. Typically, for example, such a sequence may be an IgFc domain or a tag such as a N5His tag.
  • Notch signalling can be monitored either through protein assays or through nucleic acid assays. Activation of the Notch receptor leads to the proteolytic cleavage of its cytoplasmic domain and the translocation thereof into the cell nucleus.
  • the "detectable signal" referred to herein may be any detectable manifestation attributable to the presence of the cleaved intracellular domain of Notch. Thus, increased Notch signalling can be assessed at the protein level by measuring intracellular concentrations of the cleaved Notch domain.
  • Activation of the Notch receptor also catalyses a series of downstream reactions leading to changes in the levels of expression of certain well defined genes.
  • the assay is a protein assay. In another preferred embodiment of the present invention, the assay is a nucleic acid assay.
  • nucleic acid assay The advantage of using a nucleic acid assay is that they are sensitive and that small samples can be analysed.
  • the intracellular concentration of a particular mRNA reflects the level of expression of the corresponding gene at that time.
  • levels of mRNA of downstream target genes of the Notch signalling pathway can be measured in an indirect assay of the T-cells of the immune system.
  • an increase in levels of Deltex, Hes-1 and/or IL-10 mRNA may, for instance, indicate induced anergy while an increase in levels of Dll-1 or IFN- ⁇ mRNA, or in the levels of mRNA encoding cytokines such as IL-2, IL-5 and EL- 13, may indicate improved responsiveness.
  • Narious nucleic acid assays are known. Any convention technique which is known or which is subsequently disclosed may be employed. Examples of suitable nucleic acid assay are mentioned below and include amplification, PCR, RT-PCR, R ⁇ ase protection, blotting, spectrometry, reporter gene assays, gene chip arrays and other hybridization methods.
  • gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe.
  • Southern blotting Northern blotting to quantitate the transcription of mRNA
  • dot blotting DNA or RNA analysis
  • in situ hybridisation using an appropriately labelled probe.
  • PCR was originally developed as a means of amplifying DNA from an impure sample. The technique is based on a temperature cycle which repeatedly heats and cools the reaction solution allowing primers to anneal to target sequences and extension of those primers for the 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.
  • Real-time PCR uses probes labeled with a fluorescent tag or fluorescent dyes and differs from end-point PCR for quantitative assays in that it is used to detect PCR products as they accumulate rather than for the measurement of product accumulation after a fixed number of cycles.
  • the reactions are characterized by the point in time during cycling when amplification of a target sequence is first detected through a significant increase in fluorescence.
  • the ribonuclease protection (RNase protection) assay is an extremely sensitive technique for the quantitation of specific RNAs in solution .
  • the ribonuclease protection assay can be performed on total cellular RNA or poly(A)-selected mRNA as a target.
  • the sensitivity of the ribonuclease protection assay derives from the use of a complementary in vitro transcript probe which is radiolabeled to high specific activity.
  • the probe and target RNA are hybridized in solution, after which the mixture is diluted and treated with ribonuclease (RNase) to degrade all remaining single-stranded RNA.
  • RNase ribonuclease
  • the hybridized portion of the probe will be protected from digestion and can be visualized via electrophoresis of the mixture on a denaturing polyacrylamide gel followed by autoradiography. Since the protected fragments are analyzed by high resolution polyacrylamide gel electrophoresis, the ribonuclease protection assay can be employed to accurately map mRNA features. If the probe is hybridized at a molar excess with respect to the target RNA, then the resulting signal will be directly proportional to the amount of complementary RNA in the sample.
  • Gene expression may also be detected using a reporter system.
  • a reporter system may comprise a readily identifiable marker under the control of an expression system, e.g. of the gene being monitored. Fluorescent markers, which can be detected and sorted by FACS, are preferred. Especially preferred are GFP and luciferase.
  • Another type of preferred reporter is cell surface markers, i.e. proteins expressed on the cell surface and therefore easily identifiable.
  • reporter constructs useful for detecting Notch signalling by expression of a reporter gene may be constructed according to the general teaching of Sambrook et al (1989).
  • constructs according to the invention comprise a promoter by the gene of interest, and a coding sequence encoding the desired reporter constructs, for example of GFP or luciferase.
  • Vectors encoding GFP and luciferase are known in the art and available commercially.
  • Sorting of cells may be performed by any technique known in the art, as exemplified above.
  • cells may be sorted by flow cytometry or FACS.
  • flow cytometry FACS
  • FACS Fluorescence Activated Cell Sorting
  • F.A.C.S. Fluorescence Activated Cell Sorting
  • flow cytometry Fluorescence Activated Cell Sorting
  • FACS can be used to measure gene expression in cells transfected with recombinant DNA encoding polypeptides. This can be achieved directly, by labelling of the protein product, or indirectly by using a reporter gene in the construct.
  • reporter genes are ⁇ -galactosidase and Green Fluorescent Protein (GFP).
  • ⁇ -galactosidase activity can be detected by FACS using fluorogenic substrates such as fluorescein digalactoside (FDG).
  • FDG fluorescein digalactoside
  • FDG fluorescein digalactoside
  • FDG fluorescein digalactoside
  • Mutants of GFP are available which have different excitation frequencies, but which emit fluorescence in the same channel. In a two-laser FACS machine, it is possible to distinguish cells which are excited by the different lasers and therefore assay two transfections at the same time.
  • the invention comprises the use of nucleic acid probes complementary to mRNA.
  • Such probes can be used to identify cells expressing polypeptides individually, such that they may subsequently be sorted 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 mRNA, conjugated to a fluorophore which may be used in FACS cell sorting.
  • INET In Vivo Expression Technology
  • the advantage of using a protein assay is that Notch activation can be directly measured.
  • Assay techniques that can be used to determine levels of a polypeptide are well known to those skilled in the art. Such assay methods include radioimmunoassays, competitive- binding assays, Western Blot analysis, antibody sandwich assays, antibody detection, FACS and ELISA assays.
  • the invention further provides a conjugate comprising first and second sequences, wherein the first sequence comprises an autoimmune antigen or autoimmune antigenic determinant or a polynucleotide sequence coding for such an autoimmune antigen or autoimmune antigenic determinant and the second sequence comprises a polypeptide or polynucleotide for Notch signalling modulation.
  • the conjugates of the present invention may be protein/polypeptide or polynucleotide conjugates.
  • the conjugate is a polynucleotide conjugate
  • it may suitably take the form of a polynucleotide vector such as a plasmid comprising a polynucleotide sequence coding for an autoimmune antigen or autoimmune antigenic determinant and a polynucleotide sequence coding for a modulator of the Notch signalling pathway, wherein preferably each sequence is operably linked to regulatory elements necessary for expression in eukaryotic cells.
  • a schematic representation of one such form of vector is shown in Figure 3.
  • operably linked means that the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence "operably linked" to a coding sequence is peferably ligated in such a way that expression of the coding sequence is achieved under condition compatible with the regulatory/control sequences.
  • the polynucleotide sequence coding for the modulator of the Notch signalling pathway may be a nucleotide sequence coding for a Notch ligand such as Deltal, Delta3, Delta4, Jaggedl or Jagged 2, or a biologically active fragment, derivative or homologue of such a sequence.
  • a Notch ligand such as Deltal, Delta3, Delta4, Jaggedl or Jagged 2
  • a biologically active fragment, derivative or homologue of such a sequence suitably sequences based on human sequences may be used.
  • the polynucleotide sequence coding for the modulator of the Notch signalling pathway may be a nucleotide sequence coding for a Notch ligand DSL domain and at least 1 to 20, suitably at least 2 to 15, suitably at least 2 to 10, for example at least 3 to 8 EGF-like domains.
  • the DSL and EGF-like domain sequences are or correspond to mammalian sequences.
  • the polynucleotide sequence coding for the modulator of the Notch signalling pathway may further comprise a transmembrane domain (so that the sequence may be expressed on a cell surface, as a membrane protein or polypeptide) and, suitably, a Notch ligand intracellular domain.
  • Preferred sequences include human sequences such as human Deltal, Delta3, Delta4, Jaggedl or Jagged2 sequences.
  • the polynucleotide sequence that encodes the autoantigen or bystander antigen may further include a nucleotide sequence that encodes a signal sequence which directs trafficking of the antigen or antigenic determinant within a cell to which it is administered.
  • a signal sequence may direct the antigen or antigenic determinant to be secreted or to be localized to the cytoplasm, the cell membrane, the endoplasmic reticulum, or a lysosome.
  • Regulatory elements for DNA expression include a promoter and a polyadenylation signal.
  • other elements such as a Kozak region, may also be included if desired.
  • Initiation and termination signals are regulatory elements which are often considered part of the coding sequence.
  • promoters include but are not limited to promoters from Simian Virus 40 (SN40), Mouse Mammary Tumor Virus (MMTV) promoter, Human
  • HIV Immunodeficiency Virus
  • LTR HIV Long Terminal Repeat
  • Moloney virus Moloney virus
  • ALV Cytomegalovirus
  • CMV Cytomegalovirus
  • EBV Epstein Barr Virus
  • RSV Rous Sarcoma Virus
  • Tissue-specific promoters specific for lymphocytes, dendritic cells, skin, brain cells and epithelial cells within the eye are particularly preferred, for example the CD2, CD1 lc, keratin 14, Wnt-1 and Rhodopsin promoters respectively.
  • an epithelial cell promoter such as SPC may be used.
  • suitable polyadenylation signals include but are not limited to S V40 polyadenylation signals and LTR polyadenylation signals.
  • the SV40 polyadenylation signal used in plasmid pCEP4 referred to as the SV40 polyadenylation signal, may be used.
  • additional elements include enhancers which may, for example, be selected from human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.
  • the nucleotide conjugate may for example remain present in the cell as a functioning extrachromosomal molecule and/or integrate into the cell's chromosomal DNA.
  • DNA may be introduced into cells where it remains as separate genetic material in the form of a plasmid or plasmids.
  • linear DNA which can integrate into the chromosome may be introduced into the cell.
  • reagents which promote DNA integration into chromosomes may be added.
  • DNA sequences which are useful to promote integration may also be included in the DNA molecule.
  • RNA may be administered to the cell. It is also possible, for example, to provide the conjugate in the form of a minichromosome including a centromere, telomeres and an origin of replication.
  • conjugates may be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construct in the cell.
  • mammalian origin of replication for example, plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region which produces high copy episomal replication without integration.
  • regulatory sequences may be selected which are well suited for gene expression in the type of cells the construct is to be administered to.
  • codons may be selected which are most efficiently transcribed in the cell.
  • Intracellular trafficking signals may also be included as appropriate. Such signals are well known in the art, and include the following:
  • the expressed antigen or antigenic determinant may be directed to be secreted by inclusion of an N-terminal hydrophobic sequence.
  • the hydrophobic sequence at the N terminal causes the protein to bind to the rough endoplasmic reticulum (RER).
  • RER rough endoplasmic reticulum
  • the hydrophobic sequence is subsequently clipped off by a protease and the protein is secreted.
  • the antigen or antigenic determinant may include an N terminal hydrophobic leader sequence which will direct secretion of the antigen or antigenic determinant when expressed in a cell.
  • the expressed antigen or antigenic determinant may be directed to be membrane bound by inclusion of an N-terminal hydrophobic sequence and an internal hydrophobic region.
  • the hydrophobic sequences causes the protein to bind to the RER.
  • the N terminal hydrophobic sequence is subsequently clipped off by a protease.
  • the protein follows the same secretion pathway but the internal hydrophobic sequence prevents secretion and the protein becomes membrane bound.
  • the expressed antigen or antigenic determinant may include an N terminal hydrophobic leader sequence and an internal hydrophobic sequence which will result in the antigen or antigenic determinant, or part therof, becoming membrane bound when expressed in a cell.
  • the expressed antigen or antigenic determinant may be directed to be localized in the cytosol by omitting an N-terminal hydrophobic sequence.
  • the expressed antigen or antigenic determinant is free of an N terminal hydrophobic leader sequence so that it becomes cytosolic when expressed in a cell.
  • the expressed antigen or antigenic determinant is localized in the lysosome by inclusion of a sequence (such as DKQTLL) which directs localization to lysosomes.
  • a sequence such as DKQTLL
  • the antigen or antigenic determinant may include a sequence (such as DKQTLL) so that it is directed to the lysosome when expressed in the cell.
  • expressed antigens or antigenic determinants are directed to be localized from the Golgi body back to the ER by including a sequence (such as KDEL) at the C terminal which directs localization to the ER.
  • a sequence such as KDEL
  • KDEL KDEL
  • One example of such an "ER recycling signal” is reported to be the C terminal sequence of the El 9 protein from adenovirus. That protein is localized to the ER where it binds to the MHCs and effectively keeps them from loading proteins which are presented by the MHC at the surface where they complex with T cell receptors as part of immune response induction.
  • the El 09 protein is a hexapeptide DEKKMP. Depending upon the type of immune response sought to be modulated, different intracellular localization may be desirable.
  • proteins synthesized within a cell are degraded and transported into the ER where they are loaded onto MHCs which then move to the cell surface and complex with T cell receptors of CD8 + T cells. This action encourages CTL responses.
  • proteins are complexed with antigen presenting cells (APCs) which interact with CD4 + T cells, engaging helper T cells including those associated with antibody responses.
  • APCs antigen presenting cells
  • nucleotide conjugates may code for lysosomal targeting doublets at the C terminal tail of the expressed antigen or antigenic determinant.
  • doublets LL and/or YQ and/or QY the expressed antigen or antigenic determinant is directed to a lysosome.
  • polynucleotides may be delivered in conjunction with administration of a facilitating agent.
  • Facilitating agents which are administered in conjunction with nucleic acid molecules may be administered as a mixture with the nucleic acid molecule or administered separately simultaneously, before or after administration of nucleic acid molecules.
  • Examples of facilitators include benzoic acid esters, anilides, amidines, urethans and the hydrochloride salts thereof such as those of the family of local anesthetics.
  • esters examples include: benzoic acid esters such as piperocaine, meprylcaine and isobucaine; para-aminobenzoic acid esters such as procaine, tetracaine, butethamine, propoxycaine and chloroprocaine; meta-aminobenzoic acid esters including metabuthamine and primacaine; and para-ethoxybenzoic acid esters such as parethoxycaine.
  • anilides include lidocaine, etidocaine, mepivacaine, bupivacaine, pyrrocaine and prilocaine.
  • Such compounds include dibucaine, benzocaine, dyclonine, pramoxine, proparacaine, butacaine, benoxinate, carbocaine, methyl bupivacaine, butasin picrate, phenacaine, diothan, luccaine, intracaine, nupercaine, metabutoxycaine, piridocaine, biphenamine and the botanically- derived bicyclics such as cocaine, cinnamoylcocaine, truxilline and cocaethylene and all such compounds complexed with hydrochloride.
  • the facilitating agent may be administered prior to, simultaneously with or subsequent to the genetic construct.
  • the facilitating agent and the genetic construct may be formulated in the same composition.
  • Bupivacaine-HCl is chemically designated as 2-piperidinecarboxamide, l-butyl-N-(2,6- dimethylphenyl)-monohydrochloride, monohydrate and is widely available commercially for pharmaceutical uses from many sources including from Astra Pharmaceutical Products Inc. (Westboro, Mass.) and Sanofi Winthrop Pharmaceuticals (New York, N.Y.), Eastman Kodak (Rochester, N.Y.).
  • Bupivacaine is commercially formulated with and without methylparaben and with or without epinephrine. Any such formulation may be used. It is commercially available for pharmaceutical use in concentration of 0.25%, 0.5% and 0.75% which may be used on the invention. Alternative concentrations, particularly those between 0.05% -1.0% which elicit desirable effects may be prepared if desired.
  • about 250 ⁇ g to about 10 mg of bupivacaine may be administered.
  • modulators of Notch signalling may be administered on delivery particles, preferably microparticles, preferably in combination with antigens or antigenic determinants or, preferably, nucleic acids coding for antigens or antigenic determinants, to modulate immune responses to such antigens or antigenic determinants.

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Abstract

L'invention concerne un produit comprenant : i) un modulateur de la voie de signalisation Notch ; et ii) un autoantigène ou un antigène de voisinage, ou un polynucléotide codant pour un autoantigène ou un antigène de voisinage. Ledit produit est conçu pour être utilisé sous forme de préparation combinée, simultanément, parallèlement ou séparément, pour la modulation de la réponse immunitaire.
EP04704657A 2003-01-23 2004-01-23 Traitement de maladies autoimmunes au moyen d'un activateur de la voie de signalisation notch Withdrawn EP1585543A1 (fr)

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PCT/GB2003/003285 WO2004013179A1 (fr) 2002-08-03 2003-08-01 Conjuges de modulateurs de la voie de signalisation notch et leur utilisation dans les traitements medicaux
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