EP1446424A2 - Composition comprising inhibitors of the notch signalling pathway for the modulation of the immune system - Google Patents

Composition comprising inhibitors of the notch signalling pathway for the modulation of the immune system

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Publication number
EP1446424A2
EP1446424A2 EP02779679A EP02779679A EP1446424A2 EP 1446424 A2 EP1446424 A2 EP 1446424A2 EP 02779679 A EP02779679 A EP 02779679A EP 02779679 A EP02779679 A EP 02779679A EP 1446424 A2 EP1446424 A2 EP 1446424A2
Authority
EP
European Patent Office
Prior art keywords
notch
protein
polypeptide
domain
hgand
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Withdrawn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
EP02779679A
Other languages
German (de)
French (fr)
Inventor
Mark William c/o Lorantis Limited Bodmer
Emmanuel Cyrille Pascal Lorantis Limited Briend
Brian Robert c/o Lorantis Limited Champion
Andrew Christopher c/o Lorantis Limited Lennard
Grahame James c/o Lorantis Limited McKenzie
Silvia c/o Lorantis Limited Ragno
Tamara c/o Lorantis Limited Tugal
Lesley Lynn c/o Lorantis Limited 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 GB0127267A external-priority patent/GB0127267D0/en
Priority claimed from PCT/GB2002/003426 external-priority patent/WO2003011317A1/en
Priority claimed from GB0220849A external-priority patent/GB0220849D0/en
Priority claimed from GB0220913A external-priority patent/GB0220913D0/en
Priority claimed from PCT/GB2002/004390 external-priority patent/WO2003029293A2/en
Application filed by Lorantis Ltd filed Critical Lorantis Ltd
Publication of EP1446424A2 publication Critical patent/EP1446424A2/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to the modulation of immune, function, in particular by use of a modulator of the Notch signalling pathway.
  • regulatory T cells which are able to transmit antigen-specific tolerance to other T cells, a process termed infectious tolerance (WO9S/20142).
  • infectious tolerance a process termed infectious tolerance
  • regulatory T cells can be generated by over-expression of a member of the Delta or Serrate family of Notch ligand proteins.
  • Delta or Senate induced T cells specific to one antigenic epitope are also able to transfer tolerance to T cells recognising other epitopes on the same or related antigens, a phenomenon termed "epitope spreading".
  • Notch hgand expression also plays a role in cancer. Indeed, upregulated Notch ligand expression has been observed in some tumour cells. These tumour cells are capable of rendering T cells unresponsive to restimulation with a specific antigen, thus providing a possible explanation of how tumour cells prevent normal T cell responses. By downregulating Notch signalling in vivo in T cells, it may be possible to prevent tumour cells from inducing irrununotolerance in those T cells that recognise tumour-specific antigens. In rum, this would allow the T cells to mount an immune response against the tumour cells (WO00/135990).
  • the present invention seeks to provide further methods of modulating the immune system by modification of the Notch signalling pathway, in particular for the treatment of infectious disease.
  • a product comprising: i) an inhibitor of the Notch signalling pathway or a polynucleotide coding for such an inhibitor; and ii) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
  • the agent does not act by downregulating expression of Notch or a Notch ligand.
  • a product comprising: i) an inhibitor of Notch signalling in the form of a Notch antagonist agent or a polynucleotide coding for such an agent; and ii) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
  • a product comprising: i) an inhibitor of Notch signalling in the form of an agent which inhibits Notch-Notch hgand interaction or a polynucleotide coding for such an agent; and ii) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic dete ⁇ ninant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
  • Such a product may take the form of a pharmaceutical composition or kit.
  • Such a product may take the form of a therapeutic vaccine composition or kit for treating infectious disease (including so-called “pharmaccrnes”).
  • such a product may take the form of a prophylactic vaccine composition or kit for preventing infectious disease.
  • a further aspect of the invention there is provided the use of an inhibitor of the Notch signalling pathway in the manufacture of a medicament for use as an immunostimulant.
  • the medicament is not for use in reversing bacteria, infection or tumour-induced immunosuppression or for the treatment of a tumour.
  • immunodeficiency means an agent which is capable of restoring a depressed immune function, or enhancing normal immune function, or both.
  • agent may boost a subject's immune system either generally or in respect of a specific antigen or antigenic determinant, hnmunostimulants may be used, for example, for the treatment of conditions requiring general immune stimulation including immune deficiency conditions such as Acquired Immune Deficiency Syndrome (AIDS) and Severe Combined Immunodeficiency Disease (SCJD) and in situations where antigen specific stimulation is desired, such as in vaccination.
  • AIDS Acquired Immune Deficiency Syndrome
  • SCJD Severe Combined Immunodeficiency Disease
  • an inhibitor of the Notch signalling pathway in the manufacture of a medicament for use as an adjuvant for vaccination against a pathogen.
  • pathogen means a disease causing parasite which is normally a microorganism.
  • the term includes, for example, viruses, bacteria, protozoa and fungi.
  • pathogen antigen means an antigen found on a pathogen or a fragment, variant or derivative of such an antigen comprising antigenic determinants (epitopes; preferably immunodom nant epitopes) or epitope regions (preferably immunodominant epitope regions) of such an antigen.
  • the antigen is immunogenic (an immunogen).
  • the antigen is a microbial pathogen antigen.
  • inhibitor of Notch signalling and “inhibitor of the Notch signalling pathway” as used herein include any agent which is capable of reducing any one or more of the upstream or downstream events that result in, or from, (and including) activation of the Notch receptor.
  • the inhibitor of Notch signalling does not act by do nregulating expression of Notch or a Notch hgand.
  • a method for stimulating the immune system by administering an inhibitor of the Notch signalling pathway wherein the inhibitor does not act by downregulating expression of Notch or a Notch hgand.
  • a method for stimulating the immune system to treat or prevent an infection by administering an inhibitor of the Notch signalling pathway which does not comprise reversing bacteria, infection or tumour- induced immunosuppression or treatment of a tumour According to a further aspect of the invention there is provided a method for stimulating the immune system to treat or prevent an infection by administering an inhibitor of the Notch signalling pathway wherein the inhibitor of the Notch signalling pathway does not act by downregulating expression of Notch or a Notch hgand.
  • a method for treating a chronic pathogen infection by administering an inhibitor of the Notch signalling pathway.
  • a method of increasing the immune response of a subject to a vaccine antigen or antigenic determinant comprising administering an effective amount of an inhibitor of the Notch signalling pathway to said subject simultaneously, separately or sequentially with said vaccine antigen or antigenic determinant or simultaneously, separately or sequentially with a polynucleotide coding for said vaccine antigen or antigenic determinant.
  • the inhibitor of Notch signalling inhibits Notch signalling in immune cells, such as APCs, B-cells or T-cells
  • the inhibitor of the Notch signalling pathway may be a Notch signalling repressor or an agent which increases the expression or activity of a Notch signalling repressor.
  • the inhibitor of the Notch signalling pathway is an agent capable of inhibiting the activity of a Notch receptor or a Notch hgand. - 1 -
  • the inhibitor of the Notch signalling pathway may be an agent capable of inhibiting the activity or downregulating the expression of a downstream component of the Notch signalling pathway.
  • the inhibitor of the Notch signalling pathway may be an agent which interacts with, and preferably binds to a Notch receptor or a Notch ligand so as to interfere with endogenous Notch ligand-receptor interaction (also termed "Notch-Notch hgand interaction").
  • a Notch antagonist an agent which interacts with, and preferably binds to a Notch receptor or a Notch ligand so as to interfere with endogenous Notch ligand-receptor interaction
  • the inhibitor inhibits Notch ligand-receptor interaction in immune cells such as lymphocytes and APCs, preferably in lymphocytes, preferably in T-cells.
  • the inhibitor of Notch signalling may be a protein or polypeptide or a polynucleotide which codes for such a protein or polypeptide.
  • the inhibitor of Notch signalling may comprise or codes for the extracellular domain of Delta or a fragment, derivative or homologue thereof.
  • the inhibitor of Notch signalling comprises or codes for the extracellular domain of Serrate or Jagged or a fragment, derivative or homologue thereof.
  • the inhibitor of Notch signalling comprises or codes for the extracellular domain of Notch or a fragment, derivative or homologue thereof.
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and optionally a
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and at least one
  • Notch hgand EGF-like domain ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or iii) a polynucleotide coding for such a protein or polypeptide.
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and at least two
  • Notch ligand EGF-like domains ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or iii) a polynucleotide coding for such a protein or polypeptide.
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch hgand DSL domain and either 0, 1 or 2, but no more than 2 Notch ligand EGF-like domains; h) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or iii) a polynucleotide coding for such a protein or polypeptide.
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch Hgand DSL domain having at least
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch hgand DSL domain having at least
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch EGF-like domain having at least
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch hgand DSL domain having at least
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
  • Hgand EGF-like domains having at least 30%, preferably at least 50% amino acid sequence similarity or identity to an EGF-like domain of human Jagged 1 or Jagged2;
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
  • An advantage of using a protein or polypeptide having preferably no more than two Notch ligand EGF-like domains is that it provides effective inhibition of Notch signaUing with little or no competing agonist activity, thus providing a more selective inhibitory effect.
  • Such proteins and polypeptides may also be easier to produce especially, for example, in bacterial expression systems.
  • Notch signalling inhibition is also shown by constructs having more than 2 such EGF-like repeats.
  • the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises an EGF domain having at least 70% amino acid sequence similarity or identity to EGF11 of human Notchl, Notch2, Notch3 or
  • the protein or polypeptide may be fused to a heterologous amino acid sequence, such as an HnmunoglobuHn Fc (IgFc) domain, for example a human IgGl or IgG4 Fc domain.
  • a heterologous amino acid sequence such as an HnmunoglobuHn Fc (IgFc) domain, for example a human IgGl or IgG4 Fc domain.
  • the protein or polypeptide may further comprise a Notch ligand N-terminal domain.
  • the inhibitor of Notch signaUing may comprise an antibody, antibody fragment or antibody derivative or a polynucleotide which codes for an antibody, antibody fragment or antibody derivative.
  • the antibody, antibody fragment or antibody derivative binds to a Notch receptor or a Notch Hgand so as to interfere with Notch ligand-receptor interaction.
  • the inhibitor of Notch signalling may have an ICso (preferably as measured in an assay as described herein, preferably using the Dynabeads assay of Example 12) of less than about 1000 uM, preferably less than about 100 uM, preferably less than about 10 uM, preferably less than about 1000 nM, preferably less than about 100 nM, suitably from about 0.1 to about 100 nM.
  • ICso preferably as measured in an assay as described herein, preferably using the Dynabeads assay of Example 12
  • the modulator of the Notch signaUing pathway may comprise a fusion protein comprising domains from a Notch Hgand extracellular domain and an immunoglobulin F c segment (eg IgGl Fc or IgG4 Fc, preferably human IgGl Fc or human IgG4 Fc) or a polynucleotide coding for such a fusion protein.
  • an immunoglobulin F c segment eg IgGl Fc or IgG4 Fc, preferably human IgGl Fc or human IgG4 Fc
  • Methods suitable for preparation of such fusion proteins are described, for example in Example 2 of WO 98/20142.
  • IgG fusion proteins may be prepared as well known in the art, for example, as described in US 5428130 (Genentech).
  • the modulator of the Notch signaUing pathway may be multimerised, preferably dimerised, for example by chemical cross-linking or formation of disulphide bonds between pahs of proteins or polypeptides.
  • the proteins or polypeptides comprise a heterologous amino acid sequence in the form of an immunoglobulin Fc domain, these may assemble into dimers linked by disulphide bonds formed between the Fc domains (see, for example, the schematic representations of dimeric constructs as shown in the accompanying Figures).
  • the multimerised/dhnerised form may contain more DSL and EGF domains than described in respect of the individual monomers.
  • the ratios of DSL to EGF domains will preferably remain the same, such that there wiU preferably, for example be a ratio of DSL to EGF-like domains of 1 :0, 1 :1 or 1 :2 for the multimerised aggregate as a whole.
  • the inhibitor of Notch signalling comprises a Notch hgand protein or polypeptide which consists essentiaUy of the following components: i) a Notch Hgand DSL domain;
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide or a polynucleotide coding for such a
  • Notch ligand protein or polypeptide is notched.
  • the inhibitor of Notch signaUing comprises a Notch ligand protein or polypeptide which consists essentiaUy of the foUowing components: i) a Notch ligand DSL domain;
  • H optionally aU or part of a Notch Hgand N-terminal domain; and iii) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide or a polynucleotide coding for such a
  • Notch ligand protein or polypeptide is notched.
  • the inhibitor of Notch signaUing comprises a Notch ligand protein or polypeptide which consists essentially of the foUowing components: i) a Notch ligand DSL domain; ii) one Notch Hgand EGF domain;
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide or a polynucleotide coding for such a
  • Notch ligand protein or polypeptide is notched.
  • the inhibitor of Notch signaUing comprises a Notch Hgand protein or polypeptide which consists essentiaUy of the following components: i) a Notch ligand DSL domain; H) two Notch ligand EGF domains;
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide or a polynucleotide coding for such a
  • Notch ligand protein or polypeptide is notched.
  • a binding agent which binds to a Notch ligand so as to interfere with binding of the Hgand to a Notch receptor, or a polynucleotide which codes for such a binding agent, in the manufacture of a medicament for use as an immunostimulant.
  • an antibody or antibody derivative which binds to a Notch receptor or to a Notch ligand, or a polynucleotide which codes for such an antibody or antibody derivative, in the manufacture of a medicament for use as an immunostimulant.
  • a method of increasing the immune response of a subject to a vaccine antigen or antigenic determinant comprising administering an effective amount of an inhibitor of the Notch signaUing pathway to said subject simultaneously, separately or sequentiaUy with said vaccine antigen.
  • a method for stimulating the immune system by administering a binding agent which binds to a Notch receptor or Notch ligand so as to interfere with Hgand-receptor interaction, or by administering a polynucleotide which codes for such a binding agent.
  • the binding agent may, for example, comprise one or more extraceUular domains from Notch or its ligands.
  • a method for stimulating the immune system by administering an antibody or antibody derivative which binds to a Notch receptor or to a Notch ligand, or by administering a polynucleotide which codes for such an antibody or antibody derivative.
  • an adjuvant composition comprising an inhibitor of the Notch signalling pathway.
  • a vaccine composition comprising an adjuvant composition as described above and an antigen.
  • an antigen maybe a viral, fungal, parasitic or bacterial antigen.
  • a method for modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentiaUy adnxinistering: i) an effective amount of an inhibitor of the Notch signalling pathway; and
  • H a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
  • H a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
  • an inhibitor of the Notch signaUing pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
  • H a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; in the manufacture of a medicament for modulation of the immune system.
  • a further aspect of the invention there is provided the use of .an inhibitor of the Notch signalling pathway in the manufacture of a medicament for modulation of the immune system in simultaneous, contemporaneous, separate or sequential combination with a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
  • a pharmaceutical kit comprising an inhibitor of the Notch signaUing pathway and a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
  • a conjugate comprising first and second sequences, wherein the first sequence comprises a pathogen antigen or antigenic determinant or a polynucleotide sequence coding for a pathogen antigen or antigenic deteixninant, and the second sequence comprises a polypeptide or polynucleotide for Notch signalling modulation.
  • a conjugate comprising first and second sequences, wherein the first sequence comprises a pathogen antigen or antigenic determinant or a polynucleotide sequence coding for a pathogen antigen or antigenic determinant, and the second sequence codes for an inhibitor of Notch signaUing.
  • the conjugate is in the form of a vector comprising a first polynucleotide sequence coding for a modulator of the Notch signaUing pathway and a second polynucleotide sequence coding for a pathogen antigen or antigenic determinant.
  • conjugate is in the form of an expression vector.
  • the first polynucleotide sequence codes for a Notch ligand or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • the first polynucleotide sequence of the conjugate codes for a Delta or Senate/Jagged protein or a fragment, derivative, homologue, analogue or allelic variant thereof.
  • the first polynucleotide sequence of the conjugate codes for a protein or polypeptide which comprises a Notch ligand DSL domain and optionally at least one Notch ligand EGF-like domain.
  • the first polynucleotide sequence of the conjugate codes for a protein or polypeptide which comprises a Notch ligand DSL domain and at least two Notch Hgand EGF-like domains.
  • the first polynucleotide sequence of the conjugate codes for a protein or polypeptide which comprises a Notch ligand DSL domain and lor 2 but no more than 2 Notch ligand EGF-like domains.
  • the first and second sequences of the conjugate are each operably linked to one or more promoters.
  • a method for increasing a TH2 immune response by admi stering a modulator of Notch signalling is provided.
  • a method for increasing JL-5 expression by admirtistering an inhibitor of Notch signalling there is provided a method for generating an immune stimulatory cytokine profile with reduced JL-10 expression and increased JL- 5 expression by administering an inhibitor of Notch signalling.
  • a method for generating an immune stimulatory cytokine profile with increased JL-2, IFN ⁇ , IL-5, IL-13 and TNF expression by ad ⁇ rinistering an inhibitor of Notch signalling by ad ⁇ rinistering an inhibitor of Notch signalling.
  • the cytokine profile also exhibits reduced IL-10 expression.
  • an inhibitor of Notch signalling is administered to a patient in vivo.
  • the inhibitor of Notch signaUing may be administered to a cell ex-vivo, after which the ceU may be administered to a patient.
  • the modulator of Notch signalling modifies cytokine expression in leukocytes, fibroblasts or epithehal cells.
  • the modulator of Notch signalling modifies cytokine expression in dendritic ceUs, lymphocytes or macrophages, or their progenitors or tissue-specific derivatives.
  • the inhibitor of Notch signaUing or the Notch signalling pathway for use in the present invention is an inhibitor of Notch-Notch ligand interaction.
  • an inhibitor of Notch-Notch ligand interaction is an agent which binds to a Notch receptor or Notch Hgand so as to interfere with endogenous Notch-Notch ligand interaction whUst causing less activation of the Notch receptor than would result from endogenous Notch- Notch Hgand interaction, or preferably no significant activation.
  • the inhibitor may bind to EGF-like domain 11 and/or EGF-like domain 12 of a Notch receptor or the DSL domain and/or EGF-like domain 1 and/or EGF-like domain 2 of a Notch Hgand such as Delta, Senate or Jagged.
  • the inhibitor may comprise EGF-like domains 11 and 12 of a Notch receptor.
  • the inhibitor may comprise a Notch ligand DSL domain and at least one EGF-like domain of a Notch Hgand such as Delta, Senate or Jagged.
  • the inhibitor may comprise an extraceUular domain of a Notch receptor, for example an extraceUular domain of Notchl, Notch2, Notch3 or Notch4.
  • the inhibitor may comprise an extraceUular domain of a Notch ligand such as Delta (eg a mammalian Deltal, Delta3 or Delta4), Senate or Jagged (eg a mammalian Jaggedlor Jagged2).
  • the inhibitor binds to a Notch receptor, it may bind selectively to one Notch receptor such as Notchl, or may suitably have some degree of affinity for a range of Notch receptors or substantiaUy aU of them, due to their simUar structures.
  • the inhibitor may bind selectively to one Notch Hgand such as Deltal, or may suitably have some degree of affinity for a range of Notch ligands or substantiaUy all of them, due to their similar structures.
  • the inhibitor may comprise an antibody which binds specificaUy to a Notch receptor or receptors.
  • the antibody binds to the Notch receptor in such a way as to reduce or substantiaUy prevent binding of native Notch ligands whilst the antibody is bound, or at least to reduce or substantiaUy prevent activation of the Notch receptor.
  • such an antibody may bind to EGF 11 and/or 12 of the Notch receptor (eg Notchl, Notch2, Notch3 and/or Notch4).
  • the antibody may be selective for one Notch receptor such as Notchl, or may suitably have some degree of affinity for a range of Notch receptors or substantially aU of them, due to their similar structures.
  • the inhibitor may comprise an antibody which binds specificaUy to a Notch Hgand or ligands.
  • the antibody binds to the Notch ligand Hi such a way as to reduce or substantially prevent binding of the ligand to native Notch receptors whilst the antibody is bound, or at least to reduce or substantially prevent activation of the Notch receptor.
  • such an antibody may bind to the DSL domain and/or to EGF-like domains 1 and/or 2 of a Notch Hgand (eg a mammaHan Deltal, Delta3, Delta4, Jaggedlor Jagged2).
  • the antibody may be selective for one Notch Hgand such as Deltal, or may suitably have some degree of affinity for a range of Notch ligands or substantially all of them, due to their similar structures. It will be appreciated that combinations of antibodies with complementary specificities may also be used.
  • the inhibitor of Notch signalling may be an inhibitor of Notch IC protease.
  • Notch IC protease as used herein means an enzyme or enzyme complex which acts proteolyticaUy to cleave a Notch receptor to cause the release of all or part of the intracellular (IC) domain from the Notch receptor so as to activate the Notch signaUing pathway.
  • Enzymes which are understood to participate in such cleavage include the presertilins and gamma-secretase enzymes, and presenilin-dependent garnma- secretase enzymes or complexes.
  • pre-dependent gamma-secretase as used herein means an enzyme having gamma secretase proteolytic activity which requires presenUin for activity or activation.
  • the preseniHn may for example be required as a co-activator or as part of an enzyme complex.
  • presenUin proteins which may be modulated in the present invention include Presenilin-1 (PSI) and Presenilin-2 (PS2).
  • the modulator of Notch IC protease activity will preferably be selected from polypeptides and fragments thereof, linear peptides, cycHc peptides, and nucleic acids which encode therefor, synthetic and natural compounds including low molecular weight organic or inorganic compounds and antibodies.
  • the modulator may for example be an agonist or an antagonist of presenUin or presenilin-dependent gamma-secretase, optionaUy in combination with an agent capable of respectively up-regulating or downregulating the Notch signaUing pathway respectively.
  • An example of an antagonist of presenihn which may be used in the present invention is 26S proteasome or a nucleic acid sequence which encodes therefor.
  • Synthetic inhibitors include, for example, the difluoro ketone inhibitor described in Citron et al., and Wolfe et al. having the formula:
  • the inhibitor of Notch signalling is not an inhibitor of a Notch IC protease (ie is preferably not an inhibitor of presemlins and gamma-secretase enzymes, and is preferably not an inhibitor of presenilin-dependent gamma-secretase enzymes or complexes).
  • a method for modifying an immune response by ad ⁇ mstering a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • H optionally lor 2 EGF repeat domains; Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide.
  • a method for increasing an immune response by administering a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide.
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide.
  • a method for modifying T ceU activity by administering a Notch Hgand protein or polypeptide consisting essentially of the following components: i) a Notch ligand DSL domain;
  • optionaUy all or part of a Notch Hgand N-te ⁇ ninal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide.
  • T H helper
  • T c cytotoxic
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide.
  • H optionally lor 2 EGF repeat domains; Hi) optionally all or part of a Notch Hgand N-te ⁇ ninal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide.
  • the regulatory T cells are Trl or Th3 regulatory T-ceUs.
  • Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • H optionally 1 or 2 EGF domains
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; for use to treat disease.
  • Notch ligand protein or polypeptide or polynucleotide for a use as claimed in claim 22 wherein the Notch ligand protein or polypeptide consists essentiaUy of the following components: i) a Notch ligand DSL domain;
  • H optionally aU or part of a Notch ligand N-te ⁇ ninal domain
  • Hi optionaUy one or more heterologous amino acid sequences; or wherein the polynucleotide codes for such a Notch ligand protein or polypeptide.
  • a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain; n) optionally 1 or 2 EGF domains;
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for modification of an immune response.
  • Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • H optionally 1 or 2 EGF domains; and iii) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for modification of an immune response.
  • Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for increasing an immune response.
  • a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • H optionally 1 or 2 EGF domains
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for reducing immune tolerance.
  • Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • H optionally 1 or 2 EGF domains
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multuner of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for modification of T-ceU activity.
  • Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • H optionally 1 or 2 EGF domains; iii) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionally one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for increasing helper (T H ) or cytotoxic I c ) T-ceU activity.
  • Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • H optionally 1 or 2 EGF domains
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for reducing activity of regulatory T cefls.
  • a pharmaceutical composition comprising a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • H optionally 1 or 2 EGF domains
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; optionaUy in combination with a pharmaceuticaUy acceptable carrier.
  • a pharmaceutical composition comprising a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • H optionally aU or part of a Notch ligand N-terminal domain
  • optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; optionaUy in combination with a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising a Notch ligand protein or polypeptide consisting essentially of the following components: i) a Notch Hgand DSL domain;
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; optionaUy in combination with a pharmaceuticaUy acceptable carrier.
  • a pharmaceutical composition comprising a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
  • optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; optionaUy in combination with a pharmaceuticaUy acceptable carrier.
  • Notch ligand protein or polypeptide which consists essentiaUy of the following components: i) a Notch ligand DSL domain;
  • H optionally aU or part of a Notch ligand N-terminal domain
  • Notch ligand protein or polypeptide which consists essentiaUy of the following components: i) a Notch ligand DSL domain;
  • optionaUy all or part of a Notch Hgand N-te ⁇ ninal domain
  • optionaUy one or more heterologous amino acid sequences
  • a multimer of such a protein or polypeptide wherein each monomer may be the same or different
  • a polynucleotide coding for such a Notch Hgand protein or polypeptide
  • Notch ligand protein or polypeptide which consists essentiaUy of the following components: i) a Notch ligand DSL domain;
  • Hi) optionaUy one or more heterologous amino acid sequences; or a polynucleotide sequence which codes for such a Notch Hgand protein or polypeptide.
  • the term "which consists essentiaUy of or “consisting essentiaUy of as used herein means that the construct includes the sequences and domains identified but is substantiaUy free of other sequences or domains, and in particular is substantiaUy free of any other Notch or Notch Hgand sequences or domains.
  • a vector comprising a polynucleotide coding for a Notch ligand protein or polypeptide as described above.
  • the invention also provides a host ceU transformed or transfected with such a vector.
  • a ceU displaying a Notch Hgand protein or polypeptide as described above on its surface and/or transfected with a polynucleotide coding for such a protein or polypeptide.
  • the protein or polypeptide is not bound to a ceU.
  • the protein or polypeptide may be cell-associated.
  • the protein or polypeptide may be fused to a heterologous amino acid sequence corresponding to aU or part of an immunoglobulin F c segment.
  • the heterologous amino acid sequence is not a TSST sequence, or preferably is not a superantigen sequence.
  • the protein or polypeptide comprises at least part of a mammaHan, preferably human, Notch ligand sequence.
  • the protein or polypeptide comprises Notch Hgand domains from Delta, Senate or Jagged or domains having at least 30% amino acid sequence simUarity or identity thereto.
  • the protein or polypeptide comprises Notch ligand domains from Deltal , Delta 3, Delta 4, Jagged 1 or Jagged 2 or domains having at least 30% amino acid sequence similarity thereto.
  • the protein or polypeptide inhibits a Notch receptor.
  • the protein or polypeptide is a Notch signaUing antagonist.
  • a polynucleotide coding for a protein or polypeptide as described above there is provided a vector comprising such a polynucleotide and a host ceU transformed or transfected with such a vector.
  • a cell displaying a Notch Hgand protein or polypeptide as described above on its surface and/or transfected with a polynucleotide coding for such a protein or polypeptide.
  • the modulator of the Notch signaUing pathway may comprise a fusion protein comprising domains from a. Notch ligand extraceUular domain and an immunoglobulin F c segment (eg IgGl Fc or IgG4 Fc) or a polynucleotide coding for such a fusion protein.
  • an immunoglobulin F c segment eg IgGl Fc or IgG4 Fc
  • Methods suitable for preparation of such fusion proteins are described, for example in Example 2 of WO 98/20142.
  • IgG fusion proteins may be prepared as weU known in the art, for example, as described in US 5428130 (Genentech).
  • a method for increasing TNF ⁇ expression by administering a protein, polypeptide or polynucleotide as described above.
  • a method for reducing IL- 10 expression by administering a protein, polypeptide or polynucleotide as described above.
  • the protein, polypeptide or polynucleotide modifies cytokine expression in leukocytes (such as lymphocytes or macrophages), fibrob lasts or epitheUal cells or their progenitors or tissue-specific derivatives.
  • leukocytes such as lymphocytes or macrophages
  • fibrob lasts or epitheUal cells or their progenitors or tissue-specific derivatives.
  • a method for generating an immune stimulatory cytokine profile with reduced JJ -10 expression and increased TNF ⁇ expression by administering a protein, polypeptide or polynucleotide as described above.
  • a method for generating an immune stimulatory cytokine profile with increased IL-5, JL-13 and TNF ⁇ expression by administering a protein, polypeptide or polynucleotide as described above.
  • a method for generating an immune stimulatory cytokine profile with increased IL-2, IFN ⁇ , IL-5, JL-13 and TNF ⁇ expression by administering a protein, polypeptide or polynucleotide as described above.
  • the cytokine profile also exhibits reduced IL-10 expression.
  • a method for increasing a THl immune response by administering a protein, polypeptide or polynucleotide as described above.
  • Figure 1 shows a schematic representation of Notch/Ligand interaction
  • Figure 2 shows a schematic representation of the Notch signalling pathway
  • Figure 3 shows a schematic representation of Notch 1-4
  • Figure 4 shows a schematic representation of Notch Hgands Jagged and Delta
  • Figure 5 shows ahgned amino acid sequences of DSL domains from various DrosophUa and mammahan Notch Hgands;
  • Figure 6 shows amino acid sequences of human Delta-1, Delta-3 and Delta-4;
  • Figure 7 shows amino acid sequences of human Jagged- 1 and Jagged-2
  • Figure 8 shows an amino acid sequence of human Notch-1 .
  • Figure 9 shows an amino acid sequence of human Notch-2
  • Figure 10 shows a schematic representation of Notch Hgand/IgFc fusion proteins suitable for use in the present invention
  • Figure 11 shows a schematic representation of a nucleic acid expression constract according to the present invention.
  • Figure 12 shows the amino acid sequence and domain stmcture of the fusion protein of
  • Figure 25 shows the results of Example 16 ;
  • Figure 26 shows the results of Example 17 ;
  • Figures 30 and 31 show the results of Example 21 ;
  • Figures 32 and 33 shows the results of Example 22.
  • Figure 34 shows the results of Example 23.
  • the practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skUl in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A.
  • Drosophila and vertebrate names are used interchangeably and aU homologues are included within the scope of the invention.
  • Notch signalling is synonymous with the expression “the Notch signaUing pathway” and refers to any one or more of the upstream or downstream events that result in, or from, (and including) activation of the Notch receptor.
  • Notch signalling we refer to any event directly upstream or downstream of Notch receptor activation or inhibition including activation or inhibition of Notch/Notch Hgand interactions, upregulation or downregulation of Notch or Notch Hgand expression or activity and activation or inhibition of Notch signaUing transduction including, for example, proteolytic cleavage of Notch and upregulation or downregulation of the Ras-Jnk signaUing pathway.
  • Notch signaUing we refer to the Notch signalling pathway as a signal tranducing pathway comprising elements which interact, geneticaUy and/or molecularly, with the Notch receptor protein.
  • elements which interact with the Notch protein on both a molecular and genetic basis are, by way of example only, Delta, Senate and Deltex.
  • Elements which interact with the Notch protein geneticaUy are, by way of example only, Mastermind, Hairless, Su(H) and Presenilin.
  • Notch signalling mcludes signalling events taking place extraceUularly or at the cell membrane. In a further aspect, it includes signaUing events taking place intracellularly, for example within the ceU cytoplasm or within the ceU nucleus.
  • modulate refers to a change or alteration in the biological activity of the Notch signaUing pathway or a target signaUing pathway thereof.
  • modulator preferably refers to antagonists or inhibitors of Notch signaUing, i.e. compounds which block, at least to some extent, the normal biological activity of the Notch signalling pathway. Conveniently such compounds may be refened to herein as inhibitors or antagonists.
  • the modulator is an antagonist of Notch signaUing, and preferably an antagonist of the Notch receptor (eg an antagonist of the Notchl, Notch2, Notch3 and/or Notch4 receptor).
  • An antagonist of the Notch receptor is preferably an agent which binds to the extraceUular domain of Notch to reduce or inhibit activation of signaUing.
  • an antagonist of the Notch receptor binds to Notch in immune ceUs, such as APCs, B-ceUs or T-ceUs.
  • an inhibitor of Notch signalling may bind to Notch Hgands to reduce their ability to bind to and/or activate a Notch receptor.
  • an inhibitor binds to Notch ligands in immune ceUs, such as APCs, B-ceUs or T-ceUs.
  • the active agent of the present invention may be an organic compound or other chemical.
  • a modulator wiU 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 cycHc group etc. hi addition to the possibhity of the substituents being a cyclic group, a combination of substituents may form a cyclic group. Jf 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 wUl be apparent to those skUled in the art and include, for instance, sulphur, nitrogen and oxygen.
  • the candidate modulator may comprise at least one cycHc group.
  • the cycHc group may be a polycyclic group, such as anon-fused polycycHc group.
  • the agent comprises at least the one of said cychc 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 wUl be a nucleotide sequence - which may be a sense sequence or an anti- sense sequence.
  • the modulator may also be an antibody.
  • Modulators may be synthetic compounds or natural isolated compounds.
  • a very important component of the Notch signaUing pathway is Notch receptor/Notch Hgand interaction.
  • Notch signaUing may involve changes in expression, nature, amount or activity of Notch ligands or receptors or their resulting cleavage products.
  • Notch signalling may involve changes Hi expression, nature, amount or activity of Notch signaUing pathway membrane proteins or G-proteins or Notch signaUing pathway enzymes such as proteases, kinases (e.g. serme/threorrine kinases), phosphatases, Hgases (e.g. ubiquitin Hgases) or glycosyltransferases.
  • the signalling may involve changes in expression, nature, amount or activity of DNA binding elements such as transcription factors.
  • the Notch signalling is specific signalling, meaning that the signal detected results substantiaUy or at least predominantly from the Notch signaUing pathway, and preferably from Notch Notch ligand interaction, rather than any other significant interfering or competing cause, such as for example cytokine signaUing.
  • the term "Notch signaUing" as used herein excludes cytokine signaUing.
  • the modulator or inhibitor of Notch signaUing is not a cytokine and is preferably not a mitogen.
  • the modulator of Notch signaUing is not an agent which acts primarily by inhibiting or downregulating the expression of a Notch Hgand such as Delta and/or Senate.
  • a Notch Hgand such as Delta and/or Senate.
  • the primary mode of action of the modulator of Notch signaUing is to modulate (preferably inhibit) interactions between Notch and Notch ligands which are already expressed on immune ceUs.
  • the modulator of Notch signaUing is not a Toll protein or BMP and is preferably not an agent which decreases or interferes with the production of Noggin, Chordin, FoUistatin, Xnr3, FGF or Fringe as described, for example in WO98/20142.
  • the Notch signalling pathway is described in more detail below.
  • the active agent may be a Notch Hgand, or a polynucleotide encoding a Notch ligand.
  • Notch Hgands of use in the present invention include endogenous Notch Hgands which are typically capable of binding to a Notch receptor polypeptide present in the membrane of a variety of mammalian ceUs, for example hemapoietic stem cells.
  • Notch Hgand as used herein means an agent capable of interacting with a Notch receptor to cause a biological effect.
  • the term includes naturally occurring protein ligands such as Delta and Senate, and artificial/modified constructs having equivalent activity.
  • Suitable mammalian Notch ligands identified to date include the Delta famUy, for example Delta or Delta-like 1 (Genbank Accession No. AF003522 - Homo sapiens), Delta-3 (Genbank Accession No. AFO 84576 - Rattus norvegicus) and Delta-like 3 (Mus musculus) (Genbank Accession No. NM_016941 - Homo sapiens) and US 6121045 (MUlennium), Delta-4 (Genbank Accession Nos.
  • a homologue of known mammalian Notch Hgands 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 Hgands, for example as mentioned above.
  • a homologue of a known Notch Hgand wiU be at least 20%, preferably at least 30%, identical at the amino acid level to the co ⁇ esponding 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 Hgands identified to date have a diagnostic DSL domain (D. Delta, S. Serrate, L. Lag2) comprising 20 to 22 amino acids at the amino te ⁇ ninus of the protein and up to 14 or more EGF-like repeats on the extraceUular surface. It is therefore prefened that homologues of Notch Hgands also comprise a DSL domain at the N-terminus and up to 14 or more EGF- Hke repeats on the extraceUular surface.
  • DSL domain D. Delta, S. Serrate, L. Lag2
  • homologues of Notch Hgands also comprise a DSL domain at the N-terminus and up to 14 or more EGF- Hke repeats on the extraceUular surface.
  • suitable homologues wUl 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 Hbraries with probes comprising all or part of a nucleic acid encoding a Notch Hgand 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 wiU generaUy use primers designed to target sequences within the variants and homologues encoding conserved arn no acid sequences.
  • the primers wiU contain one or more degenerate positions and wiU be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • polypeptides for Notch signalling inhibition include molecules capable of mimicking or enhancing activity or expression of any Notch signalling inhibitors.
  • the molecule wiU be a polypeptide, or a polynucleotide encoding such a polypeptide, that increases the production or activity of compounds that are capable of producing a decrease in the expression or activity of Notch, Notch Hgands, or any downstream components of the Notch signalling pathway.
  • Such molecules include the Toll-like receptor protein family, and growth factors such as the bone morphogenetic protein (BMP), BMP receptors and activins, derivatives, fragments, variants and homologues thereof.
  • BMP bone morphogenetic protein
  • BMP bone morphogenetic protein
  • a protein which is for Notch signaUing inhibition or a polynucleotide encoding such a protein we mean a molecule which is capable of inhibiting Notch, the Notch signalling pathway or any one or more of the components of the Notch signalling pathway.
  • the molecule may be capable of reducing or preventing Notch or Notch ligand expression.
  • a molecule maybe a nucleic acid sequence capable of reducing or preventing Notch or Notch ligand expression.
  • the nucleic acid sequence encodes a polypeptide selected from Toll-like receptor protein family or a growth factor such as a bone morphogenetic protein (BMP), a BMP receptor and activins.
  • BMP bone morphogenetic protein
  • the agent is a polypeptide, or a polynucleotide encoding such a polypeptide, that decreases or interferes with the production of compounds that are capable of producing an increase in the expression of Notch Hgand, such as Noggin, Chordin, FoUistatin, Xnr3, fibroblast growth factors and derivatives, fragments, variants and homologues thereof.
  • the nucleic acid sequence may be an antisense construct derived from a sense nucleotide sequence encoding a polypeptide selected from a Notch Hgand and a polypeptide capable of upregulating Notch Hgand expression, such as Noggin, Chordin, FoUistatin, Xnr3, fibroblast growth factors and derivatives, fragments, variants and homologues thereof.
  • an inhibitor of Notch signalling whlbe a molecule which is capable of inhibiting Notch-Notch Hgand interactions.
  • a molecule may be considered to modulate Notch-Notch Hgand interactions if it is capable of hihibiting the interaction of Notch with its naturally occurring Hgands, preferably to an extent sufficient to provide therapeutic efficacy.
  • Agents which modulate Notch-Notch ligand interaction may, for example be antibodies, antibody fragments or derivatives, peptides, small organic molecules, peptidomimetics or the like. Antibodies are prefened agents. Such antibodies may be polyclonal or monoclonal, intact or truncated, and may for example be xenogeneic, aUogeneic or syngeneic.
  • antibodies capable of binding to Notch receptors or Notch Hgands may be used to inhibit normal Notch-Notch ligand interactions in accordance with the present invention.
  • Notch-Notch Hgand interaction (which may be used interchangeably with the term “Notch Hgand-receptor interaction”) as used herein means the interaction between a Notch family member and a ligand capable of binding to one or more such member.
  • An agent may be considered to inhibit Notch-Notch Hgand interactions if it is capable of inhibiting the interaction of Notch with its Hgands, preferably to an extent sufficient to provide therapeutic efficacy.
  • oligopeptides and peptides may be prefened agents, other sources such as combinatorial Hbraries provide compounds other than ohgopeptides that have the necessary binding characteristics.
  • Non-peptide agents include numerous chemical types, though typically they are organic molecules, preferably small organic compounds having a molecular weight of between about 50 and about 2,500 daltons.
  • Suitable agents include functional groups necessary for stmctural interaction with proteins, particularly hydrogen bonding, and frequently include at least one group selected from, for example, an amine, carbonyl, carboxyl, hydroxyl, or sulfhydryl group, preferably at least two such functional chemical groups.
  • Compounds may, for example be cycHc or heterocychc structures and/or aromatic or polyaromatic structures substituted with one or more such functional groups.
  • the agents block binding of human Notch to human Delta and/or Senate by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
  • the receptor is activated.
  • the receptor is preferably constitutively active when expressed.
  • Inhibitors of Notch signalling also include downstream inhibitors of the Notch signalling pathway, compounds that prevent expression of Notch target genes or induce expression of genes repressed by the Notch signaUing pathway.
  • Examples of such proteins include Dsh or Numb and dominant negative versions of Notch IC or Deltex.
  • Proteins for Notch signalling inhibition wUl also include variants of the wild-type components of the Notch signaUing pathway which have been modified in such a way that then presence blocks rather than transduces the signalling pathway.
  • An example of such a compound would be a Notch receptor which has been modified such that proteolytic cleavage of its intracellular domain is no longer possible.
  • Notch signalling may also be inhibited by inhibiting Notch signaUing transduction.
  • Notch signalling pathway dkects binary ceU 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 Senate. Vertebrates express multiple Notch receptors and Hgands (discussed below). At least four Notch receptors (Notch-1, Notch-2, Notch-3 and Notch-4) have been identified to date in human ceUs (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 consistmg of a portion of the extraceUular domain, the transmembrane domain and the intracellular domain, and the other comprising the majority of the extraceUular domain.
  • the proteolytic cleavage step of Notch to activate the receptor occurs in the Golgi apparatus and is mediated by a furin-like convertase.
  • EGF epidermal growth factor
  • L/N 3 Cysteine Rich Repeats
  • the cytoplasmic domain of Notch contains six ankyrin-Hke repeats, a polyglutarnine 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 CBFl in vertebrates (Tamura K, et al. (1995) Cun. Biol. 5:1416-1423 (Tamura)).
  • the Notch ligands also display multiple EGF-like repeats in their extracellular domains together with a cysteine-rich DSL (Delta-Senate Lag2) domain that is characteristic of all Notch Hgands (Artavanis-Tsakomas et al. (1995) Science 268:225-232, Artavanis-Tsakomas et al. (1999) Science 284:770-776).
  • the Notch receptor is activated by binding of extraceUular ligands, such as Delta, Senate and Scabrous, to the EGF-like repeats of Notch's extraceUular domain.
  • Delta requires cleavage for activation. It is cleaved by the ADAM disintegrin metalloprotease Kuzbanian at the cell surface, the cleavage event releasing a soluble and active form of Delta.
  • An oncogenic variant of the human Notch-1 protein, also known as TAN-1 which has a truncated extraceUular domain, is constitutively active and has been found to be involved in T-cell lymphoblastic leukemias.
  • the cdclO/ahkyrin intraceUular-domain repeats mediate physical interaction with intraceUular signal transduction proteins. Most notably, the cdclO/ahkyrin repeats interact with Suppressor of Hairless [Su(H)].
  • Su(H) is the Drosophila homologue of C-promoter binding factor-l [CBF-1], a mammaHan DNA binding protein involved in the Epstein-Ban vims-induced immortalization of B-ceUs.
  • Su(H) associates with the cdclO/ankyrin repeats in the cytoplasm and translocates into the nucleus upon the mteraction of the Notch receptor with its Hgand Delta on adjacent ceUs.
  • Su(H) includes responsive elements found in the promoters of several genes and has been found to be a critical downstream protein in the Notch signaUing pathway. The involvement of Su(H) in transcription is thought to be modulated by Hairless.
  • the intraceUular domain of Notch also has a direct nuclear function (Lieber et al. (1993) Genes Dev 7(10):1949-65 (Lieber)). Recent studies have indeed shown that Notch activation requires that the six cdclO/ankyrin repeats of the Notch intraceUular domain reach the nucleus and participate in transcriptional activation.
  • the site of proteolytic cleavage on the intracellular taU of Notch has been identified between gly 1743 and vall744 (termed site 3, or S3) (Schroeter, E.H. et al. (1998) Nature 393f6683):382-6 (Schroeter)). It is thought that the proteolytic cleavage step that releases the cdclO/ankyrin repeats for nuclear entry is dependent on PresenUin activity.
  • the intraceUular domain has been shown to accumulate in the nucleus where it forms a transcriptional activator complex with the CSL family protein CBFl (suppressor of hairless, Su(H) in Drosophila, Lag-2 in C. elegans) (Schroeter; Struhl, G. et al. (1998) CeU 93(4):649-60 (Struhl)).
  • CSL family protein CBFl suppressor of hairless, Su(H) in Drosophila, Lag-2 in C. elegans
  • the NotchlC-CBFl complexes then activate target genes, such as the bHLH proteins HES (hairy-enhancer of split like) 1 and 5 (Weimnaster G. (2000) Cun. Opin. Genet. Dev. 10:363-369 (Weinmaster)).
  • This nuclear function of Notch has also been shown for the mammaHan Notch homologue (Lu, F. M. et al. (1996) Proc Nat
  • Fringe modifies Notch by adding ( -linked fucose groups to the EGF-like repeats (Moloney DJ, et al. (2000) Nature 406:369-375 (Moloney), Brucker K, et al. (2000) Nature 406:411-415 (Brucker)). This modification by Fringe does not prevent ligand binding, but may influence ligand induced confo ⁇ national changes in Notch. Furthermore, recent studies suggest that the action of Fringe modifies Notch to prevent it from interacting functionally with Senate/Jagged Hgands but allow it to preferentiaUy bind Delta (Panin NM, et al.
  • Notch IC proteolytic cleavage of the intraceUular domain of Notch
  • CBFl CSL farnUy protein
  • NotchlC-CBFl complexes then activate target genes, such as the bHLH proteins HES (hairy-enhancer of split like) 1 and 5.
  • Notch can also signal in a CBFl -independent manner that involves the cytoplasmic zinc finger containing protein Deltex. Unlike CBFl , Deltex does not move to the nucleus foUowing Notch activation but instead can interact with Grb2 and modulate the Ras-JNK signalling pathway.
  • Target genes of the Notch signaUing pathway include Deltex, genes of the Hes fannly (Hes-1 in particular), Enhancer of SpHt [E(spl)] complex genes, IL-10, CD-23, CD-4 and DU-1.
  • Deltex an intraceUular docking protein, replaces Su(H) as it leaves its site of interaction with the intraceUular taU of Notch.
  • Deltex is a cytoplasmic protein containing a zinc-finger (Artavanis-Tsakomas et al. (1995) Science 268:225-232; Artavanis-Tsakomas et al. (1999) Science 284:770-776; Osborne B, Miele L. (1999) Immunity 11:653-663 (Osborne)). It interacts with the ankyrin repeats of the Notch intracellular domain.
  • Deltex promotes Notch pathway activation by mteracting with Grb2 and modulating the Ras-JNK signalling pathway (Matsuno et al. (1995) Development 121(8):2633-44; Matsuno K, et al. (1998) Nat. Genet. 19:74-78). Deltex also acts as a docking protein which prevents Su(H) from binding to the intraceUular tail of Notch (Matsuno). Thus, Su(H) is released into the nucleus where it acts as a transcriptional modulator. Recent evidence also suggests that, in a vertebrate B-ceU system, Deltex, rather than the Su(H) homologue CBFl, is responsible for inhibiting E47 function (Ordentlich et al.
  • Hes-1 (Hairy-enhancer of Split-1) (Takebayashi K. et al. (1994) J Biol Chem 269(7 :150-6 (Takebayashi)) is a transcriptional factor with a basic heHx-loop-helix stmcture. It binds to an important functional site in the CD4 silencer leading to repression of CD4 gene expression. Thus, Hes-1 is strongly involved in the determination of T-ceU fate.
  • Other genes from the Hes family include Hes-5 (mammalian Enhancer of Spht homologue), the expression of which is also upregulated by Notch activation, and Hes-3. Expression of Hes- 1 is upregulated as a result of Notch activation.
  • the sequence of Mus museums Hes-1 can be found in GenBank Accession No. D16464.
  • E(spl) gene complex [E(spl)-C] (Leimeister C. et al. (1999) Mech Dev 85 (1-2): 173 -7 (Leimeister)) comprises seven genes of which only E(spl) and Groucho show visible phenotypes when mutant. E(s ⁇ l) was named after its ability to enhance SpHt mutations, Split being another name for Notch. Indeed, E(spl)-C genes repress Delta through regulation of achaete-scute complex gene expression. Expression of E(spl) is upregulated as a result of Notch activation.
  • Interleukin-10 was first characterised in the mouse as a factor produced by Th2 ceUs which was able to suppress cytokine production by Thl cells. It was then shown that IL-10 was produced by many other ceU types including macrophages, keratinocytes, B ceUs, ThO and Thl ceUs. It shows extensive homology with theEpstein-Banbcrfl gene which is now designated vkal IL-10. Although a few immunostimulatory effects have been reported, it is mainly considered as an immunosuppressive cytokine. Inhibition of T ceU responses by IL-10 is mainly mediated through a reduction of accessory functions of antigen presenting ceUs.
  • IL-10 has notably been reported to suppress the production of numerous pro-inflammatory cytokrnes by macrophages and to inhibit co-stimulatory molecules and MHC class TI expression. IL-10 also exerts anti-rnflammatory effects on other myeloid cells such as neutrophUs and eosinophUs. On B cells, JL-10 influences isotype switching and proliferation. More recently, JL-10 was reported to play a role in the induction of regulatory T ceUs and as a possible mediator of their suppressive effect. Although it is not clear whether it is a direct downstream target of the Notch signaUing pathway, its expression has been found to be strongly up-regulated coincident with Notch activation. The mRNA sequence of JL-10 may be found in GenBank ref. No. GI1041812.
  • CD-23 is the human leukocyte differentiation antigen CD23 (FCE2) which is a key molecule for B-ceU activation and growth. It is the low-affinity receptor for IgE. Furthermore, the truncated molecule can be secreted, then functioning as a potent mitogenic growth factor.
  • FCE2 human leukocyte differentiation antigen CD23
  • the sequence for CD-23 may be found in GenBank ref. No. GI1783344.
  • CTLA4 cytotoxic T-lymphocyte activated protein 4
  • CTLA4 is an accessory molecule found on the surface of T-cells which is thought to play a role in the regulation of airway inflammatory ceU recmitment and T-helper ceU differentiation after allergen inhalation.
  • the promoter region of the gene encoding CTLA4 has CBFl response elements and its expression is upregulated as a result of Notch activation.
  • the sequence of CTLA4 can be found in GenBank Accession No. L15006.
  • Dlx-1 (distaUess-1) (McGuinness T. Et al (1996) Genomics 35(3):473-85 (McGuiness)) expression is downregulated as a result of Notch activation. Sequences for Dix genes may be found in GenBank Accession Nos. U51000-3.
  • CD-4 expression is downregulated as a result of Notch activation.
  • a sequence for the CD-4 antigen may be found in GenBank Accession No. XM006966.
  • Other genes involved in the Notch signaling pathway, such as Numb, Mastermind and Dsh, and aU genes the expression of which is modulated by Notch activation, are included in the scope of this invention.
  • Notch receptor family participates in ceU-cell signalling events that influence T cell fate decisions.
  • NotchIC locahses to the nucleus and functions as an activated receptor.
  • MammaHan NotchIC interacts with the transcriptional repressor CBFl . It has been proposed that the NotchIC cdclO/ankyrin repeats are essential for this interaction.
  • Hsieh et al Hsieh et al (Hsieh et al. (1996) Molecular & CeU Biology 16(3):952-959) suggests rather that the N-terminal 114 amino acid region of mouse NotchIC contains the CBFl interactive domain.
  • NotchIC acts by targeting DNA-bound CBFl within the nucleus and aboHshing CBFl -mediated repression through masking of the repression domain.
  • Epstein Ban virus (EBN) immortalizing protein EB ⁇ A” also utilises CBFl tethering and masking of repression to upregulate expression of CBFl -repressed B-ceU genes.
  • EBN Epstein Ban virus
  • Strobl et al Strobl et al. (2000) J Nkol 74f4: 1727-35
  • EB ⁇ A2 may hence be regarded as a functional equivalent of an activated Notch receptor.
  • Other EBN proteins which fall in this category include BARF0 (Kusano and Raab-Truab (2001) J Nkol 75(1) :384-395 (Kusano and Raab-Traub)) and LMP2A.
  • 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 ceU surface, or located intracellularly.
  • smaU peptide candidate modulators or targeting molecules are synthesized on a sohd substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with a suitable target or fragment thereof and washed. Bound entities are then detected - such as by appropriately adapting methods well known hi the art.
  • a purified target can also be coated dkectly 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. CeUs can also be spread as "lawns".
  • non-neutraHsing antibodies can be used to capture the peptide and irnmobnise 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 mvention also contemplates the use of competitive drug screening assays in which neutralising antibodies capable of binding a target specificaUy compete with a test compound for binding to a target.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “protein”.
  • Protein usually refers to a short amino acid sequence that is 10 to 40 amino acids long, preferably 10 to 35 amino acids.
  • amino acid sequence may be prepared and isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
  • nucleotide sequence is synonymous with the term ' 'polynucleotide' ' .
  • the nucleotide sequence may be DNA or RNA of genomic or synthetic or of recombinant origin. They may also be cloned by standard techniques. The nucleotide sequence may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
  • RNA sequences wiU generaUy be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques.
  • This wiU involve making a pak of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the targeting sequence which it is desked to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human ceU, performing a polymerase chain reaction (PCR) under conditions which bring about amplification of the desired region, isolating the ampHfied fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • PCR polymerase chain reaction
  • primers may be designed to contain suitable restriction enzyme recognition sites so that the ampHfied DNA can be cloned into a suitable cloning vector.
  • primers wiU be produced by synthetic means, involving a step wise manufacture of the desked nucleic acid sequence one nucleotide at a time. Techniques for accompHshing this using automated techniques are readUy avaUable in the art.
  • Polynucleotide refers to a polymeric form of nucleotides of at least 10 bases in length and up to 10,000 bases or 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. GeneraUy, a nucleic acid sequence encoding the first region wUl 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 ⁇ BR322 or ⁇ UC19 (see below). Reference may be made to Molecular Cloning by Sambrook et al. (Cold Spring Harbor, 1989) or similar standard reference books for exact detaUs of the appropriate techniques.
  • Sources of nucleic acid may be ascertained by reference to published literature or databanks such as GenBank.
  • Nucleic acid encoding the desked first or second sequences may be obtained from academic or commercial sources where such sources are willing to provide the material or by synthesising or cloning the appropriate sequence where only the sequence data are avaUable. Generally this may be done by reference to literature sources which describe the cloning of the gene in question.
  • nucleic acids can be characterised as those nucleotide sequences which hybridise to the nucleic acid sequences known in the art.
  • the nucleotide sequence is DNA.
  • the nucleotide sequence is prepared by use of recombinant DNA techniques (e.g. recombinant DNA).
  • the nucleotide sequence is cDNA.
  • the nucleotide sequence may be the same as the naturally occurring form.
  • nucleic acids can be characterised as those nucleotide sequences which hybridise to the nucleic acid sequences known in the art.
  • nucleotide sequences can encode the same protein used in the present invention as a result of the degeneracy of the genetic code.
  • skilled persons may, using routine techniques, make 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 signaUing modulation of the present invention is to be expressed.
  • the present invention also encompasses the use of variants, derivatives, analogues, homologues and fragments thereof.
  • a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions.
  • a variant sequence can be modified by addition, deletion, substitution modification replacement and/or variation of at least one residue present in the naturally-occurring protein.
  • derivative as used herein, in relation to proteins or polypeptides of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide retains at least one of its endogenous functions.
  • analogue in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.
  • proteins and “polypeptides” useful in the present invention
  • the specific amino acid residues may be modified in such a manner that the protein in question retains at least one of its endogenous functions, such modified proteins are refened to as "variants”.
  • a variant protein can be modified by addition, deletion and/or substitution of at least one amino acid present in the namrally-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-naturaUy occurring analogues.
  • Proteins of use in the present invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein.
  • Dehberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubihty, hydrophobicity, hydrophihcity, 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 arginrne; and amino acids with uncharged polar head groups having similar hydrop icity values include leucine, isoleucine, valine, glycine, danine, asparagine, glutamine, serine, threonine, phenylalanine, and tyro sine.
  • 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.
  • s ⁇ bunit and domain may also refer to polypeptides and peptides having biological function.
  • a peptide useful in the invention wUl at least have a target or signalling modulation capabUity.
  • “Fragments” are also variants and the term typicaUy 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 fuU-length polypeptide, for example between about 8 and about 1500 amino acids in length, preferably 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, and even more preferably 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- dkected mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions co ⁇ esponding to the natarally-occurring sequence either side of the insertion site.
  • the flanking regions wiU contain convenient restriction sites co ⁇ esponding to sites in the natarally-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.
  • Variants of the nucleotide sequence may also be made.
  • Such variants wUl 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 thek use of the different codons. Viruses such as HTV, for instance, use a large number of rare codons.
  • the active agent is a nucleotide sequences it may suitably be codon optimised for expression in mammahan ceUs.
  • the sequence is codon optimised.
  • the sequence is codon optimised in its entkety.
  • homologous sequence wiU be taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical.
  • homology should typically he considered with respect to those regions of the sequence (such as amino acids at positions 51 , 56 and 57) known to be essential for an activity.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is prefened to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usuaUy, with the aid of readUy avaUable sequence comparison programs. These commerciaUy available computer programs can calculate % homology between two or more sequences.
  • Percent homology maybe calculated over contiguous sequences, i.e. one sequence is aHgned with the other sequence and each amino acid in one sequence is dkectly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" ahgnment. Typically, such ungapped aUgnments 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 aU six reading frames (both strands).
  • tblastx compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • BLAST uses the following search parameters:
  • HISTOGRAM - Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual).
  • DESCRIPTIONS Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter N 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 wiU not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).
  • CUTOFF - Cutoff score for reporting high-scoring segment pairs.
  • the default value is calculated from the EXPECT value (see above).
  • HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual).
  • TypicaUy significance thresholds can be more intuitively managed using EXPECT.
  • ALIGNMENTS Restricts database sequences to the number specified for which high- scoring segment paks (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 dkective in BLASTN requests returns an e ⁇ or 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.nhn.nih.gov).
  • FUtering can eliminate statisticaUy significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or prolme-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.
  • Low complexity sequence found by a filter program is substituted using the letter "N” in nucleotide sequence (e.g., "NNNNNNNNNNNNNNN”) and the letter "X” in protein sequences (e.g., "XXXXXXXXX”). Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs.
  • 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.nm.gov BLAST.
  • no gap penalties are used when determining sequence identity.
  • a scaled similarity score matrix is generaUy used that assigns scores to each pakwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is prefened to use the pub He 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 typicaUy does this as part of the sequence comparison and generates a numerical result.
  • Nucleotide sequences which are homologous to or variants of sequences of use in the present invention can be obtained in a number of ways, for example by probing DNA Hbraries made from a range of sources.
  • other vkal/bacterial, or ceUular homologues particularly ceUular homologues found in mammahan ceUs e.g. rat, mouse, bovine and primate ceUs
  • such homologues and fragments thereof in general wiU be capable of selectively hybridising to the sequences shown in the sequence Hsting herein.
  • Such sequences may be obtained by probing cDNA Hbraries made from or genomic DNA Hbraries from other animal species, and probing such Hbraries with probes comprising aU or part of the reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and aUeHc variants of the amino acid and/or nucleotide sequences useful in the present invention.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which wiU use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of use in the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence ahgnments can be performed using computer software known in the art. For example the GCG Wisconsin PUeUp program is widely used.
  • the primers used in degenerate PCR wiU contain one or more degenerate positions and wUl be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which wiU use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of use in the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence ahgnments can be performed using computer software known in the art. For example the GCG Wisconsin PfleUp program is widely used.
  • the primers used in degenerate PCR wiU contain one or more degenerate positions and wiU be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • PCR technology as described e.g. in section 14 of Sambrook et al., 1989, requires the use of oligonucleotide probes that wUl hybridise to nucleic acid. Strategies for selection of oligonucleotides are described below.
  • a probe is e.g. a single-stranded DNA or RNA that has a sequence of nucleotides that includes between 10 and 50, preferably between 15 and 30 and most preferably at least about 20 contiguous bases that are the same as (or the complement of) an equivalent or greater number of contiguous b ases.
  • the nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are minimised.
  • the nucleotide sequences are usuaUy based on conserved or highly homologous nucleotide sequences or regions of polypeptides.
  • the nucleic acids used as probes may be degenerate at one or more positions.
  • Prefened regions from which to construct probes include 5' and/or 3' coding sequences, sequences predicted to encode ligand binding sites, and the like.
  • nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridisation.
  • a suitable label means is a radiolabel.
  • the prefened method of labelling a DNA fragment is by incorporating ⁇ 32 P dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art.
  • Oligonucleotides are usually end-labelled with ⁇ 3 P-labelled ATP and polynucleotide kinase.
  • other methods e.g. non-radioactive
  • may also be used to label the fragment or oHgonucleotide including e.g. enzyme labelling, fluorescent labelling with suitable fluorophores and biotinylation.
  • prefened are such sequences, probes which hybridise under high-stringency conditions.
  • nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example sUent codon changes are requked to sequences to optimise codon preferences for a particular host ceU in which the nucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the activity of the polynucleotide or encoded polypeptide.
  • variant in relation to the nucleotide sequence used in the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for a target protein or protein for T cell signaUing modulation.
  • sequence homology preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the reference sequences. More preferably there is at least 95%, more preferably at least 98%, homology.
  • Nucleotide homology comparisons may be conducted as described above.
  • a preferred sequence comparison program is the GCG Wisconsin Bestfit program described above.
  • the default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch.
  • the default gap creation penalty is -50 and the default gap extension penalty is - 3 for each nucleotide.
  • the present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the reference sequences, or any variant, fragment or derivative thereof, or to the complement of any of the above.
  • Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
  • hybridization shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as weU 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 thek complement, wiU be generally at least 75%, preferably at least 85 or 90% and more preferably at least 95% or 98% homologous to the co ⁇ esponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
  • Prefened nucleotide sequences of the invention wiU comprise regions homologous to the nucleotide sequence, preferably at least 80 or 90% and more preferably at least 95% homologous to the nucleotide sequence.
  • the term "selectively hybridizable" means that the nucleotide sequence used as a probe is used under conditions where a target nucleotide sequence of the invention is found to hybridize to the probe at a level significantly above background.
  • the background hybridization may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened.
  • background impHes a level of signal generated by interaction between the probe and a non-specific DNA member of the Hbrary 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 l iolabeUing 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 Kim el (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
  • Maximum stringency typicaUy occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
  • 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.
  • both strands of the duplex either individuaUy or in combination, are encompassed by the present invention.
  • the nucleotide sequence is single-stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also included within the scope of the present invention.
  • Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skiU in the field. As known to those of skUl 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 which are not 100% homologous to the sequences of the present invention but faU within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA Hbraries made from a range of sources. In addition, other vkal/bacterial, or ceUular homologues particularly ceUular homologues found in mam ahan ceUs (e.g. rat, mouse, bovine and primate cells), maybe obtained and such homologues and fragments thereof in general wiU be capable of selectively hybridising to the sequences shown in the sequence Hsting herein.
  • mam ahan ceUs e.g. rat, mouse, bovine and primate cells
  • Such sequences may be obtained by probing cDNA Hbraries made from or genomic DNA Hbraries from other animal species, and probing such Hbraries with probes comprising aU or part of the reference nucleotide sequence under conditions of medium to high stringency.
  • SirnUar considerations apply to obtaining species homologues and aUehc variants of the amino acid and/or nucleotide sequences useful in the present invention.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which wiU use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserved sequences can be predicted, for example, by ahgning the amino acid sequences from several variants homologues. Sequence ahgnments can be performed using computer software known in the art. For example the GCG Wisconsin PUeUp program is widely used.
  • the primers used in degenerate PCR wUl contain one or more degenerate positions and wiU be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example sUent codon changes are requked to sequences to optimise codon preferences for a particular host ceU in which the nucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the activity of the target protein or protein for T cell signalling modulation encoded by the nucleotide sequences.
  • nucleotide sequences such as a DNA polynucleotides useful in the invention may be produced recombinantly, syntheticaUy, or by any means avaUable to those of skUl in the art. They may also be cloned by standard techniques.
  • primers wUl be produced by synthetic means, involving a step wise manufacture of the desked nucleic acid sequence one nucleotide at a time. Techniques for accompHshing this using automated techniques are readUy avaUable in the art.
  • telomere sequences wUl generaUy be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques.
  • This wiU involve making a pak of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the targeting sequence which it is desked to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human ceU, performing a polymerase chain reaction (PCR) under conditions which bring about amplification of the desked region, isolating the ampHfied fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the ampHfied DNA can be cloned into a suitable cloning vector
  • the present invention also relates to vectors which comprise a polynucleotide useful in the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides useful in the present invention by such techniques.
  • host cells can be geneticaUy engineered to incorporate expression systems or polynucleotides of the invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis et al and Sambrook et al, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid- mediated transfection, electioporation, transduction, scrape loading, baUistic introduction and infection. It wiU be appreciated that such methods can be employed in vitro or in vivo as drag delivery systems.
  • Representative examples of appropriate hosts include bacterial ceUs, such as streptococci, staphylococci, E.
  • coli streptomyces and Bacillus subtilis cells
  • fungal ceUs such as yeast ceUs and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 ceUs
  • 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.
  • vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, frombacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculo viruses, papova viruses, such as SV40, vaccinia viruses, adenovkuses, 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.
  • vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, frombacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculo viruses, papova viruses, such as
  • 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 maybe inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al.
  • secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • Proteins or polypeptides may be in the form of the "matare” 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 ohgomer, immunoglobulin Fc, glutathione S- transferase, FLAG etc) to aid in purification.
  • secretory or leader sequences or pro-sequences such as a HIS ohgomer, immunoglobulin Fc, glutathione S- transferase, FLAG etc
  • such an additional sequence may sometimes be deskable to provide added stabiHty during recombinant production. In such cases the additional sequence may be cleaved (eg chemicaUy or enzymatically) to yield the final product.
  • the additional sequence may also confer a deskable pharmacological profile (as in the case of IgFc fusion proteins) in which case it may be prefened that the additional sequence is not removed so that it is present in the final product as administered.
  • Proteins or polypeptides may be in the form of the "matare” 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 ohgomer, immunoglobulin Fc, glutathione S- transferase, FLAG etc) to aid in purification.
  • secretory or leader sequences or pro-sequences such as a HIS ohgomer, immunoglobulin Fc, glutathione S- transferase, FLAG etc
  • such an additional sequence may sometimes be deskable to provide added stabiHty during recombinant production. In such cases the additional sequence may be cleaved (eg chemicaUy or enzymatically) to yield the final product.
  • the additional sequence may also confer a deskable pharmacological profile (as in the case of IgFc fusion proteins) in which case it may be prefe ⁇ ed that the additional sequence is not removed so that it is present in the final product as administered.
  • mammalian and microbial host ceUs comprising such vectors or other polynucleotides encoding the fusion proteins, and thek production and use.
  • Active agents for use in the invention can be recovered and purified from recombinant ceU cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphoceUulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification.
  • WeU known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
  • Substances that may be used to modulate Notch signalling by inhibiting Notch ligand expression include nucleic acid sequences encoding polypeptides that affect the expression of genes encoding Notch ligands. For instance, for Delta expression, binding of extracellular BMPs (bone morphogenetic proteins, Wilson and Hemmati-Biivanlou; Hemmati-Biivanlou and Melton) to thek receptors leads to down-regulated Delta transcription due to the inhibition of the expression of transcription factors of the achaete/scute complex. This complex is beheved to be dkectly involved in the regulation of Delta expression.
  • BMPs bone morphogenetic proteins, Wilson and Hemmati-Biivanlou; Hemmati-Biivanlou and Melton
  • any polypeptide that upregulates BMP expression and/or stimulates the binding of BMPs to thek receptors may be capable of producing a decrease in the expression of Notch Hgands such as Delta and/or Senate.
  • Notch Hgands such as Delta and/or Senate. Examples may include nucleic acids encoding BMPs themselves.
  • any substance that inhibits expression of transcription factors of the achaete/scute complex may also downregulate Notch Hgand expression.
  • BMPs belong to the transforming growth factor beta (TGF-beta) superfamUy, which includes, in addition to the TGF-betas, activms/inn ⁇ bins (e.g., alpha- inhibin), muUerian inhibiting substance, and gHal cell line-derived neurotrophic factor.
  • TGF-beta transforming growth factor beta
  • polypeptides that inhibit the expression of Delta and/or Senate include the Toll-like receptor (Medzhitov) or any other receptors linked to the innate immune system (for example CD14, complement receptors, scavenger receptors or defensin proteins), and other polypeptides that decrease or interfere with the production of Noggin (Valenzuela), Chordin (Sasai), FoUistatin (Iemura), Xnr3, and derivatives and variants thereof.
  • Noggin and Chordin bind to BMPs thereby preventing activation of thek signaUing cascade which leads to decreased Delta transcription. Consequently, reducing Noggin and Chordin levels may lead to decreased Notch ligand, in particular Delta, expression.
  • the Toll transmembrane receptor plays a central role in the signaUing pathways that control amongst other things the innate nonspecific immune response.
  • This Toll-mediated immune response reflects an ancestral conserved signaUing system that has homologous components in a wide range of organisms.
  • Human ToU homologues have been identified amongst the ToU-Hke receptor (TLR) genes and ToU/mterleukin-l receptor-like (TIL) genes and contain the characteristic ToU motifs: an extraceUular leucrne-rich repeat domain and a cytoplasmic mterle ⁇ kin-1 receptor-like region.
  • TLR ToU-Hke receptor
  • TIL ToU/mterleukin-l receptor-like
  • the ToU-Hke receptor genes now include TLR4, TTL3, TTL4, and 4 other identified TLR genes.
  • Notch ligand expression examples include those encoding immune costimulatory molecules (for example CD80, CD86, ICOS, SLAM) and other accessory molecules that are associated with immune potentiation (for example CD2, LFA-1).
  • immune costimulatory molecules for example CD80, CD86, ICOS, SLAM
  • accessory molecules that are associated with immune potentiation
  • Suitable substances that may be used to downregulate Notch Hgand expression include nucleic acids that inhibit the effect of transforming growth factors such as members of the fibroblast growth factor (FGF) family.
  • the FGF may be a mammalian basic FGF, acidic FGF or another member of the FGF famUy such as an FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7.
  • the FGF is not acidic FGF (FGF-1; Zhao et al, 1995).
  • the FGF is a member of the FGF family which acts by stimulating the upregulation of expression of a Senate polypeptide on APCs. It has been shown that members of the FGF family can upregulate Senate-1 gene expression in APCs. Inhibition of Notch signaUing by use of anti-sense constructs
  • Suitable nucleic acid sequences may include anti-sense constructs, for example nucleic acid sequences encoding antisense Notch ligand constructs or antisense sequences conesponding to other components of the Notch signalling pathway as discussed above.
  • the antisense nucleic acid may be an ohgonucleoti.de such as a synthetic single-stranded DNA.
  • the antisense is an antisense RNA produced in the patient's own ceUs as a result of introduction of a genetic vector.
  • the vector is responsible for production of antisense RNA of the desked specificity on introduction of the vector into a host cell.
  • Antisense nucleic acids can be oligonucleotides that are double-stranded or single- stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences.
  • inhibitory antisense or double stranded oligonucleotides can additionaUy comprise at least one modified base moiety which is selected- from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiou ⁇ idine, 5-carboxymethylaminomethyluraci- 1, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- metnyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethylura
  • the antisense oligonucleotide may if desked comprise at least one modified phosphate backbone such as, for example, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof.
  • a modified polymeric backbone such as a modified polypeptide backbone may be used (eg protein nucleic acid: PNA).
  • the antisense oligonucleotide may be an alpha-anomeric oligonucleotide.
  • An alpha-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands nm parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).
  • the oligonucleotide may for example be a 2'-0-methyiribonucleotide (Inoue et al., 1987, Nucl. Acids Res.
  • RNA-DNA analogue a chimeric RNA-DNA analogue
  • OHgonucleotides maybe synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commerciaUy avaUable from Biosearch, Applied Biosystems, etc.).
  • phosphorothioate oligonucleotides canbe synthesized by the method of Stein et al. (1988, Nucl. Acids Res.
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
  • the nucleic acid sequence for use in the present invention is capable of inhibiting Senate and Delta, preferably Senate 1 and Senate 2 as weU as Delta 1, Delta 3 and Delta 4 expression in APCs such as dendritic ceUs.
  • the nucleic acid sequence may be capable of inhibiting Senate expression but not Delta expression, or Delta but not Senate expression in APCs or T cells.
  • the nucleic acid sequence for use in the present invention is capable of inhibiting Delta expression in T ceUs such as CD4 + helper T cells or other ceUs of the immune system that express Delta (for example in response to stimulation of cell surface receptors).
  • the nucleic acid sequence may be capable of inhibiting Delta expression but not Senate expression in T cells.
  • the nucleic acid sequence is capable of inhibiting Notch Hgand expression in both T ceUs and APC, for example Senate expression in APCs and Delta expression in T ceUs.
  • Prefened suitable substances that may be used to downregulate Notch Hgand expression include growth factors and cytokines. More preferably soluble protein growth factors may be used to inhibit Notch or Notch Hgand expression. For instance, Notch Hgand expression may be reduced or inhibited by the addition of BMPs or activins (a member of the TGF- ⁇ superfamily). In addition, T cells, APCs or tumour cells could be cultured in the presence of inflammatory type cytokines including IL-12, IFN- ⁇ , 1L-18, TNF-oc, either alone or in combination with BMPs.
  • Molecules for inhibition of Notch signalling will also include polypeptides, or polynucleotides which encode therefore, capable of modkying Notch-protein expression or presentation on the cell membrane or signalling pathways.
  • Molecules that reduce or interfere with its presentation as a fully functional ceU membrane protein may include MMP inhibitors such as hydroxymate-based inhibitors.
  • Notch Hgand derivatives would preferably have the DSL domain at the N- ternunus and between 1 to 8, suitably from 2 to 5, EGF-like repeats on the extracellular surface.
  • a peptide co ⁇ esponding to the Delta/Se ⁇ ate/LAG-2 domain of hJaggedl and supematants from COS cells expressing a soluble form of the extraceUular portion of hJaggedl was found to mimic the effect of Jaggedl in inhibiting Notchl (Li).
  • a Notch Hgand derivative maybe a fusion protein, for example, a fusion protein comprising a segment of a Notch Hgand extraceUular domain and an immunoglobulin F 0 segment such as IgGF c or IgMF 0
  • the modulator may comprise all or part of the extraceUular domain of a Notch receptor (eg Notchl, Notch2, Notch3, Notch4 or homologues thereof), which can bind to Notch Hgands and so reduce interactions with endogenous Notch receptors.
  • a modulator may comprise at least the 11th and 12th domains of Notch (EGF11 and EGF12), as these are believed to be important for Notch ligand interaction.
  • a rat Notch-1/Fc fusion protein is available from R& D Systems Inc (Minneapolis, USA and Abingdon, Oxon, UK: Catalog No 1057-TK). This comprises the 12 amino te ⁇ ninal EGF domains of rat Notch-1 (amino acid residues Met 1 to Glu 488) fused to the Fc region of human IgG (Pro 100 to Lys 330) via a polypeptide linker (TEGRMD).
  • Notch signalling pathway antagonists include antibodies which inhibit interactions between components of the Notch signalling pathway, e.g. antibodies to Notch or Notch Hgands.
  • antibody includes intact molecules as well as fragments thereof, such as Fab, Fab', F(ab') 2 , Fv and scFv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and include, for example:
  • Fab fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact Hght 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;
  • (iii) (Fab') 2 the fragment of the antibody that can be obtained by treating whole antibody with pepsin without subsequent reduction;
  • F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds;
  • Fv defined as a genetically engineered fragment containing the variable genetically fused single chain molecule
  • Antibodies maybe monoclonal or polyclonal but are preferably monoclonal.
  • the binding affinity (equUibrium association constant (Ka)) may be at least about 10 6 M “1 , at least about 10 7 M “ ⁇ at least about 10 s M “1, or at least about 10 9 M “1 .
  • the antibody, derivative or fragment binds to one or more DSL, EGF or N- terminal domains of a Notch ligand or to one or more EGF or Lin/Notch (L/N) domains of Notch (for example to EGF repeats 11 and 12 of Notch).
  • the agent may be an antibody, derivative or fragment which binds to Notch.
  • the agent may be an antibody, derivative or fragment which binds to Delta. Tn a further embodiment the agent may be an antibody, derivative or fragment which binds to Senate or Jagged.
  • Suitable antibodies for use as blocking agents are obtained by immunizing a host animal with peptides comprising aU or a portion of Notch or a Notch Hgand such as Delta or Senate/Jagged.
  • the peptide used may comprise the complete protein or a fragment or derivatives thereof.
  • Prefened irnmunogens comprise all or a part of the extracellular domain of human Notch, Delta or Senate/Jagged, where these residues contain any post-translation modifications, such as glycosylation, found in the native proteins, unmunogens comprising the extraceUular domain may be produced by a number of techniques which are weU known in the ait such as expression of cloned genes using conventional recombinant methods and/or isolation from T cells or ceU populations expressing high levels of Notch or Notch Hgands.
  • Monoclonal antibodies may be produced by means well known in the art. GeneraUy, the spleen and/or lymph nodes of an immunized host animal provide a source of plasma ceUs. The plasma cells are immortahzed by fusion with myeloma cells to produce hybridoma cells. Culture supernatant from individual hybridomas is screened using standard techniques to identify those producing antibodies with the desked specificity. The antibody may be purified from the hybridoma ceU supematants or ascites fluid by conventional techniques, such as affinity chromatography using Notch, Notch ligands or fragments thereof bound to an insoluble support, protein A sepharose, or the like.
  • WO 0020576 discloses generation of antibodies against the human Notch-1 EGF-like repeats 11 and 12.
  • WO 0020576 discloses a monoclonal antibody secreted by a hybridoma designated A6 having the ATCC Accession No.
  • HB 12654 a monoclonal antibody secreted by a hybridoma designated CU having the ATCC Accession No. HB 12656 and a monoclonal antibody secreted by a hybridoma designated F3 having the ATCC Accession No. HB 12655.
  • antibodies for use to treat human patients wUl be chimeric or humanised antibodies.
  • Antibody "humanisation” techniques are well known in the art. These techniques typically involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule.
  • WO 86/01533 discloses a process for preparing an antibody molecule having the variable domains from a mouse MAb and the constant domains from a human immunoglobulin.
  • CDRs complementarity determining regions
  • a mouse MAb is grafted onto the framework regions of the variable domains of a human immunoglobulin by site dkected mutagenesis using long oligonucleotides.
  • Such CDR-grafted humanised antibodies are much less likely to give rise to an anti-antibody response than humanised chimeric antibodies in view of the much lower proportion of non-human amino acid sequence which they contain.
  • the first criterion is to use as the human acceptor the framework from a particular human immunoglobulin that is unusuaUy homologous to the non-human donor immunoglobulin to be humanised, or to use a consensus framework from many human antibodies.
  • the second criterion is to use the donor amino acid rather than the acceptor if the human acceptor residue is unusual and the donor residue is typical for human sequences at a specific residue of the framework.
  • the thkd criterion is to use the donor framework amino acid residue rather than the acceptor at positions immediately adjacent to the CDRs.
  • the fourth criterion is to use the donor amino acid residue at framework positions at which the amino acid is predicted to have a side chain atom within about 3 A of the CDRs in a three-dimensional immunoglobuHn model and to be capable of interacting with the antigen or with the CDRs of the humanised immunoglobuHn. It is proposed that criteria two, three or four may be applied in addition or alternatively to criterion one, and may be appHed singly or in any combination.
  • isotype wiU be guided by the desked effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity.
  • Suitable isotypes include IgG 1, IgG3 and IgG4.
  • IgG 1 IgG3
  • IgG4 IgG4
  • either of the human light chain constant regions, kappa or lambda may be used.
  • ChemicaUy coupled sequences can be prepared (where requked) from individual proteins sequences and coupled using known chemicaUy coupling techniques.
  • the conjugate can be assembled using conventional solution- or sohd-phase peptide synthesis methods, affording a fully protected precursor with only the te ⁇ ninal amino group in deprotected reactive form.
  • This function can then be reacted dkectly with a protein for T ceU signaUing modulation 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.
  • a protein for T cell signaUing modulation 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.
  • Dkect linkage may occur through any convenient functional group on the protein for T ceU signaUing modulation such as a hydroxy, carboxy or amino group. Indirect linkage which is preferable, will occur through a linking moiety.
  • Suitable linking moieties include bi- and multifunctional alkyl, aryl, aralkyl or peptidic moieties, alkyl, aryl or aralkyl aldehydes acids esters and anyhdrides, sulphydryl or carboxyl groups, such as maleimido benzoic acid derivatives, maleimido proprionic acid derivatives and succinknido derivatives or may be derived from cyanuric bromide or chloride, carbonyldiknidazole, succinimidyl esters or sulphonic haHdes and the like.
  • the functional groups on the linker moiety used to form covalent bonds between linker and protein for T cell signalling modulation on the one hand, as well as linker and target protein on the other hand, may be two or more of, e.g., amino, hydrazino, hydroxyl, thiol, maleimido, carbonyl, and carboxyl groups, etc.
  • the linker moiety may include a short sequence of from 1 to 4 amino acid residues that optionaUy includes a cysteine residue through which the linker moiety bonds to the target protein. Notch ligand domains
  • natarally occurring Notch ligands typically comprise a number of distinctive domains.
  • Some predicted/potential domain locations for various natarally occurring human Notch ligands 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.
  • DSL domain may include most or all of the following consensus amino acid sequence:
  • Xaa may be any amino acid and Asx is either aspartic acid or asparagine).
  • the DSL domain used may be derived from any suitable species, including for example Drosophila, Xenopus, rat, mouse or human.
  • the DSL domain is derived from a vertebrate, preferably a mammahan, preferably a human Notch Hgand sequence.
  • DSL domain includes sequence variants, fragments, derivatives and mimetics having activity co ⁇ esponding to nataraUy occurring domains.
  • a DSL domain for use in the present invention may have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Jagged 1.
  • a DSL domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Jagged 2.
  • a DSL domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% a ino 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 mvention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Delta 4.
  • EGF-like motif has been found in a variety of proteins, as weU as EGF and Notch and Notch Hgands, including those involved in the blood clotting cascade (Furie and Furie, 1988, Cell 53: 505-518).
  • this motif has been found in extraceUular proteins such as the blood clotting factors IX and X (Rees et al, 1988, EMBO J. 7:2053- 2061; Furie and Furie, 1988, CeU 53: 505-518), in other Drosophila genes (Knust et al., 1987 EMBO J.
  • ceU- surface receptor proteins such as thrombomoduHn (Suzuki et al., 1987, EMBO J. 6:1891- 1897) and LDL receptor (Sudhof et al., 1985, Science 228:815-822).
  • a protein binding site has been mapped to the EGF repeat domain in thrombomoduHn and urokinase (Kurosawa et al., 1988, J. Biol. Chem 263:5993-5996; AppeUa et al., 1987, J. Biol. Chem.262:4437-4440).
  • 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 requked, to be a two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet.
  • Subdomains between the conserved cysteines strongly vary in length as shown in the foUowing schematic representation of a typical EGF-like domain:
  • the region between the 5th and 6th cysteines contains two conserved glycines of which at least one is normally present inmost EGF-like domains.
  • the EGF-like domain used may be derived from any suitable species, including for example DrosophUa, Xenopus, rat, mouse or human.
  • the EGF-like domain is derived from a vertebrate, preferably a mammalian, preferably a human Notch ligand sequence.
  • an EGF-like domain for use in the present invention may have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Jagged 1.
  • an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Jagged 2.
  • an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 1.
  • an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 3.
  • an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% 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 conventionaUy using known computer programs.
  • the best overaU match between a query sequence and a subject sequence also refened to as a global sequence alignment, can be dete ⁇ nined using a program such as the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245).
  • the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of the global sequence ahgnment is given as percent identity.
  • Notch ligand N-te ⁇ ninal domain means the part of a Notch ligand sequence from the N-te ⁇ ninus to the start of the DSL domain. It wUl be appreciated that this term includes sequence variants, fragments, derivatives and mimetics having activity co ⁇ esponding to natarally occurring domains.
  • 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.
  • any such heterologous amino acid sequence is not a TSST sequence, and preferably it is not a superantigen sequence.
  • Whether a substance can be used for activating Notch may be determined using suitable screening assays, for example, as described in our co-pending International Patent Application claiming priority from GB 0118153.6, and the examples herein.
  • Whether a substance can be used for modulating Notch signalling may be determined using suitable screening assays (see for example, the Examples herein)
  • 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" refened to herein may be any detectable manifestation attributable to the presence of the cleaved intraceUular 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 weU defined genes.
  • the assay is a protein assay.
  • 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 smaU samples can be analysed.
  • the intraceUular 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 signaUing 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 JL-10 mRNA may, for instance, indicate induced anergy while an increase in levels of DU-1 or IFN- ⁇ mRNA, or in the levels of mRNA encoding cytokines such as JL-2, JL-5 and IL-13, may indicate improved responsiveness.
  • nucleic acid assays are known. Any convention technique which is known or which is subsequently disclosed may be employed. Examples of suitable nucleic acid assay are mentioned below and include amplification, PCR, RT-PCR, RNase protection, blotting, spectrometry, reporter gene assays, gene chip a ⁇ ays and other hybridization methods.
  • gene presence, amplification and/or expression may be measured in a sample dkectly, 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.
  • PCR was originally developed as a means of amplifying DNA from an impure sample. The technique is based on a temperatare cycle which repeatedly heats and cools the reaction solution allowing primers to anneal to target sequences and extension of those primers for the formation of duphcate daughter strands.
  • RT-PCR uses an RNA template for generation of a first strand cDNA with a reverse transcriptase.
  • the cDNA is then ampHfied according to standard PCR protocol. Repeated cycles of synthesis and denataration result in an exponential increase in the number of copies of the target DNA produced. However, as reaction components become Hmiting, the rate of ampHfication decreases until a plateau is reached and there is Httle 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.
  • Primers can be designed using standard procedures in the art, for example the TaqmanTM technique.
  • Real-time PCR uses probes labeled with a fluorescent tag 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 ampHfication of a target sequence is first detected through a significant increase in fluorescence.
  • An advantage of real-time PCR is its accuracy in determining the amounts if target sequences in a sample. Suitable protocols are described, for example, in Meuer S. et al (2000).
  • 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 ⁇ oly(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 wiU be protected from digestion and can be visualized via electrophoresis of the mixture on a denaturing polyacrylamide gel followed by autoradiography. Since the protected fragments are analyzed by high resolution polyacrylamide gel electrophoresis, the ribonuclease protection assay can be employed to accurately map mRNA featares. If the probe is hybridized at a molar excess with respect to the target RNA, then the resulting signal wiU be dkectly 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 prefe ⁇ ed. Especially prefened are GFP and luciferase.
  • Another type of prefe ⁇ ed reporter is cell surface markers, i.e. proteins expressed on the cell surface and therefore easUy 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 desked reporter constructs, for example of GFP or luciferase.
  • Vectors encoding GFP and luciferase are known in the art and avaUable commercially.
  • ceUs may be sorted by flow cytometry or FACS.
  • flow cytometry FACS
  • FACS Fluorescence Activated Cell Sorting
  • FACS Fluorescence Activated CeU Sorting
  • PMT PhotomultipHer tubes
  • FACS can be used to measure gene expression in cells transfected with recombinant DNA encoding polypeptides. This can be achieved dkectly, by labelling of the protein product, or indkectly 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
  • CeUs expressing GFP constructs wUl fluoresce without the addition of a substrate. Mutants of GFP are available which have different excitation frequencies, but which emit fluorescence in the same channel. In a two-laser FACS machine, it is possible to distinguish cells which are excited by the different lasers and therefore assay two transfections at the same time.
  • the invention comprises the use of nucleic acid probes complementary to mRNA.
  • Such probes can be used to identify cells expressing mRNA for polypeptides individuaUy, 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 ceU sorting.
  • the advantage of using a protein assay is that Notch activation can be dkectly measured.
  • Assay techniques that can be used to determine levels of a polypeptide are well known to those skiUed in the art. Such assay methods include radioknmunoassays, competitive- binding assays, Western Blot analysis, antibody sandwich assays, antibody detection, FACS and ELISA assays.
  • the modulator of Notch signalling may also be an immune ceU which has been treated to modulate expression or interaction of Notch, a Notch ligand or the Notch signalling pathway.
  • ceUs may readily be prepared, for example, as described in WO 00/36089 in the name of Lorantis Ltd, the text of which is herein incorporated by reference.
  • active agents are administered in combination with a pharmaceuticaUy acceptable diluent, carrier, or excipient (ie as a pharmaceutical composition).
  • a pharmaceutical composition may be for human or animal usage in human and veterinary medicine.
  • Acceptable carriers or diluents for therapeutic use are weU known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Pubhshing Co. (A. R. Gennaro edit. 1985).
  • the choice of pharmaceutical carrier, excipient or dUuent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Preservatives, stabUizers, dyes and even flavoring agents may also be provided in the pharmaceutical composition as appropriate.
  • preservatives examples include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents maybe also used.
  • active agents may be administered orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixks, solutions or suspensions containing flavouring or colouring agents.
  • active agents may be administered by inhalation, intranasaUy or in the form of aerosol, or in the form of a suppository or pessary, or they may be applied topicaUy in the form of a lotion, solution, cream, ointment or dusting powder.
  • transdermal administration is by use of a skin patch.
  • they can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or Hquid paraffin. They can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabuisers and preservatives as may be requked.
  • Active agents such as polynucleotides and proteins/polypeptides may also be administered by vkal or non-vkal techniques.
  • Vkal dehvery mechanisms include but are not limited to adenovkal vectors, adeno-associated vkal (AAV) vectors, herpes vkal vectors, retrovkal vectors, lentivkal vectors, and baculovkal vectors.
  • Non-vkal delivery mechanisms include lipid mediated transfection, liposomes, immunoHpo somes, Hpoiectin, cationic facial amphiphiles (CFAs) and combinations thereof.
  • the routes for such deHvery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
  • Active agents may be adminstered by conventional DNA deHvery techniques, such as DNA vaccination etc., or injected or otherwise delivered with needleless systems, such as ballistic delivery on particles coated with the DNA for delivery to the epidermis or other sites
  • the physician wUl determine the actual dosage which will be most suitable for an individual patient and it wiU vary with the age, weight and response of the particular patient.
  • the above dosages are exemplary of the average case. There can, of course, be 101
  • treatment or therapy as used herein should be taken to encompass diagnostic and prophylatic appHcations.
  • the treatment of the present invention includes both human and veterinary applications.
  • Active agents may also be administered by any suitable means including, but not Hmited to, traditional syringes, needleless injection devices, or "microprojectUe bombardment gene guns".
  • active agents such as polynucleotides may be introduced by various means into ceUs that are removed from an individual. Such means include, for example, ex vivo transfection, electioporation, nucleoporation, rnicroinjection and microprojectUe bombardment. After an agent has been taken up by the cells, they may be reimplanted into an individual. It is also contemplated that otherwise non-knmunogenic ceUs that have gene constructs incorporated therein can be implanted into an individual even if the vaccinated ceUs were originally taken from another individual.
  • the active agent may be administered to an individual using a needleless injection device.
  • an active agent may be administered to an individual intradermally, subcutaneously and/or intramuscularly using a needleless injection device , or similarly dehvered to mucosal tissues of, for example, the respkatory, gastrointestinal or urinogenital tracts.
  • Needleless injection devices are well known and widely available. Needleless injection devices are - 102 -
  • a needleless injection device may be used to propel a liquid that contains DNA molecules toward the surface of the individual's skin.
  • the liquid is propeUed at a sufficient velocity such that upon impact with the skin the liquid penetrates the surface of the skin and permeates the skin and/or muscle tissue beneath.
  • the genetic material is simultaneously or selectively administered intradermally, subcutaneously and intramuscularly.
  • a needleless injection device may be used to deliver genetic material to tissue of other organs in order to introduce a nucleic acid molecule to ceUs of that organ.
  • the pharmaceutical preparations according to the present invention are provided sterile and pyrogen free.
  • a physician wUl determine the actaal dosage which wiU be most suitable for an individual subject and it wiU vary with the age, weight and response of the particular patient.
  • the dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
  • the therapeutic agents used in the present invention may be administered dkectly to patients in vivo.
  • the agents may be administered to immune ceUs such as T cells and/or APCs in an ex vivo manner.
  • leukocytes such as T cells or APCs may be obtained from a patient or donor in known manner, treated/incubated ex vivo in the manner of the present invention, and then administered to a patient.
  • a therapeuticaUy effective daUy dose of the conjugate of the active agent according to the invention may for example range from 0.01 to 50 mg/kg body weight of the subject to be treated, preferably 0.1 to 20 mg/kg.
  • compositions are in unit dosage form.
  • the present invention includes both human and veterinary appHcations.
  • the modulator of the Notch signalling pathway and the pathogen antigen, antigenic dete ⁇ ninant or the polynucleotide coding for the pathogen antigen or antigenic determinant are administered at substantially the same time, and preferably together in the same formulation.
  • the modulator of the Notch signaUing pathway and the pathogen antigen, antigenic determinant or the polynucleotide coding for the pathogen antigen or antigenic determinant are administered closely in time, e.g., the the pathogen antigen, antigenic determinant or the polynucleotide coding for the pathogen antigen or antigenic determinant is administered within from about one minute to within about one day before or after the modulator of the Notch signalling pathway is administered. Any contemporaneous time is useful.
  • the modulator of the Notch signalling pathway and the pathogen antigen, antigenic detenriinant or the polynucleotide coding for the pathogen antigen or antigenic determinant wUl be administered within about one minute to within about eight hours, and preferably within less than about one to about four hours.
  • the modulator of the Notch signaUing pathway and the pathogen antigen, antigenic determinant, or the polynucleotide coding for the pathogen antigen or antigenic determinant are preferably administered at the same site on the animal.
  • the term "same site" includes the exact location, but can be - 104 -
  • the term "separately" as used herein means that the modulator of the Notch signalling pathway and the pathogen antigen, antigenic determinant or the polynucleotide coding for the pathogen antigen or antigenic determinant are administered at an interval, for example at an interval of about a day to several weeks or months.
  • the active agents may be administered in either order.
  • the modulator of the Notch signalling pathway may be administered more frequently than the pathogen antigen, antigenic deteiminant or the polynucleotide coding for the pathogen antigen or antigenic determinant or vice versa.
  • the term “sequentially” as used herein means that the modulator of the Notch signalling pathway and the pathogen antigen, antigenic determinant or the polynucleotide coding for the pathogen antigen or antigenic determinant are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the active agents may be administered in a regular repeating cycle.
  • Vaccine compositions and preparations made in accordance with the present invention may be used to protect or treat a mammal susceptible to, or suffering from disease, by means of administering said vaccine via a mucosal route, such as the oral/bucal/intestinal/vaginal/rectal or nasal route.
  • a mucosal route such as the oral/bucal/intestinal/vaginal/rectal or nasal route.
  • Such administration may be in a droplet, spray, or dry powdered form.
  • Nebulised or aerosohsed vaccine formulations may also be used where appropriate.
  • Enteric formulations such as gastro resistant capsules and granules for oral administration, suppositories for rectal or vaginal administration may also be used.
  • the present invention may also be used to enhance the immunogenicity of antigens appHed to 105 -
  • the skin for example by intradermal, transdermal or transcutaneous deHvery.
  • the adjuvants of the present invention may be parentaUy delivered, for example by intramuscular or subcutaneous administration.
  • a variety of administration devices may be used.
  • a spray device such as the commercially avaUable Accuspray (Becton Dickinson) may be used.
  • Prefened spray devices for intranasal use are devices for which the performance of the device is not dependent upon the pressure appHed by the user. These devices are known as pressure threshold devices. Liquid is released from the nozzle only when a threshold pressure is attained. These devices make it easier to achieve a spray with a regular droplet size. Pressure threshold devices suitable for use with the present invention are known in the art and are described for example in WO 91/13281 and EP 311 863 B. Such devices are commercially avaUable from Pfeiffer GmbH.
  • the adjuvant formulations of the present invention may also comprise abUe acid or derivative of cholic acid.
  • the derivative of cholic acid is a salt thereof, for example a sodium salt thereof.
  • bile acids examples include cholic acid itself, deoxychohc acid, chenodeoxy colic acid, Hthocholic acid, taurodeoxycholate ursodeoxychohc acid, hyodeoxycholic acid and derivatives like glyco-, tauro-, amidopro ⁇ yl-1- propanesulfonic- and amido ⁇ ropyl-2 -hydroxy- 1 -pro anesulfonic- derivatives of the above bile acids, orN, N-bis (3DGluconoamidopropyl) deoxycholamide.
  • the adjuvant formulation of the present invention may be in the form of an aqueous solution or a suspension of non- vesicular forms.
  • Such formulations are convenient to manufacture, and also to sterilise (for example by terminal filtration through a 450 or 220 nm pore membrane).
  • the route of administration to said host is via the skin, intramuscular or via a mucosal surface such as the nasal mucosa.
  • the admixture may for example be administered as a spray.
  • the methods to enhance an immune response may be either a priming or boosting dose of the vaccine.
  • adjuvant includes an agent having the abUity to enhance the immune response of a vertebrate subject's immune system to an antigen or antigenic dete ⁇ ninant.
  • Immune response includes any response to an antigen or antigenic dete ⁇ ninant by the immune system of a subject.
  • Immune responses include for example humoral immune responses (e. g. production of antigen-specific antibodies) and cell- mediated immune responses (e. g. lymphocyte prohferation).
  • ceU-mediated immune response includes the immunological defence provided by lymphocytes, such as the defence provided by T cell lymphocytes when they come into close proximity with thek victim cells.
  • Lymphocyte prohferation When “lymphocyte prohferation” is measured, the abUity of lymphocytes to prohferate in response to specific antigen may be measured. Lymphocyte prohferation includes B ceU, T-helper cell or CTL ceU proliferation.
  • compositions of the present invention may be used to formulate vaccines containing antigens derived from a wide variety of sources.
  • antigens may include human, b cterial, or vkal nucleic acid, pathogen derived antigen or antigenic preparations, host-derived antigens, including GhRH and IgE peptides, recombinantly produced protem or peptides, and chimeric fusion proteins.
  • the vaccine formulations of the present invention contain an antigen or 107 -
  • the antigen or antigens may, for example, be pep tides/proteins, polysaccharides and lipids and may be derived from pathogens such as viruses, bacteria and parasites/fungi as follows:
  • Vkal antigens or antigenic determinants may be derived, for example, from:
  • Cytomegalo virus especiaUy Human, such as gB or derivatives thereof); Epstein Ban virus (such as gp350); flaviviruses (e. g. Yellow Fever Virus, Dengue Vkus, Tick-bome encephalitis virus, Japanese Encephalitis Vkus); hepatitis vkus such as hepatitis B virus (for example Hepatitis B Surface antigen such as the PreSl, PreS2 and S antigens described in EP-A-414 374; EP-A-0304 578, and EP-A-198474), hepatitis A virus, hepatitis C virus and hepatitis E virus; HIN-1, (such as tat, nef, gpl20 or gpl60); human herpes viruses, such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSN1 or HSN2; human papffloma viruses (for example HPN6,
  • Bacterial antigens or antigenic determinants may be derived, for example, from: 108 -
  • BacUlus spp. including B. anthracis (eg botalinum toxin); Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin, filamenteous hemagglutinin, adenylate cyclase, fimbriae); BoneHa spp., including B. burgdorferi (eg OspA, OspC, DbpA, DbpB), B. garinu (eg OspA, OspC, DbpA, DbpB), B. afzehi (eg OspA, OspC, DbpA, DbpB), B.
  • B. anthracis eg botalinum toxin
  • Bordetella spp including B. pertussis (for example pertactin, pertussis toxin, filamenteous hemagglutinin, adenylate cyclase
  • HaemophUus spp. including H. influenzae type B (eg PRP), non-typable H. influenzae, for example OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and i nbrin derived peptides (see for example US 5,843,464); Helicobacter spp, including H. pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa; Legionella spp, including L.
  • H. influenzae type B eg PRP
  • non-typable H. influenzae for example OMP26
  • high molecular weight adhesins for example OMP26
  • high molecular weight adhesins P5, P6, protein D and lipoprotein D
  • fimbrin and i nbrin derived peptides see for example US 5,843,464
  • pneumophUa pneumophUa ; Leptospka spp., including L. inte ⁇ ogans; Listeria spp., including L. monocytogenes; MoraxeUa spp, including M cata ⁇ hahs, also known as Branhamella catanhalis (for example high and low molecular weight adhesins and invasins); Morexella Cata ⁇ hahs (including outer membrane vesicles thereof, and OMP106 (see for example W097/41731)); Mycobacterium spp., including M. tuberculosis (for example ESAT6, Antigen 85 A, -B or -C), M. bovis, M. leprae, M. avium, M.
  • ESAT6 Antigen 85 A, -B or -C for example ESAT6, Antigen 85 A, -B or -C
  • M. bovis M. leprae
  • M. avium M.
  • Neisseria spp including N. gononhea and N. meningitidis (for example capsular polysaccharides and conjugates thereof, transferrin- binding proteins, lactoferrin binding proteins, PilC, adhesins); Neisseria mengitidis B (including outer membrane vesicles thereof, and NspA ( see for example WO 96/29412); 109
  • Salmonella spp including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Staphylococcus spp., including S. aureus, S. epidermidis; Streptococcus spp, including S.
  • pneumonie eg capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline-binding proteins
  • PsaA capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline-binding proteins
  • Pneumolysin Biochem Biophys Acta, 1989,67,1007; Rubins et al., Microbial Pathogenesis, 25,337-342
  • mutant detoxified derivatives thereof see for example WO 90/06951 ; WO 99/03884
  • Treponema spp. including T. pallidum (eg the outer membrane proteins), T. denticola, T. hyodysenteriae
  • Vibrio spp including V. cholera (for example cholera toxin)
  • Yersinia spp including Y. enterocolitica (for example a Yop protein), Y. pestis,
  • Parasitic/fungal antigens or antigenic determinants may be derived, for example, from:
  • Babesia spp. including B. microti; Candida spp., including C. albicans; Cryptococcus spp., including C. neoformans; Entamoeba spp., including E. histolytica; Giardia spp., including ;G. lambha; Leshmania spp., including L. major; Plasmodium.
  • Approved/Hcensed vaccines include, for example anthrax vaccines such as Biothrax (BioPort Corp); tuberculosis (BCG) vaccines such as TICE BCG (Organon Teknika Corp) and My cob ax (Aventis Pasteur, Ltd); diphtheria & tetanus toxoid and aceUular pertussis (DTP) vaccines such as Tripedia (Aventis Pasteur, Inc), fnfanrix (GlaxoSmithKline), and DAPTACEL (Aventis Pasteur, Ltd); Haemophilus b conjugate vaccines (eg diphtheria CRM197 protein conjugates such as HibTITER from Lederle Lab 110 -
  • Hepatitis A vaccines such as Havrix (Glaxo SmithKHne) and VAQTA (Merck & Co, Inc); combined Hepatitis A and Hepatitis B (recombinant) vaccines such as Twinrix (GlaxoSmithKline); recombinant Hepatitis B vaccines such as Recombivax HB (Merck & Co, Inc) and Engerix-B (GlaxoSmithKline); influenza virus vaccines such as Fluvirin (Evans Vaccine), FluShield (Wyeth Laboratories, Inc) and Fluzone (Aventis Pasteur, Inc); Japanese Encephahtis virus vaccme such as JE-Vax (Research Foundation for Microbial Diseases of Osaka
  • antigens and antigenic determinants may be used in many different forms.
  • antigens or antigenic determinants may be present as isolated proteins or peptides (for example in so-caUed "subunit vaccines") or, for example, as ceU-associated or virus-associated antigens or antigenic determinants (for example in either live or kUled pathogen strains).
  • Live pathogens wUl preferably be attenuated in known manner.
  • antigens or - Ill —
  • antigenic determinants may be generated in situ in the subject by use of a polynucleotide coding for an antigen or antigenic dete ⁇ riinant (as in so-caUed "DNA vaccination", although it will be appreciated that the polynucleotides which may be used with this approach are not limited to DNA, and may also include RNA and modified polynucleotides as discussed above).
  • the term "genetic vaccine” refers to a pharmaceutical preparation that comprises a polynucleotide (eg DNA) constmct. Genetic vaccines include pharmaceutical preparations useful to invoke a prophylactic and/or therapeutic immune response. Therapeutic vaccines may also be refe ⁇ ed to as "Pharmacines”.
  • Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al, University Park Press, Baltimore, Maryland, U. S. A. 1978.
  • the adjuvants of the present invention may further be combined with other adjuvants including, for example: Cholera toxin and its B subunit; E. Coh heat labile enterotoxin LT, its B subunit LTB and detoxified versions thereof such as mLT; immunologicaUy active saponin fractions e. g.
  • Quil A derived from the bark of the South American tree QuUlaja Saponaria Molina and derivatives thereof (for example QS21, as described in US 5,057,540); the oligonucleotide adjuvant system CpG (as described in WO 96/02555), especially 5'TCGTCGTTT TGT CGT TTT GTC GTT3 (SEQ JD NO: 1); and Monophosphoryl Lipid A and its non-toxic derivative 3-O-deacylated monophosphoryl lipid A (3D-MPL, as described in GB 2,220,211).
  • the present invention provides an increased magnitude and/or increased duration of immune response.
  • the invention provides an increased protective immune response.
  • T cells can act in different subpopulations that show different effector functions.
  • T cell responses can be pro-inflammatory T helper 1 type (Thl) characterized by the secretion of interferon gamma (IFN-gamma.) and interleukin 2 (IL-2).
  • Thl ceUs are the helper ceUs for the ceUular defence but provide Httle help for antibody secretion.
  • the other class of T cell responses is generally anti-inflammatory, and is mediated by Th2 ceUs that produce JL-4, B -5 and IL-10, but Httle or no IL-2 or IFN-gamma.
  • Th2 ceUs are the helper ceUs for antibody production.
  • CD4+ and CD 8+ cells both occur in these subpopulations: Thl/Th2:CD4, Tcl/Tc2:CD8. - 114
  • T cell response e.g., Thl vs. Th2, CD4+ vs. CD8+
  • Thl an "inappropriate” type of T ceU response
  • Thl an intracellular pathogen such as a vkus or intracellular bacterium
  • Th2 a Th2 response to clear an extracellular pathogen.
  • the present invention may be used in both so-called prophylactic and so-called therapeutic vaccines.
  • prophylactic vaccines may be used to provide protective immunity in an uninfected subject to provide protection against future establishment of infection.
  • therapeutic vaccines may be used, for example, after an infection has become established (for example as either an acute or chronic infection) in order to increase the immune response against the infection.
  • therapeutic vaccines may be used to combat chronic infections which may for example be bacterial infections (such as tuberculosis), parasitic infections such as malarial infections or vkal infections (such as HPV, HCV, HB V or HIV infections).
  • hepatitis viruses such as hepatitis A, B, C, D and E, for example hepatitis B virus (HBV) and hepatitis C virus (HCV) which cause chronic hepatitis, cirrhosis and Hver cancer (see US 5738852).
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • vkal infectious agents include those caused by the human retro viruses: human immunodeficiency viruses (HIN-1 and HTV-2), which cause acquked immune deficiency syndrome (AIDS); and human T lymphotropic viruses (HTLN-1 and HTLN-2) which cause T ceU leukemia and myelopathies.
  • HIN-1 and HTV-2 human immunodeficiency viruses
  • HTLN-1 and HTLN-2 human T lymphotropic viruses
  • human herpes viruses including the herpes simplex vkus (HSV) types 1 and 2, Epstein Ban virus (EBV), cytomegalo virus (CMV), varicella-zoster vkus (VZV) and human herpes virus 6 (HHV-6) are often not eradicated by host mechanisms, but rather become chronic and in this state may cause disease.
  • Chronic infection with human papiUoma viruses is associated with cervical carcinoma. Numerous other viruses and other infectious agents replicate intracellularly and may become chronic when host defense mechanisms faU to eliminate them.
  • pathogenic protozoa e.g., Pneumocystis carinii, Trypanosoma, Leishmania, Plasmodium (responsible for Malaria) and Toxoplasma gondii
  • bacteria e.g., mycobacteria (eg Mycobacterium tuberculosis responsible for tuberculosis), salmonella and listeria
  • fungi e.g., Candida and aspergiUus.
  • the pathogen antigen is suitably an antigen that is nataraUy encoded in the pathogen against which an enhanced or augmented immune response is desked.
  • target antigens are those which induce a T cell response, and particularly a CTL-response during infection. These may include, for example, from HBV, the core antigen (HBcAg) the E antigen, and the surface antigen (HBsAg).
  • Polynucleotide sequences for HBsAg including the pre-Sl, pre- S2 and S regions from a variety of surface antigen subtypes are well known in the art (see, for example, Okamoto et al, J. Gen. Virol., 67:1383-1389 (1986); GenBank Accession numbers D00329 and D00330).
  • the polynucleotide sequences encoding HJV glycoprotein g ⁇ l60 and other antigenic HJV regions are known in the art (Lautenberger et al, Nature, 313:277-284 (1985); Starcich et al, CeU, 45:637-648 (1986); Wiley et al., Proc. Natl. Acad. Sci. USA, 83:5038-5042 (1986); and Modrow et al., J. Virol., 61:570- 578 (1987)).
  • HXB2 Human immunodeficiency virus type 1
  • HIVl/HTLV-ITI/LAV reference genome HIVl/HTLV-ITI/LAV reference genome
  • GenBank Numerous genome sequences for HAV, HB V and HCV strains (including sequences for antigenic proteins) are provided on GenBank, for example AY057948 (Hepatitis B virus isolate kannl27, complete genome); AY057947 (Hepatitis B virus isolate kann705, complete genome); NC_003977 (Hepatitis B vkus, complete genome); NC_004102 (Hepatitis C vkus, complete genome); AF139594 (Hepatitis C virus strain HCV-N, complete genome) ; M16632 (Hepatitis A virus (HM-175 strain; attenuated)).
  • the modulator/inhibitor of Notch signaUing increases cytotoxic (CD 8+) T ceU responses to antigen.
  • the invention further provides a conjugate comprising first and second sequences, wherein the first sequence comprises a pathogen antigen or a polynucleotide sequence coding for such an antigen 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 a pathogen antigen or antigenic dete ⁇ ninant and a polynucleotide sequence coding for a modulator of the Notch signalling pathway, wherein preferably each sequence is operably finked to regulatory elements necessary for expression in eukaryotic ceUs.
  • a schematic representation of one such form of vector is shown in Figure 11. - 117 -
  • the polynucleotide sequence coding for the modulator of the Notch signaUing 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 Hgand 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-Hke domains.
  • the DSL and EGF-like domain sequences are or co ⁇ espond to mammahan sequences.
  • the polynucleotide sequence coding for the modulator of the Notch signaUing pathway may further comprise a transmembrane domain and, suitably, a Notch ligand intracellular domain.
  • Prefe ⁇ ed sequences include human sequences such as human Deltal, Delta3, Delta4, Jaggedl or Jagged2 sequences.
  • the polynucleotide sequence that encodes the pathogen antigen or antigenic determinant may further include a nucleotide sequence that encodes a signal sequence which directs trafficking of the antigen or antigenic deterrninant within a cell to which it is administered.
  • a signal sequence may dkect 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 te ⁇ nination signals are regulatory elements which are often considered part of the coding sequence.
  • suitable promoters include but are not limited to promoters from Simian Vkus 40 (S V40), Mouse Mammary Tumor Vkus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HJV Long Terminal Repeat (LTR) promoter, 118
  • S V40 Simian Vkus 40
  • MMTV Mouse Mammary Tumor Vkus
  • HV Human Immunodeficiency Virus
  • LTR HJV Long Terminal Repeat
  • Moloney virus ALV
  • Cytomegalov us CMV
  • CMV Cytomegalov us
  • EBV Epstein Barr Virus
  • RSV Rous Sarcoma Vkus
  • human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metalothionein.
  • Tissue-specific promoters specific for lymphocytes, dendritic cells, skin, brain ceUs and epithelial ceUs within the eye are particularly prefened, for example the CD2, CDllc, 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 SV40 polyadenylation signals and LTR polyadenylation signals.
  • SV40 polyadenylation signal used in plasmid pCEP4 Invitrogen, San Diego Calk
  • refened to as the SV40 polyadenylation signal may be used.
  • enhancers which may, for example, be selected from human Actin, human Myosin, human Hemoglobin, human muscle creatine and vkal enhancers such as those from CMV, RSV and EBV.
  • the nucleotide conjugate When administered to and taken up by a cell, 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 ceU.
  • 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 rephcation. - 119
  • conjugates may be provided with mammalian origin of replication in order to maintain the constract extrachromosomaUy and produce multiple copies of the constract in the cell.
  • mammalian origin of replication for example, plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calk.) contain the Epstein Ban virus origin of rephcation and nuclear antigen EBNA-1 coding region which produces high copy episomal rephcation without integration.
  • regulatory sequences may be selected which are well suited for gene expression in the type of ceUs the constract is to be administered to. Moreover, codons may be selected which are most efficiently transcribed in the cell.
  • conjugates may be used either in vivo or ex-vivo with a "genetic vaccination” approach to provide expression of both an inhibitor of Notch signalling and a pathogen antigen or antigenic determinant in cells or tissues.
  • polynucleotides may be delivered in conjunction with administration of a facihtating 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, anUides, amidines, urethans and the hydrochloride salts thereof such as those of the farnUy of local anesthetics.
  • esters include: benzoic acid esters such as piperocaine, meprylcaine and isobucarne; para-aminobenzoic acid esters such as procaine, tetracaine, butemamine, propoxycaine and chloroprocaine; meta-aminobenzoic acid esters including metabutharnine and primacaine; and para-ethoxybenzoic acid esters such as parethoxycaine.
  • benzoic acid esters such as piperocaine, meprylcaine and isobucarne
  • para-aminobenzoic acid esters such as procaine, tetracaine, butemamine, propoxycaine and chloroprocaine
  • meta-aminobenzoic acid esters including metabutharnine and primacaine
  • para-ethoxybenzoic acid esters such as parethoxycaine.
  • anUides include Hdocaine, etidocaine, mepivacaine, - 120 -
  • bupivacaine pynocaine and prilocaine.
  • Other examples of such compounds include dibucaine, benzocaine, dyclonine, pramoxine, proparacaine, butacaine, benoxinate, carbocaine, methyl bupivacaine, butas n picrate, phenacaine, diothan, luccaine, intracaine, nupercaine, metabutoxycaine, piridocaine, biphenamine and the botanically- derived bicyclics such as cocaine, cinnamoylcocaine, traxiUrne and cocaethylene and aU such compounds complexed with hydrochloride.
  • the facUitating 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 chemicaUy designated as 2-piperidinecarboxamide, l-butyl-N-(2,6- dknethyl ⁇ henyl)-monohydrochloride, monohydrate and is widely available commerciaUy 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 commerciaUy 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 deskable effects may be prepared if desked.
  • about 250 ⁇ g to about 10 mg of bupivacaine may be administered.
  • antigen-presenting cells may be "professional" antigen presenting cells or may be another cell that may be induced to present antigen to T ceUs.
  • APC precursor may be used which dkferentiates or is activated under the conditions of culture to produce an APC.
  • An APC for use in the ex vivo methods of the invention is typically isolated from a tumour or peripheral blood found within the body of - 121
  • the APC or precursor is of human origin.
  • APCs are used in preliminary in vitro screening procedures to identify and test suitable nucleic acid sequences, APCs from any suitable source, such as a healthy patient, may be used.
  • APCs include dendritic ceUs (DCs) such as interdigitating DCs or foUicular DCs, Langerhans cells, PBMCs, macrophages, B -lymphocytes, or other cell types such as epithelial cells, fibroblasts or endothelial ceUs, activated or engineered by transfection to express a MHC molecule (Class I or U) on thek surfaces.
  • DCs dendritic ceUs
  • PBMCs macrophages
  • B -lymphocytes or other cell types
  • epithelial cells fibroblasts or endothelial ceUs
  • Precursors of APCs include CD34 + cells, monocytes, fibroblasts and endothelial cells.
  • the APCs or precursors may be modified by the cultare conditions or may be geneticaUy modified, for instance by transfection of one or more genes encoding proteins which play a role in antigen presentation and/or in combination of selected cytokine genes which would promote to immune potentiation (for example IL-2, IL-12, IFN- ⁇ , TNF- ⁇ , IL-18 etc.).
  • proteins include MHC molecules (Class I or Class IT), CD80, CD86, or CD40.
  • DCs or DC-precursors are included as a source of APCs.
  • Dendritic ceUs can be isolated/prepared by a number of means, for example they can either be purified dkectly from peripheral blood, or generated from CD34 + precursor ceUs for example after mobiHsation into peripheral blood by treatment with GM-CSF, or directly from bone ma ⁇ ow. From peripheral blood, adherent precursors can be tieated with a GM-CSF/TL-4 mixture (Inaba K, et al. (1992) J. Exp. Med. 175: 1157-1167 (Inaba)), or from bone manow, non-adherent CD34 + ceUs can be treated with GM-CSF and TNF-a (Caux C, et al.
  • DCs can also be routinely prepared from the peripheral blood of human volunteers, similarly to the method of SaUusto and Lanzavecchia (Sallusto F and Lanzavecchia A (1994) J. Exp. Med. 179: 1109-1118) using purified peripheral blood mononucleocytes (PBMCs) and treating 2 hour adherent cells with GM-CSF and JL-4. If requked, these may be depleted of CD19 + B cells and CD3 + , CD2 + T cells using magnetic beads (Coffin RS, et al. (1998) Gene Therapy 5: 718-722 (Coffin)). Cultare conditions may include other cytokines such - 122 -
  • GM-CSF GM-CSF or JL-4 for the maintenance and, or activity of the dendritic cells or other antigen presenting ceUs.
  • the term "antigen presenting cell or the like" are used herein is not intended to be limited to APCs.
  • the skUled man wUl understand that any vehicle capable of presenting to the T ceU population may be used, for the sake of convenience the term APCs is used to refer to aU these.
  • suitable APCs include dendritic ceUs, L cells, hybridomas, fibroblasts, lymphomas, macrophages, B ceUs or synthetic APCs such as lipid membranes.
  • T ceUs from any suitable source may be used and may be obtained from blood or another source (such as lymph nodes, spleen, or bone ma ⁇ ow). They may optionally be enriched or purified by standard procedures.
  • the T ceUs may be used in combination with other immune cells, obtained from the same or a different individual.
  • whole blood may be used or leukocyte enriched blood or purified white blood ceUs as a source of T cells and other cell types. It is particularly prefened to use helper T cells (CD4 + ).
  • T ceUs such as CD8 + ceUs may be used. It may also be convenient to use ceU Hnes such as T ceU hybridomas.
  • the term "antigen presenting cell or the like" are used herein is not intended to be limited to APCs.
  • the skUled man wUl understand that any vehicle capable of presenting to the T ceU population may be used, for the sake of convenience the term APCs is used to refer to aU these.
  • suitable APCs include dendritic ceUs, L cells, hybridomas, fibroblasts, lymphomas, macrophages, B ceUs or synthetic APCs such as lipid membranes. - 123 -
  • T ceUs/APCs/tumour cells may be cultured as described above.
  • the APCs/T ceUs/turnour ceUs may be incubated/exposed to substances which are capable of mterterring with or downregulating Notch or Notch Hgand expression.
  • the resulting T ceUs/APCs/tumour cells that have downregulated Notch or Notch Hgand expression are now ready for use. For example, they may be prepared for administration to a patient or incubated with T cells in vitro (ex vivo).
  • tumour material may be isolated and transfected with a nucleic acid sequence which encodes for, e.g., a ToU-like receptor or BMP receptor and/or costimulatory molecules (suitable costimulants are mentioned above) and/or treated with cytokines, e.g. IFN- ⁇ , TNF- ⁇ , EL- 12, and then used in vitro to prime TRL and/or TIL ceUs.
  • a nucleic acid sequence which encodes for, e.g., a ToU-like receptor or BMP receptor and/or costimulatory molecules (suitable costimulants are mentioned above) and/or treated with cytokines, e.g. IFN- ⁇ , TNF- ⁇ , EL- 12, and then used in vitro to prime TRL and/or TIL ceUs.
  • modified cells of the present invention are preferably administered to a host by dkect injection into the lymph nodes of the patient.
  • TypicaUy from 10 4 to 10 8 treated ceUs, preferably from 10 5 to 10 7 cells, more preferably about 10 6 ceUs are administered to the patient.
  • the cells will be taken from an enriched ceU population.
  • the term "enriched" as applied to the ceU populations of the invention refers to a more homogeneous population of ceUs which have fewer other cells with which they are natarally associated.
  • An enriched population of ceUs can be achieved by several methods known in the art. For example, an enriched population of T-cells can be obtained using immunoaffinity chromatography using monoclonal antibodies specific for determinants found only on T-cells.
  • Enriched populations can also be obtained from mixed cell suspensions by positive selection (collecting only the desked cells) or negative selection (removing the undeskable ceUs).
  • positive selection collecting only the desked cells
  • negative selection removing the undeskable ceUs.
  • the technology for capturing specific cells on affinity materials is weU 124 -
  • Monoclonal antibodies against antigens specific for matare, dkferentiated cells have been used in a variety of negative selection strategies to remove undesired cells, for example, to deplete T-ceUs or mahgnant ceUs from aUogeneic or autologous ma ⁇ ow grafts, respectively (Gee, et al., J.N.C.I. 80:154, 1988).
  • Purification of human hematopoietic cells by negative selection with monoclonal antibodies and irnmunomagnetic microspheres can be accomplished using multiple monoclonal antibodies (Griffin, et al., Blood, 63:904, 1984).
  • Procedures for separation of cells may include magnetic separation, using antibodycoated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, for example, complement and cytotoxins, and "panning" with antibodies attached to a sohd matrix, for example, plate, or other convenient technique.
  • Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, for example, a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • the therapeutic agents used in the present invention may be administered dkectly to patients in vivo.
  • the agents may be administered to cells such as T cells and/or APCs in an ex vivo manner.
  • leukocytes such as T ceUs or APCs may be obtained from a patient or donor in known manner, treated/incubated ex vivo in the manner of the present invention, and then administered to a patient.
  • a combination of routes of administration may be employed if desked. For example, where appropriate one - 125 -
  • component such as the modulator of Notch signalling
  • the other may be administered in vivo, or vice versa.
  • T-ceUs and APCs as described above are cultured in a suitable culture medium such as DMEM or other defined media, optionaUy in the presence of fetal calf serum.
  • Polypeptide substances may be administered to T-ceUs and/or APCs by introducing nucleic acid constracts/vkal vectors encoding the polypeptide into ceUs under conditions that aUow for expression of the polypeptide in the T-ceU and/or APC.
  • nucleic acid constructs encoding antisense constructs may be introduced into the T-ceUs and/or APCs by transfection, viral infection or vkal transduction.
  • nucleotide sequences encoding the modulator(s) of Notch signaUing wUl be operably linked to control sequences, including promoters/enhancers and other expression regulation signals.
  • control sequences including promoters/enhancers and other expression regulation signals.
  • operably linked means that the components described are in a relationship perrnitting them to function in thek intended manner.
  • a regulatory sequence "operably linked" to a coding sequence is peferably Hgated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • the promoter is typically selected from promoters which are functional in mammalian ceUs, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used.
  • the promoter is typically derived from promoter sequences of vkal or eukaryotic genes. For example, it may be a promoter derived from the genome of a ceU in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of a-actin, b-actin, tabulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyravate kinase). Tissue-specific promoters speckle for lymphocytes, dendritic ceUs, - 126 -
  • epithelial ceU promoter SPC is used. They may also be promoters that respond to specific stirnuH, for example promoters that bind steroid hormone receptors.
  • Vkal promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalo virus (CMV) JE promoter.
  • the promoters may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the Hfe-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
  • any of the above promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences.
  • Chimeric promoters may also be used comprising sequence elements from two or more different promoters.
  • the regulatory sequences may be cell specific such that the gene of interest is only expressed in ceUs of use in the present invention.
  • ceUs include, for example, APCs and T-cells.
  • T-cells and/or APCs that comprise nucleic acid constructs capable of up- regulating Notch Hgand expression are now ready for use. If required, a smaU aliquot of ceUs may be tested for up-regulation of Notch Hgand expression as described above. The ceUs may be prepared for administration to a patient or incubated with T-ceUs in vitro (ex vivo).
  • any of the assays described above can be adapted to monitor or to detect reactivity in immune cells for use in clinical appHcations. Such assays will involve, for - 127 -
  • Immune ceU activity may be monitored by any suitable method known to those skUled in the art. For example, cytotoxic activity may be monitored. Natural kiUer (NK) ceUs wiU demonstrate enhanced cytotoxic activity after activation. Therefore any drop in or stabUisation of cytotoxicity wiUbe an indication of reduced reactivity.
  • NK Natural kiUer
  • leukocytes express a variety of new cell surface antigens.
  • NK ceUs for example, wiU express transferrin receptor, HLA-DR and the CD25 IL-2 receptor after activation. Reduced reactivity may therefore be assayed by monitoring expression of these antigens.
  • EA-1 and MLR3 are glycoproteins having major components of 28kD and 32kD.
  • EA-1 and MLR3 are not HLA class ⁇ antigens and an MLR3 Mab wUl block IL-1 binding. These antigens appear on activated T-ceUs within 18 hours and can therefore be used to monitor immune ceU reactivity.
  • leukocyte reactivity may be monitored as described in EP 0325489, which is incorporated herein by reference. Briefly this is accomplished using a monoclonal antibody ("Anti-Leu23”) which interacts with a ceUular antigen recognised by the monoclonal antibody produced by the hybridoma designated as ATCC No. HB-9627. 128
  • Anti-Leu 23 recognises a cell surface antigen on activated and antigen stimulated leukocytes. On activated NK cells, the antigen, Leu 23, is expressed within 4 hours after activation and continues to be expressed as late as 72 hours after activation. Leu 23 is a disulfide-linked homodimer composed of 24 kD subunits with at least two N-linked carbohydrates.
  • Anti-Leu 23 is useful in monitoring the reactivity of leukocytes.
  • immune cells may be used to present antigens or allergens and/or may be treated to modulate expression or interaction of Notch, a Notch Hgand or the Notch signalling pathway.
  • APCs Antigen Presenting Cells
  • a suitable cultare medium such as DMEM or other defined media, optionally in the presence of a serum such as fetal calf serum.
  • Optimum cytokine concentrations may be determined by titration.
  • One or more substances capable of up- regulating or down-regulating the Notch signaUing pathway are then typicaUy added to the cultare medium together with the antigen of interest.
  • the antigen may be added before, after or at substantially the same time as the substance(s).
  • Cells are typically incubated with the substance(s) and antigen for at least one hour, preferably at least 3 129 -
  • a smaU ahquot of cells may be tested for modulated target gene expression as described above.
  • ceU activity may be measured by the inhibition of T ceU activation by monitoring surface markers, cytokine secretion or prohferation as described in WO98/20142.
  • APCs transfected with a nucleic acid constmct directing the expression of, for example Senate, may be used as a control.
  • polypeptide substances may be administered to APCs by introducing nucleic acid constracts/vkal vectors encoding the polypeptide into cells under conditions that aUow for expression of the polypeptide in the APC.
  • nucleic acid constructs encoding antigens may be introduced into the APCs by transfection, vkal infection or vkal transduction. The resulting APCs that show increased levels of a Notch signalling are now ready for use.
  • any of the assays described above can be adapted to monitor or to detect the degree of reactivity and tolerisation in immune cells for use in clinical applications.
  • Such assays will involve, for example, detecting decreased Notch signaUing activity in host ceUs or monitoring Notch cleavage in donor cells. Further methods of monitoring immune ceU activity are set out below.
  • Immune ceU activity may be monitored by any suitable method known to those skilled in the art. For example, cytotoxic activity may be monitored. Natural kiUer (NK) ceUs wiU demonstrate enhanced cytotoxic activity after activation. Therefore any drop in or stabUisation of cytotoxicity wiU be an indication of reduced reactivity.
  • NK Natural kiUer
  • leukocytes express a variety of new cell surface antigens.
  • NK ceUs for example, wiU express transferrin receptor, HLA-DR and the CD25 IL-2 receptor after activation. Reduced reactivity may therefore be assayed by monitoring expression of these antigens.
  • EA-1 and MLR3 are glycoproteins having major components of 28kD and 32kD.
  • EA-1 and MLR3 are not HLA class II antigens and an MLR3 Mab wUl block IL-1 binding. These antigens appear on activated T-ceUs within 18 hours and can therefore be used to monitor immune cell reactivity.
  • leukocyte reactivity may be monitored as described in EP 0325489, which is incorporated herein by reference. Briefly this is accomplished using a monoclonal antibody ("Anti-Leu23”) which interacts with a ceUular antigen recognised by the monoclonal antibody produced by the hybridoma designated as ATCC No. HB-9627.
  • Anti-Leu23 a monoclonal antibody
  • ATCC No. HB-9627 a monoclonal antibody
  • Anti-Leu 23 recognises a cell surface antigen on activated and antigen stimulated leukocytes. On activated NK cells, the antigen, Leu 23, is expressed within 4 hours after activation and continues to be expressed as late as 72 hours after activation. Leu 23 is a disulfide-linked homodimer composed of 24 kD subunits with at least two N-linked carbohydrates.
  • Anti-Leu 23 is useful in monitoring the reactivity of leukocytes.
  • a fusion protein comprising the extracellular domain of human Deltal fused to the Fc domain of human IgG4 ("hDeltal-IgG4Fc") was prepared by inserting a nucleotide sequence coding for the extracellular domain of human Deltal (see, eg Genbank Accession No AF003522) into the expression vector pCON ⁇ (Lonza Biologies, Slough, UK) and expressing the resulting construct in CHO cells.
  • a 1622bp extraceUular (EC) fragment of human Delta-like ligand 1 (hECDLL-1; see GenBank Accession No AF003522) was gel purified using a Qiagen QIAquickTM Gel Extraction Kit (cat 28706) according to the manufacturer's instmctions. The fragment was then ligated into a pCR Blunt cloning vector (Invitrogen, UK) cut Hindlll - BsiWI, thus eliminating a Hindlll, BsiWI and Apal site.
  • the ligation was transformed into DH5 ⁇ ceUs, streaked onto LB + Kanamycin (30ug/ml) plates and incubated at 37°C overnight. Colonies were picked from the plates into 3ml - 132 -
  • Plasmid DNA was purified from the cultares using a Qiagen Qiaquick Spin Miniprep kit (cat 27106) according to the manufacturer's instructions, then diagnosticaUy digested with Hindlll. A clone was chosen and streaked onto an LB + Kanamycin (30ug/ml) plate with the glycerol stock of modified ⁇ CRBlunt-hECDLL-1 and incubated at 37°C overnight. A colony was picked off this plate into 60ml LB + Kanamycin (30ug/ml) and incubated at 37°C overnight.
  • the cultare was maxiprepped using a Clontech Nucleobond Maxi Kit (cat K3003-2) according to the manufacturer's instructions, and the final DNA pellet was resuspended in 300ul dH 2 O and stored at -20°C.
  • modified pCR Blunt-hECDLL-1 vector was linearised with Hindlll and partially digested with Apal.
  • the 1622bp hECDLL-1 fragment was then gel purified using a Clontech Nucleospin® Extraction Kit (K3051-1) according to the manufacturer's instructions.
  • the DNA was then passed through another Clontech Nucleospin® column and followed the isolation from PCR protocol, concentration of sample was then checked by agarose gel analysis ready for Hgation.
  • Plasmid pcon ⁇ (Lonza Biologies, UK) was cut with Hindlll - Apal and the following oligos were ligated in (SEQ ID NO: 2):
  • the Hgation was transformed into DH5 ⁇ ceUs and LB + Amp (lOOug/ml) plates were streaked with 200ul of the transformation and incubated at 37°C overnight. The following day 12 clones were picked into 2 x YT + AmpicUlin (lOOugmT 1 ) and grown up at 37°C throughout the day. Plasmid DNA was purified from the cultares using a Qiagen Qiaquick Spin Miniprep kit (cat 27106) and diagnosticaUy digested with Notl.
  • a clone (designated "pDev41") was chosen and an LB + Amp (lOOug/ml) plate was streaked with the glycerol stock of pDev41 and incubated at 37°C overnight. The following day a clone - 133
  • the polynucleotide was then cloned into the polylinker region of pEE14.4 (Lonza Biologies, UK) downstream of the strong hCMV promoter enhancer region (hCMV- MJE) and upstream of SV40 polyadenylation signal (encodes the GS gene requked for selection in glutarrHne free media; contains the GS minigene - GS cDNA which includes the last intron and polylinker adenylation signals of the wild type hamster GS gene) which is under the control of the late SV40 promoter, has the hCMV promoter to drive transcription of the desked gene.
  • 5ug of the maxiprep of pEE14.4 was digested with Hindlll - EcoRI, and the product was gel extracted and treated with alkaline phosphatase.
  • a 3 fragment Hgation was set up with pEE14.4 cut Hindlll - EcoRI, ECDLL-1 from modified pCR Blunt (Hindlll - Apal) and the IgG4Fc fragment cut from pDev41 (Apal - EcoRI). This was transformed into DH5 ceUs and LB + Amp (lOOug/ml) plates were streaked with 200ul of the transformation and incubated at 37C overnight. The foUowing day 12 clones were picked into 2 x YT + Amp (lOOug/ml) and mimpreps were grown up at 37°C throughout the day.
  • Plasmid DNA was purified from the preps using a Qiagen Qiaquick spin miniprep kit (Cat No 27106), diagnosticaUy digested (with EcoRI and HindTTT) and a clone (clone 8; designated " ⁇ Dev44") was chosen for maxiprepping.
  • the glycerol stock of pDev44 clone 8 was streaked onto an LB + Amp (lOOugml "1 ) plate and incubated at 37°C overnight. The following day a colony was picked into 60ml LB + Amp (lOOugmT 1 ) broth and incubated at 37°C overnight.
  • the plasmid DNA was isolated using a Clontech Nucleobond Maxiprep Kit (Cat K3003-2). 134
  • pDev44 was digested with Hindlll - BstBI, gel purified and treated with alkaline phosphatase. The digest was Hgated with the oligos, transformed into DH5 cells by heat shock . 200ul of each transformation were streaked onto LB + Amp plates (lOOug/ml) and incubated at 37°C overnight. Minipreps were grown up in 3 ml 2 x YT + AmpiciUin (lOOugmT 1 ). Plasmid DNA was purified from the minipreps using a Qiagen Qiaquick spin miniprep kit (Cat No 27106) and diagnosticaUy digested with Ncol. A clone (pDev46) was selected and the sequence was confirmed. The glycerol stock was streaked, broth grown up and the plasmid maxiprepped.
  • the amino acid sequence of the resulting expressed fusion protein was as foUows (SEQ ID NO: 5):
  • first underlined sequence is the signal peptide (cleaved from the mature protein) and the second underlined sequence is the IgG4 Fc sequence.
  • the protein normally exists as a dimer linked by cysteine disulphide bonds (see eg schematic - 136 -
  • Notch signaUing inhibitor enhances immune response to flu antigen
  • FlushieldTM flu vaccine (5 micrograms; Roche USA) was enrols kied in incomplete Freund's adjuvant with or without 100 micrograms of hDeltal-IgG4Fc (from Example 1 above). 6-8 weeks old B ALB/c mice (eight per group) were immunized subcutaneously at the base of the taU and 14 days later the mice were chaUenged in the right ear with 1.8 micrograms of Flushield flu vaccine in saline. Ear responses (ear thickness measured with caUipers) were measured at 1, 2 and 6 days thereafter.
  • Notch signalling inhibitor enhances immune response to KLH
  • mice 6-8 weeks old BALB/c mice (eight per group) were immunized subcutaneously at the base of the tail with keyhole limpet haemocyanin (KLH) from Pierce at 50ng or 0.5ng per mouse emulsified in incomplete Freund's adjuvant (IF A) with or without hDeltal-IgG4Fc protein from Example 1 above (100 micrograms). Some mice also received additional hDeltal-IgG4Fc (400 micrograms) at an adjacent s.c. site one day later. 14 days after the initial KLH priming, mice were chaUenged in the right ear with 20 micrograms KLH and the ear immune response was measured with calhpers as an increase in ear thickness due to the induced inflammatory reaction after 24 hours.
  • KLH keyhole limpet haemocyanin
  • IF A incomplete Freund's adjuvant
  • mice also received additional hDeltal-IgG4Fc (400 micrograms) at an
  • Notch signalling inhibitor enhances immune response to Flu vaccine
  • mice 6-8 weeks old B ALB/c mice (eight per group) were immunized subcutaneously at the base of the tail with FlushieldTM flu vaccine at 5 ⁇ g per mouse emulsified in incomplete Freund's adjuvant (JFA) with hDeltal-IgG4Fc protein from Example 1 above (100 micrograms) or isotype control hlgG4 (Sigma, UK) lOO ⁇ g/TFA control. 14 days after the initial FlushieldTM flu vaccine priming, mice were challenged in the right ear with FlushieldTM and the ear immune response was measured with callipers as an increase in ear thickness due to the induced inflammatory reaction after 24 hours.
  • FlushieldTM flu vaccine 14 days after the initial FlushieldTM flu vaccine priming, mice were challenged in the right ear with FlushieldTM and the ear immune response was measured with callipers as an increase in ear thickness due to the induced inflammatory reaction after 24 hours.
  • M450 Streptavidin DynabeadTM magnetic beads (Dynal, USA) were coated with an anti- human-IgG4 biotinylated monoclonal antibody (BD Bioscience, 555879) by rotating them in the presence of the antibody for 30 minutes at room temperatare. Beads were washed three times with PBS (1ml). They were further incubated with hDeltal-hIgG4 (see Example 1 above) for 2 hours at room temperatare and then washed three times with PBS (1ml). - 138 -
  • PBMC Human peripheral blood mononuclear ceUs
  • the CD4+ T cells were incubated in tripHcates in a 96-weU-plate (flat bottom) at 10 3 CD4/weU/200 ⁇ l in RPMI medium containing 10% FCS, glutamine, peniciUin, streptomycin and ⁇ 2 -mercaptoethanol.
  • Cytokine production was induced by stimulating the ceUs with anti-CD3/CD28 T cell expander beads from Dynal at a 1 :1 ratio (bead/ceU) in the presence of beads coated with hDeltal-IgG4Fc fusion protein (Example 1 above) at a 5:1 ratio (beads/cell). In some wells, increasing amounts of soluble hDeltal-IgG4Fc fusion protein were also added.
  • supematants were removed after 3 days of incubation at 37°C/ 5%CO 2 /humidified atmosphere and cytokine production was evaluated by ELISA using Pharm in gen kits OptEIA Set human IL10 (catalog No. 555157), OptEIA Set human IL-5 (catalog No. 555202) for IL-10 and IL-5 respectively according to the manufacturer's instructions.
  • PBMC Human peripheral blood mononuclear ceUs
  • the CD4+ T cells were incubated in tripHcates in a 96-weU-plate (flat bottom) at 10 5 CD4/weU/200 ⁇ l in RPMI medium containing 10% FCS, glutamine, peniciUin, streptomycin and ⁇ 2 -mercaptoethanol.
  • Cytokine production was induced by stimulating the cells with anti-CD3/CD28 T cell expander beads from Dynal at a 1 :1 ratio (bead/ceU) in the presence of beads coated with hDeltal-IgG4Fc fusion protein (Example 1 above) at a 5:1 ratio (beads/cell).
  • increasing amounts of soluble rat Notchl extracellular domain-hlgGl fusion protein (R&D Systems, Catalog No 1057 -TK) were also added.
  • supematants were removed after 3 days of incubation at 37°C/ 5%CO 2 /humidified atmosphere and cytokine production was evaluated by ELISA using Pharm in gen kits OptEIA Set human 1L10 (Catalog No. 555157), OptEIA Set human IL-5 (Catalog No. 555202) for IL-10 and IL-5 respectively according to the manufacturer's instructions.
  • a fusion protein capable of acting as an inhibitor of Notch signalling comprising human jaggedl sequence up to the end of EGF2 (leader sequence, amino terminal, DSL, EGF1+2) fused to the Fc domain of human IgG4 ("hJaggedl(EGFl+2)-IgG4Fc") was prepared by inserting a nucleotide sequence coding for human Jaggedl from ATG through to the end of the second EGF repeat (EGF2) into the expression vector pCON ⁇ (Lonza Biologies, Slough, UK) to add the IgG4 Fc tag. The fuU fusion protein was then shuttled into the Glutamine Synthetase (GS) selection system vector pEE14.4 (Lonza Biologies). The resulting construct was transfected and expressed in CHO-K1 cells (Lonza Biologies).
  • Plasmid pLOR47 was then modified to remove one of two Drain sites (whilst maintaining and replacing the amino acid sequence for full extracellular hJaggedl) and - 141 -
  • the resulting plasmid was named pDEV20.
  • Plasmid pLOR47 was cut with Drain. This removed a 1.7kb fragment comprising the 3' end of the extraceUular, the transmembrane and intraceUular regions of hJaggedl as well as part of the vector sequence leaving a larger fragment of 7.3kbp of the main vector backbone with almost all of the extraceUular region (EC) of hJaggedl.
  • the cut DNA was run out on an agarose gel, the larger fragment excised and gel purified using a Qiagen QIAquickTM Gel Extraction Kit (cat 28706) according to the manufacturer's instructions.
  • a pak of oHgonucleotides were ordered such that when Hgated together gave a double stranded piece of DNA that had a compatible sticky end for DraHI at the 5' end and recreated the original restriction site. This sequence was foUowed by a BsiWI site then another compatible sticky end for Drain at the 3' end that did not recreate the restriction site.
  • This oligo pak was then Hgated into the DraH cut pLOR47 thus maintaining the 5' Dram site, inserting a BsiWI and eliminating the 3'Draffl site.
  • the resulting plasmid was named pDEV20.
  • Fragment 1 EC hJagged sequence pDev 20 was cut Rsrfl - DraHI giving rise to 3 fragments; 1270 + 2459 + 3621 bp. The fragments were run out on an agarose gel, the 2459 bp band excised and the DNA gel purified using a Qiagen QIAquickTM Gel Extraction Kit (cat 28706) according to the manufacturer's instructions. This contained hJaggedl sequence - with loss of 3' sequence (up to the Rsr ⁇ site) and loss of some 5 'sequence at the end of the EC region.
  • Fragment 2 modified Kozak sequence pUC19 (Invitrogen) was modified to insert new restriction enzyme sites and also introduce a modkied Kozak with 5' hJaggedl sequence.
  • the new plasmid was named pLOR49.
  • pLOR49 was created by cutting ⁇ UC19 vector Hindm EcoRI and ligating in 4 oligonucleotides (2 ohgo pairs).
  • One pak has a Hindm cohesive end foUowed by an optimal Kozac and 5'hJagged 1 sequence followed by Rsrfl cohesive end.
  • the other pak has a cohesive Rsrfl end then Dram, Kpnl, BsiWI sites followed by a cohesive EcoRI site.
  • pLOR49 thus is a pUC19 back bone with the Hindm site followed by optimal Kozac and 5 'hJaggedl sequence and introduced unique Rsrfl, Dra m, Kpnl, BsiWI sites before recreating the Ecorl site.
  • Plasmid pLOR49 was then cut RsrH - BsiWI to give a 2.7kb ⁇ vector backbone fragment that was run out on an agarose gel, the band excised and the DNA gel purified using a Qiagen QIAquickTM Gel Extraction Kit (cat 28706) according to the manufacturer's instructions.
  • Fragment 3 generation of 3' hJaggedl EC with BsiWI site PCR fragment pLOR47 was used as a template for PCR to amplify up hJaggedl EC and add a 3' BsiWI site.
  • Fragment 1 Plasmid pDEV21-4 was cut Hindm-BglH to give 4958bp + 899bp fragments. These were ran out on an agarose gel, the smaller 889bp fragment band was excised and the DNA gel purified using a Qiagen QIAquickTM Gel Extraction Kit (cat 28706) according to the manufacturer's instructions.
  • Fragment 2 pCON ⁇ 4 (Lonza Biologies) was cut Hind m-Apal to give a 6602bp vector fragment - missing the first 5 amino acids of IgG4 FC. The fragment band was excised and the DNA gel purified using a Qiagen QIAquickTM Gel Extraction Kit (cat 28706) according to the manufacturer's instructions.
  • Fragment 3 A linker ohgonucleotide pak was ordered to give a tight junction between the end of hJaggedl EGF2 and the 3' start of IgG4 FC, with no extra amino acids introduced.
  • Ligated DNA was transformed into competent DH5alpha (Invitrogen), plated onto LB amp paltes and incubated at 37 degres overnight. A good ratio was evident between control and vector plus insert pates therefore only 8 colonies were picked into 10ml LB amp broth and incubated at 37 overnight. Glycerol broths were made and the bacterial pellets were frozen at -20degrees. Later plasmid DNA was extracted using Qiagen - 145
  • the coding sequence for hJaggedl EGFl+2 IgG4 FC fusion was shuttled out of pCON ⁇ 4 (Lonza Biologies) into pEE 14.4 (Lonza Biologies) downstream of the hCMV promoter region (hCMV-MIE) and upstream of SN40 polyadenylation signal, to enable stable ceU lines to be selected using the GS system (Lonza Biologies).
  • Plasmid ⁇ EE14.4 contains the GS minigene - (GS cD ⁇ A which includes the last intron and polylinker adenylation signals of the wild type hamster GS gene under the control of the late SV40 promoter) which encodes the GS gene requked for selection in glutamine free media.
  • pDEVIO clone 2 was cut Hindm-EcoRI giving rise to 2 fragment s 5026bp + 2497bp.
  • the 2497bp contained the coding sequence for hJaggedl EGFl+2 IgG4 FC fusion and so was excised from an agarose gel and the D ⁇ A gel purified using a Qiagen QIAquickTM Gel Extraction Kit (cat 28706) according to the manufacturer's instructions.
  • pEE14.4 (Lonza Biologies) was cut Hindm-EcoRI to remove the IgG4 FC sequence giving 2 fragments 5026bp + 1593bp. The larger 5026b ⁇ fragment was excised from an - 146
  • the pEE14.4 vector backbone and the hJaggedl EGFl+2 IgG4 FC fusion insert were Hgated to give the final transfection plasmid pDEVll.
  • the Hgation was transformed into DH5 ⁇ ceUs, streaked onto LB + AmpicUlin (lOOug/ml) plates and incubated at 37°C overnight. Colonies were picked from the plates into 7ml LB + AmpicUlin (lOOug/ml) and grown up shaking overnight at 37°C. Glycerol broths were made and the plasmid DNA was purified from the cultares using a Qiagen Qiaquick Spin Miniprep kit (cat 27106) according to the manufacturer's instractions. The DNA was then diagnosticaUy digested with Sap I.
  • a conect clone (clone 1) was chosen and lOOul of the glycerol stock was inoculated into
  • the plates showed pure growth; therefore the culture was maxi-prepped using a Clontech
  • IgG4 sandwich ELISA IgG4 sandwich ELISA. Selective media were replaced. Positive clones were identified, passaged and expanded in selective media 25um L-MSX.
  • CeUs were grown in selective DMEM (25um L-MSX) untU semi-confluent. The media was then replaced with seram free media (UltraCHO; BioWhittaker) for 3-5 days. Protein (hJaggedlEGFl+2-IgG4Fc fusion protein) was purified from the resulting media by FPLC. - 148 -
  • the protein is believed to exist as a dimer linked by cysteine disulphide bonds, with cleavage of the signal peptide.
  • PBMC Human peripheral blood mononuclear ceUs
  • CD4+ T cells were incubated in tripHeates in a 96-weU-plate (flat bottom) at 10 5 CD4/weU/200 ⁇ l in RPMI medium containing 10% FCS, glutamine, peniciUin, streptomycin and ⁇ 2 -mercaptoethanol. - 149 -
  • Cytokine production was induced by stimulating the ceUs with anti-CD3/CD28 T cell expander beads from Dynal at a T.l ratio (bead/ceU) in the presence of beads coated with hDeltal-IgG4Fc fusion protein (Example 1 above) at a 5:1 ratio (beads/cell).
  • increasing amounts of soluble Jagged-1 (2EGF)-hIgGl fusion protein hjaggedlEGFl&2 -IgG4Fc; prepared as described above
  • the supematants were removed after 3 days of incubation at 37°C/ 5%CO 2 /humidified atmosphere and cytokine production was evaluated by ELISA using Pharrningen kits OptEIA Set human IL10 (Catalog No. 555157), OptEIA Set human IL-5 (Catalog No. 555202) for JL-10 and JL-5 respectively according to the manufacturer's instructions.
  • Spleens were removed from female Balb/c mice 8-10 weeks old and passed through a 0.2 ⁇ M cell strainer into 20ml R10F medium (R10F-RPMI 1640 media (Gibco Cat No 22409) plus 2mM L-glutamine, 50 ⁇ g/ml Penicillin, 50 ⁇ g/ml Streptomycin, 5 x 10 "5 M ⁇ -mercapto-ethanol in 10% fetal calf serum). The cell suspension was spun (1150rpm 5min) and the media removed. - 150 -
  • the cells were incubated for 4 minutes with 5ml ACK lysis buffer (0.15M NELCl, 1.0M KHC0 3 , O.lmM Na EDTA in double distilled water) per spleen (to lyse red blood cells).
  • the ceUs were then washed once with R10F medium and counted.
  • CD4+ ceUs were purified from the suspensions by positive selection on a Magnetic Associated Cell Sorter (MACS) column (Miltenyi Biotec, Bisley, UK: Cat No 130-042-401) using CD4 (L3T4) beads (MUtenyi Biotec Cat No 130-049-201), according to the manufacturer's dkections.
  • MCS Magnetic Associated Cell Sorter
  • the plates were coated with DPBS plus l ⁇ g/ml anti-hamsterlgG antibody (Pharmin en Cat No 554007) plus l ⁇ g/ml anti-IgG4 antibody. lOO ⁇ l of coating mixture was added per well. Plates were incubated overnight at 4°C then washed with DPBS. Each weU then received either lOO ⁇ l DPBS plus anti-CD3 antibody (l ⁇ g/ml) or, lOO ⁇ l DPBS plus anti- CD3 antibody (l ⁇ g/ml) plus hDeltal-IgG4Fc fusion protein (lO ⁇ g/ml). The plates were incubated for 2-3 hours at 37°C then washed again with DPBS before ceUs (prepared as described above) were added.
  • Mouse CD4+T-ceUs (prepared as above) were cultured at 2 x 10 5 /weU on anti-CD3 coated plates with or without plate-bound hDeltal-IgG4Fc fusion protein (prepared as described above) and soluble anti-CD28 (Pharmingen, Cat No 553294, Clone No 37.51) at a final concentration of 2 ⁇ g/ml. Soluble hDeltal-IgG4Fc fusion protein was added into cultare at the start at the concentrations shown and IL-10 was measured in supematants on day 3 by ELBA using antibody pairs from R & D Systems (Abingdon, UK). The results (shown in Figure 19) show that the increased IL-10 release induced by - 151
  • plate-bound hDeltal-IgG4Fc fusion protein is substantiaUy reversed by all concentrations of soluble hDeltal-IgG4Fc fusion protein tested.
  • TP1 promoter sequence (TP1; equivalent to 2 CBFl repeats) with BamHl and BglH cohesive ends was generated as follows:

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Abstract

An inhibitor of the Notch signalling pathway is used in the manufacture of a medicament for use as an immunostimulant, for example as a vaccine adjuvant.

Description

Medical Treatment
Field of the invention
The present invention relates to the modulation of immune, function, in particular by use of a modulator of the Notch signalling pathway.
Background of the invention
International Patent Publication No WO 98/20142 describes how manipulation of the Notch signalling pathway can be used in irrrmunotherapy and in the prevention and/or treatment of T-cell mediated diseases. In particular, the document discusses how allergy, autoimmunity, graft rejection, tumour induced aberrations to the T-cell system and infectious diseases caused, for example, by Plasmodium species, Microfilariae, Helminths, Mycobacteria, HIN, Cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, Haemophilus influenza type B , measles, Hepatitis C or Toxicara, may be targeted.
It has also been shown that it is possible to generate a class of regulatory T cells which are able to transmit antigen-specific tolerance to other T cells, a process termed infectious tolerance (WO9S/20142). The functional activity of these cells can be mimicked by over- expression of a Notch ligand protein on their cell surfaces or on the surface of antigen presenting cells. In particular, regulatory T cells can be generated by over-expression of a member of the Delta or Serrate family of Notch ligand proteins. Delta or Senate induced T cells specific to one antigenic epitope are also able to transfer tolerance to T cells recognising other epitopes on the same or related antigens, a phenomenon termed "epitope spreading".
Notch hgand expression also plays a role in cancer. Indeed, upregulated Notch ligand expression has been observed in some tumour cells. These tumour cells are capable of rendering T cells unresponsive to restimulation with a specific antigen, thus providing a possible explanation of how tumour cells prevent normal T cell responses. By downregulating Notch signalling in vivo in T cells, it may be possible to prevent tumour cells from inducing irrununotolerance in those T cells that recognise tumour-specific antigens. In rum, this would allow the T cells to mount an immune response against the tumour cells (WO00/135990).
A description of the Notch signalling pathway and conditions affected by it may be found in our published PCT Applications PCT/GB97/03058 (filed on 6 November 1997 and claiming priority from GB 9623236.8 filed on 7 November 1996, GB 9715674.9 filed on 24 July 1997 and GB 9719350.2 filed on 11 September 1997; published as WO 98/20142) PCT/GB99/04233 (filed on 15 December 1999 and claiming priority from GB 9827604.1 filed on 15 December 1999; published as WO 00/36089) and PCT/GBOO/04391 (filed on 17 November 2000 and claiming priority from GB 9927328.6 filed on 18 November 1999; published as WO 0135990). Each of PCT/GB97/03058 (WO 98/20142), PCT/GB99/04233 (WO 00/36089) and PCT/GBOO/04391 (WO 0135990) are hereby incorporated herein by reference.
The present invention seeks to provide further methods of modulating the immune system by modification of the Notch signalling pathway, in particular for the treatment of infectious disease.
Statements of the Invention
According to a first aspect of the invention there is provided a product comprising: i) an inhibitor of the Notch signalling pathway or a polynucleotide coding for such an inhibitor; and ii) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system. Preferably the agent does not act by downregulating expression of Notch or a Notch ligand.
According to a further aspect of the invention there is provided a product comprising: i) an inhibitor of Notch signalling in the form of a Notch antagonist agent or a polynucleotide coding for such an agent; and ii) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
According to a further aspect of the invention there is provided a product comprising: i) an inhibitor of Notch signalling in the form of an agent which inhibits Notch-Notch hgand interaction or a polynucleotide coding for such an agent; and ii) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic deteπninant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
Suitably such a product may take the form of a pharmaceutical composition or kit.
Suitably such a product may take the form of a therapeutic vaccine composition or kit for treating infectious disease (including so-called "pharmaccrnes").
Alternatively such a product may take the form of a prophylactic vaccine composition or kit for preventing infectious disease. According to a further aspect of the invention there is provided the use of an inhibitor of the Notch signalling pathway in the manufacture of a medicament for use as an immunostimulant. Preferably the medicament is not for use in reversing bacteria, infection or tumour-induced immunosuppression or for the treatment of a tumour.
The term "immunostimulant'' as used herein means an agent which is capable of restoring a depressed immune function, or enhancing normal immune function, or both. The term agent may boost a subject's immune system either generally or in respect of a specific antigen or antigenic determinant, hnmunostimulants may be used, for example, for the treatment of conditions requiring general immune stimulation including immune deficiency conditions such as Acquired Immune Deficiency Syndrome (AIDS) and Severe Combined Immunodeficiency Disease (SCJD) and in situations where antigen specific stimulation is desired, such as in vaccination.
According to a further aspect of the invention there is provided the use of an inhibitor of the Notch signalling pathway in the manufacture of a medicament for use in vaccination against a pathogen.
According to a further aspect of the invention there is provided the use of an inhibitor of the Notch signalling pathway in the manufacture of a medicament for use as an adjuvant for vaccination against a pathogen.
The term "pathogen" as used herein means a disease causing parasite which is normally a microorganism.The term includes, for example, viruses, bacteria, protozoa and fungi.
The term "pathogen antigen" as used herein means an antigen found on a pathogen or a fragment, variant or derivative of such an antigen comprising antigenic determinants (epitopes; preferably immunodom nant epitopes) or epitope regions (preferably immunodominant epitope regions) of such an antigen. Preferably the antigen is immunogenic (an immunogen). Suitably the antigen is a microbial pathogen antigen. According to a further aspect of the invention there is provided a method for stimulating the immune system by administering an inhibitor of the Notch signalling pathway which preferably does not comprise reversing bacteria, infection or tumour-induced immunosuppression or treatment of a tumour.
The terms "inhibitor of Notch signalling" and "inhibitor of the Notch signalling pathway" as used herein include any agent which is capable of reducing any one or more of the upstream or downstream events that result in, or from, (and including) activation of the Notch receptor. Preferably the inhibitor of Notch signalling does not act by do nregulating expression of Notch or a Notch hgand.
According to a further aspect of the invention there is provided a method for stimulating the immune system by administering an inhibitor of the Notch signalling pathway wherein the inhibitor does not act by downregulating expression of Notch or a Notch hgand.
According to a further aspect of the invention there is provided a method for vaccination against a pathogen by administering an inhibitor of the Notch signalling pathway.
According to a further aspect of the invention there is provided a method for enhancing vaccination against a pathogen by administering an inhibitor of the Notch signalling pathway.
According to a further aspect of the invention there is provided a method for stimulating the immune system to treat or prevent an infection by administering an inhibitor of the Notch signalling pathway which does not comprise reversing bacteria, infection or tumour- induced immunosuppression or treatment of a tumour. According to a further aspect of the invention there is provided a method for stimulating the immune system to treat or prevent an infection by administering an inhibitor of the Notch signalling pathway wherein the inhibitor of the Notch signalling pathway does not act by downregulating expression of Notch or a Notch hgand.
According to a further aspect of the invention there is provided a method for treating an acute pathogen infection by administering an inhibitor of the Notch signalling pathway.
According to a further aspect of the invention there is provided a method for treating a chronic pathogen infection by administering an inhibitor of the Notch signalling pathway.
According to a further aspect of the invention there is provided a method of increasing the immune response of a subject to a vaccine antigen or antigenic determinant comprising administering an effective amount of an inhibitor of the Notch signalling pathway to said subject simultaneously, separately or sequentially with said vaccine antigen or antigenic determinant or simultaneously, separately or sequentially with a polynucleotide coding for said vaccine antigen or antigenic determinant.
Preferably the inhibitor of Notch signalling inhibits Notch signalling in immune cells, such as APCs, B-cells or T-cells
Suitably the inhibitor of the Notch signalling pathway may be a Notch signalling repressor or an agent which increases the expression or activity of a Notch signalling repressor.
Preferably the inhibitor of the Notch signalling pathway is an agent capable of inhibiting the activity of a Notch receptor or a Notch hgand. - 1 -
Alternatively or in addition the inhibitor of the Notch signalling pathway may be an agent capable of inhibiting the activity or downregulating the expression of a downstream component of the Notch signalling pathway.
Preferably the inhibitor of the Notch signalling pathway may be an agent which interacts with, and preferably binds to a Notch receptor or a Notch ligand so as to interfere with endogenous Notch ligand-receptor interaction (also termed "Notch-Notch hgand interaction"). Such an agent may be referred to as a "Notch antagonist". Preferably the inhibitor inhibits Notch ligand-receptor interaction in immune cells such as lymphocytes and APCs, preferably in lymphocytes, preferably in T-cells.
Suitably the inhibitor of Notch signalling may be a protein or polypeptide or a polynucleotide which codes for such a protein or polypeptide.
In one embodiment, for example, the inhibitor of Notch signalling may comprise or codes for the extracellular domain of Delta or a fragment, derivative or homologue thereof.
Suitably, for example, the inhibitor of Notch signalling comprises or codes for the extracellular domain of Serrate or Jagged or a fragment, derivative or homologue thereof.
Suitably, for example, the inhibitor of Notch signalling comprises or codes for the extracellular domain of Notch or a fragment, derivative or homologue thereof.
Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and optionally a
Notch ligand N-terminal domain or a heterologous amino acid sequence but which is substantially free of Notch ligand EGF-like domains; ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or iii) a polynucleotide coding for such a protein or polypeptide. Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and at least one
Notch hgand EGF-like domain; ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or iii) a polynucleotide coding for such a protein or polypeptide.
Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and at least two
Notch ligand EGF-like domains; ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or iii) a polynucleotide coding for such a protein or polypeptide.
Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch hgand DSL domain and either 0, 1 or 2, but no more than 2 Notch ligand EGF-like domains; h) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or iii) a polynucleotide coding for such a protein or polypeptide.
Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch Hgand DSL domain having at least
30%, preferably at least 50% amino acid sequence similarity or identity to the DSL domain of human Deltal , Delta3 or Delta4 and at least one Notch ligand EGF-like domain having at least 30%, preferably at least 50% amino acid sequence similarity or identity to an EGF-like domain of human Deltal, Delta3 or Delta4; ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or iii) a polynucleotide coding for such a protein or polypeptide.
Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch hgand DSL domain having at least
30%, preferably at least 50% amino acid sequence similarity or identity to the DSL domain of human Deltal , Delta3 or Delta4 and either 0, 1 or 2, but no more than 2 Notch hgand EGF-like domains having at least 30%, preferably at least 50% amino acid sequence similarity or identity to an EGF-like domain of human Deltal, Delta3 or Delta4; ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or iii) a polynucleotide coding for such a protein or polypeptide.
Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch EGF-like domain having at least
30%, preferably at least 50% amino acid sequence similarity or identity to EGF11 of human Notchl, Notch2, Notch3 or Notch4 and a Notch EGF-like domain having at least
30%, preferably at least 50% amino acid sequence similarity or identity to EGF12 of human Notchl , Notch2, Notch3 or Notch4; ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or hi) a polynucleotide coding for such a protein or polypeptide.
Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch hgand DSL domain having at least
30%, preferably at least 50% amino acid sequence similarity or identity to the DSL domain of human Jaggedl or Jagged2 and at least one Notch Hgand EGF-like domain having at least 30%, preferably at least 50% amino acid sequence similarity or identity to an EGF-like domain of human Jagged 1 or Jagged2; n) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or Hi) a polynucleotide coding for such a protein or polypeptide.
Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
30%, preferably at least 50% amino acid sequence similarity or identity to the DSL domain of human Jaggedl or Jagged2 and either 0, 1 or 2, but no more than 2 Notch
Hgand EGF-like domains having at least 30%, preferably at least 50% amino acid sequence similarity or identity to an EGF-like domain of human Jagged 1 or Jagged2;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or
Hi) a polynucleotide coding for such a protein or polypeptide.
Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
70% amino acid sequence similarity or identity to the DSL domain of human Deltal,
Delta3 or Delta4 and at least one Notch Hgand EGF-like domain having at least 70% amino acid sequence similarity or identity to an EGF-like domain of human Deltal,
Delta3 orDelta4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or
Hi) a polynucleotide coding for such a protein or polypeptide.
Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
70% amino acid sequence similarity or identity to the DSL domain of human Deltal,
Delta3 or Delta4 and either 0, 1 or 2, but no more than 2 Notch ligand EGF-like domains having at least 70% amino acid sequence similarity or identity to an EGF-like domain of human Deltal, Delta3 or Delta4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or iii) a polynucleotide coding for such a protein or polypeptide.
An advantage of using a protein or polypeptide having preferably no more than two Notch ligand EGF-like domains is that it provides effective inhibition of Notch signaUing with little or no competing agonist activity, thus providing a more selective inhibitory effect. Such proteins and polypeptides may also be easier to produce especially, for example, in bacterial expression systems.
However, it wdl be appreciated that Notch signalling inhibition is also shown by constructs having more than 2 such EGF-like repeats.
Suitably, for example, the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises an EGF domain having at least 70% amino acid sequence similarity or identity to EGF11 of human Notchl, Notch2, Notch3 or
Notch4 and an EGF domain having at least 70% amino acid sequence similarity or identity to EGF12 of human Notchl, Notch2, Notch3 or Notch4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or
Hi) a polynucleotide coding for such a protein or polypeptide.
Suitably the protein or polypeptide may be fused to a heterologous amino acid sequence, such as an HnmunoglobuHn Fc (IgFc) domain, for example a human IgGl or IgG4 Fc domain.
Suitably the protein or polypeptide may further comprise a Notch ligand N-terminal domain.
Alternatively, for example, the inhibitor of Notch signaUing may comprise an antibody, antibody fragment or antibody derivative or a polynucleotide which codes for an antibody, antibody fragment or antibody derivative. Suitably the antibody, antibody fragment or antibody derivative binds to a Notch receptor or a Notch Hgand so as to interfere with Notch ligand-receptor interaction.
Suitably for example, the inhibitor of Notch signalling may have an ICso (preferably as measured in an assay as described herein, preferably using the Dynabeads assay of Example 12) of less than about 1000 uM, preferably less than about 100 uM, preferably less than about 10 uM, preferably less than about 1000 nM, preferably less than about 100 nM, suitably from about 0.1 to about 100 nM.
In one embodiment the modulator of the Notch signaUing pathway may comprise a fusion protein comprising domains from a Notch Hgand extracellular domain and an immunoglobulin Fc segment (eg IgGl Fc or IgG4 Fc, preferably human IgGl Fc or human IgG4 Fc) or a polynucleotide coding for such a fusion protein. Methods suitable for preparation of such fusion proteins are described, for example in Example 2 of WO 98/20142. IgG fusion proteins may be prepared as well known in the art, for example, as described in US 5428130 (Genentech).
Suitably, the modulator of the Notch signaUing pathway may be multimerised, preferably dimerised, for example by chemical cross-linking or formation of disulphide bonds between pahs of proteins or polypeptides. For example, where the proteins or polypeptides comprise a heterologous amino acid sequence in the form of an immunoglobulin Fc domain, these may assemble into dimers linked by disulphide bonds formed between the Fc domains (see, for example, the schematic representations of dimeric constructs as shown in the accompanying Figures).
Where the proteins or polypeptides are multimerised or dimerised in this way, the multimerised/dhnerised form may contain more DSL and EGF domains than described in respect of the individual monomers. However, the ratios of DSL to EGF domains will preferably remain the same, such that there wiU preferably, for example be a ratio of DSL to EGF-like domains of 1 :0, 1 :1 or 1 :2 for the multimerised aggregate as a whole. Suitably, for example, the inhibitor of Notch signalling comprises a Notch hgand protein or polypeptide which consists essentiaUy of the following components: i) a Notch Hgand DSL domain;
H) optionally lor 2 EGF repeat domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide or a polynucleotide coding for such a
Notch ligand protein or polypeptide.
Suitably, for example, the inhibitor of Notch signaUing comprises a Notch ligand protein or polypeptide which consists essentiaUy of the foUowing components: i) a Notch ligand DSL domain;
H) optionally aU or part of a Notch Hgand N-terminal domain; and iii) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide or a polynucleotide coding for such a
Notch ligand protein or polypeptide.
Suitably, for example, the inhibitor of Notch signaUing comprises a Notch ligand protein or polypeptide which consists essentially of the foUowing components: i) a Notch ligand DSL domain; ii) one Notch Hgand EGF domain;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide or a polynucleotide coding for such a
Notch ligand protein or polypeptide.
Suitably, for example, the inhibitor of Notch signaUing comprises a Notch Hgand protein or polypeptide which consists essentiaUy of the following components: i) a Notch ligand DSL domain; H) two Notch ligand EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide or a polynucleotide coding for such a
Notch ligand protein or polypeptide.
According to a further aspect of the invention there is provided the use of a binding agent which binds to a Notch ligand so as to interfere with binding of the Hgand to a Notch receptor, or a polynucleotide which codes for such a binding agent, in the manufacture of a medicament for use as an immunostimulant.
According to a further aspect of the invention there is provided the use of an antibody or antibody derivative which binds to a Notch receptor or to a Notch ligand, or a polynucleotide which codes for such an antibody or antibody derivative, in the manufacture of a medicament for use as an immunostimulant.
According to a further aspect of the invention there is provided a method of increasing the immune response of a subject to a vaccine antigen or antigenic determinant comprising administering an effective amount of an inhibitor of the Notch signaUing pathway to said subject simultaneously, separately or sequentiaUy with said vaccine antigen.
According to a further aspect of the invention there is provided a method for stimulating the immune system by administering a binding agent which binds to a Notch receptor or Notch ligand so as to interfere with Hgand-receptor interaction, or by administering a polynucleotide which codes for such a binding agent. The binding agent may, for example, comprise one or more extraceUular domains from Notch or its ligands.
According to a further aspect of the invention there is provided a method for stimulating the immune system by administering an antibody or antibody derivative which binds to a Notch receptor or to a Notch ligand, or by administering a polynucleotide which codes for such an antibody or antibody derivative.
According to a further aspect of the invention there is provided an adjuvant composition comprising an inhibitor of the Notch signalling pathway.
According to a further aspect of the invention there is provided a vaccine composition comprising an adjuvant composition as described above and an antigen. Suitably the antigen maybe a viral, fungal, parasitic or bacterial antigen.
According to a further aspect of the invention there is provided a method for modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentiaUy adnxinistering: i) an effective amount of an inhibitor of the Notch signalling pathway; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
According to a further aspect of the invention there is provided a combination of: i) an inhibitor of the Notch signalling pathway; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
According to a further aspect of the invention there is provided an inhibitor of the Notch signaUing pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant. According to a further aspect of the invention there is provided the use of a combination of: i) an inhibitor of the Notch signalling pathway; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; in the manufacture of a medicament for modulation of the immune system.
According to a further aspect of the invention there is provided the use of .an inhibitor of the Notch signalling pathway in the manufacture of a medicament for modulation of the immune system in simultaneous, contemporaneous, separate or sequential combination with a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
According to a further aspect of the invention there is provided a pharmaceutical kit comprising an inhibitor of the Notch signaUing pathway and a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
According to a further aspect of the invention there is provided a conjugate comprising first and second sequences, wherein the first sequence comprises a pathogen antigen or antigenic determinant or a polynucleotide sequence coding for a pathogen antigen or antigenic deteixninant, and the second sequence comprises a polypeptide or polynucleotide for Notch signalling modulation.
According to a further aspect of the invention there is provided a conjugate comprising first and second sequences, wherein the first sequence comprises a pathogen antigen or antigenic determinant or a polynucleotide sequence coding for a pathogen antigen or antigenic determinant, and the second sequence codes for an inhibitor of Notch signaUing. Preferably the conjugate is in the form of a vector comprising a first polynucleotide sequence coding for a modulator of the Notch signaUing pathway and a second polynucleotide sequence coding for a pathogen antigen or antigenic determinant.
Preferably the conjugate is in the form of an expression vector.
Preferably in such a conjugate the first polynucleotide sequence codes for a Notch ligand or a fragment, derivative, homologue, analogue or allelic variant thereof.
Suitably the first polynucleotide sequence of the conjugate codes for a Delta or Senate/Jagged protein or a fragment, derivative, homologue, analogue or allelic variant thereof.
Suitably the first polynucleotide sequence of the conjugate codes for a protein or polypeptide which comprises a Notch ligand DSL domain and optionally at least one Notch ligand EGF-like domain.
Suitably the first polynucleotide sequence of the conjugate codes for a protein or polypeptide which comprises a Notch ligand DSL domain and at least two Notch Hgand EGF-like domains.
Suitably the first polynucleotide sequence of the conjugate codes for a protein or polypeptide which comprises a Notch ligand DSL domain and lor 2 but no more than 2 Notch ligand EGF-like domains.
Suitably the first and second sequences of the conjugate are each operably linked to one or more promoters. According to a further aspect of the invention there is provided a method for increasing a TH2 immune response by admi stering a modulator of Notch signalling.
According to a further aspect of the invention there is provided a method for increasing a TH1 immune response by administering a modulator of Notch signalling.
According to a further aspect of the invention there is provided a method for increasing JFN-γ expression by administering an inhibitor of Notch signaUing.
According to a further aspect of the invention there is provided a method for increasing IL-2 expression by administering an inhibitor of Notch signalling.
According to a further aspect of the invention there is provided a method for increasing TNFα expression by administering an inhibitor of Notch signalling.
According to a further aspect of the invention there is provided a method for increasing IL-4 expression by administering an inhibitor of Notch signalling.
According to a further aspect of the invention there is provided a method for increasing 1L-5 expression by administering an inhibitor of Notch signalling.
According to a further aspect of the invention there is provided a method for increasing J -13 expression by administering an inhibitor of Notch signaUing.
According to a further aspect of the invention there is provided a method for reducing JL- 10 expression by administering an inhibitor of Notch signalling.
According to a further aspect of the invention there is provided a method for increasing JL-5 expression by admirtistering an inhibitor of Notch signalling. According to a further aspect of the invention there is provided a method for generating an immune stimulatory cytokine profile with reduced JL-10 expression and increased JL- 5 expression by administering an inhibitor of Notch signalling.
According to a further aspect of the invention there is provided a method for generating an immune stimulatory cytokine profile with increased JL-2, IFNγ, IL-5, IL-13 and TNF expression by adπrinistering an inhibitor of Notch signalling. Suitably the cytokine profile also exhibits reduced IL-10 expression.
In one embodiment of the invention an inhibitor of Notch signalling is administered to a patient in vivo. Alternatively the inhibitor of Notch signaUing may be administered to a cell ex-vivo, after which the ceU may be administered to a patient.
Suitably the modulator of Notch signalling modifies cytokine expression in leukocytes, fibroblasts or epithehal cells. Preferably the modulator of Notch signalling modifies cytokine expression in dendritic ceUs, lymphocytes or macrophages, or their progenitors or tissue-specific derivatives.
Preferably the inhibitor of Notch signaUing or the Notch signalling pathway for use in the present invention is an inhibitor of Notch-Notch ligand interaction. Suitably such an inhibitor of Notch-Notch ligand interaction is an agent which binds to a Notch receptor or Notch Hgand so as to interfere with endogenous Notch-Notch ligand interaction whUst causing less activation of the Notch receptor than would result from endogenous Notch- Notch Hgand interaction, or preferably no significant activation. For example, the inhibitor may bind to EGF-like domain 11 and/or EGF-like domain 12 of a Notch receptor or the DSL domain and/or EGF-like domain 1 and/or EGF-like domain 2 of a Notch Hgand such as Delta, Senate or Jagged. Thus, for example, the inhibitor may comprise EGF-like domains 11 and 12 of a Notch receptor. Alternatively the inhibitor may comprise a Notch ligand DSL domain and at least one EGF-like domain of a Notch Hgand such as Delta, Senate or Jagged. Suitably, for example, the inhibitor may comprise an extraceUular domain of a Notch receptor, for example an extraceUular domain of Notchl, Notch2, Notch3 or Notch4. Alternatively the inhibitor may comprise an extraceUular domain of a Notch ligand such as Delta (eg a mammalian Deltal, Delta3 or Delta4), Senate or Jagged (eg a mammalian Jaggedlor Jagged2).
Where the inhibitor binds to a Notch receptor, it may bind selectively to one Notch receptor such as Notchl, or may suitably have some degree of affinity for a range of Notch receptors or substantiaUy aU of them, due to their simUar structures. Likewise, where the inhibitor binds to a Notch ligand, it may bind selectively to one Notch Hgand such as Deltal, or may suitably have some degree of affinity for a range of Notch ligands or substantiaUy all of them, due to their similar structures.
Alternatively the inhibitor may comprise an antibody which binds specificaUy to a Notch receptor or receptors. Preferably the antibody binds to the Notch receptor in such a way as to reduce or substantiaUy prevent binding of native Notch ligands whilst the antibody is bound, or at least to reduce or substantiaUy prevent activation of the Notch receptor. Suitably, for example, such an antibody may bind to EGF 11 and/or 12 of the Notch receptor (eg Notchl, Notch2, Notch3 and/or Notch4). The antibody may be selective for one Notch receptor such as Notchl, or may suitably have some degree of affinity for a range of Notch receptors or substantially aU of them, due to their similar structures.
Alternatively the inhibitor may comprise an antibody which binds specificaUy to a Notch Hgand or ligands. Preferably the antibody binds to the Notch ligand Hi such a way as to reduce or substantially prevent binding of the ligand to native Notch receptors whilst the antibody is bound, or at least to reduce or substantially prevent activation of the Notch receptor. Suitably, for example, such an antibody may bind to the DSL domain and/or to EGF-like domains 1 and/or 2 of a Notch Hgand (eg a mammaHan Deltal, Delta3, Delta4, Jaggedlor Jagged2). The antibody may be selective for one Notch Hgand such as Deltal, or may suitably have some degree of affinity for a range of Notch ligands or substantially all of them, due to their similar structures. It will be appreciated that combinations of antibodies with complementary specificities may also be used.
In an alternative embodiment, for example, the inhibitor of Notch signalling may be an inhibitor of Notch IC protease.
The term "Notch IC protease" as used herein means an enzyme or enzyme complex which acts proteolyticaUy to cleave a Notch receptor to cause the release of all or part of the intracellular (IC) domain from the Notch receptor so as to activate the Notch signaUing pathway. Enzymes which are understood to participate in such cleavage include the presertilins and gamma-secretase enzymes, and presenilin-dependent garnma- secretase enzymes or complexes.
The term "presenilin-dependent gamma-secretase" as used herein means an enzyme having gamma secretase proteolytic activity which requires presenUin for activity or activation. The preseniHn may for example be required as a co-activator or as part of an enzyme complex.
Examples of presenUin proteins which may be modulated in the present invention include Presenilin-1 (PSI) and Presenilin-2 (PS2).
The modulator of Notch IC protease activity will preferably be selected from polypeptides and fragments thereof, linear peptides, cycHc peptides, and nucleic acids which encode therefor, synthetic and natural compounds including low molecular weight organic or inorganic compounds and antibodies. The modulator may for example be an agonist or an antagonist of presenUin or presenilin-dependent gamma-secretase, optionaUy in combination with an agent capable of respectively up-regulating or downregulating the Notch signaUing pathway respectively. An example of an antagonist of presenihn which may be used in the present invention is 26S proteasome or a nucleic acid sequence which encodes therefor. Synthetic inhibitors include, for example, the difluoro ketone inhibitor described in Citron et al., and Wolfe et al. having the formula:
the inhibitors described in Sinha and Liederburg (2-Νaphthoyl-VF-CHO, N-(2- Naphthoyl)-Nal-phenylalaninal and Ν-Benzyloxycarbonyl-Leu-phenylalaninal Z-LF- CHO); the inhibitors described in Esler et al.; the inhibitors described in Figueiredo- Pereha et al., (Ν-Benzyloxycarbonyl-Leu-leucinal Z-LL-CHO); the inhibitors described in Higaki et al., (Ν-t αra-3,5-Dimethoxycinnamoyl)-Ue-leucinal t-3,5-DMC-TL-CHO); the inhibitors described in Murphy et al., (Boc-GNV-CHO Ν-tert-Butyloxycarbonyl-Gly- Nal-Nalinai); and the inhibitors described in Riston et al., (l-(S)-endø-Ν-(l,3,3)- Trimethylbicyclo[2.2. l]hept-2-yl)-4-fluorophenyl Sulfonamide).
In an alternative embodiment, the inhibitor of Notch signalling is not an inhibitor of a Notch IC protease (ie is preferably not an inhibitor of presemlins and gamma-secretase enzymes, and is preferably not an inhibitor of presenilin-dependent gamma-secretase enzymes or complexes).
According to a further aspect of the invention there is provided a method for modifying an immune response by adπώmstering a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally lor 2 EGF repeat domains; Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide.
According to a further aspect of the invention there is provided a method for increasing an immune response by administering a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally lor 2 EGF repeat domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide.
According to a further aspect of the invention there is provided a method for reducing immune tolerance by adπunisteiing a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally lor 2 EGF repeat domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide. According to a further aspect of the invention there is provided a method for modifying T ceU activity by administering a Notch Hgand protein or polypeptide consisting essentially of the following components: i) a Notch ligand DSL domain;
H) optionally lor 2 EGF repeat domains;
Hi) optionaUy all or part of a Notch Hgand N-teπninal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide.
According to a further aspect of the invention there is provided a method for increasing helper (TH) or cytotoxic (Tc) T-ceU activity by administering a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally lor 2 EGF repeat domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide.
According to a further aspect of the invention there is provided a method for reducing activity of regulatory T ceUs by adπώristering a Notch Hgand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally lor 2 EGF repeat domains; Hi) optionally all or part of a Notch Hgand N-teπninal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or by administering a polynucleotide coding for such a Notch ligand protein or polypeptide.
Suitably the regulatory T cells are Trl or Th3 regulatory T-ceUs.
According to a further aspect of the invention there is provided a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; for use to treat disease.
According to a further aspect of the invention there is provided a Notch ligand protein or polypeptide or polynucleotide for a use as claimed in claim 22 wherein the Notch ligand protein or polypeptide consists essentiaUy of the following components: i) a Notch ligand DSL domain;
H) optionally aU or part of a Notch ligand N-teπninal domain; and
Hi) optionaUy one or more heterologous amino acid sequences; or wherein the polynucleotide codes for such a Notch ligand protein or polypeptide.
According to a further aspect of the invention there is provided the use of a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain; n) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for modification of an immune response.
According to a further aspect of the invention there is provided the use of a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains; and iii) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for modification of an immune response.
According to a further aspect of the invention there is provided the use of a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionaUy 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for increasing an immune response. According to a further aspect of the invention there is provided the use of a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for reducing immune tolerance.
According to a further aspect of the invention there is provided the use of a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multuner of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for modification of T-ceU activity.
According to a further aspect of the invention there is provided the use of a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains; iii) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionally one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for increasing helper (TH) or cytotoxic Ic) T-ceU activity.
According to a further aspect of the invention there is provided the use of a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; in the manufacture of a medicament for reducing activity of regulatory T cefls.
According to a further aspect of the invention there is provided a pharmaceutical composition comprising a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; optionaUy in combination with a pharmaceuticaUy acceptable carrier.
According to a further aspect of the invention there is provided a pharmaceutical composition comprising a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally aU or part of a Notch ligand N-terminal domain; and
Hi) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; optionaUy in combination with a pharmaceutically acceptable carrier.
According to a further aspect of the invention there is provided a pharmaceutical composition comprising a Notch ligand protein or polypeptide consisting essentially of the following components: i) a Notch Hgand DSL domain;
H) one EGF repeat domain;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; optionaUy in combination with a pharmaceuticaUy acceptable carrier.
According to a further aspect of the invention there is provided a pharmaceutical composition comprising a Notch ligand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) two EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide; optionaUy in combination with a pharmaceuticaUy acceptable carrier.
According to a further aspect of the invention there is provided a Notch ligand protein or polypeptide which consists essentiaUy of the following components: i) a Notch ligand DSL domain;
H) optionally aU or part of a Notch ligand N-terminal domain;
Hi) an immunoglobulin Fc domain; and iv) optionaUy one or more further heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide;
According to a further aspect of the invention there is provided a Notch ligand protein or polypeptide which consists essentiaUy of the following components: i) a Notch ligand DSL domain;
H) one EGF domain;
Hi) optionaUy all or part of a Notch Hgand N-teπninal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or a polynucleotide coding for such a Notch Hgand protein or polypeptide;
According to a further aspect of the invention there is provided a Notch ligand protein or polypeptide which consists essentiaUy of the following components: i) a Notch ligand DSL domain;
H) two EGF domains; and
Hi) optionaUy one or more heterologous amino acid sequences; or a polynucleotide sequence which codes for such a Notch Hgand protein or polypeptide. The term "which consists essentiaUy of or "consisting essentiaUy of as used herein means that the construct includes the sequences and domains identified but is substantiaUy free of other sequences or domains, and in particular is substantiaUy free of any other Notch or Notch Hgand sequences or domains.
For avoidance of doubt the term "comprising" means that any additional feature or component may be present.
According to a further aspect of the invention there is provided a vector comprising a polynucleotide coding for a Notch ligand protein or polypeptide as described above. The invention also provides a host ceU transformed or transfected with such a vector. According to a further aspect of the invention there is provided a ceU displaying a Notch Hgand protein or polypeptide as described above on its surface and/or transfected with a polynucleotide coding for such a protein or polypeptide.
Suitably the protein or polypeptide is not bound to a ceU. Alternatively, the protein or polypeptide may be cell-associated.
In one embodiment the protein or polypeptide may be fused to a heterologous amino acid sequence corresponding to aU or part of an immunoglobulin Fc segment. In one embodiment, particularly where the Notch Hgand protein or polypeptide comprises only two EGF repeat domains, the heterologous amino acid sequence is not a TSST sequence, or preferably is not a superantigen sequence.
Preferably the protein or polypeptide comprises at least part of a mammaHan, preferably human, Notch ligand sequence.
Suitably the protein or polypeptide comprises Notch Hgand domains from Delta, Senate or Jagged or domains having at least 30% amino acid sequence simUarity or identity thereto. Suitably the protein or polypeptide comprises Notch ligand domains from Deltal , Delta 3, Delta 4, Jagged 1 or Jagged 2 or domains having at least 30% amino acid sequence similarity thereto.
Preferably the protein or polypeptide inhibits a Notch receptor. Suitably the protein or polypeptide is a Notch signaUing antagonist.
According to a further aspect of the invention there is provided a polynucleotide coding for a protein or polypeptide as described above. According to further aspects of the invention there are provided a vector comprising such a polynucleotide and a host ceU transformed or transfected with such a vector.
According to a further aspect of the invention there is provided a cell displaying a Notch Hgand protein or polypeptide as described above on its surface and/or transfected with a polynucleotide coding for such a protein or polypeptide.
In one embodiment the modulator of the Notch signaUing pathway may comprise a fusion protein comprising domains from a. Notch ligand extraceUular domain and an immunoglobulin Fc segment (eg IgGl Fc or IgG4 Fc) or a polynucleotide coding for such a fusion protein. Methods suitable for preparation of such fusion proteins are described, for example in Example 2 of WO 98/20142. IgG fusion proteins may be prepared as weU known in the art, for example, as described in US 5428130 (Genentech).
According to a further aspect of the invention there is provided a method for increasing TNFα expression by administering a protein, polypeptide or polynucleotide as described above. According to a further aspect of the invention there is provided a method for reducing IL- 10 expression by administering a protein, polypeptide or polynucleotide as described above.
According to a further aspect of the invention there is provided a method for increasing IL-5 expression by adnrinistering a protein, polypeptide or polynucleotide as described above.
According to a further aspect of the invention there is provided a method for increasing 1L-13 expression by administering a protein, polypeptide or polynucleotide as described above.
Suitably the protein, polypeptide or polynucleotide modifies cytokine expression in leukocytes (such as lymphocytes or macrophages), fibrob lasts or epitheUal cells or their progenitors or tissue-specific derivatives.
According to a further aspect of the invention there is provided a method for generating an immune stimulatory cytokine profile with reduced JJ -10 expression and increased TNFα expression by administering a protein, polypeptide or polynucleotide as described above.
According to a further aspect of the invention there is provided a method for generating an immune stimulatory cytokine profile with reduced IL-10 expression and increased IL- 5 expression by adnrinistering a protein, polypeptide or polynucleotide as described above.
According to a further aspect of the invention there is provided a method for generating an immune stimulatory cytokine profile with reduced IL-10 expression and increased IL- 13 expression by adnnfHstering a protein, polypeptide or polynucleotide as described above. According to a further aspect of the invention there is provided a method for generating an immune stimulatory cytokine profile with increased IL-5, JL-13 and TNFα expression by administering a protein, polypeptide or polynucleotide as described above.
According to a further aspect of the invention there is provided a method for generating an immune stimulatory cytokine profile with increased IL-2, IFNγ , IL-5, JL-13 and TNFα expression by administering a protein, polypeptide or polynucleotide as described above. Suitably the cytokine profile also exhibits reduced IL-10 expression.
According to a further aspect of the invention there is provided a method for increasing a TH2 immune response by adnrinistering a protein, polypeptide or polynucleotide as described above.
According to a further aspect of the invention there is provided a method for increasing a THl immune response by administering a protein, polypeptide or polynucleotide as described above.
Detailed description
Various preferred features and embodiments of the present invention will now be described in more detail by way of non-limiting example and with reference to the accompanying drawings, in which:
Figure 1 shows a schematic representation of Notch/Ligand interaction; Figure 2 shows a schematic representation of the Notch signalling pathway; Figure 3 shows a schematic representation of Notch 1-4; Figure 4 shows a schematic representation of Notch Hgands Jagged and Delta;
Figure 5 shows ahgned amino acid sequences of DSL domains from various DrosophUa and mammahan Notch Hgands; Figure 6 shows amino acid sequences of human Delta-1, Delta-3 and Delta-4;
Figure 7 shows amino acid sequences of human Jagged- 1 and Jagged-2;
Figure 8 shows an amino acid sequence of human Notch-1 ;
Figure 9 shows an amino acid sequence of human Notch-2;
Figure 10 shows a schematic representation of Notch Hgand/IgFc fusion proteins suitable for use in the present invention;
Figure 11 shows a schematic representation of a nucleic acid expression constract according to the present invention;
Figure 12 shows the amino acid sequence and domain stmcture of the fusion protein of
Example 1;
Figure 13 shows the results of Example 2;
Figure 14 shows the results of Example 3;
Figure 15 shows the results of Example 4;
Figure 16 shows the results of Example 5;
Figure 17 shows the results of Example 6;
Figure 18 shows the results of Example 8;
Figure 19 shows the results of Example 9;
Figure 20 shows the results of Example 10;
Figure 21 shows the results of Example 11;
Figure 21 shows the results of Example 12;
Figure 22 shows the results of Example 13;
Figure 23 shows the results of Example 14;
Figure 24 shows the results of Example 15;
Figure 25 shows the results of Example 16 ;
Figure 26 shows the results of Example 17 ;
Figure 27 shows the results of Example 18;
Figures 28 and 29 show the results of Example 19;
Figures 30 and 31 show the results of Example 21 ;
Figures 32 and 33 shows the results of Example 22; and
Figure 34 shows the results of Example 23. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skUl in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, h Press; D. M. J. LiUey and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; and J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober (1992 and periodic supplements; Current Protocols in Immunology, John WUey & Sons, New York, NY). Each of these general texts is herein incorporated by reference.
For the avoidance of doubt, Drosophila and vertebrate names are used interchangeably and aU homologues are included within the scope of the invention.
Notch signalling
As used herein, the expression "Notch signalling" is synonymous with the expression "the Notch signaUing pathway" and refers to any one or more of the upstream or downstream events that result in, or from, (and including) activation of the Notch receptor. Preferably, by "Notch signalling" we refer to any event directly upstream or downstream of Notch receptor activation or inhibition including activation or inhibition of Notch/Notch Hgand interactions, upregulation or downregulation of Notch or Notch Hgand expression or activity and activation or inhibition of Notch signaUing transduction including, for example, proteolytic cleavage of Notch and upregulation or downregulation of the Ras-Jnk signaUing pathway.
Thus, by "Notch signaUing" we refer to the Notch signalling pathway as a signal tranducing pathway comprising elements which interact, geneticaUy and/or molecularly, with the Notch receptor protein. For example, elements which interact with the Notch protein on both a molecular and genetic basis are, by way of example only, Delta, Senate and Deltex. Elements which interact with the Notch protein geneticaUy are, by way of example only, Mastermind, Hairless, Su(H) and Presenilin.
In one aspect, Notch signalling mcludes signalling events taking place extraceUularly or at the cell membrane. In a further aspect, it includes signaUing events taking place intracellularly, for example within the ceU cytoplasm or within the ceU nucleus.
Modulators of Notch signalling
The term "modulate" as used herein refers to a change or alteration in the biological activity of the Notch signaUing pathway or a target signaUing pathway thereof. The term "modulator" preferably refers to antagonists or inhibitors of Notch signaUing, i.e. compounds which block, at least to some extent, the normal biological activity of the Notch signalling pathway. Conveniently such compounds may be refened to herein as inhibitors or antagonists. . Preferably the modulator is an antagonist of Notch signaUing, and preferably an antagonist of the Notch receptor (eg an antagonist of the Notchl, Notch2, Notch3 and/or Notch4 receptor). An antagonist of the Notch receptor is preferably an agent which binds to the extraceUular domain of Notch to reduce or inhibit activation of signaUing. Preferably an antagonist of the Notch receptor binds to Notch in immune ceUs, such as APCs, B-ceUs or T-ceUs.
Alternatively, an inhibitor of Notch signalling may bind to Notch Hgands to reduce their ability to bind to and/or activate a Notch receptor. Preferably such an inhibitor binds to Notch ligands in immune ceUs, such as APCs, B-ceUs or T-ceUs.
The active agent of the present invention may be an organic compound or other chemical. In one embodiment, a modulator wiU be an organic compound comprising two or more hydrocarbyl groups. Here, the term "hydrocarbyl group" means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cycHc group etc. hi addition to the possibhity of the substituents being a cyclic group, a combination of substituents may form a cyclic group. Jf the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms wUl be apparent to those skUled in the art and include, for instance, sulphur, nitrogen and oxygen. The candidate modulator may comprise at least one cycHc group. The cycHc group may be a polycyclic group, such as anon-fused polycycHc group. For some appHcations, the agent comprises at least the one of said cychc groups linked to another hydrocarbyl group.
hi one prefened embodiment, the modulator will be an amino acid sequence or a chemical derivative thereof, or a combination thereof. In another prefened embodiment, the modulator wUl be a nucleotide sequence - which may be a sense sequence or an anti- sense sequence. The modulator may also be an antibody.
Modulators may be synthetic compounds or natural isolated compounds. A very important component of the Notch signaUing pathway is Notch receptor/Notch Hgand interaction. Thus Notch signaUing may involve changes in expression, nature, amount or activity of Notch ligands or receptors or their resulting cleavage products. In addition, Notch signalling may involve changes Hi expression, nature, amount or activity of Notch signaUing pathway membrane proteins or G-proteins or Notch signaUing pathway enzymes such as proteases, kinases (e.g. serme/threorrine kinases), phosphatases, Hgases (e.g. ubiquitin Hgases) or glycosyltransferases. Alternatively the signalling may involve changes in expression, nature, amount or activity of DNA binding elements such as transcription factors.
In a prefened form of the invention the Notch signalling is specific signalling, meaning that the signal detected results substantiaUy or at least predominantly from the Notch signaUing pathway, and preferably from Notch Notch ligand interaction, rather than any other significant interfering or competing cause, such as for example cytokine signaUing. Thus, in a preferred embodiment the term "Notch signaUing" as used herein excludes cytokine signaUing. Preferably therefore the modulator or inhibitor of Notch signaUing is not a cytokine and is preferably not a mitogen.
Preferably the modulator of Notch signaUing is not an agent which acts primarily by inhibiting or downregulating the expression of a Notch Hgand such as Delta and/or Senate. Thus, it will be appreciated that although such inhibition or downregulation may occur as a result of the main mode of action of the modulator of Notch signaUing, preferably this is not the primary mode of action of the modulator. Preferably the primary mode of action of the modulator of Notch signaUing is to modulate (preferably inhibit) interactions between Notch and Notch ligands which are already expressed on immune ceUs.
Thus, preferably the modulator of Notch signaUing is not a Toll protein or BMP and is preferably not an agent which decreases or interferes with the production of Noggin, Chordin, FoUistatin, Xnr3, FGF or Fringe as described, for example in WO98/20142.
The Notch signalling pathway is described in more detail below.
Key targets for Notch-dependent transcriptional activation are genes of the Enhancer of split complex (E[spl]). Moreover these genes have been shown to be dkect targets for binding by the Su(H) protein and to be transcriptionaUy activated in response to Notch signaUing. By analogy with EBNA2, a viral coactivator protein that interacts with a mammalian Su(H) homologue CBFl to convert it from a transcriptional repressor to a transcriptional activator, the Notch rntraceUular domain, perhaps in association with other proteins may combine with Su(H) to contribute an activation domain that aUows Su(H) to activate the transcription of E(spl) as well as other target genes. It should also be noted that Su(H) is not requked for all Notch-dependent decisions, indicating that Notch mediates some ceU fate choices by associating with other DNA-binding transcription factors or by employing other mechanisms to transduce extracellular signals.
In one embodiment, the active agent may be a Notch Hgand, or a polynucleotide encoding a Notch ligand. Notch Hgands of use in the present invention include endogenous Notch Hgands which are typically capable of binding to a Notch receptor polypeptide present in the membrane of a variety of mammalian ceUs, for example hemapoietic stem cells.
The term "Notch Hgand" as used herein means an agent capable of interacting with a Notch receptor to cause a biological effect. The term includes naturally occurring protein ligands such as Delta and Senate, and artificial/modified constructs having equivalent activity.
Particular examples of mammalian Notch ligands identified to date include the Delta famUy, for example Delta or Delta-like 1 (Genbank Accession No. AF003522 - Homo sapiens), Delta-3 (Genbank Accession No. AFO 84576 - Rattus norvegicus) and Delta-like 3 (Mus musculus) (Genbank Accession No. NM_016941 - Homo sapiens) and US 6121045 (MUlennium), Delta-4 (Genbank Accession Nos. AB043894 and AF 253468 - Homo sapiens) and the Senate family, for example Seπate-1 and Senate-2 (WO97/01571, WO96/27610 and WO92/19734), Jagged-1 (Genbank Accession No. U73936 - Homo sapiens) and Jagged-2 (Genbank Accession No. AF029778 - Homo sapiens), and LAG-2. Homology between family members is extensive.
Further homologues of known mammalian Notch Hgands may be identified using standard techniques. By 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 Hgands, for example as mentioned above. TypicaUy, a homologue of a known Notch Hgand wiU be at least 20%, preferably at least 30%, identical at the amino acid level to the coπesponding 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. Techniques and software for calculating sequence homology between two or more arnrno acid or nucleic acid sequences are well known in the art (see for example http://www.ncbi.nhn.ruh.gov and Ausubel et al., Cuπent Protocols in Molecular Biology (1995), John WUey & Sons, Inc.)
Notch Hgands identified to date have a diagnostic DSL domain (D. Delta, S. Serrate, L. Lag2) comprising 20 to 22 amino acids at the amino teπninus of the protein and up to 14 or more EGF-like repeats on the extraceUular surface. It is therefore prefened that homologues of Notch Hgands also comprise a DSL domain at the N-terminus and up to 14 or more EGF- Hke repeats on the extraceUular surface.
In addition, suitable homologues wUl 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 Hbraries with probes comprising all or part of a nucleic acid encoding a Notch Hgand under conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C). Alternatively, homologues may also be obtained using degenerate PCR which wiU generaUy use primers designed to target sequences within the variants and homologues encoding conserved arn no acid sequences. The primers wiU contain one or more degenerate positions and wiU be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
Inhibition of Notch signaUing may also be achieved by mimicking or enhancing activity or expression of inhibitors of the Notch signalling pathway. As such, polypeptides for Notch signalling inhibition include molecules capable of mimicking or enhancing activity or expression of any Notch signalling inhibitors. Preferably the molecule wiU be a polypeptide, or a polynucleotide encoding such a polypeptide, that increases the production or activity of compounds that are capable of producing a decrease in the expression or activity of Notch, Notch Hgands, or any downstream components of the Notch signalling pathway. Such molecules include the Toll-like receptor protein family, and growth factors such as the bone morphogenetic protein (BMP), BMP receptors and activins, derivatives, fragments, variants and homologues thereof.
By a protein which is for Notch signaUing inhibition or a polynucleotide encoding such a protein, we mean a molecule which is capable of inhibiting Notch, the Notch signalling pathway or any one or more of the components of the Notch signalling pathway.
hi one embodiment, the molecule may be capable of reducing or preventing Notch or Notch ligand expression. Such a molecule maybe a nucleic acid sequence capable of reducing or preventing Notch or Notch ligand expression.
Suitably the nucleic acid sequence encodes a polypeptide selected from Toll-like receptor protein family or a growth factor such as a bone morphogenetic protein (BMP), a BMP receptor and activins. Preferably the agent is a polypeptide, or a polynucleotide encoding such a polypeptide, that decreases or interferes with the production of compounds that are capable of producing an increase in the expression of Notch Hgand, such as Noggin, Chordin, FoUistatin, Xnr3, fibroblast growth factors and derivatives, fragments, variants and homologues thereof.
Alternatively, the nucleic acid sequence may be an antisense construct derived from a sense nucleotide sequence encoding a polypeptide selected from a Notch Hgand and a polypeptide capable of upregulating Notch Hgand expression, such as Noggin, Chordin, FoUistatin, Xnr3, fibroblast growth factors and derivatives, fragments, variants and homologues thereof.
Preferably, however, an inhibitor of Notch signalling whlbe a molecule which is capable of inhibiting Notch-Notch Hgand interactions. A molecule may be considered to modulate Notch-Notch Hgand interactions if it is capable of hihibiting the interaction of Notch with its naturally occurring Hgands, preferably to an extent sufficient to provide therapeutic efficacy.
Agents which modulate Notch-Notch ligand interaction may, for example be antibodies, antibody fragments or derivatives, peptides, small organic molecules, peptidomimetics or the like. Antibodies are prefened agents. Such antibodies may be polyclonal or monoclonal, intact or truncated, and may for example be xenogeneic, aUogeneic or syngeneic.
For example, antibodies capable of binding to Notch receptors or Notch Hgands may be used to inhibit normal Notch-Notch ligand interactions in accordance with the present invention.
The expression "Notch-Notch Hgand interaction" (which may be used interchangeably with the term "Notch Hgand-receptor interaction") as used herein means the interaction between a Notch family member and a ligand capable of binding to one or more such member. An agent may be considered to inhibit Notch-Notch Hgand interactions if it is capable of inhibiting the interaction of Notch with its Hgands, preferably to an extent sufficient to provide therapeutic efficacy.
Whilst oligopeptides and peptides may be prefened agents, other sources such as combinatorial Hbraries provide compounds other than ohgopeptides that have the necessary binding characteristics.
Non-peptide agents include numerous chemical types, though typically they are organic molecules, preferably small organic compounds having a molecular weight of between about 50 and about 2,500 daltons. Suitable agents include functional groups necessary for stmctural interaction with proteins, particularly hydrogen bonding, and frequently include at least one group selected from, for example, an amine, carbonyl, carboxyl, hydroxyl, or sulfhydryl group, preferably at least two such functional chemical groups. Compounds may, for example be cycHc or heterocychc structures and/or aromatic or polyaromatic structures substituted with one or more such functional groups.
Suitably the agents block binding of human Notch to human Delta and/or Senate by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
Preferably when the inhibitor is a receptor or a nucleic acid sequence encoding a receptor, the receptor is activated. Thus, for example, when the agent is a nucleic acid sequence, the receptor is preferably constitutively active when expressed.
Inhibitors of Notch signalling also include downstream inhibitors of the Notch signalling pathway, compounds that prevent expression of Notch target genes or induce expression of genes repressed by the Notch signaUing pathway. Examples of such proteins include Dsh or Numb and dominant negative versions of Notch IC or Deltex. Proteins for Notch signalling inhibition wUl also include variants of the wild-type components of the Notch signaUing pathway which have been modified in such a way that then presence blocks rather than transduces the signalling pathway. An example of such a compound would be a Notch receptor which has been modified such that proteolytic cleavage of its intracellular domain is no longer possible.
Notch signalling may also be inhibited by inhibiting Notch signaUing transduction.
Notch signalling transduction
The Notch signalling pathway dkects binary ceU 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 Senate. Vertebrates express multiple Notch receptors and Hgands (discussed below). At least four Notch receptors (Notch-1, Notch-2, Notch-3 and Notch-4) have been identified to date in human ceUs (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 consistmg of a portion of the extraceUular domain, the transmembrane domain and the intracellular domain, and the other comprising the majority of the extraceUular domain. The proteolytic cleavage step of Notch to activate the receptor occurs in the Golgi apparatus and is mediated by a furin-like convertase.
Notch receptors are inserted into the membrane as heterodimeric molecules consisting of an extracellular domain containing up to 36 epidermal growth factor (EGF)-Hke repeats [Notch 1/2 = 36, Notch 3 = 34 and Notch 4 = 29], 3 Cysteine Rich Repeats (Lin-Notch (L/N) repeats) and a transmembrane subunit that contains the cytoplasmic domain. The cytoplasmic domain of Notch contains six ankyrin-Hke repeats, a polyglutarnine 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 CBFl in vertebrates (Tamura K, et al. (1995) Cun. Biol. 5:1416-1423 (Tamura)). The Notch ligands also display multiple EGF-like repeats in their extracellular domains together with a cysteine-rich DSL (Delta-Senate Lag2) domain that is characteristic of all Notch Hgands (Artavanis-Tsakomas et al. (1995) Science 268:225-232, Artavanis-Tsakomas et al. (1999) Science 284:770-776).
The Notch receptor is activated by binding of extraceUular ligands, such as Delta, Senate and Scabrous, to the EGF-like repeats of Notch's extraceUular domain. Delta requires cleavage for activation. It is cleaved by the ADAM disintegrin metalloprotease Kuzbanian at the cell surface, the cleavage event releasing a soluble and active form of Delta. An oncogenic variant of the human Notch-1 protein, also known as TAN-1 , which has a truncated extraceUular domain, is constitutively active and has been found to be involved in T-cell lymphoblastic leukemias.
The cdclO/ahkyrin intraceUular-domain repeats mediate physical interaction with intraceUular signal transduction proteins. Most notably, the cdclO/ahkyrin repeats interact with Suppressor of Hairless [Su(H)]. Su(H) is the Drosophila homologue of C-promoter binding factor-l [CBF-1], a mammaHan DNA binding protein involved in the Epstein-Ban vims-induced immortalization of B-ceUs. It has been demonstrated that, at least in cultured ceUs, Su(H) associates with the cdclO/ankyrin repeats in the cytoplasm and translocates into the nucleus upon the mteraction of the Notch receptor with its Hgand Delta on adjacent ceUs. Su(H) includes responsive elements found in the promoters of several genes and has been found to be a critical downstream protein in the Notch signaUing pathway. The involvement of Su(H) in transcription is thought to be modulated by Hairless.
The intraceUular domain of Notch (NotchIC) also has a direct nuclear function (Lieber et al. (1993) Genes Dev 7(10):1949-65 (Lieber)). Recent studies have indeed shown that Notch activation requires that the six cdclO/ankyrin repeats of the Notch intraceUular domain reach the nucleus and participate in transcriptional activation. The site of proteolytic cleavage on the intracellular taU of Notch has been identified between gly 1743 and vall744 (termed site 3, or S3) (Schroeter, E.H. et al. (1998) Nature 393f6683):382-6 (Schroeter)). It is thought that the proteolytic cleavage step that releases the cdclO/ankyrin repeats for nuclear entry is dependent on PresenUin activity.
The intraceUular domain has been shown to accumulate in the nucleus where it forms a transcriptional activator complex with the CSL family protein CBFl (suppressor of hairless, Su(H) in Drosophila, Lag-2 in C. elegans) (Schroeter; Struhl, G. et al. (1998) CeU 93(4):649-60 (Struhl)). The NotchlC-CBFl complexes then activate target genes, such as the bHLH proteins HES (hairy-enhancer of split like) 1 and 5 (Weimnaster G. (2000) Cun. Opin. Genet. Dev. 10:363-369 (Weinmaster)). This nuclear function of Notch has also been shown for the mammaHan Notch homologue (Lu, F. M. et al. (1996) Proc Natl Acad Sci 93(11)5663-7 (Lu)).
S3 processing occurs only in response to binding of Notch ligands Delta or Senate/Jagged. The post-translational modification of the nascent Notch receptor in the Golgi (Munro S, Freeman M. (2000) Cun. Biol. 10:813-820 (Mumo); Ju BJ, et al. (2000) Nature 405:191-195 (Ju)) appears, at least in part, to control which of the two types of Hgand is expressed on a ceU surface. The Notch receptor is modified on its extracellular domain by Fringe, a glycosyl transferase enzyme that binds to the Lin/Notch motif. Fringe modifies Notch by adding ( -linked fucose groups to the EGF-like repeats (Moloney DJ, et al. (2000) Nature 406:369-375 (Moloney), Brucker K, et al. (2000) Nature 406:411-415 (Brucker)). This modification by Fringe does not prevent ligand binding, but may influence ligand induced confoπnational changes in Notch. Furthermore, recent studies suggest that the action of Fringe modifies Notch to prevent it from interacting functionally with Senate/Jagged Hgands but allow it to preferentiaUy bind Delta (Panin NM, et al. (1997) Nature 387:908-912 (Panin), Hicks C, et al. (2000) Nat. CeU. Biol.2:515-520 (Hicks)). Although Drosophila has a single Fringe gene, vertebrates are known to express multiple genes (Radical, Manic and Lunatic Fringes) (Irvine KD (1999) Cun. Opin. Genet. Devel. 9:434-441 (Irvine)).
Signal transduction from the Notch receptor can occur via two different pathways (Figure 1). The better defined pathway involves proteolytic cleavage of the intraceUular domain of Notch (Notch IC) that translocates to the nucleus and forms a transcriptional activator complex with the CSL farnUy protein CBFl (suppressor of Hairless, Su(H) in Drosophila, Lag-2 in C. elegans). NotchlC-CBFl complexes then activate target genes, such as the bHLH proteins HES (hairy-enhancer of split like) 1 and 5. Notch can also signal in a CBFl -independent manner that involves the cytoplasmic zinc finger containing protein Deltex. Unlike CBFl , Deltex does not move to the nucleus foUowing Notch activation but instead can interact with Grb2 and modulate the Ras-JNK signalling pathway.
Target genes of the Notch signaUing pathway include Deltex, genes of the Hes fannly (Hes-1 in particular), Enhancer of SpHt [E(spl)] complex genes, IL-10, CD-23, CD-4 and DU-1.
Deltex, an intraceUular docking protein, replaces Su(H) as it leaves its site of interaction with the intraceUular taU of Notch. Deltex is a cytoplasmic protein containing a zinc-finger (Artavanis-Tsakomas et al. (1995) Science 268:225-232; Artavanis-Tsakomas et al. (1999) Science 284:770-776; Osborne B, Miele L. (1999) Immunity 11:653-663 (Osborne)). It interacts with the ankyrin repeats of the Notch intracellular domain. Studies indicate that Deltex promotes Notch pathway activation by mteracting with Grb2 and modulating the Ras-JNK signalling pathway (Matsuno et al. (1995) Development 121(8):2633-44; Matsuno K, et al. (1998) Nat. Genet. 19:74-78). Deltex also acts as a docking protein which prevents Su(H) from binding to the intraceUular tail of Notch (Matsuno). Thus, Su(H) is released into the nucleus where it acts as a transcriptional modulator. Recent evidence also suggests that, in a vertebrate B-ceU system, Deltex, rather than the Su(H) homologue CBFl, is responsible for inhibiting E47 function (Ordentlich et al. (1998) Mol. Cell. Biol. 18:2230-2239 (OrdentHch)). Expression of Deltex is upregulated as a result of Notch activation in a positive feedback loop. The sequence of Homo sapiens Deltex (DTX1) mRNA may be found in GenBank Accession No. AF053700.
Hes-1 (Hairy-enhancer of Split-1) (Takebayashi K. et al. (1994) J Biol Chem 269(7 :150-6 (Takebayashi)) is a transcriptional factor with a basic heHx-loop-helix stmcture. It binds to an important functional site in the CD4 silencer leading to repression of CD4 gene expression. Thus, Hes-1 is strongly involved in the determination of T-ceU fate. Other genes from the Hes family include Hes-5 (mammalian Enhancer of Spht homologue), the expression of which is also upregulated by Notch activation, and Hes-3. Expression of Hes- 1 is upregulated as a result of Notch activation. The sequence of Mus museums Hes-1 can be found in GenBank Accession No. D16464.
The E(spl) gene complex [E(spl)-C] (Leimeister C. et al. (1999) Mech Dev 85 (1-2): 173 -7 (Leimeister)) comprises seven genes of which only E(spl) and Groucho show visible phenotypes when mutant. E(sρl) was named after its ability to enhance SpHt mutations, Split being another name for Notch. Indeed, E(spl)-C genes repress Delta through regulation of achaete-scute complex gene expression. Expression of E(spl) is upregulated as a result of Notch activation.
Interleukin-10 (IL-10) was first characterised in the mouse as a factor produced by Th2 ceUs which was able to suppress cytokine production by Thl cells. It was then shown that IL-10 was produced by many other ceU types including macrophages, keratinocytes, B ceUs, ThO and Thl ceUs. It shows extensive homology with theEpstein-Banbcrfl gene which is now designated vkal IL-10. Although a few immunostimulatory effects have been reported, it is mainly considered as an immunosuppressive cytokine. Inhibition of T ceU responses by IL-10 is mainly mediated through a reduction of accessory functions of antigen presenting ceUs. IL-10 has notably been reported to suppress the production of numerous pro-inflammatory cytokrnes by macrophages and to inhibit co-stimulatory molecules and MHC class TI expression. IL-10 also exerts anti-rnflammatory effects on other myeloid cells such as neutrophUs and eosinophUs. On B cells, JL-10 influences isotype switching and proliferation. More recently, JL-10 was reported to play a role in the induction of regulatory T ceUs and as a possible mediator of their suppressive effect. Although it is not clear whether it is a direct downstream target of the Notch signaUing pathway, its expression has been found to be strongly up-regulated coincident with Notch activation. The mRNA sequence of JL-10 may be found in GenBank ref. No. GI1041812.
CD-23 is the human leukocyte differentiation antigen CD23 (FCE2) which is a key molecule for B-ceU activation and growth. It is the low-affinity receptor for IgE. Furthermore, the truncated molecule can be secreted, then functioning as a potent mitogenic growth factor. The sequence for CD-23 may be found in GenBank ref. No. GI1783344.
CTLA4 (cytotoxic T-lymphocyte activated protein 4) is an accessory molecule found on the surface of T-cells which is thought to play a role in the regulation of airway inflammatory ceU recmitment and T-helper ceU differentiation after allergen inhalation. The promoter region of the gene encoding CTLA4 has CBFl response elements and its expression is upregulated as a result of Notch activation. The sequence of CTLA4 can be found in GenBank Accession No. L15006.
Dlx-1 (distaUess-1) (McGuinness T. Et al (1996) Genomics 35(3):473-85 (McGuiness)) expression is downregulated as a result of Notch activation. Sequences for Dix genes may be found in GenBank Accession Nos. U51000-3.
CD-4 expression is downregulated as a result of Notch activation. A sequence for the CD-4 antigen may be found in GenBank Accession No. XM006966. Other genes involved in the Notch signaling pathway, such as Numb, Mastermind and Dsh, and aU genes the expression of which is modulated by Notch activation, are included in the scope of this invention.
As described above the Notch receptor family participates in ceU-cell signalling events that influence T cell fate decisions. In this signalling NotchIC locahses to the nucleus and functions as an activated receptor. MammaHan NotchIC interacts with the transcriptional repressor CBFl . It has been proposed that the NotchIC cdclO/ankyrin repeats are essential for this interaction. Hsieh et al (Hsieh et al. (1996) Molecular & CeU Biology 16(3):952-959) suggests rather that the N-terminal 114 amino acid region of mouse NotchIC contains the CBFl interactive domain. It is also proposed that NotchIC acts by targeting DNA-bound CBFl within the nucleus and aboHshing CBFl -mediated repression through masking of the repression domain. It is known that Epstein Ban virus (EBN) immortalizing protein EBΝA" also utilises CBFl tethering and masking of repression to upregulate expression of CBFl -repressed B-ceU genes. Thus, mimicry of Notch signal transduction is involved in EBN-driven irnmortahzation. Strobl et al (Strobl et al. (2000) J Nkol 74f4): 1727-35) similarly reports that "EBΝA2 may hence be regarded as a functional equivalent of an activated Notch receptor". Other EBN proteins which fall in this category include BARF0 (Kusano and Raab-Truab (2001) J Nkol 75(1) :384-395 (Kusano and Raab-Traub)) and LMP2A.
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 ceU surface, or located intracellularly.
Techniques for drug screening maybe based on the method described in Geysen, European Patent No. 0138855, published on September 13, 1984. Th summary, large numbers of different smaU peptide candidate modulators or targeting molecules are synthesized on a sohd substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with a suitable target or fragment thereof and washed. Bound entities are then detected - such as by appropriately adapting methods well known hi the art. A purified target can also be coated dkectly 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. CeUs can also be spread as "lawns". Alternatively, non-neutraHsing antibodies can be used to capture the peptide and irnmobnise 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 mvention also contemplates the use of competitive drug screening assays in which neutralising antibodies capable of binding a target specificaUy compete with a test compound for binding to a target.
Techniques are well known in the art for the screening and development of agents such as antibodies, peptidomimetics and smaU organic molecules which are capable of binding to components of the Notch signalling pathway. These include the use of phage display systems for expressing signaUing proteins, and using a culture of transfected E. coli or other microorganism to produce the proteins for binding studies of potential binding compounds (see, for example, G. Cesarini, FEBS Letters, 307(l):66-70 (July 1992); H. Gram et al., J. Immunol. Meth., 161:169-176 (1993); and C. Summer et al., Proc. Natl. Acad. Sci., USA, 89:3756-3760 (May 1992)). Further library and screening techniques are described, for example, in US 6281344 (Phylos).
Polypeptides. Proteins and Amino Acid Sequences
As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "protein".
"Peptide" usually refers to a short amino acid sequence that is 10 to 40 amino acids long, preferably 10 to 35 amino acids.
The amino acid sequence may be prepared and isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
Nucleotide Sequences
As used herein, the term "nucleotide sequence" is synonymous with the term ' 'polynucleotide' ' .
The nucleotide sequence may be DNA or RNA of genomic or synthetic or of recombinant origin. They may also be cloned by standard techniques. The nucleotide sequence may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
Longer nucleotide sequences wiU generaUy be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This wiU involve making a pak of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the targeting sequence which it is desked to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human ceU, performing a polymerase chain reaction (PCR) under conditions which bring about amplification of the desired region, isolating the ampHfied fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the ampHfied DNA can be cloned into a suitable cloning vector. In general, primers wiU be produced by synthetic means, involving a step wise manufacture of the desked nucleic acid sequence one nucleotide at a time. Techniques for accompHshing this using automated techniques are readUy avaUable in the art.
"Polynucleotide" refers to a polymeric form of nucleotides of at least 10 bases in length and up to 10,000 bases or 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).
These may be constructed using standard recombinant DNA methodologies. The nucleic acid may be RNA or DNA and is preferably DNA. Where it is RNA, manipulations may be performed via cDNA intermediates. GeneraUy, a nucleic acid sequence encoding the first region wUl 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 ρBR322 or ρUC19 (see below). Reference may be made to Molecular Cloning by Sambrook et al. (Cold Spring Harbor, 1989) or similar standard reference books for exact detaUs of the appropriate techniques.
Sources of nucleic acid may be ascertained by reference to published literature or databanks such as GenBank. Nucleic acid encoding the desked first or second sequences may be obtained from academic or commercial sources where such sources are willing to provide the material or by synthesising or cloning the appropriate sequence where only the sequence data are avaUable. Generally this may be done by reference to literature sources which describe the cloning of the gene in question.
Alternatively, where limited sequence data is avaUable or where it is desked to express a nucleic acid homologous or otherwise related to a known nucleic acid, exemplary nucleic acids can be characterised as those nucleotide sequences which hybridise to the nucleic acid sequences known in the art.
For some appHcations, preferably, the nucleotide sequence is DNA. For some appHcations, preferably, the nucleotide sequence is prepared by use of recombinant DNA techniques (e.g. recombinant DNA). For some appHcations, preferably, the nucleotide sequence is cDNA. For some applications, preferably, the nucleotide sequence may be the same as the naturally occurring form.
Alternatively, where limited sequence data are available or where it is desked to express a nucleic acid homologous or otherwise related to a known nucleic acid, exemplary nucleic acids can be characterised as those nucleotide sequences which hybridise to the nucleic acid sequences known in the art.
It wUl be understood by a skkled 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. In addition, it is to be understood that skilled persons may, using routine techniques, make 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 signaUing modulation of the present invention is to be expressed.
Variants, Derivatives, Analogues, Homologues and Fragments
In addition to the specific amino acid sequences and nucleotide sequences mentioned herein, the present invention also encompasses the use of variants, derivatives, analogues, homologues and fragments thereof.
In the context of the present invention, a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. A variant sequence can be modified by addition, deletion, substitution modification replacement and/or variation of at least one residue present in the naturally-occurring protein. The term "derivative" as used herein, in relation to proteins or polypeptides of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide retains at least one of its endogenous functions.
The term "analogue" as used herein, in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.
Within the definitions of "proteins" and "polypeptides" useful in the present invention, the specific amino acid residues may be modified in such a manner that the protein in question retains at least one of its endogenous functions, such modified proteins are refened to as "variants". A variant protein can be modified by addition, deletion and/or substitution of at least one amino acid present in the namrally-occurring protein.
TypicaUy, 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-naturaUy occurring analogues.
Proteins of use in the present invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Dehberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubihty, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues as long as the target or modulation function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginrne; and amino acids with uncharged polar head groups having similar hydrop icity values include leucine, isoleucine, valine, glycine, danine, asparagine, glutamine, serine, threonine, phenylalanine, and tyro sine.
For ease of reference, the one and three letter codes for the main naturaUy occurring amino acids (and thek associated codons) are set out below:
Symbol 3-letter Meaning Codons
A Ala Alanine GCT,GCC,GCA,GCG
B Asp, sn Aspartic,
Asparagine GA ,GAC,AAT, AC
C Cys Cysteine TGT,TGC
D Asp Aspar ic GAT,GAC
Ξ Glu Glutamic GAA,GAG
P Phe Phenylalanine TTT,TTC
Q Gly Glycine GGT,GGC,GGA,GGG
H His Histidine CAT,CAC
I lie Isoleucine ATT,ATC,ATA
K Lys Lysine AAA,AAG
Leu Leucine TTG,TTA,CTT,CTC,CTA,CTG
M Met Methionine ATG
N Asn Asparagine AAT,AAC
P Pro Proline CCT,CCC,CCA,CCG
Q Qln Glutamine CAA,CAG
R Arg Arginine CGT,CGC,CGA,CQG,AGA,AGO
S Ser Serine TCT,TCC,TCA,TCG,AGT,AGC
T Thr Threonine ACT,ACC,ACA,ACG
V Val Valine GT ,GTC,GTA,GTG w Trp Tryptophan TGG
X Xxx Unknown
Y Tyr Tyrosine TAT, TAC z Glu,Gin Glutamic,
Glutamine GAA,GAG,CAA,CAG
* End Terminator TAA,TAG,TGA
Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the thkd column may be substituted for each other:
As used herein, the term "protein" includes single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the terms "polypeptide" and "peptide" refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. The terms sύbunit and domain may also refer to polypeptides and peptides having biological function.
A peptide useful in the invention wUl at least have a target or signalling modulation capabUity. "Fragments" are also variants and the term typicaUy 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 fuU-length polypeptide, for example between about 8 and about 1500 amino acids in length, preferably 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, and even more preferably 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- dkected mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions coπesponding to the natarally-occurring sequence either side of the insertion site. The flanking regions wiU contain convenient restriction sites coπesponding to sites in the natarally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only iUustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.
Variants of the nucleotide sequence may also be made. Such variants wUl 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 thek use of the different codons. Viruses such as HTV, for instance, use a large number of rare codons. By changing a nucleotide sequence such that rare codons are replaced by the corresponding commonly used mammalian codons, increased expression of the sequences in mammahan target ceUs can be achieved. Codon usage tables are known in the art for mammahan ceUs, as well as for a variety of other organisms.
Where the active agent is a nucleotide sequences it may suitably be codon optimised for expression in mammahan ceUs. Preferably, at least part of the sequence is codon optimised. Even more preferably, the sequence is codon optimised in its entkety.
Sequence Homology. Similarity and Identity
As used herein, the term "homology" can be equated with "identity". An homologous sequence wiU be taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical. In particular, homology should typically he considered with respect to those regions of the sequence (such as amino acids at positions 51 , 56 and 57) known to be essential for an activity. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is prefened to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usuaUy, with the aid of readUy avaUable sequence comparison programs. These commerciaUy available computer programs can calculate % homology between two or more sequences.
Percent homology maybe calculated over contiguous sequences, i.e. one sequence is aHgned with the other sequence and each amino acid in one sequence is dkectly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" ahgnment. Typically, such ungapped aUgnments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fads to take into consideration that, for example, in an otherwise identical pak of sequences, one insertion or deletion wUl cause the foUowing amino acid residues to be put out of ahgnment, thus potentiaUy resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence ahgnment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - wiU achieve a higher score than one with many gaps. "AJfine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaUer penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties wiU of course produce optimised alignments with fewer gaps. Most ahgnment programs allow the gap penalties to be modified. However, it is prefened to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
Calculation of maximum % homology therefor firstly requkes the production of an optimal ahgnment, taking into consideration gap penalties. A suitable computer program for caπying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410 (Atschul)) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are avaUable for offline and online searching (see Ausubel et al, 1999 ibid, pages 7-58 to 7-60). However it is prefened to use the GCG Bestfit program.
The five BLAST programs available at http://www.ncbi.nhn.nih.gov perform the foUowing tasks:
blastp - compares an amino acid query sequence against a protein sequence database.
blastn - compares a nucleotide query sequence against a nucleotide sequence database.
blastx - compares the six-frame 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 aU six reading frames (both strands). tblastx - compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
BLAST uses the following search parameters:
HISTOGRAM - Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual).
DESCRIPTIONS - Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter N 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 wiU not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).
CUTOFF - Cutoff score for reporting high-scoring segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual). TypicaUy, significance thresholds can be more intuitively managed using EXPECT. ALIGNMENTS - Restricts database sequences to the number specified for which high- scoring segment paks (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 dkective in BLASTN requests returns an eπor response.
STRAND - Restrict a TBLASTN search to just the top or bottom strand of the database sequences; or restrict a BLASTN, BLASTX or TBLASTX search to just reading frames on the top or bottom strand of the query sequence.
FILTER - Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17:149-163, or segments consisting of short-periodicity internal repeats, as determined by the XNU program of Claverie & States (1993) Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman (see http://www .ncbi.nhn.nih.gov). FUtering can eliminate statisticaUy significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or prolme-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.
Low complexity sequence found by a filter program is substituted using the letter "N" in nucleotide sequence (e.g., "NNNNNNNNNNNNN") and the letter "X" in protein sequences (e.g., "XXXXXXXXX"). Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs.
It is not unusual for nothing at aU to be masked by SEG, XNU, or both, when appHed to sequences in SWISS-PROT, so filtering should not be expected to always yield an effect. Furthermore, in some cases, sequences are masked in thek entkety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect.
NCBI-gi - Causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.
Most preferably, sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nm.gov BLAST.
In some aspects of the present invention, no gap penalties are used when determining sequence identity.
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an aU-or-nothing pak comparison. Instead, a scaled similarity score matrix is generaUy used that assigns scores to each pakwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is prefened to use the pub He default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typicaUy does this as part of the sequence comparison and generates a numerical result.
Nucleotide sequences which are homologous to or variants of sequences of use in the present invention can be obtained in a number of ways, for example by probing DNA Hbraries made from a range of sources. In addition, other vkal/bacterial, or ceUular homologues particularly ceUular homologues found in mammahan ceUs (e.g. rat, mouse, bovine and primate ceUs), may be obtained and such homologues and fragments thereof in general wiU be capable of selectively hybridising to the sequences shown in the sequence Hsting herein. Such sequences may be obtained by probing cDNA Hbraries made from or genomic DNA Hbraries from other animal species, and probing such Hbraries with probes comprising aU or part of the reference nucleotide sequence under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and aUeHc variants of the amino acid and/or nucleotide sequences useful in the present invention.
Variants and strain/species homologues may also be obtained using degenerate PCR which wiU use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of use in the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence ahgnments can be performed using computer software known in the art. For example the GCG Wisconsin PUeUp program is widely used. The primers used in degenerate PCR wiU contain one or more degenerate positions and wUl be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
Variants and strain/species homologues may also be obtained using degenerate PCR which wiU use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of use in the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence ahgnments can be performed using computer software known in the art. For example the GCG Wisconsin PfleUp program is widely used. The primers used in degenerate PCR wiU contain one or more degenerate positions and wiU be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
PCR technology as described e.g. in section 14 of Sambrook et al., 1989, requires the use of oligonucleotide probes that wUl hybridise to nucleic acid. Strategies for selection of oligonucleotides are described below.
As used herein, a probe is e.g. a single-stranded DNA or RNA that has a sequence of nucleotides that includes between 10 and 50, preferably between 15 and 30 and most preferably at least about 20 contiguous bases that are the same as (or the complement of) an equivalent or greater number of contiguous b ases. The nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are minimised. The nucleotide sequences are usuaUy based on conserved or highly homologous nucleotide sequences or regions of polypeptides. The nucleic acids used as probes may be degenerate at one or more positions.
Prefened regions from which to construct probes include 5' and/or 3' coding sequences, sequences predicted to encode ligand binding sites, and the like. For example, either the full-length cDNA clone disclosed herein or fragments thereof can be used as probes. Preferably, nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridisation. For example, a suitable label means is a radiolabel. The prefened method of labelling a DNA fragment is by incorporating α32P dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art. Oligonucleotides are usually end-labelled with γ3 P-labelled ATP and polynucleotide kinase. However, other methods (e.g. non-radioactive) may also be used to label the fragment or oHgonucleotide, including e.g. enzyme labelling, fluorescent labelling with suitable fluorophores and biotinylation. Prefened are such sequences, probes which hybridise under high-stringency conditions.
Alternatively, such nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example sUent codon changes are requked to sequences to optimise codon preferences for a particular host ceU in which the nucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the activity of the polynucleotide or encoded polypeptide.
In general, the terms "variant", "homologue" or "derivative" in relation to the nucleotide sequence used in the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for a target protein or protein for T cell signaUing modulation.
As indicated above, with respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the reference sequences. More preferably there is at least 95%, more preferably at least 98%, homology. Nucleotide homology comparisons may be conducted as described above. A preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty is - 3 for each nucleotide.
Hybridisation
The present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the reference sequences, or any variant, fragment or derivative thereof, or to the complement of any of the above. Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as weU 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 thek complement, wiU be generally at least 75%, preferably at least 85 or 90% and more preferably at least 95% or 98% homologous to the coπesponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides. Prefened nucleotide sequences of the invention wiU comprise regions homologous to the nucleotide sequence, preferably at least 80 or 90% and more preferably at least 95% homologous to the nucleotide sequence.
The term "selectively hybridizable" means that the nucleotide sequence used as a probe is used under conditions where a target nucleotide sequence of the invention is found to hybridize to the probe at a level significantly above background. The background hybridization may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened. In this event, background impHes a level of signal generated by interaction between the probe and a non-specific DNA member of the Hbrary 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 l iolabeUing the probe, e.g. with 32P.
Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kim el (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below. Maximum stringency typicaUy occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm. As wiU be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.
In a prefened aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention under stringent conditions (e.g. 65°C and O.lxSSC { lxSSC = 0.15 M NaCl, 0.015 M Na3 Citrate pH 7.0). Where the nucleotide sequence of the invention is double-stranded, both strands of the duplex, either individuaUy or in combination, are encompassed by the present invention. Where the nucleotide sequence is single-stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also included within the scope of the present invention.
Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skiU in the field. As known to those of skUl in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrid which decreases approximately 1 to 1.5°C with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridisation reaction is performed under conditions of higher stringency, followed by washes of varying stringency.
As used herein, high stringency preferably refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65-68 °C. High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6x SSC, 5x Denhardt's, 1 % SDS (sodium dodecyl sulphate), 0.1 Na+ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific competitor. Following hybridisation, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 - O.lx SSC, 0.1 % SDS.
It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g. formamide-based buffers, and temperatures. Denhardt's solution and SSC are well known to those of skiU in the art as are other suitable hybridisation buffers (see, e.g. Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds. (1990) Cuπent Protocols in Molecular Biology, John Wiley & Sons, Inc.). Optimal hybridisation conditions have to be determined empirically, as the length and the GC content of the hybridising pak also play a role.
Cloning and Expression
Nucleotide sequences which are not 100% homologous to the sequences of the present invention but faU within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA Hbraries made from a range of sources. In addition, other vkal/bacterial, or ceUular homologues particularly ceUular homologues found in mam ahan ceUs (e.g. rat, mouse, bovine and primate cells), maybe obtained and such homologues and fragments thereof in general wiU be capable of selectively hybridising to the sequences shown in the sequence Hsting herein. Such sequences may be obtained by probing cDNA Hbraries made from or genomic DNA Hbraries from other animal species, and probing such Hbraries with probes comprising aU or part of the reference nucleotide sequence under conditions of medium to high stringency. SirnUar considerations apply to obtaining species homologues and aUehc variants of the amino acid and/or nucleotide sequences useful in the present invention.
Variants and strain/species homologues may also be obtained using degenerate PCR which wiU use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by ahgning the amino acid sequences from several variants homologues. Sequence ahgnments can be performed using computer software known in the art. For example the GCG Wisconsin PUeUp program is widely used. The primers used in degenerate PCR wUl contain one or more degenerate positions and wiU be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
Alternatively, such nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example sUent codon changes are requked to sequences to optimise codon preferences for a particular host ceU in which the nucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the activity of the target protein or protein for T cell signalling modulation encoded by the nucleotide sequences.
The nucleotide sequences such as a DNA polynucleotides useful in the invention may be produced recombinantly, syntheticaUy, or by any means avaUable to those of skUl in the art. They may also be cloned by standard techniques.
In general, primers wUl be produced by synthetic means, involving a step wise manufacture of the desked nucleic acid sequence one nucleotide at a time. Techniques for accompHshing this using automated techniques are readUy avaUable in the art.
Longer nucleotide sequences wUl generaUy be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This wiU involve making a pak of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the targeting sequence which it is desked to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human ceU, performing a polymerase chain reaction (PCR) under conditions which bring about amplification of the desked region, isolating the ampHfied fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the ampHfied DNA can be cloned into a suitable cloning vector
The present invention also relates to vectors which comprise a polynucleotide useful in the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides useful in the present invention by such techniques.
For recombinant production, host cells can be geneticaUy engineered to incorporate expression systems or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis et al and Sambrook et al, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid- mediated transfection, electioporation, transduction, scrape loading, baUistic introduction and infection. It wiU be appreciated that such methods can be employed in vitro or in vivo as drag delivery systems. Representative examples of appropriate hosts include bacterial ceUs, such as streptococci, staphylococci, E. coli, streptomyces and Bacillus subtilis cells; fungal ceUs, such as yeast ceUs and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 ceUs; animal cells such as CHO, COS, NSO, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.
A great variety of expression systems can be used to produce a polypeptide useful in the present invention. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, frombacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculo viruses, papova viruses, such as SV40, vaccinia viruses, adenovkuses, 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. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence maybe inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular env onment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
Proteins or polypeptides may be in the form of the "matare" protein or may be a part of a larger protein such as a fusion protein or precursor. For example, it is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences or pro-sequences (such as a HIS ohgomer, immunoglobulin Fc, glutathione S- transferase, FLAG etc) to aid in purification. Likewise such an additional sequence may sometimes be deskable to provide added stabiHty during recombinant production. In such cases the additional sequence may be cleaved (eg chemicaUy or enzymatically) to yield the final product. In some cases, however, the additional sequence may also confer a deskable pharmacological profile (as in the case of IgFc fusion proteins) in which case it may be prefened that the additional sequence is not removed so that it is present in the final product as administered.
Proteins or polypeptides may be in the form of the "matare" protein or may be a part of a larger protein such as a fusion protein or precursor. For example, it is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences or pro-sequences (such as a HIS ohgomer, immunoglobulin Fc, glutathione S- transferase, FLAG etc) to aid in purification. Likewise such an additional sequence may sometimes be deskable to provide added stabiHty during recombinant production. In such cases the additional sequence may be cleaved (eg chemicaUy or enzymatically) to yield the final product. In some cases, however, the additional sequence may also confer a deskable pharmacological profile (as in the case of IgFc fusion proteins) in which case it may be prefeπed that the additional sequence is not removed so that it is present in the final product as administered.
Also included within the invention are mammalian and microbial host ceUs comprising such vectors or other polynucleotides encoding the fusion proteins, and thek production and use.
Active agents for use in the invention can be recovered and purified from recombinant ceU cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphoceUulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. WeU known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
Various prefened features and embodiments of the present invention will now be described in more detail by way of non-limiting examples.
Substances that may be used to modulate Notch signalling by inhibiting Notch ligand expression include nucleic acid sequences encoding polypeptides that affect the expression of genes encoding Notch ligands. For instance, for Delta expression, binding of extracellular BMPs (bone morphogenetic proteins, Wilson and Hemmati-Biivanlou; Hemmati-Biivanlou and Melton) to thek receptors leads to down-regulated Delta transcription due to the inhibition of the expression of transcription factors of the achaete/scute complex. This complex is beheved to be dkectly involved in the regulation of Delta expression. Thus, any polypeptide that upregulates BMP expression and/or stimulates the binding of BMPs to thek receptors may be capable of producing a decrease in the expression of Notch Hgands such as Delta and/or Senate. Examples may include nucleic acids encoding BMPs themselves. Furthermore, any substance that inhibits expression of transcription factors of the achaete/scute complex may also downregulate Notch Hgand expression.
Members of the BMP family include BMP1 to BMP6, BMP7 also caUed OP1, OP2 (BMP8) and others. BMPs belong to the transforming growth factor beta (TGF-beta) superfamUy, which includes, in addition to the TGF-betas, activms/innϊbins (e.g., alpha- inhibin), muUerian inhibiting substance, and gHal cell line-derived neurotrophic factor.
Other examples of polypeptides that inhibit the expression of Delta and/or Senate include the Toll-like receptor (Medzhitov) or any other receptors linked to the innate immune system (for example CD14, complement receptors, scavenger receptors or defensin proteins), and other polypeptides that decrease or interfere with the production of Noggin (Valenzuela), Chordin (Sasai), FoUistatin (Iemura), Xnr3, and derivatives and variants thereof. Noggin and Chordin bind to BMPs thereby preventing activation of thek signaUing cascade which leads to decreased Delta transcription. Consequently, reducing Noggin and Chordin levels may lead to decreased Notch ligand, in particular Delta, expression.
hi more detaU, in Drosopmla, the Toll transmembrane receptor plays a central role in the signaUing pathways that control amongst other things the innate nonspecific immune response. This Toll-mediated immune response reflects an ancestral conserved signaUing system that has homologous components in a wide range of organisms. Human ToU homologues have been identified amongst the ToU-Hke receptor (TLR) genes and ToU/mterleukin-l receptor-like (TIL) genes and contain the characteristic ToU motifs: an extraceUular leucrne-rich repeat domain and a cytoplasmic mterleιιkin-1 receptor-like region. The ToU-Hke receptor genes (including TIL genes) now include TLR4, TTL3, TTL4, and 4 other identified TLR genes.
Other suitable sequences that may be used to downregulate Notch ligand expression include those encoding immune costimulatory molecules (for example CD80, CD86, ICOS, SLAM) and other accessory molecules that are associated with immune potentiation (for example CD2, LFA-1).
Other suitable substances that may be used to downregulate Notch Hgand expression include nucleic acids that inhibit the effect of transforming growth factors such as members of the fibroblast growth factor (FGF) family. The FGF may be a mammalian basic FGF, acidic FGF or another member of the FGF famUy such as an FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7. Preferably the FGF is not acidic FGF (FGF-1; Zhao et al, 1995). Most preferably, the FGF is a member of the FGF family which acts by stimulating the upregulation of expression of a Senate polypeptide on APCs. It has been shown that members of the FGF family can upregulate Senate-1 gene expression in APCs. Inhibition of Notch signaUing by use of anti-sense constructs
Suitable nucleic acid sequences may include anti-sense constructs, for example nucleic acid sequences encoding antisense Notch ligand constructs or antisense sequences conesponding to other components of the Notch signalling pathway as discussed above. The antisense nucleic acid may be an ohgonucleoti.de such as a synthetic single-stranded DNA. However, more preferably, the antisense is an antisense RNA produced in the patient's own ceUs as a result of introduction of a genetic vector. The vector is responsible for production of antisense RNA of the desked specificity on introduction of the vector into a host cell.
Antisense nucleic acids can be oligonucleotides that are double-stranded or single- stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences.
For example, as described in US -A-20020119540 inhibitory antisense or double stranded oligonucleotides can additionaUy comprise at least one modified base moiety which is selected- from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouιidine, 5-carboxymethylaminomethyluraci- 1, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- metnyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracU, 5-methoxyamoinomethyl-2-thiouracU, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracU, 5-methoxyuracU, 2-methylthio- N6-isopentenyladenine, uracU-5-oxy acetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracU, 2-thiouracU, 4-thiouracil, 5 -methyluracil, uracil-5-oxyacetic acid methylester, uracU-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3 -amino-3 -N-2-carboxypropyl) uracU, (acp3)w, and 2,6-diaminopurine. An antisense oligonucleotide may also comprise one or more modified sugar moieties such as, for example, arabrnose, 2-fluoroarabinose, xylulose, orhexose.
hi yet another embodiment, the antisense oligonucleotide may if desked comprise at least one modified phosphate backbone such as, for example, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof. Alternatively another polymeric backbone such as a modified polypeptide backbone may be used (eg protein nucleic acid: PNA).
In yet another embodiment, the antisense oligonucleotide may be an alpha-anomeric oligonucleotide. An alpha-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands nm parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide may for example be a 2'-0-methyiribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al, 1987, FEBS Lett. 215:327-330). OHgonucleotides maybe synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commerciaUy avaUable from Biosearch, Applied Biosystems, etc.). Merely as examples, phosphorothioate oligonucleotides canbe synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
Preferably, the nucleic acid sequence for use in the present invention is capable of inhibiting Senate and Delta, preferably Senate 1 and Senate 2 as weU as Delta 1, Delta 3 and Delta 4 expression in APCs such as dendritic ceUs. In particular, the nucleic acid sequence may be capable of inhibiting Senate expression but not Delta expression, or Delta but not Senate expression in APCs or T cells. Alternatively, the nucleic acid sequence for use in the present invention is capable of inhibiting Delta expression in T ceUs such as CD4+ helper T cells or other ceUs of the immune system that express Delta (for example in response to stimulation of cell surface receptors). In particular, the nucleic acid sequence may be capable of inhibiting Delta expression but not Senate expression in T cells. In a particularly prefeπed embodiment, the nucleic acid sequence is capable of inhibiting Notch Hgand expression in both T ceUs and APC, for example Senate expression in APCs and Delta expression in T ceUs.
Prefened suitable substances that may be used to downregulate Notch Hgand expression include growth factors and cytokines. More preferably soluble protein growth factors may be used to inhibit Notch or Notch Hgand expression. For instance, Notch Hgand expression may be reduced or inhibited by the addition of BMPs or activins (a member of the TGF-β superfamily). In addition, T cells, APCs or tumour cells could be cultured in the presence of inflammatory type cytokines including IL-12, IFN-γ, 1L-18, TNF-oc, either alone or in combination with BMPs.
Molecules for inhibition of Notch signalling will also include polypeptides, or polynucleotides which encode therefore, capable of modkying Notch-protein expression or presentation on the cell membrane or signalling pathways. Molecules that reduce or interfere with its presentation as a fully functional ceU membrane protein may include MMP inhibitors such as hydroxymate-based inhibitors.
Other substances which may be used to reduce interaction between Notch and Notch Hgands are exogenous Notch or Notch ligands or functional derivatives thereof. For example, Notch Hgand derivatives would preferably have the DSL domain at the N- ternunus and between 1 to 8, suitably from 2 to 5, EGF-like repeats on the extracellular surface. A peptide coπesponding to the Delta/Seπate/LAG-2 domain of hJaggedl and supematants from COS cells expressing a soluble form of the extraceUular portion of hJaggedl was found to mimic the effect of Jaggedl in inhibiting Notchl (Li). In one embodiment a Notch Hgand derivative maybe a fusion protein, for example, a fusion protein comprising a segment of a Notch Hgand extraceUular domain and an immunoglobulin F0 segment such as IgGFc or IgMF0
Alternatively, the modulator may comprise all or part of the extraceUular domain of a Notch receptor (eg Notchl, Notch2, Notch3, Notch4 or homologues thereof), which can bind to Notch Hgands and so reduce interactions with endogenous Notch receptors. Preferably, such a modulator may comprise at least the 11th and 12th domains of Notch (EGF11 and EGF12), as these are believed to be important for Notch ligand interaction.
For example, a rat Notch-1/Fc fusion protein is available from R& D Systems Inc (Minneapolis, USA and Abingdon, Oxon, UK: Catalog No 1057-TK). This comprises the 12 amino teπninal EGF domains of rat Notch-1 (amino acid residues Met 1 to Glu 488) fused to the Fc region of human IgG (Pro 100 to Lys 330) via a polypeptide linker (TEGRMD).
Other Notch signalling pathway antagonists include antibodies which inhibit interactions between components of the Notch signalling pathway, e.g. antibodies to Notch or Notch Hgands.
The term "antibody" includes intact molecules as well as fragments thereof, such as Fab, 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 Hght chain and a portion of one heavy chain;
(ii) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
(iii) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with pepsin without subsequent reduction; F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds;
(iv) Fv, defined as a genetically engineered fragment containing the variable genetically fused single chain molecule; and
(v) fragments consisting of essentiaUy only a variable (VH or V ), antigen-binding domain of the antibody (so-caUed "domain antibodies").
General methods of making antibodies are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), the text of which is incorporated herein by reference). Antibodies maybe monoclonal or polyclonal but are preferably monoclonal.
Suitably, the binding affinity (equUibrium association constant (Ka)) may be at least about 106 M"1, at least about 107M"\ at least about 10s M"1, or at least about 109 M"1.
Suitably the antibody, derivative or fragment binds to one or more DSL, EGF or N- terminal domains of a Notch ligand or to one or more EGF or Lin/Notch (L/N) domains of Notch (for example to EGF repeats 11 and 12 of Notch).
In one embodiment the agent may be an antibody, derivative or fragment which binds to Notch.
In a further embodiment the agent may be an antibody, derivative or fragment which binds to Delta. Tn a further embodiment the agent may be an antibody, derivative or fragment which binds to Senate or Jagged.
Suitable antibodies for use as blocking agents are obtained by immunizing a host animal with peptides comprising aU or a portion of Notch or a Notch Hgand such as Delta or Senate/Jagged.
The peptide used may comprise the complete protein or a fragment or derivatives thereof. Prefened irnmunogens comprise all or a part of the extracellular domain of human Notch, Delta or Senate/Jagged, where these residues contain any post-translation modifications, such as glycosylation, found in the native proteins, unmunogens comprising the extraceUular domain may be produced by a number of techniques which are weU known in the ait such as expression of cloned genes using conventional recombinant methods and/or isolation from T cells or ceU populations expressing high levels of Notch or Notch Hgands.
Monoclonal antibodies may be produced by means well known in the art. GeneraUy, the spleen and/or lymph nodes of an immunized host animal provide a source of plasma ceUs. The plasma cells are immortahzed by fusion with myeloma cells to produce hybridoma cells. Culture supernatant from individual hybridomas is screened using standard techniques to identify those producing antibodies with the desked specificity. The antibody may be purified from the hybridoma ceU supematants or ascites fluid by conventional techniques, such as affinity chromatography using Notch, Notch ligands or fragments thereof bound to an insoluble support, protein A sepharose, or the like.
For example, antibodies against Notch and Notch Hgands are described in US 5648464, US 5849869 and US 6004924 (Yale University/Imperial Cancer Technology), the texts of which are herein incoφorated by reference. Antibodies generated against the Notch receptor are also described in WO 0020576 (the text of which is also incorporated herein by reference). For example, this document discloses generation of antibodies against the human Notch-1 EGF-like repeats 11 and 12. For example, in particular embodiments, WO 0020576 discloses a monoclonal antibody secreted by a hybridoma designated A6 having the ATCC Accession No. HB 12654, a monoclonal antibody secreted by a hybridoma designated CU having the ATCC Accession No. HB 12656 and a monoclonal antibody secreted by a hybridoma designated F3 having the ATCC Accession No. HB 12655.
Preferably, antibodies for use to treat human patients wUl be chimeric or humanised antibodies. Antibody "humanisation" techniques are well known in the art. These techniques typically involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule.
As described in US5859205 early methods for humanising monoclonal antibodies (Mabs) involved production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody is linked to constant domains derived from another antibody. Such chimerisation procedures are described in EP-A-0120694 (CeUtech Limited), EP-A-0125023 (Genentech Inc. and City of Hope), EP-A-0 171496 (Res. Dev. Corp. Japan), EP-A-0 173 494 (Stanford University), and WO 86/01533 (CeUtech Limited). For example, WO 86/01533 discloses a process for preparing an antibody molecule having the variable domains from a mouse MAb and the constant domains from a human immunoglobulin.
In an alternative approach, described in EP-A-0239400 (Winter), the complementarity determining regions (CDRs) of a mouse MAb are grafted onto the framework regions of the variable domains of a human immunoglobulin by site dkected mutagenesis using long oligonucleotides. Such CDR-grafted humanised antibodies are much less likely to give rise to an anti-antibody response than humanised chimeric antibodies in view of the much lower proportion of non-human amino acid sequence which they contain. Examples in which a mouse MAb recognising lysozyme and a rat MAb recognising an antigen on human T-cells were humanised by CDR-grafting have been described by Verhoeyen et al (Science, 239, 1534-1536, 1988) and Riechmann et al (Nature, 332, 323-324, 1988) respectively. The preparation of CDR-grafted antibody to the antigen on human T cells is also described in WO 89/07452 (Medical Research CouncU).
Th WO 90/07861 Queen et al propose four criteria for designing humanised immunoglobuHns. The first criterion is to use as the human acceptor the framework from a particular human immunoglobulin that is unusuaUy homologous to the non-human donor immunoglobulin to be humanised, or to use a consensus framework from many human antibodies. The second criterion is to use the donor amino acid rather than the acceptor if the human acceptor residue is unusual and the donor residue is typical for human sequences at a specific residue of the framework. The thkd criterion is to use the donor framework amino acid residue rather than the acceptor at positions immediately adjacent to the CDRs. The fourth criterion is to use the donor amino acid residue at framework positions at which the amino acid is predicted to have a side chain atom within about 3 A of the CDRs in a three-dimensional immunoglobuHn model and to be capable of interacting with the antigen or with the CDRs of the humanised immunoglobuHn. It is proposed that criteria two, three or four may be applied in addition or alternatively to criterion one, and may be appHed singly or in any combination.
The choice of isotype wiU be guided by the desked effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Suitable isotypes include IgG 1, IgG3 and IgG4. Suitably, either of the human light chain constant regions, kappa or lambda, may be used.
Chemical linking
ChemicaUy coupled sequences can be prepared (where requked) from individual proteins sequences and coupled using known chemicaUy coupling techniques. The conjugate can be assembled using conventional solution- or sohd-phase peptide synthesis methods, affording a fully protected precursor with only the teπninal amino group in deprotected reactive form. This function can then be reacted dkectly with a protein for T ceU signaUing modulation or a suitable reactive derivative thereof. Alternatively, this amino group may be converted into a different functional group suitable for reaction with a cargo moiety or a linker. Thus, e.g. reaction of the amino group with succinic anhydride wUl provide a selectively addressable carboxyl group, while further peptide chain extension with a cysteine derivative will result in a selectively addressable thiol group. Once a suitable selectively addressable functional group has been obtained in the dehvery vector precursor, a protein for T cell signaUing modulation 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.
As discussed above the target protein and protein for T cell signalling modulation maybe linked dkectly or indirectly via a cleavable linker moiety. Dkect linkage may occur through any convenient functional group on the protein for T ceU signaUing modulation such as a hydroxy, carboxy or amino group. Indirect linkage which is preferable, will occur through a linking moiety. Suitable linking moieties include bi- and multifunctional alkyl, aryl, aralkyl or peptidic moieties, alkyl, aryl or aralkyl aldehydes acids esters and anyhdrides, sulphydryl or carboxyl groups, such as maleimido benzoic acid derivatives, maleimido proprionic acid derivatives and succinknido derivatives or may be derived from cyanuric bromide or chloride, carbonyldiknidazole, succinimidyl esters or sulphonic haHdes and the like. The functional groups on the linker moiety used to form covalent bonds between linker and protein for T cell signalling modulation on the one hand, as well as linker and target protein on the other hand, may be two or more of, e.g., amino, hydrazino, hydroxyl, thiol, maleimido, carbonyl, and carboxyl groups, etc. The linker moiety may include a short sequence of from 1 to 4 amino acid residues that optionaUy includes a cysteine residue through which the linker moiety bonds to the target protein. Notch ligand domains
As discussed above, natarally occurring Notch ligands typically comprise a number of distinctive domains. Some predicted/potential domain locations for various natarally occurring human Notch ligands (based on amino acid numbering in the precursor proteins) are shown below:
Human Delta 1
Component Amino acids Proposed function/doi
SIGNAL 1-17 SIGNAL
CHAIN 18-723 DELTA-LIKE PROTEIN 1
DOMAIN 18-545 EXTRACELLULAR
TBANSMEM 546- 568 TRANSMEMBRANE
DOMAIN 569-723 CYTOPLASMIC
DOMAIN 159-221 DSL
DOMAIN 226-254 EGF-LIKE 1
DOMAIN 257-285 EGF-LIKE 2
DOMAIN 292-325 EGF-LIKE 3
DOMAIN 332-363 EGF-LIKE 4
DOMAIN 370-402 EGF-LIKE 5
DOMAIN 409-440 EGF-LIKE 6
DOMAIN 447-478 EGF-LIKE 7
DOMAIN 485-516 EGF-LIKE 8
Human Delta 3
Component Amino acids Proposed fl
DOMAIN 158-248 DSL
DOMAIN 278-309 EGF-LIKE 1
DOMAIN 316-350 EGF-LIKE 2
DOMAIN 357-388 EGF-LIKE 3
DOMAIN 395-426 EGF-LIKE 4
DOMAIN 433-464 EGF-LIKE 5
Human Delta 4
Component Amino acids Proposed function/domain
SIGNAL 1-26 SIGNAL
CHAIN 27 - 685 DELTA- LIKE PROTEIN 4
DOMAIN 27 -529 EXTRACELLULAR
TRANSMEM 530- 550 TRANSMEMBRANE
DOMAIN 551- 685 CYTOPLASMIC
DOMAIN 155-217 DSL
DOMAIN 218- 251 EGF-LIKE 1
DOMAIN 252-282 EGF-LIKE 2
DOMAIN 284-322 EGF-LIKE 3
DOMAIN 324- 360 EGF-LIKE 4
DOMAIN 362-400 EGF-LIKE 5
DOMAIN 402-438 EGF-LIKE 6
DOMAIN 440-476 EGF-LIKE 7
DOMAIN 480-518 EGF-LIKE 8
Human Jagged 1
Component Amino acids Proposed function/domain
SIGNAL 1-33 SIGNAL
CHAIN 34-1218 JAGGED 1
DOMAIN 34-1067 EXTRACELLULAR
TRANSMEM 1068-1093 TRANSMEMBRANE
DOMAIN 1094-1218 CYTOPLASMIC
DOMAIN 167-229 DSL
DOMAIN 234-262 EGF-LIKE 1
DOMAIN 265-293 EGF-LIKE 2
DOMAIN 300-333 EGF-LIKE 3
DOMAIN 340-371 EGF-LIKE 4
DOMAIN 378-409 EGF-LIKE 5
DOMAIN 416-447 EGF-LIKE 6
DOMAIN 454-484 EGF-LIKE 7
DOMAIN 491-522 EGF-LIKE 8
DOMAIN 529-560 EGF-LIKE 9
DOMAIN 595-626 EGF-LIKE 10
DOMAIN 633-664 EGF-LIKE 11
DOMAIN 671-702 EGF-LIKE 12
DOMAIN 709-740 EGF-LIKE 13
DOMAIN 748-779 EGF-LIKE 14
DOMAIN 786-817 EGF-LIKE 15
DOMAIN 824-855 EGF-LIKE 16
DOMAIN 863-917 VON WILLEBRAND FACTOR C Human Tagged 2
Component Amino acids Proposed function/domain
SIGNAL 1-26 SIGNAL
CHAIN 27-1238 JAGGED 2
DOMAIN 27-1080 EXTRACELLULAR
TRANSMEM 1081-1105 TRANSMEMBRANE
DOMAIN 1106-1238 CYTOPLASMIC
DOMAIN 178-240 DSL
DOMAIN 249-273 EGF-LIKE 1
DOMAIN 276-304 EGF-LIKE 2
DOMAIN 311-344 EGF-LIKE 3
DOMAIN 351-382 EGF-LIKE 4
DOMAIN 389-420 EGF-LIKE 5
DOMAIN 427-458 EGF-LIKE 6
DOMAIN 465-495 EGF-LIKE 7
DOMAIN 502-533 EGF-LIKE 8
DOMAIN 540-571 EGF-LIKE 9
DOMAIN 602-633 EGF-LIKE 10
DOMAIN 640-671 EGF-LIKE 11
DOMAIN 678-709 EGF-LIKE 12
DOMAIN 716-747 EGF-LIKE 13
DOMAIN 755-786 EGF-LIKE 14
DOMAIN 793-824 EGF-LIKE 15
DOMAIN 831-862 EGF-LIKE 16
DOMAIN 872-949 VON WILLEBRAND FACTOR C
DSL domain
A typical DSL domain may include most or all of the following consensus amino acid sequence:
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
Preferably the DSL domain may include most or all of the following consensus amino acid sequence:
Cys Xaa Xaa Xaa ARO ARO Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys BAS NOP BAS ACM ACM Xaa ARO NOP ARO Xaa Xaa Cys Xaa Xaa Xaa NOP Xaa Xaa Xaa Cys Xaa Xaa NOP ARO Xaa NOP Xaa Xaa Cys wherein:
ARO is an aromatic amino acid residue, such as tyrosine, phenylalanine, tryptophan or histidine; NOP is a non-polar amino acid residue such as glycine, alanine, proline, leucine, isoleucine or valine;
BAS is a basic amino acid residue such as arginine or lysine; and
ACM is an acid or amide amino acid residue such as aspartic acid, glutamic acid, asparagine or glutamine.
Preferably the DSL domain may include most or all of the following consensus amino acid sequence:
Cys Xaa Xaa Xaa Tyr Tyr Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Arg Pro Arg Asx Asp Xaa Phe Gly His Xaa Xaa Cys Xaa Xaa Xaa Gly Xaa Xaa Xaa Cys Xaa Xaa Gly Trp Xaa Gly Xaa Xaa Cys
(wherein Xaa may be any amino acid and Asx is either aspartic acid or asparagine).
An alignment of DSL domains from Notch ligands from various sources is shown in Figure 3.
The DSL domain used may be derived from any suitable species, including for example Drosophila, Xenopus, rat, mouse or human. Preferably the DSL domain is derived from a vertebrate, preferably a mammahan, preferably a human Notch Hgand sequence.
It wUl be appreciated that the term "DSL domain" as used herein includes sequence variants, fragments, derivatives and mimetics having activity coπesponding to nataraUy occurring domains.
Suitably, for example, a DSL domain for use in the present invention may have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Jagged 1.
Alternatively a DSL domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Jagged 2.
Alternatively a DSL domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% a ino acid sequence identity to the DSL domain of human Delta 1.
Alternatively a DSL domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Delta 3.
Alternatively a DSL domain for use in the present mvention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to the DSL domain of human Delta 4.
EGF-like domain
The EGF-like motif has been found in a variety of proteins, as weU as EGF and Notch and Notch Hgands, including those involved in the blood clotting cascade (Furie and Furie, 1988, Cell 53: 505-518). For example, this motif has been found in extraceUular proteins such as the blood clotting factors IX and X (Rees et al, 1988, EMBO J. 7:2053- 2061; Furie and Furie, 1988, CeU 53: 505-518), in other Drosophila genes (Knust et al., 1987 EMBO J. 761-766; Rothberg et al, 1988, CeU 55:1047-1059), and in some ceU- surface receptor proteins, such as thrombomoduHn (Suzuki et al., 1987, EMBO J. 6:1891- 1897) and LDL receptor (Sudhof et al., 1985, Science 228:815-822). A protein binding site has been mapped to the EGF repeat domain in thrombomoduHn and urokinase (Kurosawa et al., 1988, J. Biol. Chem 263:5993-5996; AppeUa et al., 1987, J. Biol. Chem.262:4437-4440).
As reported by PROSITE a typical EGF domain may include six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds. The main structure is proposed, but not necessarily requked, to be a two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet. Subdomains between the conserved cysteines strongly vary in length as shown in the foUowing schematic representation of a typical EGF-like domain:
x(4)-C-x(0,48)-C-x(3,12)-C-x(l,70)-C-x(:L,S)-C-x(2)-a-a-x(0,21)-3-x(2)-C-x I i ************************************
+ + wherein:
'C: conserved cysteine involved in a disulfide bond.
'G': often conserved glycine
'a': often conserved aromatic amino acid
'*': position of both patterns.
'x': any residue
The region between the 5th and 6th cysteines contains two conserved glycines of which at least one is normally present inmost EGF-like domains.
The EGF-like domain used may be derived from any suitable species, including for example DrosophUa, Xenopus, rat, mouse or human. Preferably the EGF-like domain is derived from a vertebrate, preferably a mammalian, preferably a human Notch ligand sequence.
It wUl be appreciated that the term ΕGF domain" as used herein includes sequence variants, fragments, derivatives and mimetics having activity coπesponding to nataraUy occurring domains. Suitably, for example, an EGF-like domain for use in the present invention may have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Jagged 1.
Alternatively an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Jagged 2.
Alternatively an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 1.
Alternatively an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 3.
Alternatively an EGF-like domain for use in the present invention may, for example, have at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity to an EGF-like domain of human Delta 4.
As a practical matter, whether any particular amino acid sequence is at least X% identical to another sequence can be determined conventionaUy using known computer programs. For example, the best overaU match between a query sequence and a subject sequence, also refened to as a global sequence alignment, can be deteπnined using a program such as the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence ahgnment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of the global sequence ahgnment is given as percent identity.
The term "Notch ligand N-teπninal domain" means the part of a Notch ligand sequence from the N-teπninus to the start of the DSL domain. It wUl be appreciated that this term includes sequence variants, fragments, derivatives and mimetics having activity coπesponding to natarally occurring domains.
The term "heterologous amino acid sequence" or "heterologous nucleotide sequence" as used herein means a sequence which is not found in the native sequence (eg in the case of a Notch ligand sequence is not found in the native Notch ligand sequence) or its coding sequence. Preferably any such heterologous amino acid sequence is not a TSST sequence, and preferably it is not a superantigen sequence.
Whether a substance can be used for activating Notch may be determined using suitable screening assays, for example, as described in our co-pending International Patent Application claiming priority from GB 0118153.6, and the examples herein.
Screening Assays
Whether a substance can be used for modulating Notch signalling may be determined using suitable screening assays (see for example, the Examples herein)
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" refened to herein may be any detectable manifestation attributable to the presence of the cleaved intraceUular 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 weU defined genes. Thus, increased Notch signaUing can be assessed at the nucleic acid level by say measuring intraceUular concentrations of specific mRNAs. In one prefened embodiment of the present invention, the assay is a protein assay. In another prefeπed embodiment of the present invention, the assay is a nucleic acid assay.
The advantage of using a nucleic acid assay is that they are sensitive and that smaU samples can be analysed.
The intraceUular concentration of a particular mRNA, measured at any given time, reflects the level of expression of the corresponding gene at that time. Thus, levels of mRNA of downstream target genes of the Notch signaUing pathway can be measured in an indirect assay of the T-cells of the immune system. In particular, an increase in levels of Deltex, Hes-1 and/or JL-10 mRNA may, for instance, indicate induced anergy while an increase in levels of DU-1 or IFN-γ mRNA, or in the levels of mRNA encoding cytokines such as JL-2, JL-5 and IL-13, may indicate improved responsiveness.
Various nucleic acid assays are known. Any convention technique which is known or which is subsequently disclosed may be employed. Examples of suitable nucleic acid assay are mentioned below and include amplification, PCR, RT-PCR, RNase protection, blotting, spectrometry, reporter gene assays, gene chip aπays and other hybridization methods.
In particular, gene presence, amplification and/or expression may be measured in a sample dkectly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe. Those skilled in the art wiU readily envisage how these methods may be modified, if desked. PCR was originally developed as a means of amplifying DNA from an impure sample. The technique is based on a temperatare cycle which repeatedly heats and cools the reaction solution allowing primers to anneal to target sequences and extension of those primers for the formation of duphcate daughter strands. RT-PCR uses an RNA template for generation of a first strand cDNA with a reverse transcriptase. The cDNA is then ampHfied according to standard PCR protocol. Repeated cycles of synthesis and denataration result in an exponential increase in the number of copies of the target DNA produced. However, as reaction components become Hmiting, the rate of ampHfication decreases until a plateau is reached and there is Httle 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. Primers can be designed using standard procedures in the art, for example the Taqman™ technique.
Real-time PCR uses probes labeled with a fluorescent tag 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 ampHfication of a target sequence is first detected through a significant increase in fluorescence. An advantage of real-time PCR is its accuracy in determining the amounts if target sequences in a sample. Suitable protocols are described, for example, in Meuer S. et al (2000).
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 ρoly(A)-selected mRNA as a target. The sensitivity of the ribonuclease protection assay derives from the use of a complementary in vitro transcript probe which is radiolabeled to high specific activity. The probe and target RNA are hybridized in solution, after which the mixture is diluted and treated with ribonuclease (RNase) to degrade all remaining single-stranded RNA. The hybridized portion of the probe wiU be protected from digestion and can be visualized via electrophoresis of the mixture on a denaturing polyacrylamide gel followed by autoradiography. Since the protected fragments are analyzed by high resolution polyacrylamide gel electrophoresis, the ribonuclease protection assay can be employed to accurately map mRNA featares. If the probe is hybridized at a molar excess with respect to the target RNA, then the resulting signal wiU be dkectly proportional to the amount of complementary RNA in the sample.
Gene expression may also be detected using a reporter system. Such a reporter system may comprise a readily identifiable marker under the control of an expression system, e.g. of the gene being monitored. Fluorescent markers, which can be detected and sorted by FACS, are prefeπed. Especially prefened are GFP and luciferase. Another type of prefeπed reporter is cell surface markers, i.e. proteins expressed on the cell surface and therefore easUy identifiable.
In general, reporter constructs useful for detecting Notch signalling by expression of a reporter gene may be constructed according to the general teaching of Sambrook et al (1989). Typically, constructs according to the invention comprise a promoter by the gene of interest, and a coding sequence encoding the desked reporter constructs, for example of GFP or luciferase. Vectors encoding GFP and luciferase are known in the art and avaUable commercially.
Sorting of ceUs, based upon detection of expression of genes, may be performed by any technique known in the art, as exempHfied above. For example, ceUs may be sorted by flow cytometry or FACS. For a general reference, see Flow Cytometry and Cell Sorting: A Laboratory Manual (1992) A. Radbruch (Ed.), Springer Laboratory, New York.
How cytometry is a powerful method for studying and purifying ceUs. It has found wide apphcation, particularly in immunology and cell biology: however, the capabiHties of the FACS can be appHed in many other fields of biology. The acronym F. A.C.S. stands for Fluorescence Activated CeU Sorting, and is used interchangeably with "flow cytometry". The principle of FACS is that individual cells, held in a thin stream of fluid, are passed through one or more laser beams, causing Hght to be scattered and fluorescent dyes to emit light at various frequencies. PhotomultipHer tubes (PMT) convert Hght to electrical signals, which are interpreted by software to generate data about the ceUs. Sub- populations of cells with defined characteristics can be identified and automatically sorted from the suspension at very high purity (-100%).
FACS can be used to measure gene expression in cells transfected with recombinant DNA encoding polypeptides. This can be achieved dkectly, by labelling of the protein product, or indkectly by using a reporter gene in the construct. Examples of reporter genes are β-galactosidase and Green Fluorescent Protein (GFP). β-galactosidase activity can be detected by FACS using fluorogenic substrates such as fluorescein digalactoside (FDG). FDG is introduced into ceUs by hypotonic shock, and is cleaved by the enzyme to generate a fluorescent product, which is trapped within the cell. One enzyme can therefore generate a large amount of fluorescent product. CeUs expressing GFP constructs wUl fluoresce without the addition of a substrate. Mutants of GFP are available which have different excitation frequencies, but which emit fluorescence in the same channel. In a two-laser FACS machine, it is possible to distinguish cells which are excited by the different lasers and therefore assay two transfections at the same time.
Alternative means of cell sorting may also be employed. For example, the invention comprises the use of nucleic acid probes complementary to mRNA. Such probes can be used to identify cells expressing mRNA for polypeptides individuaUy, such that they may subsequently be sorted either manually, or using FACS sorting. Nucleic acid probes complementary to mRNA may be prepared according to the teaching set forth above, using the general procedures as described by Sambrook et al (1989).
In a prefened embodiment, the invention comprises the use of an antisense nucleic acid molecule, complementary to a mRNA, conjugated to a fluorophore which may be used in FACS ceU sorting.
Methods have also been described for obtaining information about gene expression and identity using so-caUed gene chip aπays or high density DNA anays (Chee). These high density anays are particularly useful for diagnostic and prognostic purposes. Use may also be made of In Vivo Expression Technology (JNET) (CamUH). IVET identifies genes upregulated during say treatment or disease when compared to laboratory culture.
The advantage of using a protein assay is that Notch activation can be dkectly measured. Assay techniques that can be used to determine levels of a polypeptide are well known to those skiUed in the art. Such assay methods include radioknmunoassays, competitive- binding assays, Western Blot analysis, antibody sandwich assays, antibody detection, FACS and ELISA assays.
The modulator of Notch signalling may also be an immune ceU which has been treated to modulate expression or interaction of Notch, a Notch ligand or the Notch signalling pathway. Such ceUs may readily be prepared, for example, as described in WO 00/36089 in the name of Lorantis Ltd, the text of which is herein incorporated by reference.
Pharmaceutical Compositions
Suitably active agents are administered in combination with a pharmaceuticaUy acceptable diluent, carrier, or excipient (ie as a pharmaceutical composition). The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
Acceptable carriers or diluents for therapeutic use are weU known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Pubhshing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or dUuent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Preservatives, stabUizers, dyes and even flavoring agents may also be provided in the pharmaceutical composition as appropriate. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents maybe also used. For some applications, active agents may be administered orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixks, solutions or suspensions containing flavouring or colouring agents.
Alternatively or in addition, active agents may be administered by inhalation, intranasaUy or in the form of aerosol, or in the form of a suppository or pessary, or they may be applied topicaUy in the form of a lotion, solution, cream, ointment or dusting powder. An alternative means of transdermal administration is by use of a skin patch. For example, they can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or Hquid paraffin. They can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabuisers and preservatives as may be requked.
Active agents such as polynucleotides and proteins/polypeptides may also be administered by vkal or non-vkal techniques. Vkal dehvery mechanisms include but are not limited to adenovkal vectors, adeno-associated vkal (AAV) vectors, herpes vkal vectors, retrovkal vectors, lentivkal vectors, and baculovkal vectors. Non-vkal delivery mechanisms include lipid mediated transfection, liposomes, immunoHpo somes, Hpoiectin, cationic facial amphiphiles (CFAs) and combinations thereof. The routes for such deHvery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes. Active agents may be adminstered by conventional DNA deHvery techniques, such as DNA vaccination etc., or injected or otherwise delivered with needleless systems, such as ballistic delivery on particles coated with the DNA for delivery to the epidermis or other sites such as mucosal surfaces.
TypicaUy, the physician wUl determine the actual dosage which will be most suitable for an individual patient and it wiU vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be 101
be noted that whilst the above-mentioned dosages are exemplary of the average case there can, of course, be individual instances where higher or lower dosage ranges are merited and such dose ranges are within the scope of this invention.
The routes of administration and dosages described are intended only as a guide since a skUled practitioner wUl be able to determine readily the optimum route of administration and dosage for any particular patient depending on, for example, the age, weight and condition of the patient.
The term treatment or therapy as used herein should be taken to encompass diagnostic and prophylatic appHcations.
The treatment of the present invention includes both human and veterinary applications.
Active agents may also be administered by any suitable means including, but not Hmited to, traditional syringes, needleless injection devices, or "microprojectUe bombardment gene guns". Alternatively, active agents such as polynucleotides may be introduced by various means into ceUs that are removed from an individual. Such means include, for example, ex vivo transfection, electioporation, nucleoporation, rnicroinjection and microprojectUe bombardment. After an agent has been taken up by the cells, they may be reimplanted into an individual. It is also contemplated that otherwise non-knmunogenic ceUs that have gene constructs incorporated therein can be implanted into an individual even if the vaccinated ceUs were originally taken from another individual.
According to some prefened embodiments of the present invention, the active agent may be administered to an individual using a needleless injection device. For example, an active agent may be administered to an individual intradermally, subcutaneously and/or intramuscularly using a needleless injection device , or similarly dehvered to mucosal tissues of, for example, the respkatory, gastrointestinal or urinogenital tracts. Needleless injection devices are well known and widely available. Needleless injection devices are - 102 -
especiaUy well suited to dehver genetic material to tissues. They are particularly useful to deliver genetic material to skin and muscle cells. In some embodiments, for example, a needleless injection device may be used to propel a liquid that contains DNA molecules toward the surface of the individual's skin. The liquid is propeUed at a sufficient velocity such that upon impact with the skin the liquid penetrates the surface of the skin and permeates the skin and/or muscle tissue beneath. Thus, the genetic material is simultaneously or selectively administered intradermally, subcutaneously and intramuscularly. In some embodiments, a needleless injection device may be used to deliver genetic material to tissue of other organs in order to introduce a nucleic acid molecule to ceUs of that organ.
Preferably the pharmaceutical preparations according to the present invention are provided sterile and pyrogen free.
Pharmaceutical Adrmnistration
TypicaUy, a physician wUl determine the actaal dosage which wiU be most suitable for an individual subject and it wiU vary with the age, weight and response of the particular patient. The dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
It will be appreciated that in one embodiment the therapeutic agents used in the present invention may be administered dkectly to patients in vivo. Alternatively or in addition, the agents may be administered to immune ceUs such as T cells and/or APCs in an ex vivo manner. For example, leukocytes such as T cells or APCs may be obtained from a patient or donor in known manner, treated/incubated ex vivo in the manner of the present invention, and then administered to a patient. - 103
In general, a therapeuticaUy effective daUy dose of the conjugate of the active agent according to the invention may for example range from 0.01 to 50 mg/kg body weight of the subject to be treated, preferably 0.1 to 20 mg/kg.
A skilled practitioner will be able to deteπnine readUy the optimum route of administration and dosage for any particular patient depending on, for example, the age, weight and condition of the patient. Preferably the pharmaceutical compositions are in unit dosage form. The present invention includes both human and veterinary appHcations.
By "simultaneously" is meant that the modulator of the Notch signalling pathway and the pathogen antigen, antigenic deteπninant or the polynucleotide coding for the pathogen antigen or antigenic determinant are administered at substantially the same time, and preferably together in the same formulation.
By "contemporaneously" it is meant that the modulator of the Notch signaUing pathway and the pathogen antigen, antigenic determinant or the polynucleotide coding for the pathogen antigen or antigenic determinant are administered closely in time, e.g., the the pathogen antigen, antigenic determinant or the polynucleotide coding for the pathogen antigen or antigenic determinant is administered within from about one minute to within about one day before or after the modulator of the Notch signalling pathway is administered. Any contemporaneous time is useful. However, it wUl often be the case that when not adrnrnistered simultaneously, the modulator of the Notch signalling pathway and the pathogen antigen, antigenic detenriinant or the polynucleotide coding for the pathogen antigen or antigenic determinant wUl be administered within about one minute to within about eight hours, and preferably within less than about one to about four hours. When administered contemporaneously, the modulator of the Notch signaUing pathway and the pathogen antigen, antigenic determinant, or the polynucleotide coding for the pathogen antigen or antigenic determinant are preferably administered at the same site on the animal. The term "same site" includes the exact location, but can be - 104 -
within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters.
The term "separately" as used herein means that the modulator of the Notch signalling pathway and the pathogen antigen, antigenic determinant or the polynucleotide coding for the pathogen antigen or antigenic determinant are administered at an interval, for example at an interval of about a day to several weeks or months. The active agents may be administered in either order.
Likewise, the modulator of the Notch signalling pathway may be administered more frequently than the pathogen antigen, antigenic deteiminant or the polynucleotide coding for the pathogen antigen or antigenic determinant or vice versa.
The term "sequentially" as used herein means that the modulator of the Notch signalling pathway and the pathogen antigen, antigenic determinant or the polynucleotide coding for the pathogen antigen or antigenic determinant are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the active agents may be administered in a regular repeating cycle.
Vaccine Compositions
Vaccine compositions and preparations made in accordance with the present invention may be used to protect or treat a mammal susceptible to, or suffering from disease, by means of administering said vaccine via a mucosal route, such as the oral/bucal/intestinal/vaginal/rectal or nasal route. Such administration may be in a droplet, spray, or dry powdered form. Nebulised or aerosohsed vaccine formulations may also be used where appropriate.
Enteric formulations such as gastro resistant capsules and granules for oral administration, suppositories for rectal or vaginal administration may also be used. The present invention may also be used to enhance the immunogenicity of antigens appHed to 105 -
the skin, for example by intradermal, transdermal or transcutaneous deHvery. In addition, the adjuvants of the present invention may be parentaUy delivered, for example by intramuscular or subcutaneous administration.
Depending on the route of administration, a variety of administration devices may be used. For example, for intranasal administration a spray device such as the commercially avaUable Accuspray (Becton Dickinson) may be used.
Prefened spray devices for intranasal use are devices for which the performance of the device is not dependent upon the pressure appHed by the user. These devices are known as pressure threshold devices. Liquid is released from the nozzle only when a threshold pressure is attained. These devices make it easier to achieve a spray with a regular droplet size. Pressure threshold devices suitable for use with the present invention are known in the art and are described for example in WO 91/13281 and EP 311 863 B. Such devices are commercially avaUable from Pfeiffer GmbH.
For certain vaccine formulations, other vaccine components may be included in the formulation. For example the adjuvant formulations of the present invention may also comprise abUe acid or derivative of cholic acid. Suitably the derivative of cholic acid is a salt thereof, for example a sodium salt thereof. Examples of bile acids include cholic acid itself, deoxychohc acid, chenodeoxy colic acid, Hthocholic acid, taurodeoxycholate ursodeoxychohc acid, hyodeoxycholic acid and derivatives like glyco-, tauro-, amidoproρyl-1- propanesulfonic- and amidoρropyl-2 -hydroxy- 1 -pro anesulfonic- derivatives of the above bile acids, orN, N-bis (3DGluconoamidopropyl) deoxycholamide.
Suitably, the adjuvant formulation of the present invention may be in the form of an aqueous solution or a suspension of non- vesicular forms. Such formulations are convenient to manufacture, and also to sterilise (for example by terminal filtration through a 450 or 220 nm pore membrane). - 106 -
Suitably, the route of administration to said host is via the skin, intramuscular or via a mucosal surface such as the nasal mucosa. When the admixture is administered via the nasal mucosa, the admixture may for example be administered as a spray. The methods to enhance an immune response may be either a priming or boosting dose of the vaccine.
The term "adjuvant" as used herein includes an agent having the abUity to enhance the immune response of a vertebrate subject's immune system to an antigen or antigenic deteπninant.
The term "immune response" includes any response to an antigen or antigenic deteπninant by the immune system of a subject. Immune responses include for example humoral immune responses (e. g. production of antigen-specific antibodies) and cell- mediated immune responses (e. g. lymphocyte prohferation).
The term "ceU-mediated immune response" includes the immunological defence provided by lymphocytes, such as the defence provided by T cell lymphocytes when they come into close proximity with thek victim cells.
When "lymphocyte prohferation" is measured, the abUity of lymphocytes to prohferate in response to specific antigen may be measured. Lymphocyte prohferation includes B ceU, T-helper cell or CTL ceU proliferation.
Compositions of the present invention may be used to formulate vaccines containing antigens derived from a wide variety of sources. For example, antigens may include human, b cterial, or vkal nucleic acid, pathogen derived antigen or antigenic preparations, host-derived antigens, including GhRH and IgE peptides, recombinantly produced protem or peptides, and chimeric fusion proteins.
Preferably the vaccine formulations of the present invention contain an antigen or 107 -
antigenic composition capable of eliciting an immune response against a human pathogen. The antigen or antigens may, for example, be pep tides/proteins, polysaccharides and lipids and may be derived from pathogens such as viruses, bacteria and parasites/fungi as follows:
Vkal antigens
Vkal antigens or antigenic determinants may be derived, for example, from:
Cytomegalo virus ( especiaUy Human, such as gB or derivatives thereof); Epstein Ban virus (such as gp350); flaviviruses (e. g. Yellow Fever Virus, Dengue Vkus, Tick-bome encephalitis virus, Japanese Encephalitis Vkus); hepatitis vkus such as hepatitis B virus (for example Hepatitis B Surface antigen such as the PreSl, PreS2 and S antigens described in EP-A-414 374; EP-A-0304 578, and EP-A-198474), hepatitis A virus, hepatitis C virus and hepatitis E virus; HIN-1, (such as tat, nef, gpl20 or gpl60); human herpes viruses, such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSN1 or HSN2; human papffloma viruses (for example HPN6, 11, 16, 18); Influenza virus (whole Hve or inactivated virus, spht influenza vkus, grown in eggs or MDCK cells, or Vero cells or whole flu vkosomes (as described by Gluck, Vaccine, 1992,10, 915-920) or purified or recombinant proteins thereof, such as ΝP, ΝA, HA, or M proteins); measles virus; mumps virus; parainfluenza virus; rabies virus; Respkatory Syncytial virus (such as F and G proteins); rotavirus (including Hve attenuated viruses); smaUpox virus; Varicella Zoster Virus (such as gpl, II and JE63); and the HPV viruses responsible for cervical cancer (for example the early proteins E6 or E7 in fusion with a protein D carrier to form Protein D-E6 or E7 fusions from HPV 16, or combinations thereof; or combinations of E6 or E7 with L2 (see for example WO 96/26277).
Bacterial antigens
Bacterial antigens or antigenic determinants may be derived, for example, from: 108 -
BacUlus spp., including B. anthracis (eg botalinum toxin); Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin, filamenteous hemagglutinin, adenylate cyclase, fimbriae); BoneHa spp., including B. burgdorferi (eg OspA, OspC, DbpA, DbpB), B. garinu (eg OspA, OspC, DbpA, DbpB), B. afzehi (eg OspA, OspC, DbpA, DbpB), B. andersonii (eg OspA, OspC, DbpA, DbpB), B. hermsii; Campylobacter spp, including C. jejuni (for example toxins, adhesins and invasins) and C. coh; Chlamydia spp., including C. trachomatis (eg MOMP, hep arm-binding proteins), C. pneumonie (eg MOMP, heparin-binding proteins), C. psittaci; Clostridium spp., including C. tetani (such as tetanus toxin), C. botuhnum (for example botalinum toxin), C. difficile (eg clostridium toxins A or B); Corynebacterium spp., including C. diphtheriae (eg diphtheria toxin); EhrHchia spp., including E. equi and the agent of the Human Granulocytic EhrUchiosis; Rickettsia spp, including R.rickettsii; Enterococcus spp., including E. faecahs, E. faecium; Escherichia spp, including enterotoxic E. coli (for example colonization factors, heat-labile toxin or derivatives thereof, or heat-stable toxin), enterohemoπagic E. coh, enteropathogenic E. coh (for example shiga toxin-like toxin); HaemophUus spp., including H. influenzae type B (eg PRP), non-typable H. influenzae, for example OMP26, high molecular weight adhesins, P5, P6, protein D and lipoprotein D, and fimbrin and i nbrin derived peptides (see for example US 5,843,464); Helicobacter spp, including H. pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa; Legionella spp, including L. pneumophUa ; Leptospka spp., including L. inteπogans; Listeria spp., including L. monocytogenes; MoraxeUa spp, including M cataπhahs, also known as Branhamella catanhalis (for example high and low molecular weight adhesins and invasins); Morexella Cataπhahs (including outer membrane vesicles thereof, and OMP106 (see for example W097/41731)); Mycobacterium spp., including M. tuberculosis (for example ESAT6, Antigen 85 A, -B or -C), M. bovis, M. leprae, M. avium, M. parataberculosis, M. smegmatis; Neisseria spp, including N. gononhea and N. meningitidis (for example capsular polysaccharides and conjugates thereof, transferrin- binding proteins, lactoferrin binding proteins, PilC, adhesins); Neisseria mengitidis B (including outer membrane vesicles thereof, and NspA ( see for example WO 96/29412); 109
Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Staphylococcus spp., including S. aureus, S. epidermidis; Streptococcus spp, including S. pneumonie (eg capsular polysaccharides and conjugates thereof, PsaA, PspA, streptolysin, choline-binding proteins) and the protein antigen Pneumolysin (Biochem Biophys Acta, 1989,67,1007; Rubins et al., Microbial Pathogenesis, 25,337-342), and mutant detoxified derivatives thereof (see for example WO 90/06951 ; WO 99/03884); Treponema spp., including T. pallidum (eg the outer membrane proteins), T. denticola, T. hyodysenteriae; Vibrio spp, including V. cholera (for example cholera toxin); and Yersinia spp, including Y. enterocolitica (for example a Yop protein), Y. pestis, Y. pseudotuberculosis.
Parasite/Fungal antigens
Parasitic/fungal antigens or antigenic determinants may be derived, for example, from:
Babesia spp., including B. microti; Candida spp., including C. albicans; Cryptococcus spp., including C. neoformans; Entamoeba spp., including E. histolytica; Giardia spp., including ;G. lambha; Leshmania spp., including L. major; Plasmodium. faciparum (MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin, PfEMPl, Pf332, LSA1, LSA3, STARP, SALSA, PfEXPl, Pfs25, Pfs28, PFS27/25, Pfsl6, Pfs48/45, Pfs230 and thek analogues in Plasmodium spp.); Pneumocystis spp., including P. ;carinii; Schisostoma spp., including S. mansoni; Trichomonas spp., including T. vaginahs; Toxoplasma spp., including T. gondii (for example SAG2, S AG3, Tg34); Trypanosoma spp., including T. cruzi.
Approved/Hcensed vaccines include, for example anthrax vaccines such as Biothrax (BioPort Corp); tuberculosis (BCG) vaccines such as TICE BCG (Organon Teknika Corp) and My cob ax (Aventis Pasteur, Ltd); diphtheria & tetanus toxoid and aceUular pertussis (DTP) vaccines such as Tripedia (Aventis Pasteur, Inc), fnfanrix (GlaxoSmithKline), and DAPTACEL (Aventis Pasteur, Ltd); Haemophilus b conjugate vaccines (eg diphtheria CRM197 protein conjugates such as HibTITER from Lederle Lab 110 -
Div, American Cyanamid Co; meningococcal protein conjugates such as PedvaxHTB from Merck & Co, Inc; and tetanus toxoid conjugates such as ActHIB from Aventis Pasteur, SA); Hepatitis A vaccines such as Havrix (Glaxo SmithKHne) and VAQTA (Merck & Co, Inc); combined Hepatitis A and Hepatitis B (recombinant) vaccines such as Twinrix (GlaxoSmithKline); recombinant Hepatitis B vaccines such as Recombivax HB (Merck & Co, Inc) and Engerix-B (GlaxoSmithKline); influenza virus vaccines such as Fluvirin (Evans Vaccine), FluShield (Wyeth Laboratories, Inc) and Fluzone (Aventis Pasteur, Inc); Japanese Encephahtis virus vaccme such as JE-Vax (Research Foundation for Microbial Diseases of Osaka University); Measles virus vaccines such as Attenuvax (Merck & Co, Inc); measles and mumps vkus vaccines such as M-M-Vax (Merck & Co, Inc); measles, mumps, and rubeUa virus vaccines such as M-M-R II (Merck & Co, Inc); meningococcal polysaccharide vaccines (Groups A, C, Y and W-135 combined) such as Menomune-A/C/Y/W-135 (Aventis Pasteur, Inc); mumps virus vaccines such as Mumpsvax (Merck & Co, Inc); pneumococcal vaccines such as Pneumovax (Merck & Co, Inc) and Pnu-Jmune (Lederle Lab Div, American Cyanamid Co); Pneumococcal 7- valent conjugate vaccines (eg diphtheria CRM197 Protein conjugates such as Prevnar from Lederle Lab Div, American Cyanamid Co); polio vkus vaccines such as Poliovax (Aventis Pasteur, Ltd); poliovkus vaccines such as IPOL (Aventis Pasteur, SA); rabies vaccines such as hnovax (Aventis Pasteur, SA) and RabAvert (Chkon Behring GmbH & Co); rubella vkus vaccines such as Meruvax II (Merck & Co, Inc); Typhoid Vi polysaccharide vaccines such as TYPHJM Vi (Aventis Pasteur, S A); VariceUa virus vaccines such as Varivax (Merck & Co, Inc) and Yellow Fever vaccines such as YF-Vax (Aventis Pasteur, Inc).
It wUlbe appreciated that in accordance with this aspect of the present invention antigens and antigenic determinants may be used in many different forms. For example, antigens or antigenic determinants may be present as isolated proteins or peptides (for example in so-caUed "subunit vaccines") or, for example, as ceU-associated or virus-associated antigens or antigenic determinants (for example in either live or kUled pathogen strains). Live pathogens wUl preferably be attenuated in known manner. Alternatively, antigens or - Ill —
antigenic determinants may be generated in situ in the subject by use of a polynucleotide coding for an antigen or antigenic deteπriinant (as in so-caUed "DNA vaccination", although it will be appreciated that the polynucleotides which may be used with this approach are not limited to DNA, and may also include RNA and modified polynucleotides as discussed above).
As used herein, the term "genetic vaccine" refers to a pharmaceutical preparation that comprises a polynucleotide (eg DNA) constmct. Genetic vaccines include pharmaceutical preparations useful to invoke a prophylactic and/or therapeutic immune response. Therapeutic vaccines may also be refeπed to as "Pharmacines".
As discussed, for example, in US 6025341 and elsewhere, dkect injection of polynucleotides such as DNA is a promising method for delivering antigens for irnmunization (Barry, et al., Bio Techniques, 1994, 16, 616-619; Davis, et al., Hum. Mol. Genet., 1993, 11, 1847-1851; Tang, et al., Nature, 1992, 356, 152-154; Wang, et al., J. Virol., 1993, 67, 3338-3344; and Wolff, et al., Science, 1990, 247, 1465-1468). This approach has been successfully used to generate protective immunity against influenza virus in mice and chickens, against bovine herpes virus 1 in mice and cattle and against rabies vkus in mice (Cox, et al, J. Virol., 1993, 67, 5664-5667; Fynan, et al., DNA and Cell Biol, 1993, 12, 785-789; Ulmer, et al., Science, 1993, 259, 1745-1749; and Xiang, et al., Virol, 1994, 199, 132-140). In most cases, strong, yet highly variable, antibody and cytotoxic T-cell responses were associated with control of infection. Indeed, the potential to generate long-lasting memory CTLs without using a Hver vector makes this approach particularly attractive compared with those involving kiUed-vkus vaccines and generating a CTL response that not only protects against acute infection but also may have benefits in eradicating persistent vkal infection (Wolff, et al., Science, 1990, 247, 1465-1468; Wolff, et al., Hum. Mol. Genet., 1992, 1, 363-369; Manthorpe, et al., Human Gene Therapy, 1993, 4, 419-431; Ulmer, et al., Science, 1993, 259, 1745-1749; Yankauckas, et al., DNA and Cell Biol, 1993, 12, 777-783; Montgomery, et al., DNA and CeU Biol, 1993, 12, 777-783; Fynan, et al, DNA and Cell Biol., 1993, 12, 785-789; 113 -
treating a mammal susceptible to or suffering from an infectious disease. In a further aspect of the present invention there is provided an adjuvant combination and a vaccine as herein described for use in medicine. Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al, University Park Press, Baltimore, Maryland, U. S. A. 1978.
It wUl be appreciated that the adjuvants of the present invention may further be combined with other adjuvants including, for example: Cholera toxin and its B subunit; E. Coh heat labile enterotoxin LT, its B subunit LTB and detoxified versions thereof such as mLT; immunologicaUy active saponin fractions e. g. Quil A derived from the bark of the South American tree QuUlaja Saponaria Molina and derivatives thereof (for example QS21, as described in US 5,057,540); the oligonucleotide adjuvant system CpG (as described in WO 96/02555), especially 5'TCGTCGTTT TGT CGT TTT GTC GTT3 (SEQ JD NO: 1); and Monophosphoryl Lipid A and its non-toxic derivative 3-O-deacylated monophosphoryl lipid A (3D-MPL, as described in GB 2,220,211).
The present invention provides an increased magnitude and/or increased duration of immune response. Preferably the invention provides an increased protective immune response.
The present invention also contemplates generating selective Thl or Th2 immunity. In general, T cells can act in different subpopulations that show different effector functions. T cell responses can be pro-inflammatory T helper 1 type (Thl) characterized by the secretion of interferon gamma (IFN-gamma.) and interleukin 2 (IL-2). Thl ceUs are the helper ceUs for the ceUular defence but provide Httle help for antibody secretion. The other class of T cell responses is generally anti-inflammatory, and is mediated by Th2 ceUs that produce JL-4, B -5 and IL-10, but Httle or no IL-2 or IFN-gamma. Th2 ceUs are the helper ceUs for antibody production. CD4+ and CD 8+ cells both occur in these subpopulations: Thl/Th2:CD4, Tcl/Tc2:CD8. - 114
For each type of pathogen infection there maybe an "appropriate" (and different) type of T cell response (e.g., Thl vs. Th2, CD4+ vs. CD8+) that combats the infectious agent but does not cause excessive tissue damage in the subject. It may be detrimental to the subject if an "inappropriate" type of T ceU response is induced (Thl instead of Th2, or vice versa). Generally, one would want to induce the Thl response to clear an intracellular pathogen such as a vkus or intracellular bacterium and a Th2 response to clear an extracellular pathogen.
It wUl be appreciated that the present invention may be used in both so-called prophylactic and so-called therapeutic vaccines.
For example, prophylactic vaccines may be used to provide protective immunity in an uninfected subject to provide protection against future establishment of infection.
Conversely, therapeutic vaccines may be used, for example, after an infection has become established (for example as either an acute or chronic infection) in order to increase the immune response against the infection. Suitably, therapeutic vaccines may be used to combat chronic infections which may for example be bacterial infections (such as tuberculosis), parasitic infections such as malarial infections or vkal infections (such as HPV, HCV, HB V or HIV infections).
Examples of chronic infections associated with significant morbidity and early death include human hepatitis viruses such as hepatitis A, B, C, D and E, for example hepatitis B virus (HBV) and hepatitis C virus (HCV) which cause chronic hepatitis, cirrhosis and Hver cancer (see US 5738852).
Additional examples of chronic infections caused by vkal infectious agents include those caused by the human retro viruses: human immunodeficiency viruses (HIN-1 and HTV-2), which cause acquked immune deficiency syndrome (AIDS); and human T lymphotropic viruses (HTLN-1 and HTLN-2) which cause T ceU leukemia and myelopathies. Many - 115 -
other infections such as human herpes viruses including the herpes simplex vkus (HSV) types 1 and 2, Epstein Ban virus (EBV), cytomegalo virus (CMV), varicella-zoster vkus (VZV) and human herpes virus 6 (HHV-6) are often not eradicated by host mechanisms, but rather become chronic and in this state may cause disease. Chronic infection with human papiUoma viruses is associated with cervical carcinoma. Numerous other viruses and other infectious agents replicate intracellularly and may become chronic when host defense mechanisms faU to eliminate them. These include pathogenic protozoa (e.g., Pneumocystis carinii, Trypanosoma, Leishmania, Plasmodium (responsible for Malaria) and Toxoplasma gondii), bacteria (e.g., mycobacteria (eg Mycobacterium tuberculosis responsible for tuberculosis), salmonella and listeria), and fungi (e.g., Candida and aspergiUus).
The pathogen antigen is suitably an antigen that is nataraUy encoded in the pathogen against which an enhanced or augmented immune response is desked.
The nucleotide sequences of a large number of bacteria, protozoans and viruses, including different species, strains, and isolates are known in the art (see, for example Levy, Microbiological Reviews, 57:183-289 (1993) (HIV); and Choo et al., Seminars in Liver Disease, 12:279-288 (1992) (HCV)). Particularly suitable target antigens are those which induce a T cell response, and particularly a CTL-response during infection. These may include, for example, from HBV, the core antigen (HBcAg) the E antigen, and the surface antigen (HBsAg). Polynucleotide sequences for HBsAg including the pre-Sl, pre- S2 and S regions from a variety of surface antigen subtypes are well known in the art (see, for example, Okamoto et al, J. Gen. Virol., 67:1383-1389 (1986); GenBank Accession numbers D00329 and D00330). The polynucleotide sequences encoding HJV glycoprotein gρl60 and other antigenic HJV regions are known in the art (Lautenberger et al, Nature, 313:277-284 (1985); Starcich et al, CeU, 45:637-648 (1986); Wiley et al., Proc. Natl. Acad. Sci. USA, 83:5038-5042 (1986); and Modrow et al., J. Virol., 61:570- 578 (1987)). - 116
For example, the genome for Human immunodeficiency virus type 1 (HXB2; HIVl/HTLV-ITI/LAV reference genome) is provided at GenBank Accession No K03455, which reports sequences for various HJV antigenic proteins.
Numerous genome sequences for HAV, HB V and HCV strains (including sequences for antigenic proteins) are provided on GenBank, for example AY057948 (Hepatitis B virus isolate Tibetl27, complete genome); AY057947 (Hepatitis B virus isolate Tibet705, complete genome); NC_003977 (Hepatitis B vkus, complete genome); NC_004102 (Hepatitis C vkus, complete genome); AF139594 (Hepatitis C virus strain HCV-N, complete genome) ; M16632 (Hepatitis A virus (HM-175 strain; attenuated)).
fh one embodiment the modulator/inhibitor of Notch signaUing increases cytotoxic (CD 8+) T ceU responses to antigen.
Conjugates
As noted above, the invention further provides a conjugate comprising first and second sequences, wherein the first sequence comprises a pathogen antigen or a polynucleotide sequence coding for such an antigen 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.
Where 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 a pathogen antigen or antigenic deteπninant and a polynucleotide sequence coding for a modulator of the Notch signalling pathway, wherein preferably each sequence is operably finked to regulatory elements necessary for expression in eukaryotic ceUs. A schematic representation of one such form of vector is shown in Figure 11. - 117 -
Suitably the polynucleotide sequence coding for the modulator of the Notch signaUing 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. Where intended for human therapy, suitably sequences based on human sequences may be used.
Preferably the polynucleotide sequence coding for the modulator of the Notch signalling pathway may be a nucleotide sequence coding for a Notch Hgand 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-Hke domains. Suitably the DSL and EGF-like domain sequences are or coπespond to mammahan sequences. Suitably the polynucleotide sequence coding for the modulator of the Notch signaUing pathway may further comprise a transmembrane domain and, suitably, a Notch ligand intracellular domain. Prefeπed sequences include human sequences such as human Deltal, Delta3, Delta4, Jaggedl or Jagged2 sequences.
ff desked, the polynucleotide sequence that encodes the pathogen antigen or antigenic determinant may further include a nucleotide sequence that encodes a signal sequence which directs trafficking of the antigen or antigenic deterrninant within a cell to which it is administered. For example, such a signal sequence may dkect 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. In addition, other elements, such as a Kozak region, may also be included if desired. Initiation and teπnination signals are regulatory elements which are often considered part of the coding sequence.
Examples of suitable promoters include but are not limited to promoters from Simian Vkus 40 (S V40), Mouse Mammary Tumor Vkus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HJV Long Terminal Repeat (LTR) promoter, 118
Moloney virus, ALV, Cytomegalov us (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Vkus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metalothionein. Tissue-specific promoters specific for lymphocytes, dendritic cells, skin, brain ceUs and epithelial ceUs within the eye are particularly prefened, for example the CD2, CDllc, keratin 14, Wnt-1 and Rhodopsin promoters respectively. Suitably an epithelial cell promoter such as SPC may be used.
Examples of suitable polyadenylation signals include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals. For example, the SV40 polyadenylation signal used in plasmid pCEP4 (Invitrogen, San Diego Calk), refened to as the SV40 polyadenylation signal, may be used.
In addition to the regulatory elements requked for DNA expression, other elements may also be included in the conjugate. Such additional elements include enhancers which may, for example, be selected from human Actin, human Myosin, human Hemoglobin, human muscle creatine and vkal enhancers such as those from CMV, RSV and EBV.
When administered to and taken up by a cell, 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. Alternatively, linear DNA which can integrate into the chromosome may be introduced into the ceU. When introducing DNA into the ceU, 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. Alternatively, 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 rephcation. - 119
If desked, conjugates may be provided with mammalian origin of replication in order to maintain the constract extrachromosomaUy and produce multiple copies of the constract in the cell. For example, plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calk.) contain the Epstein Ban virus origin of rephcation and nuclear antigen EBNA-1 coding region which produces high copy episomal rephcation without integration.
In order to maximize protein production, regulatory sequences may be selected which are well suited for gene expression in the type of ceUs the constract is to be administered to. Moreover, codons may be selected which are most efficiently transcribed in the cell.
Such conjugates may be used either in vivo or ex-vivo with a "genetic vaccination" approach to provide expression of both an inhibitor of Notch signalling and a pathogen antigen or antigenic determinant in cells or tissues.
Facilitating Agents
In some embodiments, polynucleotides may be delivered in conjunction with administration of a facihtating 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, anUides, amidines, urethans and the hydrochloride salts thereof such as those of the farnUy of local anesthetics.
Examples of esters include: benzoic acid esters such as piperocaine, meprylcaine and isobucarne; para-aminobenzoic acid esters such as procaine, tetracaine, butemamine, propoxycaine and chloroprocaine; meta-aminobenzoic acid esters including metabutharnine and primacaine; and para-ethoxybenzoic acid esters such as parethoxycaine. Examples of anUides include Hdocaine, etidocaine, mepivacaine, - 120 -
bupivacaine, pynocaine and prilocaine. Other examples of such compounds include dibucaine, benzocaine, dyclonine, pramoxine, proparacaine, butacaine, benoxinate, carbocaine, methyl bupivacaine, butas n picrate, phenacaine, diothan, luccaine, intracaine, nupercaine, metabutoxycaine, piridocaine, biphenamine and the botanically- derived bicyclics such as cocaine, cinnamoylcocaine, traxiUrne and cocaethylene and aU such compounds complexed with hydrochloride.
The facUitating 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 chemicaUy designated as 2-piperidinecarboxamide, l-butyl-N-(2,6- dknethylρhenyl)-monohydrochloride, monohydrate and is widely available commerciaUy 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 commerciaUy 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 deskable effects may be prepared if desked. Suitably, for example, about 250μg to about 10 mg of bupivacaine may be administered.
Antigen Presenting Cells
Where requked, antigen-presenting cells (APCs) may be "professional" antigen presenting cells or may be another cell that may be induced to present antigen to T ceUs. Alternatively a APC precursor may be used which dkferentiates or is activated under the conditions of culture to produce an APC. An APC for use in the ex vivo methods of the invention is typically isolated from a tumour or peripheral blood found within the body of - 121
a patient. Preferably the APC or precursor is of human origin. However, where APCs are used in preliminary in vitro screening procedures to identify and test suitable nucleic acid sequences, APCs from any suitable source, such as a healthy patient, may be used.
APCs include dendritic ceUs (DCs) such as interdigitating DCs or foUicular DCs, Langerhans cells, PBMCs, macrophages, B -lymphocytes, or other cell types such as epithelial cells, fibroblasts or endothelial ceUs, activated or engineered by transfection to express a MHC molecule (Class I or U) on thek surfaces. Precursors of APCs include CD34+ cells, monocytes, fibroblasts and endothelial cells. The APCs or precursors may be modified by the cultare conditions or may be geneticaUy modified, for instance by transfection of one or more genes encoding proteins which play a role in antigen presentation and/or in combination of selected cytokine genes which would promote to immune potentiation (for example IL-2, IL-12, IFN-γ, TNF-α, IL-18 etc.). Such proteins include MHC molecules (Class I or Class IT), CD80, CD86, or CD40. Most preferably DCs or DC-precursors are included as a source of APCs.
Dendritic ceUs (DCs) can be isolated/prepared by a number of means, for example they can either be purified dkectly from peripheral blood, or generated from CD34+ precursor ceUs for example after mobiHsation into peripheral blood by treatment with GM-CSF, or directly from bone maπow. From peripheral blood, adherent precursors can be tieated with a GM-CSF/TL-4 mixture (Inaba K, et al. (1992) J. Exp. Med. 175: 1157-1167 (Inaba)), or from bone manow, non-adherent CD34+ ceUs can be treated with GM-CSF and TNF-a (Caux C, et al. (1992) Nature 360: 258-261 (Caux)). DCs can also be routinely prepared from the peripheral blood of human volunteers, similarly to the method of SaUusto and Lanzavecchia (Sallusto F and Lanzavecchia A (1994) J. Exp. Med. 179: 1109-1118) using purified peripheral blood mononucleocytes (PBMCs) and treating 2 hour adherent cells with GM-CSF and JL-4. If requked, these may be depleted of CD19+ B cells and CD3+, CD2+ T cells using magnetic beads (Coffin RS, et al. (1998) Gene Therapy 5: 718-722 (Coffin)). Cultare conditions may include other cytokines such - 122 -
as GM-CSF or JL-4 for the maintenance and, or activity of the dendritic cells or other antigen presenting ceUs.
Thus, it wUl be understood that the term "antigen presenting cell or the like" are used herein is not intended to be limited to APCs. The skUled man wUl understand that any vehicle capable of presenting to the T ceU population may be used, for the sake of convenience the term APCs is used to refer to aU these. As indicated above, prefeπed examples of suitable APCs include dendritic ceUs, L cells, hybridomas, fibroblasts, lymphomas, macrophages, B ceUs or synthetic APCs such as lipid membranes.
T cells
Where requked, T ceUs from any suitable source, such as a healthy patient, may be used and may be obtained from blood or another source (such as lymph nodes, spleen, or bone maπow). They may optionally be enriched or purified by standard procedures. The T ceUs may be used in combination with other immune cells, obtained from the same or a different individual. Alternatively whole blood may be used or leukocyte enriched blood or purified white blood ceUs as a source of T cells and other cell types. It is particularly prefened to use helper T cells (CD4+). Alternatively other T ceUs such as CD8+ ceUs may be used. It may also be convenient to use ceU Hnes such as T ceU hybridomas.
Thus, it wUl be understood that the term "antigen presenting cell or the like" are used herein is not intended to be limited to APCs. The skUled man wUl understand that any vehicle capable of presenting to the T ceU population may be used, for the sake of convenience the term APCs is used to refer to aU these. As indicated above, prefeπed examples of suitable APCs include dendritic ceUs, L cells, hybridomas, fibroblasts, lymphomas, macrophages, B ceUs or synthetic APCs such as lipid membranes. - 123 -
Exposure of agent to APCs and T cells
T ceUs/APCs/tumour cells may be cultured as described above. The APCs/T ceUs/turnour ceUs may be incubated/exposed to substances which are capable of mterterring with or downregulating Notch or Notch Hgand expression. The resulting T ceUs/APCs/tumour cells that have downregulated Notch or Notch Hgand expression are now ready for use. For example, they may be prepared for administration to a patient or incubated with T cells in vitro (ex vivo).
For example, tumour material may be isolated and transfected with a nucleic acid sequence which encodes for, e.g., a ToU-like receptor or BMP receptor and/or costimulatory molecules (suitable costimulants are mentioned above) and/or treated with cytokines, e.g. IFN-γ, TNF-α, EL- 12, and then used in vitro to prime TRL and/or TIL ceUs.
Where treated ex-vivo, modified cells of the present invention are preferably administered to a host by dkect injection into the lymph nodes of the patient. TypicaUy from 104 to 108 treated ceUs, preferably from 105 to 107 cells, more preferably about 106 ceUs are administered to the patient. Preferably, the cells will be taken from an enriched ceU population.
As used herein, the term "enriched" as applied to the ceU populations of the invention refers to a more homogeneous population of ceUs which have fewer other cells with which they are natarally associated. An enriched population of ceUs can be achieved by several methods known in the art. For example, an enriched population of T-cells can be obtained using immunoaffinity chromatography using monoclonal antibodies specific for determinants found only on T-cells.
Enriched populations can also be obtained from mixed cell suspensions by positive selection (collecting only the desked cells) or negative selection (removing the undeskable ceUs). The technology for capturing specific cells on affinity materials is weU 124 -
known in the art (Wigzel, et al., J. Exp. Med., 128:23, 1969; Mage, et al, J. Imnmunol. Meth., 15:47, 1977; Wysocki, et al, Proc. Natl. Acad. Sci. U.S.A., 75:2844, 1978; Schrempf-Decker, et al, J. Immunol Meth., 32:285, 1980; Muller-Sieburg, et al., Cell, 44:653, 1986).
Monoclonal antibodies against antigens specific for matare, dkferentiated cells have been used in a variety of negative selection strategies to remove undesired cells, for example, to deplete T-ceUs or mahgnant ceUs from aUogeneic or autologous maπow grafts, respectively (Gee, et al., J.N.C.I. 80:154, 1988). Purification of human hematopoietic cells by negative selection with monoclonal antibodies and irnmunomagnetic microspheres can be accomplished using multiple monoclonal antibodies (Griffin, et al., Blood, 63:904, 1984).
Procedures for separation of cells may include magnetic separation, using antibodycoated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, for example, complement and cytotoxins, and "panning" with antibodies attached to a sohd matrix, for example, plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, for example, a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
It wUl be appreciated that in one embodiment the therapeutic agents used in the present invention may be administered dkectly to patients in vivo. Alternatively or in addition, the agents may be administered to cells such as T cells and/or APCs in an ex vivo manner. For example, leukocytes such as T ceUs or APCs may be obtained from a patient or donor in known manner, treated/incubated ex vivo in the manner of the present invention, and then administered to a patient. In addition, it will be appreciated that a combination of routes of administration may be employed if desked. For example, where appropriate one - 125 -
component (such as the modulator of Notch signalling) may be administered ex-vivo and the other may be administered in vivo, or vice versa.
Introduction of nucleic acid sequences into APCs and T-cells
T-ceUs and APCs as described above are cultured in a suitable culture medium such as DMEM or other defined media, optionaUy in the presence of fetal calf serum.
Polypeptide substances may be administered to T-ceUs and/or APCs by introducing nucleic acid constracts/vkal vectors encoding the polypeptide into ceUs under conditions that aUow for expression of the polypeptide in the T-ceU and/or APC. Similarly, nucleic acid constructs encoding antisense constructs may be introduced into the T-ceUs and/or APCs by transfection, viral infection or vkal transduction.
In a prefened embodiment, nucleotide sequences encoding the modulator(s) of Notch signaUing wUl be operably linked to control sequences, including promoters/enhancers and other expression regulation signals. . The term "operably linked" means that the components described are in a relationship perrnitting them to function in thek intended manner. A regulatory sequence "operably linked" to a coding sequence is peferably Hgated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
The promoter is typically selected from promoters which are functional in mammalian ceUs, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used. The promoter is typically derived from promoter sequences of vkal or eukaryotic genes. For example, it may be a promoter derived from the genome of a ceU in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of a-actin, b-actin, tabulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyravate kinase). Tissue-specific promoters speckle for lymphocytes, dendritic ceUs, - 126 -
skin, brain ceUs and epithelial cells within the eye are particularly prefened, for example the CD2, CDllc, keratin 14, Wht-1 and Rhodopsin promoters respectively. Preferably the epithelial ceU promoter SPC is used. They may also be promoters that respond to specific stirnuH, for example promoters that bind steroid hormone receptors. Vkal promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalo virus (CMV) JE promoter.
It may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the Hfe-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
Any of the above promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences. Chimeric promoters may also be used comprising sequence elements from two or more different promoters.
Alternatively (or in addition), the regulatory sequences may be cell specific such that the gene of interest is only expressed in ceUs of use in the present invention. Such ceUs include, for example, APCs and T-cells.
The resulting T-cells and/or APCs that comprise nucleic acid constructs capable of up- regulating Notch Hgand expression are now ready for use. If required, a smaU aliquot of ceUs may be tested for up-regulation of Notch Hgand expression as described above. The ceUs may be prepared for administration to a patient or incubated with T-ceUs in vitro (ex vivo).
Any of the assays described above (see "Assays") can be adapted to monitor or to detect reactivity in immune cells for use in clinical appHcations. Such assays will involve, for - 127 -
example, detecting Notch-ligand activity in host ceUs or monitoring Notch cleavage in donor cells. Further methods of monitoring immune ceU activity are set out below.
Immune ceU activity may be monitored by any suitable method known to those skUled in the art. For example, cytotoxic activity may be monitored. Natural kiUer (NK) ceUs wiU demonstrate enhanced cytotoxic activity after activation. Therefore any drop in or stabUisation of cytotoxicity wiUbe an indication of reduced reactivity.
Once activated, leukocytes express a variety of new cell surface antigens. NK ceUs, for example, wiU express transferrin receptor, HLA-DR and the CD25 IL-2 receptor after activation. Reduced reactivity may therefore be assayed by monitoring expression of these antigens.
Hara et al. Human T-ceU Activation: in, Rapid Induction of a Phosphorylated 28 kD/32kD Disulfide linked Early Activation Antigen (EA-1) by 12-0-tetradecanoyl Phorbol-13 -Acetate, Mitogens and Antigens, J. Exp. Med., 164:1988 (1986), and Cosulich et al. Functional Characterization of an Antigen (MLR3) Involved in an Early Step of T-Cell Activation, PNAS, 84:4205 (1987), have described cell surface antigens that are expressed on T-cells shortly after activation. These antigens, EA-1 and MLR3 respectively, are glycoproteins having major components of 28kD and 32kD. EA-1 and MLR3 are not HLA class π antigens and an MLR3 Mab wUl block IL-1 binding. These antigens appear on activated T-ceUs within 18 hours and can therefore be used to monitor immune ceU reactivity.
Additionally, leukocyte reactivity may be monitored as described in EP 0325489, which is incorporated herein by reference. Briefly this is accomplished using a monoclonal antibody ("Anti-Leu23") which interacts with a ceUular antigen recognised by the monoclonal antibody produced by the hybridoma designated as ATCC No. HB-9627. 128
Anti-Leu 23 recognises a cell surface antigen on activated and antigen stimulated leukocytes. On activated NK cells, the antigen, Leu 23, is expressed within 4 hours after activation and continues to be expressed as late as 72 hours after activation. Leu 23 is a disulfide-linked homodimer composed of 24 kD subunits with at least two N-linked carbohydrates.
Because the appearance of Leu 23 on NK cells conelates with the development of cytotoxicity and because the appearance of Leu 23 on certain T-ceUs conelates with stimulation of the T-cell antigen receptor complex, Anti-Leu 23 is useful in monitoring the reactivity of leukocytes.
Further detaUs of techniques for the monitoring of immune ceU reactivity may be found in: 'The Natural KUler Cell' Lewis C. E. and J. O'D. McGee 1992. Oxford University Press; Trincbieri G. 'Biology of Natural KiUer CeUs' Adv. hnmunol. 1989 vol 47 ppl 87-376; 'Cytokines of the Immune Response' Chapter 7 in "Handbook of Tmmune Response Genes". Mak T.W. and J.J.L. Simard 1998, which are incorporated herein by reference.
Preparation of Primed APCs and Lymphocytes
According to one aspect of the invention immune cells may be used to present antigens or allergens and/or may be treated to modulate expression or interaction of Notch, a Notch Hgand or the Notch signalling pathway. Thus, for example, Antigen Presenting Cells (APCs) may be cultured in a suitable cultare medium such as DMEM or other defined media, optionally in the presence of a serum such as fetal calf serum. Optimum cytokine concentrations may be determined by titration. One or more substances capable of up- regulating or down-regulating the Notch signaUing pathway are then typicaUy added to the cultare medium together with the antigen of interest. The antigen may be added before, after or at substantially the same time as the substance(s). Cells are typically incubated with the substance(s) and antigen for at least one hour, preferably at least 3 129 -
hours, at 37°C. If required, a smaU ahquot of cells may be tested for modulated target gene expression as described above. Alternatively, ceU activity may be measured by the inhibition of T ceU activation by monitoring surface markers, cytokine secretion or prohferation as described in WO98/20142. APCs transfected with a nucleic acid constmct directing the expression of, for example Senate, may be used as a control.
As discussed above, polypeptide substances may be administered to APCs by introducing nucleic acid constracts/vkal vectors encoding the polypeptide into cells under conditions that aUow for expression of the polypeptide in the APC. Similarly, nucleic acid constructs encoding antigens may be introduced into the APCs by transfection, vkal infection or vkal transduction. The resulting APCs that show increased levels of a Notch signalling are now ready for use.
Tolerisation assays
Any of the assays described above (see "Assays") can be adapted to monitor or to detect the degree of reactivity and tolerisation in immune cells for use in clinical applications. Such assays will involve, for example, detecting decreased Notch signaUing activity in host ceUs or monitoring Notch cleavage in donor cells. Further methods of monitoring immune ceU activity are set out below.
Immune ceU activity may be monitored by any suitable method known to those skilled in the art. For example, cytotoxic activity may be monitored. Natural kiUer (NK) ceUs wiU demonstrate enhanced cytotoxic activity after activation. Therefore any drop in or stabUisation of cytotoxicity wiU be an indication of reduced reactivity.
Once activated, leukocytes express a variety of new cell surface antigens. NK ceUs, for example, wiU express transferrin receptor, HLA-DR and the CD25 IL-2 receptor after activation. Reduced reactivity may therefore be assayed by monitoring expression of these antigens. - 130 -
Hara et al. Human T-ceU Activation: JJJ, Rapid Induction of a Phosphorylated 28 kD/32kD Disulfide linked Early Activation Antigen (EA-1) by 12-0-tetradecanoyl Phorbol-13-Acetate, Mitogens and Antigens, J. Exp. Med., 164:1988 (1986), and Cosulich et al. Functional Characterization of an Antigen (MLR3) Involved in an Early Step of T-Cell Activation, PNAS, 84:4205 (1987), have described cell surface antigens that are expressed on T-cells shortly after activation. These antigens, EA-1 and MLR3 respectively, are glycoproteins having major components of 28kD and 32kD. EA-1 and MLR3 are not HLA class II antigens and an MLR3 Mab wUl block IL-1 binding. These antigens appear on activated T-ceUs within 18 hours and can therefore be used to monitor immune cell reactivity.
Additionally, leukocyte reactivity may be monitored as described in EP 0325489, which is incorporated herein by reference. Briefly this is accomplished using a monoclonal antibody ("Anti-Leu23") which interacts with a ceUular antigen recognised by the monoclonal antibody produced by the hybridoma designated as ATCC No. HB-9627.
Anti-Leu 23 recognises a cell surface antigen on activated and antigen stimulated leukocytes. On activated NK cells, the antigen, Leu 23, is expressed within 4 hours after activation and continues to be expressed as late as 72 hours after activation. Leu 23 is a disulfide-linked homodimer composed of 24 kD subunits with at least two N-linked carbohydrates.
Because the appearance of Leu 23 on NK cells conelates with the development of cytotoxicity and because the appearance of Leu 23 on certain T-ceUs conelates with stimulation of the T-cell antigen receptor complex, Anti-Leu 23 is useful in monitoring the reactivity of leukocytes.
Further detaUs of techniques for the monitoring of immune ceU reactivity may be found in: 'The Natural KUler Cell' Lewis C. E. and J. O'D. McGee 1992. Oxford University 131
Press; Tikichieri G. 'Biology of Nataral KiUer CeUs' Adv. Immunol. 1989 vol 47 ppl 87-376; 'Cytokines of the Immune Response' Chapter 7 in "Handbook of Immune Response Genes". Mak T.W. and J.J.L. Simard 1998, which are incorporated herein by reference.
Various prefeπed featares and embodiments of the present invention will now be described in more detail by way of non-limiting examples.
Example 1
Preparation of inhibitor of Notch signalling (hDeltal-IgG4Fc Fusion Protein)
A fusion protein comprising the extracellular domain of human Deltal fused to the Fc domain of human IgG4 ("hDeltal-IgG4Fc") was prepared by inserting a nucleotide sequence coding for the extracellular domain of human Deltal (see, eg Genbank Accession No AF003522) into the expression vector pCONγ (Lonza Biologies, Slough, UK) and expressing the resulting construct in CHO cells.
i) Cloning
A 1622bp extraceUular (EC) fragment of human Delta-like ligand 1 (hECDLL-1; see GenBank Accession No AF003522) was gel purified using a Qiagen QIAquick™ Gel Extraction Kit (cat 28706) according to the manufacturer's instmctions. The fragment was then ligated into a pCR Blunt cloning vector (Invitrogen, UK) cut Hindlll - BsiWI, thus eliminating a Hindlll, BsiWI and Apal site.
The ligation was transformed into DH5α ceUs, streaked onto LB + Kanamycin (30ug/ml) plates and incubated at 37°C overnight. Colonies were picked from the plates into 3ml - 132 -
LB + Kanamycin (30ugml_1) and grown up overnight at 37°C. Plasmid DNA was purified from the cultares using a Qiagen Qiaquick Spin Miniprep kit (cat 27106) according to the manufacturer's instructions, then diagnosticaUy digested with Hindlll. A clone was chosen and streaked onto an LB + Kanamycin (30ug/ml) plate with the glycerol stock of modified ρCRBlunt-hECDLL-1 and incubated at 37°C overnight. A colony was picked off this plate into 60ml LB + Kanamycin (30ug/ml) and incubated at 37°C overnight. The cultare was maxiprepped using a Clontech Nucleobond Maxi Kit (cat K3003-2) according to the manufacturer's instructions, and the final DNA pellet was resuspended in 300ul dH2O and stored at -20°C.
5ug of modified pCR Blunt-hECDLL-1 vector was linearised with Hindlll and partially digested with Apal. The 1622bp hECDLL-1 fragment was then gel purified using a Clontech Nucleospin® Extraction Kit (K3051-1) according to the manufacturer's instructions. The DNA was then passed through another Clontech Nucleospin® column and followed the isolation from PCR protocol, concentration of sample was then checked by agarose gel analysis ready for Hgation.
Plasmid pconγ (Lonza Biologies, UK) was cut with Hindlll - Apal and the following oligos were ligated in (SEQ ID NO: 2):
agcttgcggc cgcgggccca gcggtggtgg acctcactga gaagctagag gcttccacca aaggcc acgccg gcgcccgggt cgccaccacc tggagtgact cttcgatctc cgaaggtggt tt
The Hgation was transformed into DH5α ceUs and LB + Amp (lOOug/ml) plates were streaked with 200ul of the transformation and incubated at 37°C overnight. The following day 12 clones were picked into 2 x YT + AmpicUlin (lOOugmT1) and grown up at 37°C throughout the day. Plasmid DNA was purified from the cultares using a Qiagen Qiaquick Spin Miniprep kit (cat 27106) and diagnosticaUy digested with Notl. A clone (designated "pDev41") was chosen and an LB + Amp (lOOug/ml) plate was streaked with the glycerol stock of pDev41 and incubated at 37°C overnight. The following day a clone - 133
was picked from this plate into 60ml LB + Amp (lOOug/ml) and incubated with shaking at 37°C overnight. The clone was maxiprepped using a Clontech Nucleobond Maxi Kit (cat K3003-2) according to the manufacturer's instructions and stored at -20°C. The pDev41 clone 5 maxiprep was then digested with Apal - EcoRI to generate the IgG4Fc fragment (1624bρ). The digest was purified on a 1% agarose gel and the main band was cut out and purified using a Clontech Nucleospin Extraction Kit (K3051-1).
The polynucleotide was then cloned into the polylinker region of pEE14.4 (Lonza Biologies, UK) downstream of the strong hCMV promoter enhancer region (hCMV- MJE) and upstream of SV40 polyadenylation signal (encodes the GS gene requked for selection in glutarrHne free media; contains the GS minigene - GS cDNA which includes the last intron and polylinker adenylation signals of the wild type hamster GS gene) which is under the control of the late SV40 promoter, has the hCMV promoter to drive transcription of the desked gene. 5ug of the maxiprep of pEE14.4 was digested with Hindlll - EcoRI, and the product was gel extracted and treated with alkaline phosphatase.
u) Generation of Expression Constructs
A 3 fragment Hgation was set up with pEE14.4 cut Hindlll - EcoRI, ECDLL-1 from modified pCR Blunt (Hindlll - Apal) and the IgG4Fc fragment cut from pDev41 (Apal - EcoRI). This was transformed into DH5 ceUs and LB + Amp (lOOug/ml) plates were streaked with 200ul of the transformation and incubated at 37C overnight. The foUowing day 12 clones were picked into 2 x YT + Amp (lOOug/ml) and mimpreps were grown up at 37°C throughout the day. Plasmid DNA was purified from the preps using a Qiagen Qiaquick spin miniprep kit (Cat No 27106), diagnosticaUy digested (with EcoRI and HindTTT) and a clone (clone 8; designated "ρDev44") was chosen for maxiprepping. The glycerol stock of pDev44 clone 8 was streaked onto an LB + Amp (lOOugml"1) plate and incubated at 37°C overnight. The following day a colony was picked into 60ml LB + Amp (lOOugmT1) broth and incubated at 37°C overnight. The plasmid DNA was isolated using a Clontech Nucleobond Maxiprep Kit (Cat K3003-2). 134
Hi) Addition of optimal KOZAK Sequence
A Kozak sequence was inserted into the expression constract as follows. Oligonucleotides were kinase treated and annealed to generate the foUowing sequences:
AGCTTGCCGCCACCATGGGCAGTCGGTGCGCGCTGGCCCTGGCGGTGCTC
ACGGCOGTGGTACCCGTCAGCCACGCGCGACCGGGACCGC (SEQ ID NO : 3)
TCGGCCTTGCTGTGTCAGGTCTGGAGCTCTGGGGTGTT CACGAGAGCCGGAACGACACAGTCCAGACCTCGAGACCCCACAAGC (SEQ ID NO : 4)
pDev44 was digested with Hindlll - BstBI, gel purified and treated with alkaline phosphatase. The digest was Hgated with the oligos, transformed into DH5 cells by heat shock . 200ul of each transformation were streaked onto LB + Amp plates (lOOug/ml) and incubated at 37°C overnight. Minipreps were grown up in 3 ml 2 x YT + AmpiciUin (lOOugmT1). Plasmid DNA was purified from the minipreps using a Qiagen Qiaquick spin miniprep kit (Cat No 27106) and diagnosticaUy digested with Ncol. A clone (pDev46) was selected and the sequence was confirmed. The glycerol stock was streaked, broth grown up and the plasmid maxiprepped.
iv) Transfection
Approx lOOug ρDev46 Clone 1 DNA was Hnearised with restriction enzyme Pvu L The resulting DNA preparation was cleaned up using phenol/chloroform/IAA extraction foUowed by ethanol wash and precipitation. The peUets were resuspended in sterile water and linearisation and quantification was checked by agarose gel electrophoresis and UV spectrophotometry.
40ug linearised DNA (pDev46 Clone 1) and 1 x 107 CHO-K1 cells were mixed in serum free DMEM in a 4mm cuvette, at room temp. The cells were then electroporated at 975uF 135
280 volts, washed out into non-selective DMEM, diluted into 96 well plates and incubated. After 24 hours media were removed and replaced with selective media (25uM L-MSX). After 6 weeks media were removed and analysed by IgG4 sandwich ELISA. Selective media were replaced. Positive clones were identified and passaged in selective media 25um L-MSX.
v Expression
Cells were grown in selective DMEM (25um L-MSX) untU semi-confluent. The media was then replaced with serum free media (UltraCHO) for 3-5 days. Protein (hDeltal- IgG4Fc fusion protein) was purified from the resulting media by HPLC.
The amino acid sequence of the resulting expressed fusion protein was as foUows (SEQ ID NO: 5):
MGSRCALALAVLSALLCOVWSSGVFELKLOEFVNKKGLLGNRNCCRGGAGPPP
CACRTFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPI ETFGFTWPGTFSLΠEALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLH
SSGRTDLKYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWK
GPYCTEPICLPGCDEQHGFCDKPGECKCRVGWQGRYCDECIRYPGCLHGTCQQP
WQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGA
TCELGΓDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNG
GRCSDSPDGGYSCRCPVGYSGFNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCQ
AGFSGRHCDDNVDDCASSPCANGGTCRDGVNDFSCTCPPGYTGRNCSAPVSRCE
HAPCHNGATCHERGHGYVCECARGYGGPNCQFLLPELPPGPAVVDLTEKLEAST
KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLO
SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEF
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSOEDPEVOFNWΎVDGVEVHNAK
TKPREEOFNSTYRVVSVLTVLHODWLNGKEYKCKVSNKGLPSSIEKTISKAKGO
PREPOVYTLPPSOEEMTKNOVSLTCLVKGFYPSDIAVEWΈSNGOPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWOEGNVFSCSVMHEALHNHYTOKSLSLSLGK
Wherein the first underlined sequence is the signal peptide (cleaved from the mature protein) and the second underlined sequence is the IgG4 Fc sequence. The protein normally exists as a dimer linked by cysteine disulphide bonds (see eg schematic - 136 -
representation in Figure 10). The domain stmcture of the expressed fusion protein is shown in more detaU in Figure 12.
Example 2
Notch signaUing inhibitor enhances immune response to flu antigen
Flushield™ flu vaccine (5 micrograms; Roche USA) was enrols kied in incomplete Freund's adjuvant with or without 100 micrograms of hDeltal-IgG4Fc (from Example 1 above). 6-8 weeks old B ALB/c mice (eight per group) were immunized subcutaneously at the base of the taU and 14 days later the mice were chaUenged in the right ear with 1.8 micrograms of Flushield flu vaccine in saline. Ear responses (ear thickness measured with caUipers) were measured at 1, 2 and 6 days thereafter.
Results expressed as increase (right ear - left ear) in ear swelling are shown in Figure 13.
Example 3
Notch signalling inhibitor enhances immune response to KLH
6-8 weeks old BALB/c mice (eight per group) were immunized subcutaneously at the base of the tail with keyhole limpet haemocyanin (KLH) from Pierce at 50ng or 0.5ng per mouse emulsified in incomplete Freund's adjuvant (IF A) with or without hDeltal-IgG4Fc protein from Example 1 above (100 micrograms). Some mice also received additional hDeltal-IgG4Fc (400 micrograms) at an adjacent s.c. site one day later. 14 days after the initial KLH priming, mice were chaUenged in the right ear with 20 micrograms KLH and the ear immune response was measured with calhpers as an increase in ear thickness due to the induced inflammatory reaction after 24 hours.
Results are shown in Figure 14. - 137 -
Example 4
Notch signalling inhibitor enhances immune response to Flu vaccine
6-8 weeks old B ALB/c mice (eight per group) were immunized subcutaneously at the base of the tail with Flushield™ flu vaccine at 5 μg per mouse emulsified in incomplete Freund's adjuvant (JFA) with hDeltal-IgG4Fc protein from Example 1 above (100 micrograms) or isotype control hlgG4 (Sigma, UK) lOOμg/TFA control. 14 days after the initial Flushield™ flu vaccine priming, mice were challenged in the right ear with Flushield™ and the ear immune response was measured with callipers as an increase in ear thickness due to the induced inflammatory reaction after 24 hours.
Results are shown in Figure 15.
Example 5
The modulation of cytokine production induced by Deltal beads is inhibited by the addition of soluble hPeltal-IgG4Fc
j) Preparation of beads coated with hDeltal-IgG4Fc fusion proteins
M450 Streptavidin Dynabead™ magnetic beads (Dynal, USA) were coated with an anti- human-IgG4 biotinylated monoclonal antibody (BD Bioscience, 555879) by rotating them in the presence of the antibody for 30 minutes at room temperatare. Beads were washed three times with PBS (1ml). They were further incubated with hDeltal-hIgG4 (see Example 1 above) for 2 hours at room temperatare and then washed three times with PBS (1ml). - 138 -
u) Investigation of Notch signalling by ELISA
Human peripheral blood mononuclear ceUs (PBMC) were purified from blood using Ficoll-Paque separation medium (Pharmacia). Briefly, 28 ml of blood were overlaid on 21 ml of Ficoll-Paque separation medium and centrifuged at 18-20°C for 40 minutes at 400g. PBMC were recovered from the interface and washed 3 times before use for CD4+ T cell purification.
The CD4+ T cells were incubated in tripHcates in a 96-weU-plate (flat bottom) at 103 CD4/weU/200μl in RPMI medium containing 10% FCS, glutamine, peniciUin, streptomycin and β2-mercaptoethanol.
Cytokine production was induced by stimulating the ceUs with anti-CD3/CD28 T cell expander beads from Dynal at a 1 :1 ratio (bead/ceU) in the presence of beads coated with hDeltal-IgG4Fc fusion protein (Example 1 above) at a 5:1 ratio (beads/cell). In some wells, increasing amounts of soluble hDeltal-IgG4Fc fusion protein were also added.
The supematants were removed after 3 days of incubation at 37°C/ 5%CO2/humidified atmosphere and cytokine production was evaluated by ELISA using Pharm in gen kits OptEIA Set human IL10 (catalog No. 555157), OptEIA Set human IL-5 (catalog No. 555202) for IL-10 and IL-5 respectively according to the manufacturer's instructions.
Results showing the effect of increasing concentrations of added soluble hDeltal- IgG4Fc are shown in Figure 16.
As can be seen from these results, bead-immobUised human Deltal enhances IL-10 production by activated human CD4+ T cells. This effect was inhibited when soluble hDeltal-IgG4Fc was added into the cultare medium. 139 -
Example 6
The modulation of cytokine production induced by Deltal beads is inhibited by the addition of soluble Notchl EC domain/Fc Fusion Protein.
Human peripheral blood mononuclear ceUs (PBMC) were purified from blood using Ficoll-Paque separation medium (Pharmacia). Briefly, 28 ml of blood were overlaid on 21 ml of Ficoll-Paque separation medium and centrifuged at 18-20°C for 40 minutes at 400g. PBMC were recovered from the interface and washed 3 times before use for CD4+ T cell purification.
The CD4+ T cells were incubated in tripHcates in a 96-weU-plate (flat bottom) at 105 CD4/weU/200μl in RPMI medium containing 10% FCS, glutamine, peniciUin, streptomycin and β2-mercaptoethanol.
Cytokine production was induced by stimulating the cells with anti-CD3/CD28 T cell expander beads from Dynal at a 1 :1 ratio (bead/ceU) in the presence of beads coated with hDeltal-IgG4Fc fusion protein (Example 1 above) at a 5:1 ratio (beads/cell). In some wells, increasing amounts of soluble rat Notchl extracellular domain-hlgGl fusion protein (R&D Systems, Catalog No 1057 -TK) were also added.
The supematants were removed after 3 days of incubation at 37°C/ 5%CO2/humidified atmosphere and cytokine production was evaluated by ELISA using Pharm in gen kits OptEIA Set human 1L10 (Catalog No. 555157), OptEIA Set human IL-5 (Catalog No. 555202) for IL-10 and IL-5 respectively according to the manufacturer's instructions.
Results showing the effect of increasing concentrations of added soluble rat Notchl EC- gGlFc fusion protein are shown in Figure 17. 140 -
As can be seen from these results, bead-irnmobilised human Deltal -Fc enhances IL-10 production by activated human CD4+ T cells. This effect was inhibited when soluble rat Notchl - gGlFc was added into the culture medium. ,
Example 7
Preparation of inhibitor of Notch signalling: truncated human Jaggedl fusion protein 0ιiaggedlEGFl&2 -IgG4Fc)
A fusion protein capable of acting as an inhibitor of Notch signalling comprising human jaggedl sequence up to the end of EGF2 (leader sequence, amino terminal, DSL, EGF1+2) fused to the Fc domain of human IgG4 ("hJaggedl(EGFl+2)-IgG4Fc") was prepared by inserting a nucleotide sequence coding for human Jaggedl from ATG through to the end of the second EGF repeat (EGF2) into the expression vector pCONγ (Lonza Biologies, Slough, UK) to add the IgG4 Fc tag. The fuU fusion protein was then shuttled into the Glutamine Synthetase (GS) selection system vector pEE14.4 (Lonza Biologies). The resulting construct was transfected and expressed in CHO-K1 cells (Lonza Biologies).
1. Cloning
i) Preparation of DNA - pDEV 47 and pDEV20
Human Jaggedl was cloned into pcDNA3.1 (Invitrogen) to give plasmid pLOR47. The Jagged 1 sequence from ρLOR47 was aHgned against full length human jaggedl (GenBank U61276) and found to have only a small number of apparently silent changes.
Plasmid pLOR47 was then modified to remove one of two Drain sites (whilst maintaining and replacing the amino acid sequence for full extracellular hJaggedl) and - 141 -
add a BsiWI site after for ease of subsequent cloning. The resulting plasmid was named pDEV20.
Plasmid pLOR47 was cut with Drain. This removed a 1.7kb fragment comprising the 3' end of the extraceUular, the transmembrane and intraceUular regions of hJaggedl as well as part of the vector sequence leaving a larger fragment of 7.3kbp of the main vector backbone with almost all of the extraceUular region (EC) of hJaggedl. The cut DNA was run out on an agarose gel, the larger fragment excised and gel purified using a Qiagen QIAquick™ Gel Extraction Kit (cat 28706) according to the manufacturer's instructions. A pak of oHgonucleotides were ordered such that when Hgated together gave a double stranded piece of DNA that had a compatible sticky end for DraHI at the 5' end and recreated the original restriction site. This sequence was foUowed by a BsiWI site then another compatible sticky end for Drain at the 3' end that did not recreate the restriction site.
ie Dram BsiWI Drain gtg ctg tta ccc gta egg ta gaa cac gac aat ggg cat gc (SEQ ID NO: 6)
This oligo pak was then Hgated into the DraH cut pLOR47 thus maintaining the 5' Dram site, inserting a BsiWI and eliminating the 3'Draffl site. The resulting plasmid was named pDEV20.
H) Preparing hJaggedl IgG4 FC fusion DNA:
A three fragment Hgation was necessary to reassemble full hJaggedl EC sequence with addition of amodkιed 5' Kozak sequence and 5' end repak together with repak of 3 'end. - 142 -
Fragment 1 : EC hJagged sequence pDev 20 was cut Rsrfl - DraHI giving rise to 3 fragments; 1270 + 2459 + 3621 bp. The fragments were run out on an agarose gel, the 2459 bp band excised and the DNA gel purified using a Qiagen QIAquick™ Gel Extraction Kit (cat 28706) according to the manufacturer's instructions. This contained hJaggedl sequence - with loss of 3' sequence (up to the Rsrπ site) and loss of some 5 'sequence at the end of the EC region.
Fragment 2: modified Kozak sequence pUC19 (Invitrogen) was modified to insert new restriction enzyme sites and also introduce a modkied Kozak with 5' hJaggedl sequence. The new plasmid was named pLOR49. pLOR49 was created by cutting ρUC19 vector Hindm EcoRI and ligating in 4 oligonucleotides (2 ohgo pairs).
One pak has a Hindm cohesive end foUowed by an optimal Kozac and 5'hJagged 1 sequence followed by Rsrfl cohesive end.
ie Hindm optimal Kozak + 5' hJaggedl sequence Rsrπ ag ctt gcc gcc ace atg ggt tec cca egg aca cgc ggc eg a egg egg tgg tac cca agg ggt gcc tgt gcg ccg gcc ag (SEQ JD NO:7)
The other pak has a cohesive Rsrfl end then Dram, Kpnl, BsiWI sites followed by a cohesive EcoRI site.
ie Rsrπ Dram Kpnl BsiWI EcoRI gtc cgc ace ttg tgg gta ccc gta egg gcg tgg aac ace cat ggg cat gcc tta a (SEQ ID NO: 8) - 143 -
pLOR49 thus is a pUC19 back bone with the Hindm site followed by optimal Kozac and 5 'hJaggedl sequence and introduced unique Rsrfl, Dra m, Kpnl, BsiWI sites before recreating the Ecorl site.
Plasmid pLOR49 was then cut RsrH - BsiWI to give a 2.7kbρ vector backbone fragment that was run out on an agarose gel, the band excised and the DNA gel purified using a Qiagen QIAquick™ Gel Extraction Kit (cat 28706) according to the manufacturer's instructions.
Fragment 3: generation of 3' hJaggedl EC with BsiWI site PCR fragment pLOR47 was used as a template for PCR to amplify up hJaggedl EC and add a 3' BsiWI site.
5' primer from RsrH site of hJagged I
3' site up to end of hJaggedl EC with BsiWI site stitched on 3' The resulting fragment was cut with DraHI and BsiWI to give a fragment around 600bp. This was run out on an agarose gel, the band excised and the DNA gel purified using a Qiagen QIAquick™ Gel Extraction Kit (cat 28706) according to the manufacturer's instructions.
The three fragments described above;
1) 2459bp h Jaggedl fragment from pDev 20 cut RsrH - Drain
2) 2.7kbp optimised Kozak and 5' hJaggedl from Lor 49 cut Rsrπ - BsiWI
3) 600bp 3 'EC hJaggedl PCR fragment cut Dram- BsiWI
were then Hgated together to give plasmid pDEV21.
Hi) Further Hgation (pDEVIO):
To exclude any extraneous sequences a further 3 fragment Hgation was carried out to drop straight into the vector pCONγ 4 (Lonza Biologies, Slough, UK). - 144 -
Fragment 1: Plasmid pDEV21-4 was cut Hindm-BglH to give 4958bp + 899bp fragments. These were ran out on an agarose gel, the smaller 889bp fragment band was excised and the DNA gel purified using a Qiagen QIAquick™ Gel Extraction Kit (cat 28706) according to the manufacturer's instructions.
Fragment 2: pCONγ 4 (Lonza Biologies) was cut Hind m-Apal to give a 6602bp vector fragment - missing the first 5 amino acids of IgG4 FC. The fragment band was excised and the DNA gel purified using a Qiagen QIAquick™ Gel Extraction Kit (cat 28706) according to the manufacturer's instructions.
Fragment 3: A linker ohgonucleotide pak was ordered to give a tight junction between the end of hJaggedl EGF2 and the 3' start of IgG4 FC, with no extra amino acids introduced.
ie Bgiπ D L A S T K G Apal DL = hJaggedl sequence gat etc get tec ace aag ggc c remainder = IgG4 FC sequence ag cga agg tgg ttc (SEQ JD NO:9)
The three fragments described above;
1. 899bρ hJaggedl fragment pDEV21-4 cut HindlH-Bgin
2. 6602bp pConGamma vector backbone cut Hindm Apal
3. ohgo linker BglH- Apal were Hgated together to give plasmid pDEVlO.
Ligated DNA was transformed into competent DH5alpha (Invitrogen), plated onto LB amp paltes and incubated at 37 degres overnight. A good ratio was evident between control and vector plus insert pates therefore only 8 colonies were picked into 10ml LB amp broth and incubated at 37 overnight. Glycerol broths were made and the bacterial pellets were frozen at -20degrees. Later plasmid DNA was extracted using Qiagen - 145
miniprep spin kit and were diagnosticaUy digested with Seal . Clones 2,4, and 5 looked conect so clone 2 was steaked onto LB Amp plates and inoculate 1/100 into 120ml LB + amp broth. Plates and broths were incubated at 37 degrees overnight. Glycerol broths were made from the broths and pellets frozen to maxiprep later. Plasmid DNA was extracted Clontech Maxiprep, diagnostic digests were set up with Seal and the DNA was diluted for quantification and quahty check by UV spectropho tome try.
iv) pDevll cloning:
The coding sequence for hJaggedl EGFl+2 IgG4 FC fusion was shuttled out of pCONγ 4 (Lonza Biologies) into pEE 14.4 (Lonza Biologies) downstream of the hCMV promoter region (hCMV-MIE) and upstream of SN40 polyadenylation signal, to enable stable ceU lines to be selected using the GS system (Lonza Biologies).
Plasmid ρEE14.4 contains the GS minigene - (GS cDΝA which includes the last intron and polylinker adenylation signals of the wild type hamster GS gene under the control of the late SV40 promoter) which encodes the GS gene requked for selection in glutamine free media.
v) Insert:
pDEVIO clone 2 was cut Hindm-EcoRI giving rise to 2 fragment s 5026bp + 2497bp. The 2497bp contained the coding sequence for hJaggedl EGFl+2 IgG4 FC fusion and so was excised from an agarose gel and the DΝA gel purified using a Qiagen QIAquick™ Gel Extraction Kit (cat 28706) according to the manufacturer's instructions.
vi) Vector:
pEE14.4 (Lonza Biologies) was cut Hindm-EcoRI to remove the IgG4 FC sequence giving 2 fragments 5026bp + 1593bp. The larger 5026bρ fragment was excised from an - 146
agarose gel and the DNA gel purified using a Qiagen QIAquick™ Gel Extraction Kit (cat 28706) according to the manufacturer's instructions.
The pEE14.4 vector backbone and the hJaggedl EGFl+2 IgG4 FC fusion insert were Hgated to give the final transfection plasmid pDEVll.
The Hgation was transformed into DH5α ceUs, streaked onto LB + AmpicUlin (lOOug/ml) plates and incubated at 37°C overnight. Colonies were picked from the plates into 7ml LB + AmpicUlin (lOOug/ml) and grown up shaking overnight at 37°C. Glycerol broths were made and the plasmid DNA was purified from the cultares using a Qiagen Qiaquick Spin Miniprep kit (cat 27106) according to the manufacturer's instractions. The DNA was then diagnosticaUy digested with Sap I.
vii) Maxiprep for transfection:
A conect clone (clone 1) was chosen and lOOul of the glycerol stock was inoculated into
100ml LB + AmpicUlin (lOOug/ml), and also streaked out onto LB + AmpicUlin
(lOOug/ml) plates. Both plate and broth were incubated at 37°C overnight.
The plates showed pure growth; therefore the culture was maxi-prepped using a Clontech
Nucleobond Maxi Kit (cat K3003-2) according to the manufacturer's instructions.The final DNA pellet was resuspended in 500ul dH O.
A sample of pLOR 11 clonel DNA was then diluted and the concentration and quahty of
DNA assessed by UV spectrophotometry. A sample was also diagnosticaUy digested with
Sap I, and gave bands of the conect size.
viH) Linearisation of DNA:
Approx lOOug pDevll Clone 1 DNA was linearised with restriction enzyme Pvu L The resulting DNA preparation was cleaned up using phenoVchloroform/IAA extraction foUowed by ethanol wash and precipitation inside a laminar flow hood. The pellets were resuspended in sterile water. Linearisation was checked by agarose gel electrophoresis - 147 -
while quantification and quality were assessed by UV spectrophotometry at 260 and 280nm.
2. Transfection
40ug linearised DNA (pDevll Clone 1) and 1 x 107 CHO-K1 cells (Lonza) were mixed in 500ul of serum free DMEM in a 4mm cuvette, at room temp. The ceUs were then electroporated at 975uF 280 volts, washed out into 60ml of non-selective DMEM
(DMEM/glut 10%FCS).
From this dilution 6 x 96 weU pates were inoculated with 50ul per well. A lA dilution of the original stock was made and from this 8 x 96 well pates were inoculated with 50ul per well. A further 1/10 dUution was made from the second stock, and from this 12 x 96 well pates were inoculated with 50ul per weU.
Plates were incubated at 37 degrees C 5% CO2 overnight. After 24 hours the media was removed and replaced with 200ul of selective media (25uM L-MSX).
Between 4-6 weeks post transfection media was removed from the plates for analysis by
IgG4 sandwich ELISA. Selective media were replaced. Positive clones were identified, passaged and expanded in selective media 25um L-MSX.
3. Expression
CeUs were grown in selective DMEM (25um L-MSX) untU semi-confluent. The media was then replaced with seram free media (UltraCHO; BioWhittaker) for 3-5 days. Protein (hJaggedlEGFl+2-IgG4Fc fusion protein) was purified from the resulting media by FPLC. - 148 -
Amino acid sequence of the expressed fusion protein fbJaggedl EGFl+2 IgG4 FC):
1 mrsprtrgrs grplslllal lσalrakvσg asgqfeleil smqnvngelq ngnccggarn
61 pg r ctr e σdtyfkvclk eyqsrvtagg pcsfgsgstp viggntfnlk asrgndpnri
121 vlpfsfawpr sytllveawd ssndtvqpds iiekashsgm inpsr wqtl kqntgva fe
181 yqirvtσddy yygfgcnkfσ rprddffghy acdqngnktσ megwmgpecn raicrqgcsp
241 khgsσklpgd σrcqyg qgl ycdkcip pg σvhgicnep qclcatn gg qlcdkdlvra
301 stkgpsvfpl apcsrstses taalgclvkd yfpepytvsw nsgaltsgvh tfpayl ssg
361 lyslsswtv pssslgtkty tcnvdhkpsn tkvdkrvesk ygppcpscpa peflggpsvf
421 lfppkpkdtl misrtpevtc vwdvsqedp evqfnwyvdg vevhnaktkp reeqfnstyr
481 vvsyltyl q dwlngkeykc kvsnkglpss. iektiskakg qprepqvytl ppsqeemtkn
541 qvsltclvkg fypsdiave esngqperaxy kttppyldsd gsfflysrlt vdksrwqegn
601 vfscsvmhea l n ytqksl slslgk
(SEQ ID NO: 10) Bold = hJaggedl extracellular domain leader sequence, amino terrrrinal region, DSL and EGF 1+2, Underlined = IgG4 Fc sequence
The protein is believed to exist as a dimer linked by cysteine disulphide bonds, with cleavage of the signal peptide.
Example 8
The modulation of cytokine production induced by Deltal beads is inhibited by the addition of soluble Jaggedl (2EGF truncation) /Fc Fusion Protein.
Human peripheral blood mononuclear ceUs (PBMC) were purified from blood using Ficoll-Paque separation medium (Pharmacia). Briefly, 28 ml of blood were overlaid on 21 ml of Ficoll-Paque separation medium and centrifuged at 18-20°C for 40 minutes at 400g. PBMC were recovered from the interface and washed 3 times before use for CD4+ T cell purification.
The CD4+ T cells were incubated in tripHeates in a 96-weU-plate (flat bottom) at 105 CD4/weU/200μl in RPMI medium containing 10% FCS, glutamine, peniciUin, streptomycin and β2-mercaptoethanol. - 149 -
Cytokine production was induced by stimulating the ceUs with anti-CD3/CD28 T cell expander beads from Dynal at a T.l ratio (bead/ceU) in the presence of beads coated with hDeltal-IgG4Fc fusion protein (Example 1 above) at a 5:1 ratio (beads/cell). In some wells, increasing amounts of soluble Jagged-1 (2EGF)-hIgGl fusion protein (hjaggedlEGFl&2 -IgG4Fc; prepared as described above) were also added.
The supematants were removed after 3 days of incubation at 37°C/ 5%CO2/humidified atmosphere and cytokine production was evaluated by ELISA using Pharrningen kits OptEIA Set human IL10 (Catalog No. 555157), OptEIA Set human IL-5 (Catalog No. 555202) for JL-10 and JL-5 respectively according to the manufacturer's instructions.
Results showing the effect of increasing concentrations of added soluble hjaggedlEGFl&2 -IgG4Fc are shown in Figure 18.
As can be seen from these results, bead-irnmobiHsed human Deltal -Fc enhances IL-10 production by activated human CD4+ T cells. This effect was inhibited when soluble hjaggedlEGFl&2 -IgG4Fc fusion protein (hJlE2Fc) was added into the cultare medium.
Example 9
ELISA Assay Method For Detecting Notch Signalling Modulator Activity in Mouse CD4+ cells
Ti) CD4+ cell purification
Spleens were removed from female Balb/c mice 8-10 weeks old and passed through a 0.2μM cell strainer into 20ml R10F medium (R10F-RPMI 1640 media (Gibco Cat No 22409) plus 2mM L-glutamine, 50μg/ml Penicillin, 50μg/ml Streptomycin, 5 x 10"5 M β-mercapto-ethanol in 10% fetal calf serum). The cell suspension was spun (1150rpm 5min) and the media removed. - 150 -
The cells were incubated for 4 minutes with 5ml ACK lysis buffer (0.15M NELCl, 1.0M KHC03, O.lmM Na EDTA in double distilled water) per spleen (to lyse red blood cells). The ceUs were then washed once with R10F medium and counted. CD4+ ceUs were purified from the suspensions by positive selection on a Magnetic Associated Cell Sorter (MACS) column (Miltenyi Biotec, Bisley, UK: Cat No 130-042-401) using CD4 (L3T4) beads (MUtenyi Biotec Cat No 130-049-201), according to the manufacturer's dkections.
(Ii) Antibody Coating
The following protocol was used for coating 96 well fiat-bottomed plates with antibodies.
The plates were coated with DPBS plus lμg/ml anti-hamsterlgG antibody (Pharmin en Cat No 554007) plus lμg/ml anti-IgG4 antibody. lOOμl of coating mixture was added per well. Plates were incubated overnight at 4°C then washed with DPBS. Each weU then received either lOOμl DPBS plus anti-CD3 antibody (lμg/ml) or, lOOμl DPBS plus anti- CD3 antibody (lμg/ml) plus hDeltal-IgG4Fc fusion protein (lOμg/ml). The plates were incubated for 2-3 hours at 37°C then washed again with DPBS before ceUs (prepared as described above) were added.
Hi") Investigation of Notch Signaling Inhibition
Mouse CD4+T-ceUs (prepared as above) were cultured at 2 x 105/weU on anti-CD3 coated plates with or without plate-bound hDeltal-IgG4Fc fusion protein (prepared as described above) and soluble anti-CD28 (Pharmingen, Cat No 553294, Clone No 37.51) at a final concentration of 2μg/ml. Soluble hDeltal-IgG4Fc fusion protein was added into cultare at the start at the concentrations shown and IL-10 was measured in supematants on day 3 by ELBA using antibody pairs from R & D Systems (Abingdon, UK). The results (shown in Figure 19) show that the increased IL-10 release induced by - 151
plate-bound hDeltal-IgG4Fc fusion protein is substantiaUy reversed by all concentrations of soluble hDeltal-IgG4Fc fusion protein tested.
Example 10
CHO-N2 (N27) Luciferase Reporter Assay
A) Construction of Luciferase Reporter Plasmid lOxCBFl-Luc (pLOR91)
An adenovkus major late promoter TATA-box motk with BglH and Hindm cohesive ends was generated as foUows:
Bgiπ Hindm
GATCTGGGGGGCTATAAAAGGGGGTA
ACCCCCCGATATTTTCCCCCATTCGA
(SEQ ID NO:l l) This was cloned into plasmid pGL3-Basic (Promega) between the Bgiπ and Hindm sites to generate plasmid pGL3-AdTATA.
A TP1 promoter sequence (TP1; equivalent to 2 CBFl repeats) with BamHl and BglH cohesive ends was generated as follows:
BamHl BglH
5 ' GATCCCGACTCGTGGGAAAATGGGCGGAAGGGCACCGTGGGAAAATAGTA 3 '
3 ' GGCTGAGCACCCTTTTACCCGCCTTCCCGTGGCACCCTTTTATCATCTAG 5 '
(SEQ ID NO: 12)
This sequence was pentamerised by repeated insertion into a Bgin site and the resulting TP1 pentamer (equivalent to 10 CBFl repeats) was inserted into pGL3-AdTATA at the BglH site to generate plasmid pLOR91.
B) Generation of a stable CHO ceU reporter cell line expressing fuU length Notch2 and - 152 -
the lOxCBFl-Luc reporter cassette
A cDNA clone spanning the complete coding sequence of the human Notch2 gene (see, eg GenBank Accession No AF315356) was constructed as follows. A 3' cDNA fragment encoding the entke intraceUular domain and a portion of the extracellular domain was isolated from a human placental cDNA library (OriGene Technologies Ltd., USA) using a PCR-based screening strategy. The remaining 5' coding sequence was isolated using a RACE (Rapid AmpHfication of cDNA Ends) strategy and Hgated onto the existing 3' fragment using a unique restriction site common to both fragments (Cla I). The resulting full-length cDNA was then cloned into the marnmahan expression vector pcDNA3.1-V5- HisA (Invitrogen) without a stop codon to generate plasmid pLOR92. When expressed in mammalian ceUs, pLOR92 thus expresses the full-length human Notch2 protein with V5 and His tags at the 3' end of the intraceUular domain.
WUd-type CHO-Kl cells (eg see ATCC No CCL 61) were transfected with ρLOR92 (pcDNA3.1-FLNotch2-V5-His) using Lipfectamine 2000™ (Invitrogen) to generate a stable CHO cell clone expressing full length human Notch2 (N2). Transfectant clones were selected in Dulbecco's Modified Eagle Medium (DMEM) plus 10% heat inactivated fetal calf serum ((HI)FCS) plus glutamine plus PenicUlin-Streptomycin (P/S) plus 1 mg/ml G418 (Geneticin™ - Invitrogen) in 96-weU plates using limiting dUution. Individual colonies were expanded in DMEM plus 10%(HI)FCS plus glutamine plus P/S plus 0.5 mg/ml G418. Clones were tested for expression of N2 by Western blots of ceU lysates using an anti-V5 monoclonal antibody (Invitrogen). Positive clones were then tested by transient transfection with the reporter vector pLOR91 (lOxCBFl-Luc) and co- culture with a stable CHO cell clone (CHO-Delta) expressing full length human delta-like ligand 1 (DLL1; eg see GenBank Accession No AF196571). CHO-Delta ceUs were prepared in the same way as the CHO Notch 2 clone, but with human DLL1 used in place of Notch 2. A strongly positive clone was selected by Western blots of cell lysates with anti-V5 mAb. 153 -
One CHO-N2 stable clone, N27, was found to give high levels of induction when transiently transfected with pLOR91 (lOxCBFl-Luc) and co-cultared with the stable CHO cell clone expressing fuU length human DLL1 (CHO-Deltal). A hygromycin gene cassette (obtainable from pcDNA3.1/hygro, Invitrogen) was inserted into ρLOR91 (lOxCBFl-Luc) using BamHl and Sail and this vector (lOxCBFl-Luc-hygro) was transfected into the CHO-N2 stable clone (N27) using Lipfectamine 2000 (Invitrogen). Transfectant clones were selected in DMEM plus 10%(HI)FCS plus glutamine plus P/S plus 0.4 mg/ml hygromycin B (Invitrogen) plus 0.5 mg/ml G418 (Invitrogen) in 96-weU plates using limiting dUution. Individual colonies were expanded in DMEM plus 10%(HJ)FCS plus glutamine plus P/S + 0.2 mg/ml hygromycin B plus 0.5 mg/ml G418 (Invitrogen).
Clones were tested by co-culture with a CHO Delta (expressing full length human Deltal (DLL1)). Three stable reporter cell lines were produced N27#ll, N27#17 and N27#36. N27#ll was selected for further use because of its low background signal in the absence of Notch signalling, and hence high fold induction when signalling is initiated. Assays were set up in 96-well plates with 2 x 104 N27#l 1 cells per well in 100 μl per well of DMEM plus 10%(ffl)FCS plus glutamine plus P/S.
CHO-Delta ceUs (as described above) were maintained in DMEM plus 10% (HT)FCS plus glutamine plus P/S plus 0.5 mg/ml G418. Just prior to use the cells were removed from a T80 flask using 0.02% EDTA solution (Sigma), spun down and resuspended in 10 ml DMEM plus 10%(HJ)FCS plus glutamine plus P/S. lOμl of ceUs were counted and the ceU density was adjusted to 5.0 x 105 cells/ml with fresh DMEM plus 10%(HI)FCS plus glutamine plus P/S.
To set up the CHO-Delta antagonist assay, N27#ll cells (T80 flask) were removed using 0.02% EDTA solution (Sigma), spun down and resuspended in 10 ml DMEM plus 10%(HJ)FCS plus glutamine plus P/S. 10 μl of ceUs were counted and the cell density was adjusted to 2.0 x 105 cells/ml with fresh DMEM plus 10%(HI)FCS plus glutamine 154 -
plus P/S. The reporter cells were plated out at 100 μl per well of a 96-well plate (i.e. 2 x 104 cells per well) and were placed in an incubator to settle down for at least 30 mrnutes.
hDeltal-IgG4Fc (soluble ligand inhibitor of Notch signaUing) prepared as described above was diluted in complete DMEM to 5 x final concentration requked in the assay and 50 μl of diluted Hgand was added to the 100 μl of N27#ll cells in a 96-well plate. Then 100 μl of CHO-Delta ceUs at 5 x 105 ceUs/ml was added to initiate the signaUing - giving a final volume of 250 μl in each well. The plate was then placed at 37 °C in an incubator overnight.
The following day 150 μl of supernatant was then removed from all the wells, 100 μl of SteadyGlo™ luciferase assay reagent (Promega) was added and the resulting mixture left at room temperature for 5 mrnutes. The mixture was then pipetted up and down 2 times to ensure cell lysis and the contents from each weU were transfeπed to a white 96-weU plate (Nunc). Luminescence was then read in a TopCount™ (Packard) counter. Identical assays were performed using IgG4 as a control.
Results are shown in Figure 20.
Example 11
Soluble h.Taggedir2EGF1-IgG4Fc Antagonizes Notch Activation in CHO-N2 Cells
Antagonist assay of Notch signalling from CHO-Delta cells
The procedure of Example 8 was repeated with use hjaggedlEGFl&2 -IgG4Fc in place of hDeltal-IgG4Fc. Coπesponding experiments were performed using hDeltal-IgG4Fc for comparison. - 155 -
Results are shown in Figure 21. It can be seen that the truncated Jagged protein with just 2 EGF repeats (hjaggedlEGFl&2 -IgG4Fc) provided substantiaUy the same inhibition of Notch signalling as a coπesponding protein comprising a full length human Deltal extraceUular domain (hDeltal-IgG4Fc).
Example 12
Antagonist assays of Notch signalling from mDLLl-Fc-coated Dynabeads
A fusion protein was prepared conesponding to hDeltal-IgG4Fc as described above but using mouse Deltal instead of human Deltal ("mDeltal-IgG4Fc").
Fc tagged Notch signalling modulators were immobilised on Streptavidin-Dynabeads (CELLection Biotin Binder Dynabeads [Cat. No. 115.21] at 4.0 x 108 beads/ml from Dynal (UK) Ltd; 'beads") in combination with biotinylated -IgG-4 (clone JDC14 at 0.5 mg/ml from Pharmingen [Cat. No. 555879]) as foUows:
A volume of Dynabeads beads conesponding to the total number requked was removed from a stock of beads at 4.0 x 108 beads/ml. This was washed twice with 1 ml of PBS, and resuspended in a final volume of 100 μl of PBS containing a biotinylated anti-IgG4 antibody (clone JDC14 at 0.5 mg/ml from Pharmingen [Cat. No. 555879]) hi a sterile Eppendorf tube and placed on shaker at room temperature for 30 rninutes. The amount of biotinylated anti-IgG4 antibody needed to coat the beads was calculated relative to the fact that 1 x 107 streptavidin Dynabeads bind a maximum of 2 μg of antibody.
After coating the beads with antibody they were washed 3 times with 1 ml of PBS and finaUy resuspended in mDeltal-IgG4Fc protein dUuted in PBS. Beads were coated in a solution of 2 ug/ml protein (usually 5 μg of mDeltal-IgG4Fc protein was added per 107 beads to be coated) and the ligand was allowed to bind to the beads in a 1 ml volume for 2 h at room temperatare (or 4 °C overnight) on a rotary shaker to keep the beads in - 156 -
suspension. After coating the beads with mDeltal-IgG4Fc the beads were washed 3 times with 1 ml of PBS and finaUy resuspended complete DMEM at 2 x 107 beads per ml so that addition of 100 μl of this to a well of 2 x 104 reporter ceUs gave a ratio of 100 beads :cell.
To set up the bead antagonist assay, N27#ll ceUs (T8o flask) were removed using 0.02% EDTA solution (Sigma), spun down and resuspended in 10 ml DMEM plus 10%(HT) FCS plus glutamine plus P/S. Ten μl of ceUs were counted and the cell density was • adjusted to 2.0 x 105 cells/ml with fresh DMEM plus 10%(HI) FCS plus glutamine plus P/S. The reporter cells were plated out at 100 μl per weU of a 96-well plate (i.e. 2 x 104 ceUs per well) and were placed in an incubator to settle down for at least 30 minutes.
Purified mDeltal-IgG4Fc was dUuted in complete DMEM to 5 x final concentration requked in the assay and 50 μl of dUuted ligand was added to the 100 μl of N27#ll cells in a 96-weU plate. Then 100 μl of mDeltal-IgG4Fc Dynabeads at 2 x 107 beads/ml was added to initiate the signalling - giving a final volume of 250 μl in each well. The plate was then placed at 37 °C in an incubator overnight.
The following day 150 μl of supernatant was then removed from all the wells, 100 μl of SteadyGlo™ luciferase assay reagent (Promega) was added and the resulting mixture left at room temperature for 5 minutes. The mixture was then pipetted up and down 2 times to ensure cell lysis and the contents from each weU were transfened to a 96 well plate (with V-shaped wells) and spun in a plate holder for 5 ininutes at 1000 rpm at room temperature. The cleared supernatant was then transfened to a white 96-weU plate (Nunc) leaving the beads pellet behind. Luminescence was then read in a TopCount™ (Packard) counter. Results are shown in Figure 22. - 157 -
Example 13
Soluble hiaggedlEGFl&2 -IgG4Fc Antagonizes Notch Activation in CHO-N2 Cells
Antagonist assay of Notch signaUing from Delta Beads
The procedure of Example 8B was repeated with use of hjaggedlEGFl&2 -IgG4Fc in place of mDeltal-IgG4Fc. Coπesponding experiments were performed using hDeltal- IgG4Fc for comparison and using IgG4Fc as a control.
Results are shown in Figure 23. It can be seen that the truncated Jagged protein with just 2 EGF repeats (hjaggedlEGFl&2 -IgG4Fc) provided substantiaUy the same inhibition of Notch signalling as a coπesponding protein comprising a fuU length human Deltal extraceUular domain (hDeltal-IgG4Fc). fn both cases there was significant inhibition compared to control.
Example 14
Reporter Assay using Jurkat cell line
As Jurkat ceUs cannot be cloned by simple limiting dilution a methylceUulose-containing medium (ClonaCelϊ™ TCS) was used with these ceUs.
Jurkat E6.1 ceUs (lymphoblast ceU line; ATCC No TJJB-152) were cloned using ClonaCeU™ Transfected Cell Selection (TCS) medium (StemCeU Technologies, Vancouver, Canada and Meylan, France) according to the manufacturer's guidelines.
Plasmid pLOR92 (prepared as described above) was electroporated into the Jurkat E6.1 ceUs with a Biorad Gene Pulser H electroporator as follows: 158 -
Actively dividing ceUs were spun down and resuspended in ice-cold RPMI medium containing 10% heat-inactivated FCS plus glutamine plus penicillin/streptomycin (complete RPMI) at 2.0 x 107 ceUs per ml. After 10 min on ice, 0.5 ml of ceUs (ie 1 x 107 ceUs) was placed into a pie-cooled 4 mm electroporation cuvette containing 20 μg of plasmid DNA (Endo-free Maxiprep DNA dissolved in sterile water). The cells were electroporated at 300 v and 950 μF and then quickly removed into 0.5 ml of warmed complete RPMI medium in an Eppendorf tube. The ceUs were spun for at 3000 rpm for 1 min in a microfuge and placed at 37 °C for 15 min to recover from being electroporated. The supernatant was then removed and the cells were plated out into a weU of a 6-weU dish in 4 ml of complete RPMI and left at 37 °C for 48 h to allow for expression of the antibiotic resistance marker.
After 48 h the ceUs were spun down and resupended in to 10 ml fresh complete RPMI. This was then divided into 10 x 15 ml Falcon tabes and 8 ml of pre-warmed ClonaCell- TCS medium was added followed by 1 ml of a 10 x final concentration of the antibiotic being used for selection. For G418 selection the final concentration of G418 was 1 mg/ml so a 10 mg/ml solution in RPMI was prepared and 1 ml of this was added to each tube. The tabes were mixed well by inversion and allowed to settle for 15 min at room temperature before being plated out into 10 cm tissue cultare dishes. These were then placed in a CO2 incubator for 14 days when that were examined for visible colonies.
Macroscopic aUy visible colonies were picked off the plates and these colonies were expanded through 96-weU plates to 24-weU plates to T25 flasks.
A clone was selected and transiently transfected with ρLOR91 reporter contract using Lipofectarnine 2000 reagent and then plated out onto a 96-well plate containing plate- bound immobilised hDLLl-Fc (plates were coated by adding 10 μg of purified Notch Hgand protein to each plate in sterile PBS; sealing the lid of the plate with parafilm and incubating at 4 °C overnight or at 37 °C for 2 hours and washing the plate with 200 μl of PBS before use). - 159
Luckerase assays were then conducted generally as described above. Results are shown in Figure 24.
Example 15
Antagonism of A20-Delta and A20-.Tagged Notch signalling with Soluble hDLL-1 Fc
A20-Delta and A20-Jagged ceUs
The INS, IRES, Neo and pA elements were removed from plasmid ρIRESneo2 (Clontech, USA) and inserted into a pUC cloning vector downstream of a chicken beta- actin promoter (eg see GenBank Accession No E02199). Mouse Delta-1 cDNA (eg see GenBank Accession No NM_007865) was inserted between the actin promoter and IVS elements and a sequence with multiple stop codons in aU three reading frames was inserted between the Delta and IVS elements.
The resulting construct was transfected into A20 cells using electroporation and G418 to provide AZO cells expressing mouse Deltal on thek surfaces (A20-Delta).
Conesponding ceUs (A20-Jagged) were prepared using human Jaggedl cDNA (see eg GenBank Accession No U61276).
The procedure of Example was repeated using A20-Delta or A20- Jagged cells (1 x 105 per well) in place of CHO-Delta cells. IgG4 was used as a control. Results are shown in Figure 25. The results show that hDeltal-IgG4Fc was able to inhbit Notch signalling from Jaggedl as weU as from Delta. - 160 -
Example 16
A fusion protein was prepared conesponding to hDeltal-IgG4Fc as described above but using human Jaggedl instead of human Deltal (hJaggedl -IgG4Fc).
The procedure of Example 8 was repeated using hJaggedl -IgG4Fc instead of hDeltal- IgG4Fc, and a coπesponding repeat experiment was performed using hDeltal-IgG4Fc for comparison. Results are shown in Figure 26.
Example 17
Notch signaUing inhibitor reduces induction of tolerance to KLH
B ALB/c mice (eight per group) were treated intranasaUy with i) PBS , ii) KLH (lOmg) alone or Hi) KLH (lOmg) plus hDeltal-IgG4Fc (lOOmg). After 14 days, the mice were given KLH 50mg/TFA s.c. 28 days after the initial KLH priming, mice were chaUenged in the ear with KLH 50mg/IFA s.c and the ear immune response was measured with caUipers as an increase in ear thickness due to the induced inflammatory reaction after 48 hours.
Results are shown in Figure 27.
Example 18
Modulation of cytokine production by v-secretase inhibitor in human CD4+ T cells
Human peripheral blood mononuclear ceUs (PBMC) were purified from blood using Ficoll-Paque separation medium (Pharmacia). Briefly, 28 ml of blood were overlaid on 21 ml of Ficoll-Paque separation medium and centrifuged at 18-20°C for 40 minutes at 400g. PBMC were recovered from the interface and washed 3 times before use for CD4+ T cell purification. - 161 -
Human CD4+ T cells were isolated by positive selection using anti-CD4 microbeads from Miltenyi Biotech according to the manufacturer's instructions. The CD4+ T cells were incubated in tripHcates in a 96-well-plate (flat bottom) at 105 CD4/weU/200ml in RPMI medium containing 10% FCS, glutamine, penicUlin, streptomycin and b -mercaptoethanol.
Cytokine production was induced by stimulating the ceUs with anti-CD3/CD28 T cell expander beads from Dynal at a 1 :1 ratio (bead/ceU). Dynal beads coated with hDeltal- IgG4Fc fusion protein or control beads were added in some of the weUs at a 5:1 ratio (beads/cell) and the γ-secretase inhibitor MW 167 (Calbiochem γ-secretase inhibitor H, Cat. No. 565755) was added variously (in DMSO) to final concentrations of 0, 0.4 mM, 2 mM and 10 mM.
The supematants were removed after 3 days of incubation at 37°C/ 5%CO2/humidified atmosphere and cytokine production was evaluated by ELISA using Pharmingen kits OptEIA Set human IL10 (catalog No. 555157), OptEIA Set human IL-5 (catalog No. 555202) for IL-10 and IL-5 respectively according to the manufacturer's instructions.
Results are shown in Figure 22 from which it can be seen that the γ-secretase inhibitor substantially reversed a Delta-mediated increase in IL-10 expression and also substantially reversed a Delta-mediated reduction in IL-5 expression.
Example 19
Effect of y-secretase inhibitor on Delta-mediated activation of Notch signalling in .Turkat-N2 cells
As Jurkat ceUs cannot be cloned by simple limiting dilution a methylceUulose-containing ' medium (ClonaCell™ TCS) was used with these ceUs. 162 -
Jurkat E6.1 ceUs (lymphoblast ceU line; ATCC No TH3-152) were cloned using ClonaCeU™ Transfected Cell Selection (TCS) medium (StemCeU Technologies, Vancouver, Canada and Meylan, France) according to the manufacturer's guidelines.
Plasmid ρLOR92 (prepared as described above) was electroporated into the Jurkat E6.1 cells with a Biorad Gene Pulser H electroporator as follows:
Actively dividing ceUs were spun down and resuspended in ice-cold RPMI medium containing 10% heat-inactivated FCS plus glutamine plus peniciUin/streptomycin (complete RPMI) at 2.0 x 107 ceUs per ml. After 10 min on ice, 0.5 ml of ceUs (ie 1 x 107 ceUs) was placed into a pre-cooled 4 mm electroporation cuvette containing 20 μg of plasmid DNA (Endo-free Maxiprep DNA dissolved in sterile water). The ceUs were electroporated at 300 v and 950 μF and then quickly removed into 0.5 ml of warmed complete RPMI medium in an Eppendorf tube. The ceUs were spun for at 3000 rpm for 1 min in a microfuge and placed at 37 °C for 15 min to recover from being electroporated. The supernatant was then removed and the cells were plated out into a weU of a 6-weU dish in 4 ml of complete RPMI and left at 37 °C for 48 h to aUow for expression of the antibiotic resistance marker.
After 48 h the ceUs were spun down and resupended into 10 ml fresh complete RPML This was then divided into 10 x 15 ml Falcon tubes and 8 ml of pre-warmed ClonaCell- TCS medium was added followed by 1 ml of a 10 x final concentration of the antibiotic being used for selection. For G418 selection the final concentration of G418 was 1 mg/ml so a 10 mg/ml solution in RPMI was prepared and 1 ml of this was added to each tube. The tabes were mixed well by inversion and allowed to settle for 15 min at room temperature before being plated out into 10 cm tissue cultare dishes. These were then placed in a CO2 incubator for 14 days when that were examined for visible colonies. - 163
MacroscopicaUy visible colonies were picked off the plates and these colonies were expanded through 96-well plates to 24-weU plates to T25 flasks - in complete RPMI containing 1 mg/ml G418.
The resulting clones were each transiently transfected with pLOR91 using Lipofectarnine 2000 reagent (according to manufacturer's protocol) and then plated out onto a 96-well plate containing plate-bound immobilised hDeltal-IgG4Fc (prepared as described below). A weU-perfor ing clone (#24) was selected and used for further study.
10 μg of purified hDeltal-IgG4Fc fusion protein was added to sterile PBS in a sterile Eppendorf tube to give a final volume of 1 ml and 100 μl was added to wells of a 96-weU tissue cultare plate. The lid of the plate was sealed with parafilm and the plate was left at 4 °C overnight or at 37 °C for 2 hours. The protein was then removed and the plate was washed twice with 200 μl of PBS.
Assays were set up in the coated 96-weU plates with 2 x 105 Jurkat ceUs per weU in 100 μl per well of DMEM plus 10%(HI)FCS plus glutamine plus P/S. MW167 was dUuted to 20 μM final concentration in complete RPMI from a 10 mM stock solution in DMSO. Control wells were set up with an equivalent dilution of DMSO alone. Plates were left in a CO2 incubator overnight.
Supernatant was removed from all wells leaving 100 μl of ceUs plus medium behind and 100 μl of SteadyGlo™ luciferase assay reagent (Promega) was added and the cells were left at room temperature for 5 minutes. The mixture was pipetted up and down 2 times to ensure cell lysis and contents from each well were transfened into a white 96-weU OptiPlate™ (Packard). Luminescence was measured in a TopCount™ counter (Packard). - 164
Results of sample assays using the Jurkat ceUs described above with plate-immobiHsed hDeltal-IgG4Fc fusion protein, are shown in Figure 29 (expressed as fold activation of reporter activity compared to ceUs cultured in the absence of Delta).
Example 20
Preparation of Notch inhibitor construct with human agged 1 DSL domain plus
A human Jagged 1 (JAG-1) deletion coding for the DSL domain and the first two only of the natarally occurring EGF repeats (ie omitting EGF repeats 3 to 16 inclusive) was generated by PCR from a JAG-1 clone (for the sequence of the human JAG-1 see Figure 4 and, for example, Genbank Accession No. U73936) using a primer pak as follows:
EN0102f: CCAGGCAAGCTTATGGGTTCCCCACGGACGCGC (SEQ ID NO: 13) and
JlE2Fc4rev: CAGCTCTGTGACAAAGATCTCAATTACCTCGAGATCG (SEQ ID NO:14)
These primers generate a sequence that changes aa. 2 of the leader peptide region from R to G.
PCR conditions were:
1 cycle at 95°C/2 minutes;
18 cycles of (95°C/30 seconds, 60°C/30 seconds, 72°C/ll/2 minutes); and
1 cycle at 72°C/10 minutes.
The DNA was then isolated from a 1% agarose gel in 1 x TBE (Tris/borate/EDTA) buffer. - 165 -
pCONγ (Lonza Biologies, UK) was cut with Hindm and Apal and the following adaptor oligonucleotide sequence was Hgated to introduce a Xhol site then subsequently cloned in DH5α ceUs :
AGCTTTCAGTTCTCGAGGGATCGGCTTCCACCAAGGGCC (SEQ ID NO: 15)
pCONγX was cut with Hindm and Xhol then treated with shrimp alkaline phosphatase (Roche) and gel purified. The purified JAG-1 PCR product was cut with Hindm and Xhol and Hgated into restricted pCONγX then subsequently cloned in DH5α ceUs (InVitrogen). Plasmid DNA was generated using a Qiagen Minprep kit (QIAprep™) according to the manufacturer's instructions and the identity of the PCR product was corrfirmed by sequencing.
The resulting constract (pCONγ hJlE2) coded for the following JAG-1 amino acid sequence (SEQ ED NO: 16) fused to the IgGFc domain encoded by the pCONγ vector.
MGSPRTRGRSGRPLS LLA LCALRAKVCGASGQFELEILSMQ VNGELQNGNCCGGAR
NPGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRN
RIV PFSFA PR5YTL VEAWDS5NDTVQPDSIIEKASHSGMINPSRQ QTLKQNTGVA
HFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGITYACT
GCSPKHGSCKLPGDCRCOYG OGLYCDKCIPHPGCVHGICNEPWQC CETN GGQLCDK
DLNYEGS
(wherein the emboldened portion of the sequence which is single underlined is the DSL domain and the emboldened portions of the sequence which are double underlined are EGF repeats 1 and 2 respectively and the linker/hinge in italic). 166 -
DNA encoding the JlE2.Fc4 sequence was excised with EcoRI and Hindu! and Hgated into EcoRI and Hindm restricted ρEE14.4. The resulting plasmid, pEEl4.JlE2.Fc4, was cloned in DH5α (Invitrogen). Plasmid DNA was generated using a Qiagen Endofree Maxiprep kit (QIAprep™) according to the manufacturer's instractions and the identity of the product was confirmed by sequencing.
Example 21
A series of truncations based on human Deltal comprising varying numbers of EGF repeats was prepared as follows:
A Delta 1 DSL domain plus EGF repeats 1-2
A human Delta 1 (DLL-1) deletion coding for the DSL domain and the fkst two only of the naturally occurring EGF repeats (ie omitting EGF repeats 3 to 8 inclusive) was generated by PCR from a DLL-1 extraceUular (EC) domain/ V5His clone (for the sequence of the human DLL-1 EC domain see Figures and, for example, Genbank Accession No. AF003522) using a primer pak as foUows:
DLacB: CACCAT GGGCAG TCGGTG CGCGCT GG
(SEQ JD NO: 17) and
DLLld3-8: GTAGTT CAGGTC CTGGTT GCAG (SEQ JD NO: 18)
PCR conditions were:
1 cycle at 95°C/3 minutes;
18 cycles of (95°C/1 minute, 60°C/1 minute, 72°C/2 minutes); and
1 cycle at 72°C/2 minutes. - 167 -
The DNA was then isolated from a 1% agarose gel in 1 x U/V-Safe TAE (Tris/acetate/EDTA) buffer (MWG-Biotech, Ebersberg, Germany) and used as a template for PCR with the following primers:
FcDL.4: CACCAT GGGCAG TCGGTG CGCGCT GG (SEQ JD NO: 19) and
FcDLLd3-8:
GGATAT GGGCCC TTGGTG GAAGCG TAGTTC AGGTCC TGGTTG CAG
(SEQ ID NO: 20)
PCR conditions were:
1 cycle at 94°C/3 minutes;
18 cycles of (94°C/1 minute, 68°C/1 minute, 72°C/2 minutes); and
1 cycle at 72°C/10 minutes.
The fragment was Hgated into pCRbluntπ.TOPO (Invitrogen) and cloned in TOP10 ceUs (Invitrogen). Plasmid DNA was generated using a Qiagen Minprep kit (QIAprep™) according to the manufacturer's instructions and the identity of the PCR products was corrfirmed by sequencing.
An IgFc fusion vector pCONγ (Lonza Biologies, UK) was cut with Apal and Hindm then treated with shrimp alkaline phosphatase (Roche) and gel purified.
The DLL-1 deletions cloned in pCRbluntH were cut with Hindm (and EcoRV to aid later selection of the desked DNA product) foUowed by Apal partial restriction. The sequences were then gel purified and Hgated into the pCONγ vector which was cloned into TOP10 ceUs.
Plasmid DNA was generated using a Qiagen Minprep kit (QIAprep™') according to the manufacturer's instructions. 168
The resulting construct (pCONγ HDLLl EGFl-2) coded for the following DLL-1 amino acid sequence (SEQ ID NO: 21) fused to the IgG Fc domain encoded by the pCONγ vector.
MGSRCALA AVLSAL CQVWSSGVFELKLQEFV KKGL GNR CCRGGAGPPPCACR TFFRVCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGF T PGTFS IIEALHTDSPDD ATENPERLISR ATQRH TVGEEWSQD HSSGRTD KYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLP GCDEQHGFCDKPGECKCRVG QGRYCDECIRYPGCLHGTCQQPWQCNCQEG GGLFC NQD NY
(wherein the emboldened portion of the sequence which is single underlined is the DSL domain and the emboldened portions of the sequence which are double underlined are EGF repeats 1 and 2 respectively).
B) Delta 1 DSL domain plus EGF repeats 1-3
A human Delta 1 (DLL-1) deletion coding for the DSL domain and the fkst three only of the naturaUy occurring EGF repeats (ie omitting EGF repeats 4 to 8 inclusive) was generated by PCR from a DLL-1 DSL plus EGF repeats 1-4 clone using a primer pak as foUows:
DLacB: CACCATGGGCAGTCGGTGCGCGCTGG (SEQ ID NO: 22) and
FcDLLd4-8: GGA TAT GGG CCC TTG GTG GAA GCC TCG TCA ATC CCC AGC TCG CAG (SEQ ID NO: 23)
PCR conditions were: lcycle at 94°C/3 minutes; - 169 -
18 cycles of (94°C/1 minute, 68°C/1 minute, 72°C/2.5 minutes); and 1 cycle at 72°C/10 minutes
The DNA was then isolated from a 1 % agarose gel in 1 x U/V-Safe TAE (Tris/acetate/EDTA) buffer (MWG-Biotech, Ebersberg, Germany) and Hgated into pCRbluntJLTOPO and cloned in TOP10 cells (Invitrogen). Plasmid DNA was generated using a Qiagen Minprep kit (QIAprep™) according to the manufactarer's instructions and the identity of the PCR products was confirmed by sequencing.
An IgFc fusion vector pCONγ (Lonza Biologies, UK) was cut with Apal and Hindm then treated with shrimp alkaline phosphatase (Roche) and gel purified.
The DLL-1 deletions cloned in pCRbluntπ were cut with Hindm followed by Apal partial restriction. The sequences were then gel purified and Hgated into the pCONγ vector which was cloned into TOP10 ceUs.
Plasmid DNA was generated using a Qiagen Minprep kit (QIAprep™) according to the manufacturer's instructions and the identity of the PCR products was confirmed by sequencing.
The resulting construct (pCONγ HDLLl EGF1-3) coded for the following DLL-1 sequence (SEQ ID NO: 24) fused to the IgG Fc domain coded by the pCONγ vector.
MGSRCA ALAV SAL CQVWSSGVFELK QEFV KKGLLGNRNCCRGGAGPPPCACR TFFRVC KHYQASVSPEPPCTYGSAVTPV GVDSFS PDGGGADSAFSNPIRFPFGF TWPGTFSLIIEA HTDSPDDLATENPERLISR ATQRHLTVGEEWSQDUISSGRTD KYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPIC P GCDEQHGFCDKPGECKCRVGWOGRYCDECIRYPGCLHGTCQQPWQCNCOEG GGLFC 170 -
NODLNYCTHHKPCKNGATCTNTGQGSYTCSCRPGYTGATCE GIDE
(wherein the emboldened portion of the sequence which is single underlined is the DSL domain and the emboldened portions of the sequence which are double underlined are EGF repeats 1 to 3 respectively).
C) Delta 1 DSL domain plus EGF repeats 1-4
A human Delta 1 (DLL-1) deletion coding for the DSL domain and the fkst four only of the natarally occurring EGF repeats (ie omitting EGF repeats 5 to 8 inclusive) was generated by PCR from a DLL-1 EC domain/V5His clone using a primer pak as foUows:
DLacB: CACCAT GGGCAGTCGGTGCGCGCT GG (SEQ ID NO: 25)and
DLLld5-8: GGTCAT GGCACT CAATTC ACAG (SEQ ID NO: 26)
PCR conditions were:
1 cycle at 95°C/3 minutes;
18 cycles of (95°C/1 minute, 60°C/1 minute, 72°C/2.5 nrinutes); and
1 cycle at 72°C/10 minutes.
The DNA was then isolated from a 1% agarose gel in 1 x U/N-Safe TAE (Tris/acetate EDTA) buffer (MWG-Biotech, Ebersberg, Germany) and used as a template for PCR using the following primers:
FcDL.4:
CACCAT GGGCAGTCGGTGCGCGCT GG
(SEQ ID NO: 27); and
FcDLLd5-8:
GGATAT GGGCCCTTGGTGGAAGCG GTCATGGCACTCAATTCACAG 171 -
(SEQ ID NO: 28)
PCR conditions were:
1 cycle at 94°C/3 minutes;
18 cycles of (94°C/1 minute, 68°C/1 minute, 72°C/2.5 nknutes); and
1 cycle at 72°C/10 πunutes.
The fragment was Hgated into pCRbluntJITOPO and cloned in TOP10 cells (Invitrogen). Plasmid DNA was generated using a Qiagen Minprep kit (QIAprep™) according to the manufacturer's instractions and the identity of the PCR products was confirmed by sequencing.
An IgFc fusion vector pCONγ (Lonza Biologies, UK) was cut with Apal and Hindm then treated with shrimp alkaline phosphatase (Roche) and gel purified.
The DLL-1 deletions cloned in pCRbluntH were cut with Hindm (and EcoRN to aid later selection of the desired DΝA product) foUowed by Apal partial restriction. The sequences were then gel purified and Hgated into the pCOΝγ vector which was cloned into TOP10 ceUs.
Plasmid DΝA was generated using a Qiagen Minprep kit (QIAprep™) according to the manufacturer's instructions and the identity of the PCR products was confirmed by sequencing.
The resulting construct (pCOΝγ HDLLl EGF1-4) coded for the following DLL-1 sequence (SEQ ID NO: 29) fused to the IgGFc domain coded by the pCONγ vector. - 172 -
MGSRCA A AV SAL CQVWSSGVFELK QEFVNKKGLLGNRNCCRGGAGPPPCACR TFFRVC KHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGF PGTFS IIEA HTDSPDD ATENPER ISRLATQRHLTVGEEWSQD HSSGRTD KYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLP GCDEQHGFCDKPGECKCRVG OGRYCDECIRYPGC HGTCOOPWOCNCQEG GG FC NODL YCTHHKPCK GATCTNTGOGSYTCSCRPGYTGATCE GIDECDPSPCKNGGS CTD ENSYSCTCPPGFYGKICE SAMT
(wherein the emboldened portion of the sequence which is single underlined is the DSL domain and the emboldened portions of the sequence which are double underlined are EGF repeats 1 to 4 respectively).
D) Delta 1 DSL domain plus EGF repeats 1-7
A human Delta 1 (DLL-1) deletion coding for the DSL domain and the fkst seven of the naturally occurring EGF repeats (ie omitting EGF repeat 8) was generated by PCR from a DLL-1 EC domain/N5His clone using a primer pak as follows:
DLacB: CACCAT GGGCAG TCGGTG CGCGCT GG (SEQ ID NO: 30); and
DLLld8 : CCTGCT GACGGG GGCACT GCAGTT C (SEQ ID NO: 31)
PCR conditions were:
1 cycle at 95°C/3 minutes;
18 cycles of (95°C/1 minute, 68°C/1 minute, 72°C/3 minutes); and
1 cycle at 72°C/10 minutes. 173
The DNA was then isolated from a 1% agarose gel in 1 x U V-Safe TAE (Tris/acetate/EDTA) buffer (MWG-Biotech, Ebersberg, Germany) and used as a template for PCR using the following primers:
FcDL.4: CACCAT GGGCAG TCGGTG CGCGCT GG (SEQ ID NO: 32); and
FCDLLd8:
GGATAT GGGCCC TTGGTG GAAGCC CTGCTG ACGGGG GCACTG CAGTTC
(SEQ ID NO: 33)
PCR conditions were:
1 cycle at 94°C/3 minutes;
18 cycles of (940C/lπHnute, 68°C/lminute, 72°C/3minutes); and
1 cycle at 72°C/10 minutes.
The fragment was Hgated into pCRbluntH.TOPO and cloned in TOP10 cells (Invitrogen). Plasmid DNA was generated using a Qiagen Minprep kit (QIAprep™) according to the manufactarer's instructions and the identity of the PCR products was confirmed by sequencing.
An IgFc fusion vector pCONγ (Lonza Biologies, UK) was cut with Apal and Hindm then treated with shrimp alkaline phosphatase (Roche) and gel purified.
The DLL-1 deletions cloned in pCRbluntπ were cut with Hindm (and EcoRV to aid later selection of the desked DNA product) foUowed by Apal partial restriction. The sequences were then gel purified and Hgated into the pCONγ vector which was cloned into TOP10 ceUs.
Plasmid DNA was generated using a Qiagen Minprep kit (QIAprep™1) according to the manufacturer's instructions and the PCR products were sequenced. 174 -
The resulting constract (pCONγ hDLLl EGF1-7) coded for the following DLL-1 sequence (SEQ ID NO: 34) fused to the IgG Fc domain coded by the pCONγ vector.
MGSRCALA AVLSAL CQλMSSGVFELKLQEFVNKKGLLG RNCCRGGAGPPPCACR TFFRVC KHYQASVSPEPPCTYGSAVTPV GVDSFSLPDGGGADSAFSNPIRFPFGF TWPGTFSLIIEA HTDSPDD ATENPER ISRLATQRHLTVGEEWSQD HSSGRTDL KYSYRFVCDEHYYGEGCSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLP GCDEQHGFCDKPGECKCRVG QGRYCDECIRYPGCLHGTCQOPWOCNCQEGWGG FC NODLNYCTHHKPCKNGATCTNTGOGSYTCSCRPGYTGATCELGIDECDPSPCKNGGS CTDLENSYSCTCPPGFYGKICELSAMTCADGPCFNGGRCSDSPDGGYSCRCPVGYSG FNCEKKIDYCSSSPCSNGAKCVDLGDAYLCRCOAGFSGRHCDDNVDDCASSPCANGG TCRDGVMDFSCTCPPGYTGRNCSAPVSR
(wherein the emboldened portion of the sequence which is single underlined is the DSL domain and the emboldened portions of the sequence which are double underlined are EGF repeats 1 to 7 respectively).
E) Transfection and Expression
i) Transfection and expression of constructs of constructs A. C and D
Cos 1 cells were separately transfected with each of the expression constructs from Examples 1, 3 and 4 above (viz pCONγ hDLLl EGFl-2, pCONγ hDLLl EGF1-4, pCONγ hDLLl EGF1-7) and pCONγ control as foUows: 175 ■
In each case 3xl06 ceUs were plated in a 10cm dish in Dulbecco's Modified Eagle's Medium (DMEM) + 10% Fetal Calf Serum (FCS) and cells were left to adhere to the plate overnight. The cell monolayer was washed twice with 5 ml phosphate-buffered saline (PBS) and ceUs left in 8 ml OPTIMEM ™ medium (Gibco/Invitrogen). 12 μg of the relevant constmct DNA was dUuted into 810 μl OPTIMEM medium and 14 μl Lipofectamine2000™ cationic Hpid transfection reagent (Invitrogen) was diluted in 810 μl OPTIMEM medium. The DNA-containing and Lipofectamfne2000 reagent- containing solutions were then mixed and incubated at room temperatare for a minimum of 20 minutes, and then added to the ceUs ensuring an even distribution of the transfection mix within the dish. The cells were incubated with the transfection reagent for 6 hours before the media was removed and replaced with 20 ml DMEM + 10% FCS. Supernatant containing secreted protein was collected from the ceUs after 5 days and dead ceUs suspended in the supernatant were removed by centrifugation (4,500 rpm for 5 minutes). The resulting expression products were designated: hDLLl EGFl-2 Fc (from pCONγ hDLLl EGFl-2), hDLLl EGF1-4 Fc (from pCONγ hDLLl EGF1-4) and hDLLl EGF1-7 Fc (from pCONγ hDLLl EGF1-7).
Expression of the Fc fusion proteins was assessed by western blot. The protein in 10 μl of supernatant was separated by 12% SDS-PAGE and blotted by semi dry apparatus on to Hybond™-ECL (Amersham Pharmacia Biotech) nitrocellulose membrane (17 N for 28 minutes). The presence of Fc fusion proteins was detected by Western blot using JDC14 anti-human IgG4 antibody dUuted 1:500 in blocking solution (5% non-fat Milk solids in Tris-buffered saline with Tween 20 surfactant; TBS-T). The blot was incubated in this solution for 1 hour before being washed in TBS-T. After 3 washes of5 rninutes each, the presence of mouse anti-human ϊgG4 antibodies was detected using anti mouse IgG- HPRT conjugate antisemm diluted 1:10,000 in blocking solution. The blot was incubated in this solution for 1 hour before being washed in TBS-T (3 washes of 5 minutes each). The presence of Fc fusion proteins was then visuahsed using ECL™ detection reagent (Amersham Pharmacia Biotech). - 176 -
The amount of protein present in 10 ml supematant was assessed by comparing to Kappa chain standards containing 10 ng (7), 30ng (8) and 100 ng (9) protein.
The blot results are shown in Figure 30.
H) Transfection and expression of constructs of construct B
Cos 1 cells were transfected with the expression construct from Example 2 above (viz pCONγ hDLLl EGF1-3) as foUows:
7.1xl05 ceUs were plated in a T25 flask in Dulbecco's Modified Eagle's Medium (DMEM) + 10% Fetal Calf Serum (FCS) and ceUs were left to adhere to the plate overnight. The ceU monolayer was washed twice with 5 ml phosphate-buffered saline (PBS) and cells left in 1.14 ml OPTIMEM™ medium (Gibco/ nvitrogen). 2.85 μg of the relevant constmct DNA was dUuted into 143 μl OPTIMEM medium and 14.3 μl Lipofectamine2000™ cationic lipid transfection reagent (Invitrogen) was diluted in 129 μl OPTIMEM medium and incubated at room temperature for 45 minutes. The DNA- containing and Lipofectarnine2000 reagent-containing solutions were then mixed and incubated at room temperatare for 15 minutes, and then added to the cells ensuring an even distribution of the transfection mix within the flask. The cells were incubated with the transfection reagent for 18 hours before the media was removed and replaced with 3 ml DMEM + 10% FCS. Supematant containing secreted protein was coUected from the cells after 4 days and dead cells suspended in the supernatant were removed by centrifugation (1 ,200 rpm for 5 minutes). The resulting expression product was designated: hDLLl EGF1-3 Fc (from pCONγ hDLLl EGF1-3).
F) Luciferase Reporter Assay
The Fc-tagged Notch ligand expression products from A to D above (hDLLl EGFl-2 Fc, hDLLl EGF1-4 Fc and hDLLl EGF1-7 Fc) were each separately immobilised on 177 -
Streptavidin-Dynabeads (CELLection Biotin Binder Dynabeads [Cat. No. 115.21] at 4.0 x 108 beads/ml from Dynal (UK) Ltd; 'beads") in combination with biotinylated -IgG-4 (clone JDC14 at 0.5 mg/ml from Pharmingen [Cat. No. 555879]) as follows:
1 x 107 beads (25 μl of beads at 4.0 x 108 beads/ml) and 2 μg biotinylated -IgG4 was used for each sample assayed. PBS was added to the beads to 1 ml and the mixture was spun down at 13,000 rpm for 1 minute. FoUowing wasMng with a further 1 ml of PBS the mixture was spun down again. The beads were then resuspended in a final volume of 100 μl of PBS containing the biotinylated α-IgG4 in a sterile Eppendorf tube and placed on shaker at room temperature for 30 minutes. PBS to was added to 1 ml and the mixture was spun down at 13,000 rpm for 1 rrHnute and then washed twice more with 1 ml of PBS.
The mixture was then spun down at 13,000 rpm for 1 minute and the beads were resupsended in 50 μl PBS per sample. 50 μl of biotinylated -IgG4 -coated beads were added to each sample and the mixture was incubated on a rotary shaker at 4 °C ovemight. The tube was then spun at 1000 rpm for 5 minutes at room temperature. The beads then were washed with 10 ml of PBS, spun down, resupended in 1 ml of PBS, transferred to a sterile Eppendorfta.be, washed with a further 2 x 1 ml of PBS, spun down and resuspended in a final volume of 100 μl of DMEM plus 10%(Hι)FCS plus glutamine plus P/S, i.e. at 1.0 x 105beads/μl.
Stable N27#ll ceUs (T80 flask)were removed using 0.02% EDTA solution (Sigma), spun down and resuspended in 10 ml DMEM plus 10%(HI)FCS plus glutamine plus P/S. 10 μl of cells were counted and the cell density was adjusted to 1.0 x 105 cells/ml with fresh DMEM plus 10%(HI)FCS plus glutamine plus P/S. 1.0 x 105 of the cells were plated out per weU of a 24-weU plate in a 1 ml volume of DMEM plus 10%(HI)FCS plus glutamine plus P/S and cells were placed in an incubator to settle down for at least 30 minutes. - 178
20 μl of beads were then added in duplicate to a pak of weUs to give 2.0 x 106 beads / well (100 beads / cell). The plate was left in a CO2 incubator overnight.
Supernatant was then removed from all the wells, 100 μl of SteadyGIo™ luciferase assay reagent (Promega) was added and the resulting mixture left at room temperatare for 5 minutes.
The mixture was then pipetted up and down 2 times to ensure ceU lysis and the contents from each well were transfened to a 96 weU plate (with N-shaped wells) and spun in a plate holder for 5 minutes at 1000 rpm at room temperature.
175 μl of cleared supernatant was then transfened to a white 96-weU plate (Νunc) leaving the beads pellet behind.
Luminescence was then read in a TopCount™ (Packard) counter. Results are shown in Figure 31 (where activity from fusion protein comprising a fuU Dill EC domain φDeltal-IgG4Fc) is also shown for comparison).
Example 22
agged truncations
A similar series of truncations based on human Jaggedl comprising varying numbers of EGF repeats was prepared as follows:
In a similar manner to that described in Example 21, nucleotide sequences coding for the human Jaggedl (hJagl) DSL domain and the fkst two, three, four and sixteen respectively of the natarally occurring Jagged EGF repeats were generated by PCR from a human Jagged-1 (see eg GenBank Accession No U61276) cDNA. The sequences were then purified, Hgated into a pCONγ expression vector coding for an immunogolbulin Fc - 179
domain, expressed and coated onto micro-beads. The expressed proteins comprised the DSL domain and the first two (hJagl EGFl-2), three (hJagl EGF1-3), four (hJagl EGF1- 4) and sixteen (hJagl EGF1-16) respectively of the Jagged EGF repeats fused to the IgG Fc domain encoded by the pCONγ vector.
Beads coated with each of the expressed proteins were then tested for activity in the Notch signalling reporter assay as described above (Example 21). The activity data obtained is shown in Figure 32 .
Similar assays were conducted with expressed Jagged proteins alongside coπesponding Delta proteins, for more ready comparison. Results are shown in Figure 33.
Example 23
Assay of Jagged EGFl-2 with increased sensitivity
In a further experiment purified protein comprising human Jaggedl DSL domain plus the fkst two EGF repeats (hjaggedlEGFl&2 -IgG4Fc) from Example 7 was coated onto beads and tested for activity in a Notch reporter assay as described above, at a higher protein load, to give greater sensitivity. The activity data obtained is shown in Figure 34 (activity from a fusion protein comprising a full Dill EC domain (hDeltal-IgG4Fc) is also shown for comparison).
AU publications are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention wiU be apparent to those skUled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific prefened embodiments, it should be understood that the invention as claimed should not be limited to such specific embodiments. Indeed, numerous modifications of the described modes for caπying out the invention which will be obvious to those skiUed in 180 -
biochemistry and biotechnology or related fields are intended to be within the scope of the foUowing claims.
References (incorporated herein by reference)
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Panin VM, et al. (1997) Nature 387:908-912. Sasai et al. (1994) CeU 79:779-790. Schroeter, E.H. et al. (1998) Nature 393{6683}:382-6. Struhl G, Adachi A. (1998) Cell 93:649-660. Takebayashi K. et al. (1994) J Biol Chem 269(7):150-6. Tamura K, et al. (1995) Cun. Biol. 5:1416-1423. Valenzuela et al. (1995) J. Neurosci. 15:6077-6084. Weimnaster G. (2000) Cun. Opin. Genet. Dev. 10:363-369. WUson and Hemmati-Brivanlou (1997) Neuron 18:699-710. Zhao et al. (1995) J. Jmmunol. 155:3904-3911.

Claims

182 -Claims
1. A product comprising: i) an inhibitor of the Notch signalling pathway or a polynucleotide coding for such an inhibitor; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic deterniinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
2. A product as claimed in claim 1 wherein the inhibitor of Notch signaUing does not act by downregulating expression of Notch or a Notch Hgand.
3. A product comprising : i) an inhibitor of Notch signalling in the form of a Notch antagonist or a polynucleotide coding for such an antagonist; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
4. A product comprising: i) an inhibitor of Notch signaUing in the form of an agent which inhibits Notch-Notch
Hgand interaction or a polynucleotide coding for such an agent; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic deterniinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system. 183
5. A product as claimed in claim 4 wherein the inhibitor of Notch signaUing binds to a Notch ligand or Notch receptor so as to interfere with Notch-Notch ligand interaction.
6. A product as claimed in any one of the preceding claims in the form of a pharmaceutical composition or kit.
7. A product as claimed in any one of the preceding claims in the form of a therapeutic vaccine composition or kit for treating infectious disease.
8. A product as claimed in any one of claims 1 to 6 in the form of a prophylactic vaccine composition or kit for preventing infectious disease.
9. A product as claimed in any one of the preceding claims wherein the inhibitor of Notch signaUing is an agent capable of inhibiting the activity of a Notch receptor or a Notch ligand.
10. A product as claimed in any one of the preceding claims wherein the inhibitor of Notch signaUing is an agent capable of inhibiting the activity or downregulating the expression of a downstream component of the Notch signaUing pathway.
11. A product as claimed in any one of the preceding claims wherein the inhibitor of Notch signalling is a protein or polypeptide or a polynucleotide which codes for such a protein or polypeptide.
12. A product as claimed in any one of the preceding claims wherein the inhibitor of Notch signalling comprises or codes for the extraceUular domain of Delta or a fragment thereof.
12. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises or codes for the extraceUular domain of Senate or Jagged or a fragment thereof. - 184 -
13. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises or codes for the extracellular domain of Notch or a fragment thereof.
14. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and optionaUy a
Notch ligand N-terminal domain or a heterologous amino acid sequence but which is substantially free of Notch ligand EGF-like domains;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
15. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and at least one
Notch Hgand EGF-like domain;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dtfferent); or iii) a polynucleotide coding for such a protein or polypeptide.
16. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch Hgand DSL domain and at least two
Notch ligand EGF-like domains; k) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide. - 185
17. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch Hgand DSL domain and either 1 or
2, but no more than 2 Notch Hgand EGF-like domains;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or
Hi) a polynucleotide coding for such a protein or polypeptide.
18. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch Hgand DSL domain having at least
50% amino acid sequence identity to the DSL domain of human Deltal, Delta3 or Delta4 and at least one Notch ligand EGF-like domain having at least 50% aπiino acid sequence identity to an EGF-like domain of human Deltal, Delta3 or Delta4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
19. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch Hgand DSL domain having at least 50% amino acid sequence identity to the DSL domain of human Deltal, Delta3 or Delta4 and either 1 or 2, but no more than 2 Notch ligand EGF-like domains having at least 50% amino acid sequence identity to an EGF-like domain of human Deltal, Delta3 or Delta4; H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or Hi) a polynucleotide coding for such a protein or polypeptide.
20. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: 186 -
i) a protein or polypeptide which comprises a Notch EGF-like domain having at least
50% amino acid sequence identity to EGF11 of human Notchl, Notch2, Notch3 or
Notch4 and a Notch EGF-like domain having at least 50% amino acid sequence identity to EGF12 of human Notchl, Notchfi, Notch3 or Notch4; ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same ordkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
21. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
50% amino acid sequence identity to the DSL domain of human Jaggedl or Jagged2 and at least one Notch ligand EGF-like domain having at least 50% amino acid sequence identity to an EGF-like domain of human Jagged 1 or Jagged2;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
22. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least 50% amino acid sequence identity to the DSL domain of human Jaggedl or Jagged2 and either 0, 1 or 2, but no more than 2 Notch ligand EGF-like domains having at least 50% amino acid sequence identity to an EGF-like domain of human Jagged 1 or Jagged2; H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or Hi) a polynucleotide coding for such a protein or polypeptide.
23. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: 187 -
i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
70% amino acid sequence identity to the DSL domain of human Deltal, Delta3 or Delta4 and at least one Notch ligand EGF-like domain having at least 70% amino acid sequence identity to an EGF-like domain of human Deltal , Delta3 or Delta4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
24. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
70% amino acid sequence identity to the DSL domain of human Deltal, Delta3 or Delta4 and either 0, 1 or 2, but no more than 2 Notch ligand EGF-like domains having at least
70% amino acid sequence identity to an EGF-like domain of human Deltal, Delta3 or
Delta4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same ordkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
25. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises an EGF domain having at least 70% amino acid sequence identity to EGF11 of human Notchl, Notch2, Notch3 orNotch.4 and an
EGF domain having at least 70% a ino acid sequence identity to EGF12 of human
Notchl, Notch2, Notch3 or Notch4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same ordkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide. - 188 -
26. A product as claimed in any one of claims 12 to 25 wherein the protein or polypeptide is fused to a heterologous amino acid sequence.
27. A product as claimed in claim 26 wherein the protein or polypeptide is fused to an immunoglobulin Fc (IgFc) domain.
28. A product as claimed in claim 27 wherein the IgFc domain is a human IgGl or IgG4 Fc domain.
29. A product as claimed in any one of claims 12 to 28 wherein the protein or polypeptide further comprises a Notch Hgand N-terminal domain.
30. A product as claimed in any one of claims 1 to 11 wherein the inhibitor of Notch signaUing is an antibody, antibody fragment or antibody derivative or a polynucleotide which codes for an antibody, antibody fragment or antibody derivative.
31. A product as claimed in claim 30 wherein the antibody, antibody fragment or antibody derivative binds to a Notch receptor or a Notch Hgand so as to interfere with Notch ligand-receptor interaction.
32. The use of an inhibitor of the Notch signalling pathway in the manufacture of a medicament for use as an immunostimulant wherein the medicament is not for use in reversing bacteria, infection or tumour-induced immunosuppression or for the treatment of a tumour.
33. The use of an inhibitor of the Notch signalling pathway in the manufacture of a medicament for use as an inrmunostimulant wherein the inhibitor does not act by downregulating expression of Notch or a Notch Hgand. - 189 -
34. The use of an inhibitor of the Notch signalling pathway in the manufacture of a medicament for use in vaccination against a pathogen.
35. The use of an inhibitor of the Notch signalling pathway in the manufacture of a medicament for use as an adjuvant for vaccination against a pathogen.
36. A use as claimed in any one of claims 30 to 35 wherein the inhibitor of the Notch signaUing pathway is a Notch signaUing repressor or an agent which increases the expression or activity of a Notch signalling repressor.
37. A use as claimed in any one of claims 30 to 35 wherein the inhibitor of the Notch signaUing pathway is an agent capable of inhibiting the activity of a Notch receptor or a Notch ligand.
38. A use as claimed in any one of claims 30 to 35 wherein the inhibitor of the Notch signaUing pathway is an agent capable of inMbiting the activity or downregulating the expression of a downstream component of the Notch signaUing pathway.
39. A use as claimed in any one of claims 30 to 35 wherein the inhibitor of the Notch signaUing pathway is an agent which binds to a Notch receptor or to a Notch ligand so as to interfere with Notch Hgand-receptor interaction.
40. A use as claimed in any one of claims 30 to 39 wherein the inhibitor of the Notch signaUing pathway is a protein or polypeptide or a polynucleotide which codes for such a protein or polypeptide.
41. A use as claimed in claim 40 wherein the agent comprises or codes for the extraceUular domain of Delta or a fragment thereof. 190 -
42. A use as claimed in claim 40 wherein the agent comprises or codes for the extraceUular domain of Senate or Jagged or a fragment thereof.
43. A use as claimed in claim 40 wherein the agent comprises or codes for the extraceUular domain of Notch or a fragment thereof.
44. A use as claimed in any one of claims 30 to 40 wherein the inhibitor of the Notch signaUing pathway comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and at least one
Notch ligand EGF-like domain; ii) a multimer of such a protein or polypeptide; or
Hi) a polynucleotide coding for such a protein or polypeptide.
45. A use as claimed in any one of claims 30 to 40 wherein the inhibitor of the Notch signaUing pathway comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and at least two
Notch ligand EGF-like domains;
H) a multimer of such a protein or polypeptide; or
Hi) a polynucleotide coding for such a protein or polypeptide.
46. A use as claimed in any one of claims 30 to 40 wherein the inhibitor of the Notch signaUing pathway comprises: i) a protein or polypeptide which comprises a Notch Hgand DSL domain and either 0, 1 or 2, but no more than 2 Notch ligand EGF-like domains;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
47. A use as claimed in any one of claims 30 to 40 wherein the inhibitor of the Notch signaUing pathway comprises: 191 -
i) a protein or polypeptide which comprises a Notch Hgand DSL domain having at least
50% amino acid sequence identity to the DSL domain of human Deltal, Delta3 or Delta4 and at least one Notch ligand EGF-like domain having at least 50% amino acid sequence identity to an EGF-like domain of human Deltal, Delta3 or Delta4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
48. A use as claimed in any one of claims 30 to 40 wherein the inhibitor of the Notch signaUing pathway comprises: i) a protein or polypeptide which comprises a Notch Hgand DSL domain having at least 50% a ino acid sequence identity to the DSL domain of human Deltal , Delta3 or Delta4 and either 1 or 2, but no more than 2 Notch ligand EGF-like domains having at least 50% amino acid sequence identity to an EGF-like domain of human Deltal, Delta3 or Delta4; u) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or ki) a polynucleotide coding for such a protein or polypeptide.
49. A use as claimed in any one of claims 30 to 40 wherein the inhibitor of the Notch signaUing pathway comprises: i) a protein or polypeptide which comprises an EGF domain having at least 50% a ino acid sequence identity to EGF11 of human Notchl, Notch2, Notch3 or Notch4 and an
EGF domain having at least 50% amino acid sequence identity to EGF12 of human
Notchl, Notch2, Notch3 or Notch4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
50. A use as claimed in any one of claims 30 to 40 wherein the inbibitoi of the Notch signaUing pathway comprises: 192 -
i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
50% amino acid sequence identity to the DSL domain of human Jaggedl or Jagged2 and at least one Notch ligand EGF-like domain having at least 50% amino acid sequence identity to an EGF-like domain of human Jagged 1 or Jagged2; ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same ordkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
51. A use as claimed in any one of claims 30 to 40 wherein the inhibitor of the Notch signaUing pathway comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least 50% amino acid sequence identity to the DSL domain of human Jaggedl or Jagged2 and either 1 or 2, but no more than 2 Notch ligand EGF-like domains having at least 50% amino acid sequence identity to an EGF-like domain of human Jagged 1 or Jagged2; H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or Hi) a polynucleotide coding for such a protein or polypeptide.
52. Ause as claimed in any one of claims 30 to 40 wherein the inhibitor of the Notch signaUing pathway comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
70% amino acid sequence identity to the DSL domain of human Deltal, Delta3 or Delta4 and at least one Notch ligand EGF-like domain having at least 70% amino acid sequence identity to an EGF-like domain of human Deltal, Delta3 or Delta4; ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
53. Ause as claimed in any one of claims 30 to 40 wherein the inhibitor of the Notch signaUing pathway comprises: - 193 -
i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least 70% amino acid sequence identity to the DSL domain of human Deltal, Delta3 or Delta4 and either 1 or 2, but no more than 2 Notch ligand EGF-like domains having at least 70% amino acid sequence identity to an EGF-like domain of human Deltal, Delta3 or Delta4; H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or Hi) a polynucleotide coding for such a protein or polypeptide.
54. A use as claimed in any one of claims 30 to 40 wherein the inhibitor of the Notch signaUing pathway comprises: i) a protein or polypeptide which comprises an EGF domain having at least 70% amino acid sequence identity to EGF11 of human Notchl, Notch2, Notch3 or Notch4 and an
EGF domain having at least 70% amino acid sequence identity to EGF12 of human
Notchl, Notch2, Notch3 or Notch4; n) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
55. A use as claimed in any one of claims 40 to 54 wherein the protein or polypeptide is fused to a heterologous amino acid sequence.
56. A use as claimed in claim 55 wherein the protein or polypeptide is fused to an irnmunoglobulin Fc (IgFc) domain.
57. A use as claimed in claim 56 wherein the IgFc domain is a human IgGl or IgG4 Fc domain.
58. A use as claimed in any one of claims 32 to 39 wherein the inhibitor of the Notch signaUing pathway is an antibody, antibody fragment or antibody derivative or a polynucleotide which codes for an antibody, antibody fragment or antibody derivative. 194
59. Ause as claimed in claim 58 wherein the antibody, antibody fragment or antibody derivative binds to a Notch receptor or a Notch ligand so as to interfere with Notch Hgand-receptor interaction.
60. The use of a binding agent which binds to a Notch Hgand so as to interfere with binding of the Hgand to a Notch receptor, or a polynucleotide which codes for such a binding agent, in the manufacture of a medicament for use as an immunostimulant.
61. The use of an antibody or antibody derivative which binds to a Notch receptor or to a Notch ligand, or a polynucleotide which codes for such an antibody or antibody derivative, in the manufacture of a medicament for use as an immunostimulant.
62. A method for stimulating the immune system by administering an inhibitor of the Notch signalling pathway which does not comprise reversing bacteria, infection or tumour- induced immunosuppression or treatment of a tumour.
63. A method for stimulating the immune system by administering an inhibitor of the Notch signaUing pathway wherein the inhibitor does not act by downregulating expression of Notch or a Notch Hgand.
64. A method for stimulating the immune system to treat or prevent an infection by administering an inhibitor of the Notch signaUing pathway which does not comprise reversing bacteria, infection or tumour-induced immunosuppression or treatment of a tumour.
65. A method for stimulating the immune system to treat or prevent an infection by admim^tering an inhibitor of the Notch signaUing pathway wherein the inhibitor of the Notch signalling pathway does not act by downregulating expression of Notch or a Notch Hgand. 195
66. A method for vaccination against a pathogen by administering an inhibitor of the Notch signalling pathway.
67. A method for enhancing vaccination against a pathogen by administering an inhibitor of the Notch signalling pathway.
68. A method for treating a chronic pathogen infection by adπiinistering an inhibitor of the Notch signalling pathway.
69. A method of increasing the immune response of a subject to a vaccine antigen or antigenic determinant comprising administering an effective amount of an inhibitor of the Notch signaUing pathway to said subject simultaneously, separately or sequentially with said vaccine antigen or antigenic determinant or simultaneously, separately or sequentially with a polynucleotide coding for said vaccine antigen or antigenic determinant.
70. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of the Notch signalling pathway comprises a protein or polypeptide or a polynucleotide which codes for such a protein or polypeptide.
71. A method as claimed in any one of claims 62 to 69 wherein the agent comprises or codes for the extraceUular domain of Delta or a fragment thereof.
72. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of the Notch signalling pathway comprises or codes for the extraceUular domain of Senate or Jagged or a fragment thereof. 196
73. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of the Notch signalling pathway comprises or codes for the extraceUular domain of Notch or a fragment thereof.
74. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and at least one
Notch ligand EGF-like domain;
H) a multimer of such a protein or polypeptide; or
Hi) a polynucleotide coding for such a protein or polypeptide.
75. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain and at least two
Notch ligand EGF-like domains;
H) a multimer of such a protein or polypeptide; or
Hi) a polynucleotide coding for such a protein or polypeptide.
76. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch Hgand DSL domain and either 0, 1 or 2, but no more than 2 Notch ligand EGF-like domains;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
77. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch Hgand DSL domain having at least 50% amino acid sequence identity to the DSL domain of human Deltal, Delta3 or Delta4 - 197
and at least one Notch ligand EGF-like domain having at least 50% amino acid sequence identity to an EGF-like domain of human Deltal, Delta3 or Delta4; k) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or ki) a polynucleotide coding for such a protein or polypeptide.
78. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch Hgand DSL domain having at least
50% amino acid sequence identity to the DSL domain of human Deltal, Delta3 or Delta! and either 0, 1 or 2, but no more than 2 Notch Hgand EGF-like domains having at least
50% amino acid sequence identity to an EGF-like domain of human Deltal, Delta3 or
Delta4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or
Hi) a polynucleotide coding for such a protein or polypeptide.
79. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises an EGF domain having at least 50% amino acid sequence identity to EGF11 of human Notchl, Notch2, Notch3 or Notch4 and an
EGF domain having at least 50% amino acid sequence identity to EGF12 of human
Notchl, Notch2, Notch3 or Notch4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
80. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of Notch signaUing comprises: 198
i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
50% amino acid sequence identity to the DSL domain of human Jaggedl or Jagged2 and at least one Notch ligand EGF-like domain having at least 50% amino acid sequence identity to an EGF-like domain of human Jagged 1 or Jagged2; ii) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
81. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least 50% amino acid sequence identity to the DSL domain of human Jaggedl or Jagged2 and either 0, 1 or 2, but no more than 2 Notch ligand EGF-like domains having at least 50% amino acid sequence identity to an EGF-like domain of human Jagged 1 or Jagged2; H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or Hi) a polynucleotide coding for such a protein or polypeptide.
82. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of Notch signalling comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
70% amino acid sequence identity to the DSL domain of human Deltal, Delta3 or Delta4 and at least one Notch ligand EGF-like domain having at least 70% amino acid sequence identity to an EGF-like domain of human Deltal, Delta3 or Delta4; k) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or
Hi) a polynucleotide coding for such a protein or polypeptide. 199 -
83. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises a Notch ligand DSL domain having at least
70% amino acid sequence identity to the DSL domain of human Deltal, Delta3 or Delta4 and either 1 or 2, but no more than 2 Notch ligand EGF-like domains having at least 70% amino acid sequence identity to an EGF-like domain of human Deltal, Delta3 or Delta4; n) a multimer of such a protein or polypeptide (wherein each monomer may be the same or different); or
Hi) a polynucleotide coding for such a protein or polypeptide.
84. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of Notch signaUing comprises: i) a protein or polypeptide which comprises an EGF domain having at least 70% amino acid sequence identity to EGF11 of human Notchl, Notch2, Notch3 or Notch4 and an
EGF domain having at least 70% amino acid sequence identity to EGF12 of human
Notchl , Notch2, Notch3 or Notch4;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same or dkferent); or iii) a polynucleotide coding for such a protein or polypeptide.
85. A method as claimed in any one of claims 62 to 69 wherein the protein or polypeptide is fused to a heterologous amino acid sequence.
86. A method as claimed in claim 85 wherein the protein or polypeptide is fused to an immunoglobuHn Fc (IgFc) domain.
87. A method as claimed in claim 86 wherein the IgFc domain is a human IgG4 Fc domain. 200 -
88. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of the Notch signalling pathway is a Notch signaUing repressor or an agent which increases the expression or activity of a Notch signalling repressor.
89. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of the Notch signaUing pathway is an agent capable of inhibiting the activity of a Notch receptor or a Notch Hgand.
90. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of the Notch signalling pathway is an agent capable of inhibiting the activity or downregulating the expression of a downstream component of the Notch signalling pathway.
91. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of the Notch signalling pathway is an agent which binds to a Notch receptor or a Notch Hgand so as to interfere with Notch-Notch Hgand interaction.
92. A method as claimed in claim 91 wherein the agent is a protein or polypeptide or a polynucleotide which codes for such a protein or polypeptide.
93. A method as claimed in any one of claims 62 to 69 wherein the inhibitor of the Notch signaUing pathway is an antibody, antibody fragment or antibody derivative or a polynucleotide which codes for an antibody, antibody fragment or antibody derivative.
94. A method as claimed in claim 93 wherein the antibody, antibody fragment or antibody derivative binds to a Notch receptor or a Notch Hgand so as to interfere with Notch-Notch ligand interaction.
95. A method for stimulating the immune system by administering an antibody or antibody derivative which binds to a Notch receptor or to a Notch Hgand, or by administering a polynucleotide which codes for such an antibody or antibody derivative. 201
96. An adjuvant composition comprising an inhibitor of the Notch signalling pathway.
97. A composition as claimed in claim 96 wherein the inhibitor of the Notch signaUing pathway is a Notch signaUing repressor or an agent which increases the expression or activity of a Notch signalling repressor.
98. A composition as claimed in claim 96 wherein the inhibitor of the Notch signaUing pathway is an agent capable of inhibiting the activity of a Notch receptor or a Notch ligand.
99. A composition as claimed in claim 96 wherein the inhibitor of the Notch signaUing pathway is an agent capable of inhibiting the activity or downregulating the expression of a downstream component of the Notch signaUing pathway.
100. A composition as claimed in claim 96 wherein the inhibitor of the Notch signaUing pathway is an agent which binds to a Notch receptor or a Notch Hgand so as to interfere with Notch-Notch ligand interaction.
101. A composition as claimed in claim 96 wherein the agent is a protein or polypeptide or a polynucleotide which codes for such a protein or polypeptide.
102. A composition as claimed in claim 101 wherein the inhibitor of the Notch signaUing pathway is an antibody, antibody fragment or antibody derivative or a polynucleotide which codes for an antibody, antibody fragment or antibody derivative.
103. A composition as claimed in claim 102 wherein the antibody, antibody fragment or antibody derivative binds to a Notch receptor or a Notch ligand so as to interfere with Notch-Notch ligand interaction. • 202 -
104. A composition as claimed in claim 96 wherein the agent comprises or codes for the extraceUular domain of Delta or a fragment thereof.
105. A composition as claimed in claim 96 wherein the agent comprises or codes for the extracellular domain of Senate or Jagged or a fragment thereof.
106. A composition as claimed in claim 96 wherein the agent comprises or codes for the extraceUular domain of Notch or a fragment thereof.
107. A vaccine composition comprising an adjuvant composition as claimed in any one of claims 94 to 106 and a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic deteiminant.
108. A vaccine composition as claimed in claim 107 comprising a vkal, fungal, parasitic or bacterial antigen or antigenic determinant or a polynucleotide coding for a vkal, fungal, parasitic or bacterial antigen or antigenic determinant.
109. A product comprising : i) an inhibitor of the Notch signalling pathway; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system.
110. A product comprising: i) a Notch antagonist; and ii) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; as a combined preparation for simultaneous, contemporaneous, separate or sequential use for modulation of the immune system. 203 -
111. A product as claimed in claim 109 or claim 110 for increasing effector T ceU activity.
112. A method for modulating the immune system in a mammal comprising simultaneously, contemporaneously, separately or sequentiaUy adnunistering: i) an effective amount of an inhibitor of the Notch signalling pathway; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
113. A combination of: i) an inhibitor of the Notch signalling pathway; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic deterniinant; for simultaneous, contemporaneous, separate or sequential use in modulating the immune system.
114. An inhibitor of the Notch signaUing pathway for use in modulating the immune system in simultaneous, contemporaneous, separate or sequential combination with a pathogen antigen or antigenic deterniinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
115. The use of a combination of: i) an inhibitor of the Notch signalling pathway; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant; in the manufacture of a medicament for modulation of the immune system.
116. The use of an inhibitor of the Notch signalling pathway in the manufacture of a medicament for modulation of the immune system in simultaneous, contemporaneous, 204 -
separate or sequential combination with a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
117. A pharmaceutical kit comprising an inhibitor of the Notch signalling pathway and a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
118. A conjugate comprising first and second sequences, wherein the first sequence comprises a pathogen antigen or antigenic determinant or a polynucleotide sequence coding for a pathogen antigen or antigenic determinant, and the second sequence comprises a polypeptide or polynucleotide for Notch signaUing modulation.
119. A conjugate as claimed in claim 118 in the form of a vector comprising a first polynucleotide sequence coding for a modulator of the Notch signalling pathway and a second polynucleotide sequence coding for a pathogen antigen or antigenic determinant.
120. A conjugate as claimed in claim 119 in the form of an expression vector.
121. A conjugate as claimed in any one of claims 118 to 120 wherein the fkst polynucleotide sequence codes for a Notch ligand or a fragment, derivative, homologue, analogue or allehc variant thereof.
122. A conjugate as claimed in claim 121 wherein the first polynucleotide sequence codes for a Delta or Senate/Jagged protein or a fragment, derivative, homologue, analogue or allehc variant thereof.
123. A conjugate as claimed in any one of claims 118 to 122 wherein the first polynucleotide sequence codes for a protein or polypeptide which comprises a Notch Hgand DSL domain and at least one Notch ligand EGF-like domain. - 205 -
124. A conjugate as claimed in claim 123 wherein the first polynucleotide sequence codes for a protein or polypeptide which comprises a Notch Hgand DSL domain and at least two Notch Hgand EGF-like domains.
125. A conjugate as claimed in claim 123 wherein the first polynucleotide sequence codes for a protein or polypeptide which comprises a Notch Hgand DSL domain and lor 2 but no more than 2 Notch Hgand EGF-like domains.
126. A conjugate as claimed in any one of claims 118 to 125 wherein the fkst and second sequences are operably linked to one or more promoters.
127. A method for increasing the immune response to a pathogen antigen or antigenic determinant by administering in any order: i) an inhibitor of the Notch signalling pathway; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
128. A method for increasing the immune response to a pathogen antigen or antigenic deterniinant by adnrinistering in any order: i) an agent which binds to Notch or a Notch Hgand to inhibit Notch-Notch Hgand interactions; and
H) a pathogen antigen or antigenic determinant or a polynucleotide coding for a pathogen antigen or antigenic determinant.
129. A method as claimed in claim 127 or claim 128 for treatment of an infection
130. A method as claimed in claim 127 or claim 128 for treatment of a chronic infection.
131. A method as claimed in claim 127 or claim 128 for prophylactic vaccination. 206
132. A method as claimed in claim 131 which confers protective immunity.
133. A pharmaceutical composition comprising: i) a protein or polypeptide which comprises a Notch Hgand DSL domain and either 0, 1 or 2, but no more than 2 Notch ligand EGF-like domains;
H) a multimer of such a protein or polypeptide (wherein each monomer may be the same ordkferent); or
Hi) a polynucleotide coding for such a protein or polypeptide.
134. Amethod for modkying an immune response by adπiinistering a Notch ligand protein or polypeptide consisting essentiaUy of the following components: i) a Notch Hgand DSL domain;
H) optionally lor 2 EGF repeat domains;
Hi) optionaUy all or part of a Notch Hgand N-terπiinal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by adrninistering a polynucleotide coding for such a Notch Hgand protein or polypeptide.
135. Amethod for increasing an immune response by administering a Notch ligand protein or polypeptide consisting essentiaUy of the following components: i) a Notch Hgand DSL domain;
H) optionally lor 2 EGF repeat domains; iii) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a polynucleotide coding for such a Notch Hgand protein or polypeptide. 207 -
136. Amethod for reducing immune tolerance by adniinistering a Notch Hgand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally lor 2 EGF repeat domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a polynucleotide coding for such a Notch Hgand protein or polypeptide.
137. Amethod for modkying T ceU activity by administering a Notch ligand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally lor 2 EGF repeat domains;
Hi) optionaUy all or part of a Notch Hgand N-terrninal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by adrninistering a polynucleotide coding for such a Notch Hgand protein or polypeptide.
138. Amethod for increasing helper (TH) or cytotoxic (Tc) T-ceU activity by adrninistering a Notch ligand protein or polypeptide consisting essentiaUy of the following components: i) a Notch Hgand DSL domain;
H) optionally lor 2 EGF repeat domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a polynucleotide coding for such a Notch Hgand protein or polypeptide.
139. A method for reducing activity of regulatory T ceUs by administering a Notch ligand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch Hgand DSL domain; 208 -
H) optionaUy lor 2 EGF repeat domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or by administering a polynucleotide coding for such a Notch Hgand protein or polypeptide.
140. A method as claimed in claim 138 or claim 139 wherein the regulatory T ceUs are Trl regulatory T-cells.
141. A method as claimed in any one of the preceding claims which comprises admkύstering a Notch ligand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch ligand DSL domain;
H) optionally all or part of a Notch ligand N-terminal domain; and
Hi) optionaUy one or more heterologous amino acid sequences; or which comprises administering a polynucleotide coding for such a Notch Hgand protein or polypeptide.
142. A method as claimed in any one of claims 134 to 141 which comprises admimstering a Notch ligand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch ligand DSL domain;
H) one Notch ligand EGF domain;
Hi) optionally all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or which comprises admimstering a polynucleotide coding for such a Notch Hgand protein or polypeptide. 209
143. Amethod as claimed in any one of claims 134 to 142 which comprises adrninistering a Notch ligand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch ligand DSL domain;
H) two Notch ligand EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or which comprises adnrinistering a polynucleotide coding for such a Notch Hgand protein or polypeptide.
144. Amethod as claimed in any one of claims 134 to 143 comprising adirunistering a Notch ligand protein or polypeptide which is not bound to a cell or part of a cell.
145. Amethod as claimed in any of claims 134 to 143 comprising administering a Notch Hgand protem or polypeptide which is bound to a cell or part of a cell.
146. Amethod as claimed in any one of claims 134 to 145 wherein the Notch Hgand protein or polypeptide is a Notch receptor antagonist.
147. A method as claimed in any one of claims 134 to 146 wherein the Notch Hgand protein, polypeptide or polynucleotide comprises or codes for a heterologous amino acid sequence corresponding to aU or part of an immunoglobuHn Fc domain.
148. Amethod as claimed in any one of claims 134 to 147 wherein the Notch Hgand protein, polypeptide or polynucleotide comprises or codes for at least part of a mammalian Notch ligand sequence.
149. A method as claimed in any one of claims 134 to 148 wherein the Notch Hgand protein, polypeptide or polynucleotide comprises or codes for at least part of a human Notch ligand sequence. 210
150. Amethod as claimed in any one of claims 134 to 149 wherein the Notch Hgand protein, polypeptide or polynucleotide comprises or codes for Notch Hgand domains from Delta, Senate or Jagged or domains having at least 30% amino acid sequence similarity or identity thereto.
151. A method as claimed in any one of claims 134 to 150 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for Notch Hgand domains from Deltal, Delta 3, Delta 4, Jagged 1 or Jagged 2 or domains having at least 30% amino acid sequence similarity or identity thereto.
152. Amethod as claimed in any one of claims 134 to 151 wherein the protein, polypeptide or polynucleotide is adπiinistered to a patient in vivo.
153. Amethod as claimed in any of claims 134 to 151 wherein the protein, polypeptide or polynucleotide is administered to cells from a patient ex vivo.
154. A method as claimed in claim 153 wherein the cells are administered to a patient after administration of the protein, polypeptide or polynucleotide.
155. A Notch ligand protein or polypeptide consisting essentiaUy of the following components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, for use to treat disease. 211 -
156. A Notch Hgand protein or polypeptide or polynucleotide for a use as claimed in claim 155 wherein the Notch ligand protein or polypeptide consists essentiaUy of the foUowing components: i) a Notch ligand DSL domain;
H) optionally aU or part of a Notch ligand N-terminal domain; and
Hi) optionaUy one or more heterologous amino acid sequences; or wherein the polynucleotide codes for such a Notch ligand protein or polypeptide.
157. A Notch Hgand protein or polypeptide or polynucleotide for a use as claimed in claim 155 wherein the Notch Hgand protein or polypeptide consists essentiaUy of the foUowing components: i) a Notch ligand DSL domain; k) one Notch ligand EGF domain;
Hi) optionaUy all or part of a Notch Hgand N-terrninal domain; and iv) optionaUy one or more heterologous amino acid sequences; or wherein the polynucleotide codes for such a Notch ligand protein or polypeptide.
158. A Notch Hgand protein or polypeptide or polynucleotide for a use as claimed in claim 155 wherein the Notch ligand protein or polypeptide consists essentiaUy of the foUowing components: i) a Notch ligand DSL domain;
H) two Notch Hgand EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-teiniinal domain; and iv) optionaUy one or more heterologous amino acid sequences; or wherein the polynucleotide codes for such a Notch ligand protein or polypeptide.
159. A Notch Hgand protein or polypeptide or polynucleotide for a use as claimed in any one of claims 155 to 158 wherein the Notch Hgand protein or polypeptide is not bound to a ceU or part of a ceU. ■ 212 -
160. A Notch Hgand protein or polypeptide or polynucleotide for a use as claimed in any one of claims 155 to 158 wherein the Notch Hgand protein or polypeptide is bound to a cell or part of a ceU.
161. A Notch Hgand protein or polypeptide or polynucleotide for a use as claimed in any one of claims 155 to 160 wherein the Notch ligand protein or polypeptide activates a Notch receptor.
162. A Notch Hgand protein or polypeptide or polynucleotide for a use as claimed in any one of claims 155 to 161 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for a heterologous amino acid sequence conesponding to aU or part of an iinmunoglobiilin F0 segment.
163. A Notch Hgand protein or polypeptide or polynucleotide for a use as claimed in any one of claims 155 to 162 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for at least part of a mammahan Notch ligand sequence.
164. A Notch Hgand protein or polypeptide or polynucleotide for a use as claimed in any one of claims 155 to 163 wherein the Notch Hgand protein, polypeptide or polynucleotide comprises or codes for at least part of a human Notch ligand sequence.
165. A Notch ligand protein or polypeptide or polynucleotide for a use as claimed in any one of claims 155 to 164 wherein the Notch Hgand protein, polypeptide or polynucleotide comprises or codes for Notch Hgand domains from Delta, Senate or Jagged or domains having at least 30% amino acid sequence similarity thereto.
166. A Notch Hgand protein or polypeptide or polynucleotide for a use as claimed in any one of claims 155 to 165 wherein the Notch Hgand protein, polypeptide or polynucleotide comprises or codes for Notch Hgand domains from Deltal, Delta 3, Delta 4, Jagged 1 or Jagged 2 or domains having at least 30% amino acid sequence similarity thereto. 213 -
167. The use of a Notch ligand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, in the manufacture of a medicament for modification of an immune response.
168. The use of a Notch Hg and protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains; and
Hi) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, in the manufacture of a medicament for modification of an immune response.
169. The use of a Notch Hgand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, in the manufacture of a medicament for increasing an immune response.
170. The use of a Notch ligand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch Hgand DSL domain; 214
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, in the manufacture of a medicament for reducing immune tolerance.
171. The use of a Notch ligand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, in the manufacture of a medicament for modification of T-ceU activity.
172. The use of a Notch Hgand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, in the manufacture of a medicament for increasing helper (TH) or cytotoxic (Tc ) T-ceU activity.
173. The use of a Notch ligand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains; ki) optionaUy all or part of a Notch Hgand N-terminal domain; and - 215 -
iv) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, in the manufacture of a medicament for reducing activity of regulatory T ceUs.
174. Ause as claimed in claim 173 for reducing activity of Trl or Th3 regulatory T-ceUs.
175. Ause as claimed in any one of claims 167 to 174 wherein the Notch ligand protein or polypeptide consists essentially of the following components: i) a Notch ligand DSL domain;
H) optionally aU or part of a Notch ligand N-terminal domain; and
Hi) optionaUy one or more heterologous amino acid sequences; or wherein the polynucleotide codes for such a Notch ligand protein or polypeptide.
176. A use as claimed in any one of claims 167 to 174 wherein the Notch ligand protein or polypeptide consists essentially of the following components: i) a Notch ligand DSL domain;
H) one EGF domain; ki) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or wherein the polynucleotide codes for such a Notch Hgand protein or polypeptide.
177. Ause as claimed in any one of claims 167 to 174 wherein the Notch ligand protein or polypeptide consists essentially of the following components: i) a Notch ligand DSL domain;
H) two EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or wherein the polynucleotide codes for such a Notch ligand protein or polypeptide. 216 -
178. A use as claimed in any one of claims 167 to 177 wherein the Notch ligand protein or polypeptide is not bound to a cell or part of a cell.
179. Ause as claimed in any one of claims 167 to 177 wherein the Notch ligand protein or polypeptide is bound to a cell or part of a cell.
180. A use as claimed in any one of claims 167 to 179 wherein the Notch ligand protein or polypeptide inhibits a Notch receptor.
181. A use as claimed in any one of claims 167 to 180 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for a heterologous amino acid sequence corresponding to aU or part of an immunoglobuHn Fc segment.
182. A use as claimed in any one of claims 167 to 181 wherein the Notch Hgand protein, polypeptide or polynucleotide comprises or codes for at least part of a mammalian Notch Hgand sequence.
183. Ause as claimed in any one of claims 167 to 182 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for at least part of a human Notch Hgand sequence.
184. A use as claimed in any one of claims 167 to 183 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for Notch ligand domains from Delta, Senate or Jagged or domains having at least 30% aπiino acid sequence similarity or identity thereto.
185. A use as claimed in any one of claims 167 to 184 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for Notch ligand domains from Deltal, Delta 3 , Delta 4, Jagged 1 or Jagged 2 or domains having at least 30% amino acid sequence similarity or identity thereto. ■ 217
186. A pharmaceutical composition comprising a Notch Hgand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally 1 or 2 EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-teπninal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, optionaUy in combination with a pharmaceutically acceptable carrier.
187. A pharmaceutical composition comprising a Notch Hgand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) optionally aU or part of a Notch ligand N-terniinal domain; and
Hi) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, optionaUy in combination with a pharmaceutically acceptable carrier.
188. A pharmaceutical composition comprising a Notch Hgand protein or polypeptide consisting essentially of the foUowing components: i) a Notch Hgand DSL domain;
H) one EGF repeat domain;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, optionaUy in combination with a pharmaceutically acceptable carrier.
189. A pharmaceutical composition comprising a Notch Hgand protein or polypeptide consisting essentiaUy of the foUowing components: i) a Notch Hgand DSL domain; 218
H) two EGF domains;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a polynucleotide coding for such a Notch Hgand protein or polypeptide, optionaUy in combination with a pharmaceutically acceptable carrier.
190. A pharmaceutical composition as claimed in any of claims 186 to 189 wherein the Notch ligand protein or polypeptide is not bound to a cell or part of a ceU.
191. A pharmaceutical composition as claimed in any of claims 186 to 189 wherein the Notch ligand protein or polypeptide is bound to a cell or part of a cell.
192. A pharmaceutical composition as claimed in any of claims 186 to 191 wherein the Notch ligand protein or polypeptide inhibits a Notch receptor.
193. A pharmaceutical composition as claimed in any of claims 186 to 192 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for a heterologous amino acid sequence conesponding to aU or part of an immunoglobuHn Fc segment.
194. A pharmaceutical composition as claimed in any of claims 186 to 193 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for at least part of a mammahan Notch ligand sequence.
195. A pharmaceutical composition as claimed in any of claims 186 to 194 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for at least part of a human Notch ligand sequence.
196. A pharmaceutical composition as claimed in any of claims 186 to 195 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for Notch ligand 219 -
domains from Delta, Senate or Jagged or domains having at least 30% amino acid sequence similarity or identity thereto.
197. A pharmaceutical composition as claimed in any of claims 186 to 196 wherein the Notch ligand protein, polypeptide or polynucleotide comprises or codes for Notch ligand domains from Deltal, Delta 3, Delta 4, Jagged 1 or Jagged 2 or domains having at least 30% amino acid sequence similarity thereto.
198. A Notch Hgand protein or polypeptide which consists essentiaUy of the following components: i) a Notch ligand DSL domain; k) optionally aU or part of a Notch ligand N-terminal domain; ki) an immunoglobulin Fc domain; and iv) optionaUy one or more further heterologous amino acid sequences; or a polynucleotide which codes for such a Notch ligand protein or polypeptide.
199. A Notch Hgand protein or polypeptide which consists essentiaUy of the following components: i) a Notch ligand DSL domain;
H) one EGF domain;
Hi) optionaUy all or part of a Notch Hgand N-terminal domain; and iv) optionaUy one or more heterologous amino acid sequences; or a polynucleotide which codes for such a Notch ligand protein or polypeptide.
200. A Notch Hgand protein or polypeptide which consists essentiaUy of the following components: i) a Notch Hgand DSL domain;
H) two EGF domains; and
Hi) optionaUy one or more heterologous amino acid sequences; or a polynucleotide sequence which codes for such a Notch Hgand protein or polypeptide. 220 -
201. A Notch Hgand protein or polypeptide as claimed in any of claims 198 to 200 which is not bound to a ceU or part of a ceU.
202. A Notch Hgand protein or polypeptide as claimed in any of claims 198 to 200 which is bound to a cell or part of a cell.
203. A Notch Hgand protein or polypeptide or polynucleotide as claimed in any one of claims 198 to 202 wherein the Notch Hgand protein or polypeptide activates a Notch receptor.
204. A Notch Hgand protein or polypeptide or polynucleotide as claimed in any one of claims 198 to 203 which comprises or codes for a heterologous amino acid sequence conesponding to aU or part of an irnmunoglobulin F0 segment.
205. A Notch Hgand protein or polypeptide or polynucleotide as claimed in any one of claims 198 to 204 which comprises or codes for at least part of a mammalian Notch Hgand sequence.
206. A Notch Hgand protein or polypeptide or polynucleotide as claimed in any one of claims 198 to 205 which comprises or codes for at least part of a human Notch Hgand sequence.
207. A Notch Hgand protein or polypeptide or polynucleotide as claimed in any one of claims 198 to 206 which comprises or codes for Notch Hgand domains from Delta, Senate or Jagged or domains having at least 30% amino acid sequence similarity thereto.
208. A Notch Hgand protein or polypeptide or polynucleotide as claimed in any one of claims 196 to 207 which comprises or codes for Notch Hgand domains from Deltal, - 221 -
Delta 3 , Delta 4, Jagged 1 or Jagged 2 or domains having at least 30% amino acid sequence similarity thereto.
209. A vector comprising a polynucleotide coding for a Notch ligand protein or polypeptide as claimed in any one of claims 196 to 208.
210. A host ceU transformed or transfected with a vector as claimed in claim 209.
211. A cell displaying a Notch ligand protein or polypeptide as claimed in any one of claims 196 to 208 on its surface and/or transfected with a polynucleotide coding for such a protein or polypeptide.
EP02779679A 2001-11-14 2002-11-13 Composition comprising inhibitors of the notch signalling pathway for the modulation of the immune system Withdrawn EP1446424A2 (en)

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
GB0127267 2001-11-14
GB0127267A GB0127267D0 (en) 2001-11-14 2001-11-14 Medical treatment
WOPCT/GB02/03426 2002-07-25
PCT/GB2002/003426 WO2003011317A1 (en) 2001-07-25 2002-07-25 Modulators of notch signalling for use in immunotherapy
GB0220849A GB0220849D0 (en) 2002-09-07 2002-09-07 Medical treatment
GB0220849 2002-09-07
GB0220913A GB0220913D0 (en) 2002-09-10 2002-09-10 Medical treatment
GB0220913 2002-09-10
PCT/GB2002/004390 WO2003029293A2 (en) 2001-09-28 2002-09-27 Modulators of notch ic protease activity for use in immunotherapy
WOPCT/GB20/04390 2002-09-27
PCT/GB2002/005137 WO2003041735A2 (en) 2001-11-14 2002-11-13 Composotion comprising inhibitors of the notch signalling pathway for the modulation of the immune system

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JP (1) JP2005518785A (en)
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WO (1) WO2003041735A2 (en)

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