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Definitions

• the present invention relates to use of therapeutic compounds in the modification of T-cell activation.
• it relates to their use in modulating the interaction between Notch protein family members and their ligands and to the use of such compounds in the therapy of conditions such as graft rejection, autoirnmunity, allergy, asthma, infectious diseases and tumours.
• T cells and between antigen presenting cells (APC) and T cells are vital to the function of the human immune system.
• APC antigen presenting cells
• T cell or T cell-APC interaction in conditions such as autoirnmunity, allergy and graft rejection it is desirable to induce the downregulation of an immune response by stimulation of negative T cell or T cell-APC interaction.
• Models of "infectious tolerance” and "linked suppression” suggest that tolerance may be induced in a small number of T cells and that these T cells then transmit this tolerance to other T cells thus preventing an effective immunological attack.
• immunosuppression is a common feature. In such circumstances it would therefore be desirable to inhibit the T cell interactions passing on the infectious tolerance.
• WO 92/19734 purports to disclose the nucleotide sequences of the human Notch and Delta genes and amino acid sequences of their encoded proteins. The disclosure shows that the Notch gene family has been well characterised as essential to the correct embryological cell lineage development of insects such as Drosophila.
• Proteins belonging to the Notch family are transmembrane receptors that contain several conserved peptide motifs. Each protein within the family displays characteristic extracellular EGF (epidermal growth factor)-like repeats and a juxtamembrane Lin-12/Notc 7 motif. In addition each protein has 6-8 ankyrin repeat motifs on the cytoplasmic tail together with a PEST sequence.
• the Notch ligands have a diagnostic DSL domain (J_.Delta, _, Serrate, L,Lag2) comprising 20-22 amino acids at the amino terminus of the protein and between 3-8 EGF-like repeats on the extracellular surface.
• the proteins have a short cytoplasmic tail with no conserved functional domains.
• immunisation with a peptide fragment of antigen under selected conditions may inhibit specific responses not only to itself but to other regions in the same molecule provided the intact protein is used for the challenge immunisation (linked suppression; Hoyne GF, et al. J. Exp Med. 1993; 178: 183. and Metzler B, Wraith DC. Int. Immunol. 1993 ;5: 1159).
• the present invention arises from the discovery that the Notch receptor family and its ligands. Delta and Serrate, are expressed on the cell surface of normal adult cells of the peripheral immune system.
• Notch signalling may play a central role in the induction of peripheral unresponsiveness (tolerance or anergy) and may provide a physical explanation for linked suppression and infectious tolerance.
• the present invention further relates to the use of a JVotc/z-ligand in the treatment of a T cell mediated reaction.
• a JVotc/z-ligand in the treatment of a T cell mediated reaction.
• the invention also relates to the use of a vVotc z-ligand in affecting linked tolerance and/or bystander tolerance (also known in the art as infectious tolerance).
• a further embodiment of the invention relates to the use of a N ⁇ tc/2-ligand in the modulation of the expression of a functional Notc j-protein or Notch signalling pathways.
• the NotcA-ligand may be exposed to the T cells by incubating/mixing the T cells with antigen presenting cells (APCs) or the like, that express or overexpress a N ⁇ tc 2-ligand in the presence of an allergen or antigen.
• APCs antigen presenting cells
• the (over)expression of the M?tc/?-ligand gene may be brought about by the APCs being transfected with a gene capable of expressing a N ⁇ tc/j-ligand or by the APCs being exposed to an agent capable of up-regulating expression of endogenous JV ⁇ tc/z-ligand gene(s).
• Suitable agents that influence expression of Notcft-ligands include agents that affect the expression of Delta and/or Serrate genes.
• any agent that inhibits the binding of BMPs to their receptors is capable of producing an increase in the expression of Delta and/or Serrate.
• Such agents include Noggin.
• Chordin Follistatin, Xnr3, FGFs, Fringe and derivatives and variants thereof (see references for noggin-Smith W. C. and Harland, R.M Cell 70:829-840, chordin-Sasai, Yet al. , 1994 Cell 79: 779-790).
• Noggin and Chordin bind to BMPs thereby preventing activation of their signalling cascade which leads to decreased Delta transcription.
• the body may be immuno-compromised and it may be desirable to downregulate the expression of Delta and/or Serrate in order to overcome the imposed immunosuppression.
• Agents that inhibit the expression of Delta and/or Serrate may be used in such circumstances.
• agents that inhibit the expression of Delta and/or Serrate include the Toll protein (Medzhitov, R.et al. , 1997 Nature 388: 394-397) BMPs and other agents that decrease or interfere with the production of Noggin, Chordin, Follistatin, Xnr3, FGFs and Fringe.
• the invention further relates to the use of an agent capable of downregulating the expression of Delta or Serrate proteins in the manufacture of a medicament for use in reversing bacteria infection or tumour-induced immunosuppression.
• the invention also relates to the modification of Notch- protein expression or presentation on the cell membrane or signalling pathways. These have been shown to be involved in T-cell mediated responses that participate in the induction of tolerance (linked and/or bystander).
• Agents that enhance the presentation of a fully functional /Votc z-protein on the cell surface include matrix metalloproteinases such as the product of the Kuzbanian gene of Drosophila (Dkuz, Pan, D and Rubin, G.M. 1997 Cell 90: 271-280) and other ADAMALYSIN gene family members.
• Agents that reduce or interfere with its presentation as a fully functional cell membrane protein may include MMP inhibitors such as hydroxymate-based inhibitors.
• APC antigen presenting cell or the like
• suitable APCs include dendritic cells. L cells, hybridomas, lymphomas, macrophages, B cells or synthetic APCs such as lipid membranes.
• the transfection may be brought about by a virus such as a retrovirus or adenovirus. or by any other vehicle or method capable of delivering a gene to the cells.
• viruses such as a retrovirus or adenovirus.
• vehicle or method capable of delivering a gene to the cells.
• retroviruses include any vehicles or methods shown to be effective in gene therapy and include retroviruses, liposomes, electroporation, other viruses such as adenovirus, adeno-associated virus, herpes virus, vaccinia, calcium phosphate precipitated DNA, DEAE dextran assisted transfection, microinjection, polyethylene glycol, protein-DNA complexes.
• a virus may be used in combination with liposomes in order to increase the efficiency of DNA uptake.
• the site specificity of the delivery may be achieved by the inclusion of specific proteins (eg a single chain antibody reactive with CDl lc for dendritic cells/macrophages) in the viral coat or liposome.
• constructs through which expression of the the gene eg serrate
• APCs (over)expressing Serrate would therefore also express high levels of the relevant antigen and so preferentially interact with T cells of the appropriate specificity.
• a further embodiment of the present invention relates to a molecule comprising a N ⁇ tc z-ligand moiety operably linked to a T cell allergen or antigen moiety such that upon exposure to T cells both moieties are capable of binding to their respective sites.
• a molecule is capable of rendering an antigen/allergen specific T cell tolerant to the allergen or antigen upon which the allergen or antigen moiety is based, as the specificity required of the N ⁇ tc/z-ligand moiety is provided by the close proximity of the allergen or antigen moiety.
• the antigen or allergen moiety may be, for example, a synthetic MHC-peptide complex. That is a fragment of the MHC molecule bearing the antigen groove bearing an element of the antigen. Such complexes have been described in Altman JD et al Science 1996 274: 94-6.
• the compound is a fusion protein comprising a segment of Notch or Notch ligand extracellular domain and an immunoglobulin F c segment, preferably IgGF c or IgMF c .
• the N ⁇ tc/z-ligand is of the Serrate family of proteins or Delta family of proteins, derivatives, fragments or analogs thereof.
• T cells Diseased or infectious states that may be described as being mediated by T cells include any one or more of asthma, allergy, graft rejection, autoimmunity, tumour induced abberations to the T cell system and infectious diseases such as those caused by Plasmodium species, Microfilariae, Helminths, Mycobacteria, HIV, Cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, Haemophilus influenza type B, measles, Hepatitis C or Toxicara.
• infectious diseases such as those caused by Plasmodium species, Microfilariae, Helminths, Mycobacteria, HIV, Cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, Haemophilus influenza type B, measles, Hepatitis C or Toxicara.
• a Notch- ligand may be used in accordance with the present invention to treat the said disease or infection.
• the invention also provides a method for detecting immune suppression induced by an invading organism.
• Such organisms may generate soluble forms of family members of Serrate, Notch and/or Delta or derivatives thereof or proteins that affect their expression in vivo, thus inducing infectious tolerance immunosuppression.
• the method comprises an assay for the presence of in vivo non-membrane bound Serrate, Delta, Notch or derivatives thereof and preferably comprises an antibody to Serrate, Delta or Notch or their derivatives.
• Methods of use in screening assays for the detection of increased or decreased Notch, Delta/Serrate expression and/or processing include:
• RT-PCR quantitative - reverse transcriptase-polymerase chain reaction
• This construct may be modified as new ligand members are identified.
• the invention also relates to compounds, or N ⁇ tc z-ligands detectable by these assay methods.
• the invention also includes an assay method comprising contacting (a) Notch protein and a ligand capable of binding to the Notch protein with (b) a compound: and determining if the compound affects binding of the ligand to the Notch protein preferably wherein the Notch protein is associated with a T cell.
• the N ⁇ tc/z-ligands of use in the present invention are preferably Delta or Serrate family member proteins or polypeptides or derivatives thereof. These are preferably obtained using standard techniques of recombinant technology well known to the person skilled in the art. Appropriate gene sequences for use to generate such compounds of the present invention may be obtained from publications such as WO97/01571, WO 96/27610 and WO 92/19734. The invention is not however in any way limited by the Notch, Delta and Serrate invention is not however in any way limited by the Notch, Delta and Serrate sequences disclosed in these publications.
• Notch ligand further includes any ligand or ligand family member that interacts with a Notch protein family member and includes the group of proteins referred to as "toporythmic proteins” i.e. the protein product of the Delta, Serrate, Deltex and Enhancer of split genes as well as other members of this gene family identifiable by virtue of the ability of their gene sequences to hybridize to, or their homology with Notch, Delta or Serrate proteins, or the ability of their genes to display phenotypic interactions.
• Notch Delta and Serrate were first described in Drosophila and therefore represent prototypic proteins of the Notch receptor and Notch-ligand family members respectively. Multiple Notch proteins and ligands have now been described in many invertebrate and vertebrate species but their nomenclature may differ from that used in the fly. For example Notch is a homolog of Lin 12 and Glp 1, Serrate/Delta are homologs of Jagged, Apxl and Lag-2.
• compositions of the present invention may be formulated according to principles well known in the art. Thus the nature of the excipient and the amount of activity will depend upon the compound of the present invention which is to be formulated.
• compositions are in unit dosage form. Dosages of compounds of the present invention, to be administered to a patient in the form of a pharmaceutical formulation, could be determined by a suitable physician.
• the preferred administration route of a formulation of the present invention is any one of the usual methods of administration including intra-muscular and intra-peritoneal, intravenous injection, intranasal inhalation, lung inhalation subcutaneous, intradermal, intra-articular, intrathecal, topical, and via the alimentary tract (for example, via the Peyers patches).
• derivative in relation to proteins or polypeptides of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein on polypeptide possesses the capability of modulating Notch-Notch ligand interactions.
• variant in relation to proteins or polypeptides of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein on polypeptide possesses the capability of modulating Notch-Notch ligand interactions.
• analog in relation to proteins or polypeptides of the present invention includes any peptidomimetic, that is, a chemical compound that possesses the capability of modulating Notch-Notch ligand interactions in a similar manner to the parent protein or polypeptide.
• peptidomimetic that is, a chemical compound that possesses the capability of modulating Notch-Notch ligand interactions in a similar manner to the parent protein or polypeptide.
• These include compounds that may agonise or antagonise the expression or activity of a N ⁇ tc ⁇ -protein or N ⁇ tc z-ligand.
• a compound may be considered to modulate Notch-Notch ligand interactions if it is capable of either inhibiting or enhancing the interaction of Notch with its ligands, preferably to an extent sufficient to provide therapeutic efficacy.
• Notch-Notch ligand means the interaction between a Notch family member and a ligand capable of binding to one or more such member.
• therapy should be taken to encompass diagnostic and prophylactic applications.
• protein and polypeptide may be assumed to be synonymous, protein merely being used in a general sense to indicate a relatively longer amino acid sequence than that present in a polypeptide.
• Figure 1 shows the results of in ___ hybridisations carried out as described in Example 1 herein;
• Figure 2 shows the results of the experiment described in Example 4.
• FIG. 3 shows the results of the experiment described in Example 5.
• Figure 4 shows the results of the experiment described in Example 6.
• FIG. 5 shows the results of the experiment described in Example 7.
• Figure 6 shows the results of the experiment described in Example 8.
• FIG. 7 shows the results of the experiment described in Example 9.
• Figure 8a and 8b show d e results of the experiment described in Example 10.
• Figure 9 shows the results of the experiment described in Example 11.
• Antisense RNA probes specific for Notch I, Delta 1 and Serrate I were synthesized and incorporated with digoxigenin labelled-UTP. Each probe was dissolved in hybridisation buffer, heated to 70°C for 5-10 minutes and added to TESPA coated slides containing 10mm sections of spleen or thymus that had been previously fixed with 4% paraformaldehyde + PBS. Slides were hybridized overnight at 65 °C. The following day, slides were washed twice at 65°C. and twice at room temperature (RT) with lxSSC/50% Formamide/0.1 %Tween 20.
• RT room temperature
• the pIG-1 [D. Simmons, "Cloning cell surface molecules by transient surface expression in mammalian cells” pp 93-128, Cellular Interactions in Development Ed. D. Hartley, pub. Ox. Uni. Press (1993)] expression vector allows production of a fusion protein that contains the extracellular portion of Delta 1 linked to the human IgGl-F c domain. A restriction enzyme fragment that contained only the extracellular domain of the Delta 1 protein was cloned into the pIG-1 vector. The resultant plasmid was tranformed into E. Coli MC 1061, and grown in SOB containing lO ⁇ g/ml tetracycline/ampicillin. Purified vector was used to transfect COS cells in vitro.
• COS cells were grown to 50- 75% confluency and transfected with 10 ⁇ g of plasmid DNA per dish by DEAE-dextran method.
• the culture medium was replaced with culture medium containing 1 % FCS and cells were cultured for a further 3-6 days in vitro. Cells were spun for 5mins/5000 rpm to pellet cells and debris, the supernatant was removed and stored until required.
• the Delta- Fc fusion was purified from culture supernatants by adding 2ml of 50% slurry of protein a Sepharose (Phamacia) and rotated overnight at 4°C.
• Sepharose beads were isolated by passing culture supernatants through a 0.45 mm filter, washed and transferred to a 10 ml plastic column.
• the Delta-Fc fusion construct was eluted with 2 ml of elution buffer pH 4.0. The eluate was neutralised by the addition of 1M Tris base.
• Peripheral tolerance to self antigens can be analysed in T cell receptor (TCR) transgenic mice in which the TCR ligand is expressed as a self antigen only in the periphery.
• TCR T cell receptor
• Peripheral tolerance to transplantation antigens can be induced in several ways including recipient pre-treatment with T cell antibodies or blockade of costimulation. It is thereby possible to demonstrate both linked suppression and infectious tolerance.
• Peripheral tolerance to allergens may be induced by the intranasal delivery of allergen derived peptides.
• Notch-Notch ligands The expression of Notch-Notch ligands is measured on cells recruited into the airways and/or lymphoid tissues following allergen inhalation and modifications in tolerance demonstrated. Furthermore, in experimental models of infections with infectious agents, the expression of Notch-Notch ligands can be measured on the organism (pathogen) and immunocompetent cells in the host.
• Delta expressing hybridomas can inhibit the responses of antigen primed lymphocytes.
• LNCs lymph node cells
• T cell hybridomas were transfected with either full length Delta or a control plasmid, such that delta was expressed as a membrane protein. After two days in culture the hybridomas were irradiated to prevent them from proliferating or from producing cytokines. Therefore, the only response which was measured in the assay comes from the lymph node cells alone.
• the irradiated hybridomas were added in increasing numbers to cultures containing the primed LNCs.
• the appropriate antigen i.e. pi 10-131 or OVA
• Supernatent fluids were then collected and assayed for IL-2 (a major T cell growth factor) content. Proliferative responses of the lymph node cells after 72 hours were also measured.
• LNC lymph node cells
• 2BB11 and 2BC3 are two different Der pi reactive hybridomas.
• Dendritic cells are the primary antigen presenting cell in the immune system and are critical for stimulating T cell responses.
• DCs were obtained from the spleen and transfected with either a retrovirus allowing expression of the full length Serrate protein or a control retrovirus.
• the DCs were also pulsed with the HDM peptide pi 10-131 for 3 hours in vitro at 37°C. The DCs were then washed and used to immunise naive mice subcutaneously using 10 5 cells/mouse. After 7 days the draining LNCs were recovered and restimulated in culture with peptide at 4x10 cells/well.
• FIG. 3 shows the data presented as cpm of LNCs 72 hours after culture from animals immunised with control transfected (open circles) or serrated transfected (open squares) dendritic cells (DCs).
• mice with DCs expressing Serrate resulted in a 10 fold decrease in the number of cells recovered from lymph nodes when compared to immunisation with control DCs.
• LNCs from mice immunised with DCs + Serrate failed to proliferate (93% reduction on control values, Figure 3) or secrete IL-2 when compared to cells from mice immunised with control DCs.
• T cell hybridomas are able to inhibit the development of immunity to Der p 1 antigen in animals.
• T cell hybridomas reactive with Der p i
• C57 BL mice were injected with 10 million irradiated hybridomas i.p. and immunised with 50 microgram Der p 1 emulsified in Complete Freunds Adjuvant (CFA) sub-cutaneously.
• CFA Complete Freunds Adjuvant
• Delta expressing human T cells can block the response of normal T cells.
• HA 1.7 An influenza-reactive human T cell clone (HA 1.7) was transfected with mouse Delta using a retroviral construct to allow cell surface expression of the Delta protein. Mixing of this cell population with normal HA1.7 prevented subsequent reactivity of these normal HA1.7 with peptide HA306-318 and antigen presenting cells. 5 x 10 5 HA1.7 were mixed with 1 x 10 irradiated
• PBMC blood mononuclear cells
• EXAMPLE 8 Serrate expression by antigen presenting cells prevents T cell responses.
• Clone HA1.7 was mixed with peptide HA306-318 (1.0 microgram/ml) in the presence of L cells expressing HLA-DRBl*0101(as antigen presenting cells), using 2x10 4 of each cell type.
• the L cells were transfected with either control (puro) or serrate (serrate L cells) expressing retrovirus.
• the proliferative response was measured after 72 hours following addition of tritiated thymidine for the last 8 hours of culture.
• Serrate expressing antigen presenting cells induce regulatory T cells.
• Clone HA1.7 was mixed with peptide HA306-318 and L cells (expressing DRB1*0101 as antigen presenting cells) in the presence of 2% IL-2.
• the L cells were transfected with either control or serrate expressing retrovirus. After 7 days in culture, the HA 1.7 were harvested washed and irradiated before being mixed with fresh HA1.7 (using 2xl0 4 each population). Cells were cultured for a further 2 days before being stimulated with peptide (1.0 microgram/ml) + normal antigen presenting cells (DRB1*0101 PBMCs).
• the regulation induced by delta expressing T cells is antigen specific.
• mice were injected with 10 T cell hybridoma cells transduced with either delta or control retrovirus.
• the hybridoma was 2BC3 which has specificity for peptide 110-131 of Der pi .
• animals received either Der pi or ovalbumin emulsified in complete Freund's adjuvant. 7 days later, lymph node cells were removed and 4xl0 3 cells were cultured in the presence of Der pi or ovalbumin (using the same antigen that they had already been immunised with). The production of IL-2 was measured 24 hours after the start of culture.
• Stimulation indexes for IL-2 production are shown in Figures 8a (responses of animals immunised with Der pi) and 8b (responses of animals immunised with
• OVA OVA for animals injected with hybridomas transfected with control (puro) or Delta (delta) retrovirus.
• C57 BL/6J mice were treated intranasally with either 100 microgram Der pi peptide 110-131 or a control solution (phosphate buffered saline, PBS) on three consecutive days. Intranasal adminstration of antigen in this way is known to induce tolerance to the antigen. Some animals were then rested for 2 weeks before being rechallenged with antigen by injection of 50 ⁇ g Der pl/CFA subcutaneously into the base of the tail. The superficial lymph nodes and spleens of animals were harvested at various time points thereafter (dO being the first day of intranasal treatment or antigen rechallenge) and processed for immunohistology or in situ hybridisation.
• PBS phosphate buffered saline
• Data shown in tables 1 and 2 are for immunohistology and in situ hybridisation respectively. Data represent the analysis of tissues from 5 separate mice after intranasal peptide alone (PBS/p 110-131 primary) or intranasal and subcutaneous antigen (PBS/pl 10-131 rechallenge.

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