USRE39433E1 - Therapeutic agents as cytokine antagonists and agonists - Google Patents

Therapeutic agents as cytokine antagonists and agonists Download PDF

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USRE39433E1
USRE39433E1 US10/386,813 US38681398A USRE39433E US RE39433 E1 USRE39433 E1 US RE39433E1 US 38681398 A US38681398 A US 38681398A US RE39433 E USRE39433 E US RE39433E
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Angel Lopez
Christopher Bagley
Joanna Woodcock
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Medvet Science Pty Ltd
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7153—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for colony-stimulating factors [CSF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to antagonists and agonists of cytokines, the therapeutic use of such antagonists and agonists, and method of isolation of such antagonists and agonists.
  • GM-CSF Human granulocyte-macrophage colony-stimulating factor
  • IL-3 and IL-5 are cytokines involved in hemopoietic cell survival, production and function (reviewed in Lopez et al, 1992). Because of these properties, IL-3 and GM-CSF are currently being used clinically for bone marrow reconstitution following chemotherapy and radiotherapy (Groopman et al, 1987). However, it is also becoming apparent that excessive or aberrant production of GM-CSF, IL-3 and IL-5 can lead to disease states.
  • GM-CSF GM-CSF
  • IL-3 GM-CSF
  • IL-5 IL-5
  • GM-CSF can stimulate the proliferation of leukaemic cells (Young et al, 1986) and IL-3 has been shown to be produced by follicular B cell lymphomas in an autocrine fashion resulting in the proliferation of these cells in an IL-3 dependent manner (Clayberger et al, 1991).
  • eosinophils which are believed to be the major cell type involved in allergy can be maintained in numbers and be stimulated by either IL-3, GM-CSF or IL-5 (Lopez et al, 1989).
  • Antagonism of all three cytokines may thus be necessary to inhibit the actions of eosinophils and basophils.
  • basophils which are also believed to play an effector role in allergy can be stimulated by either IL-3, GM-CSF or IL-5 (Lopez et al., 1990)
  • Antagonism of GM-CSF, IL-3 and IL-3 may be accomplished by the concomitant administration of specific antagonists for each different cytokine. Though feasible, this approach has the disadvantage of having to administer up to three different proteins which is not only convenient but which also increases the risk of immunogenicity and other side-effects.
  • GM-CSF has also been detected in the lungs of asthmatics. Indeed in one study of sputum cytokines, GM-CSF appeared to have the dominant effect on eosinophil survival (Adachi et al., 1995).
  • IL-5 also activates eosinophils to express the EG2 epitope.
  • increased IL-5 levels were associated with EG2 + eosinophils in the lung (Fukuda et al., 1994; Bentley et al., 1996).
  • tissue-specific activation of eosinophils In one experiment, activation of eosinophils in the lung was compared to those in the blood of the same patient. Activation was assessed by cell surface expression of CR-3, p150/95, CD67, CD63 and loss of L-selecting.
  • mice in which the IL-5 gene was genetically ablated not only had no detectable IL-5 and significant reduction in eosinophil numbers but also developed significantly less severe asthma (as manifest by airways hyperreactivity and lung damage) than IL-5 gene positive littermates (Foster et al., 1996).
  • IL-5 deficient mice were given IL-5 expressing-, but not control-, vaccinia virus infections (Foster et al., 1996).
  • GM-CSF GM-CSF
  • IL-3 Human interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5 exert their biological effect by binding to specific surface receptors on the surface of cells (Bagley et al, 1995; Elliott et al, 1989; Park et al, 1989; Lopez et al, 1991).
  • the receptors are heterodimers comprising an a chain which is specific for each ligand, and a ⁇ chain ( ⁇ c ) which is shared between the three receptors (Lopez et al, 1992; Kitamura et al, 1991).
  • ⁇ c contains four extracellular domains (Goodall et al, 1993) with domain 1 representing the most N-terminal domain and domain 4 representing the most membrane proximal domain. Each domain consists of seven ⁇ strands linked together by intervening loops. The loop of interest, the F′-G′ loop, is located in domain 4 of ⁇ c . The residues of interest are numbered from the N-terminus at primary translation terminus with the initiation methionine being Met 1 .
  • a further aspect of this invention arises as a result of the surprising identification of a single amino acid in the receptor oc chain for GM-CSF, IL-3 and IL-5 which is necessary for the high affinity binding of all of these three ligands to their surface receptors and for signalling and the fact that all three of these cytokines require the F′-G′ loop for high affinity conversion. It is believed that compounds that bind to Tyr 421 or inhibit the binding of ligand to Tyr 421 will behave as generic antagonists of IL-3, GM-CSF and IL-5. It is though that compounds that bind to the F′-G′ loop will sterically inhibit this binding.
  • cytokine receptor superfamily some of which are shared subunits in a given subfamily (that is they bind several cytokines), and some which are ligand specific and bind to only one cytokine.
  • the receptor ⁇ -chains for GM-CSF, IL-3 and IL-5 and ⁇ c belong to the rapidly expanding cytokine receptor superfamily.
  • gpl130 acts as an affinity converter and signal transducer for IL-6 (Hibi et al., 1990; Taga et al., 1992), IL-11 (Hilton et al., 1994), oncostatin M (Liu et al., 1995), ciliary neutrotrophic factor, leukaemia inhibitory factor (LIF) (Ip et a., 1992) and cardiotrophin-1 (Pennica et al., 1995); the LIF receptor (LIFR) also binds ciliary neurotrophic factor (Davis et al., 1993), cardiotrophin-1 (Pennica et al., 1995) and oncostatin M in addition to LIF (Gearing et al., 1994); IL-2R ⁇ supports affinity conversion and signalling of IL-2 and IL-15 (Giri et al.,
  • members of the cytokine receptor family may use a structurally conserved receptor framework to display a series of loops that through predominantly hydrophobic interactions support the binding of specific ligands.
  • Support for this notion also stems from mutational analysis of the IL-6R which showed that a mutant in which both Glu 297 and Phe 298 in the F′-G′ loop were substituted exhibited loss of IL-6 binding (Yawata et al., 1993).
  • the GHR is a homodimer.
  • the two identical subunits of GHR bind growth hormone sequentially and in a non-equivalent manner.
  • the two growth hormone-binding proteins are correspondingly identified as GHbpI and GHbpII.
  • the initial interaction of GHbpI utilises sites I contacts in growth hormone and is relatively strong with a dissociation constant of 600 pM (Cunningham et al., 1991).
  • the invention could therefore be said to reside in a therapeutic agent capable of binding to the F′-G′ loop of domain 4 of the common ⁇ c chain of the receptor for GM-CSF, IL-3 and IL-5, or to an analogous loop of another cytokine receptor the binding of which agent has an antagonist or agonist effect on the respective cytokine or one of the cytokines.
  • the cytokine receptor is a common signal transducing receptor, which may be one selected from the group comprising ⁇ c , gp130, LIFR, IL-2R ⁇ IL-2R ⁇ , and IL-4R/IL-13R.
  • the receptor is ⁇ c .
  • the agent has the antagonist or agonist effect on all respective cytokines that bind to the receptor.
  • the F′-G′ loop includes a hydrophobic amino acid.
  • the aromatic hydrophobic amino acid for the respective receptor may be as indicated in Table 3 and the invention may be restricted to the receptor listed therein, or may be to a tyrosine and the invention may be restricted to receptors with a tyrosine present in the F′-G′ loop.
  • the receptor may alternatively be restricted to those receptors that also interact with ⁇ c .
  • the cytokine receptor is a signalling receptor, which in one more specific form is specific for a single cytokine, and may be selected from the group comprising EPOR, TPOR and OBR.
  • the common signal transducing receptor is ⁇ c .
  • the amino acid specificity at position 421 is quite stringent and the therapeutic agent may need to be identified as requiring the presence of Tyr 421 for binding, or of sterically hindering the normal interaction that Tyr 421 has.
  • the agent may be capable of alleviating the symptoms of asthma.
  • the therapeutic agent may take the form of any one of a number of classes of compounds and may be selected from a group comprising, antibodies, peptides, oligosaccharides, oligonucleotides, or other organic or inorganic compounds.
  • a second aspect of the invention could be said to reside in a method of isolating a therapeutic agent capable of binding to the F′-G′ loop of domain 4 of the common ⁇ c chain of the receptor for GM-CSF, IL-3 and IL-5, or to an analogous loop of another cytokine receptor, said method including the steps of screening candidate molecules for their capacity to bind to the said loop.
  • the step of screening candidate molecules for their capacity to bind to said loop comprises screening for the capacity to bind to a sequence selected from the group comprising SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO.
  • loop to be screened may be selected from those derived from a common signal transducing receptor, or as a second alternative from those receptors having a hydrophobic amino acid residue, or as a third alternative from those receptors that are signal transducing receptors.
  • the invention could be said to reside in a method of isolating a therapeutic agent, by screening for the ability of compounds to bind to Tyr 421 of the common ⁇ c chain of the receptor for GM-CSF, IL-3 and IL-5, or the corresponding aromatic hydrophobic residue of gp130 or to an analogous aromatic hydrophobic residue of another common signal transducing receptor or of a specific receptor responsive to other cytokines.
  • the agent may be an antibody or fragment thereof and the method of isolating such an antibody will be understood to further including the steps of inoculating an animal with a peptide molecule having the F′-G′ loop, fusing antibody producing cells with a myeloma cell line and screening for a cell line that produces an antibody reactive with said F′-G′ loop, and harvesting antibodies from said cell line, testing for inhibition of high affinity binding and testing for inhibition or excitation of function. This may further include making small figments of antibodies produced by the said cell line capable of binding said the loop.
  • the cell line may conveniently be a mouse cell line and the method may include the further step of humanising the said antibody fragments by replacing mouse sequences with human sequences in the non-binding regions.
  • the invention could be said to reside in a method of treating an human or an animal for a condition, said method including the step of administering a therapeutic agent as identified above in a pharmaceutically acceptable carrier and in a therapeutically effective dose.
  • the treatment may be aimed at being preventative by reducing the risk of contracting the condition, or the treatment may be used to alleviate or obviate the condition.
  • the administration of the therapeutic agent can be any pharmaceutically acceptable form and in a suitable carrier.
  • gpl130 is functionally analogous to ⁇ c in the GM-CSF/IL-3/IL-5 receptor system, in that it is a common binding sub-unit and signal transducer for the IL-6, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF) and IL-11, it is suggested that targeting/blocking of this tyrosine will lead to antagonism of the IL-6, LIF, OSM, CNTF and IL-11. Antagonism of this receptor system will be useful in inflammation, leukaemia and lymphoma.
  • OSM oncostatin M
  • CNTF ciliary neurotrophic factor
  • LIF leukaemia inhibitory factor
  • IL-11 it is suggested that targeting/blocking of this tyrosine will lead to antagonism of the IL-6, LIF, OSM, CNTF and IL-11.
  • Antagonism of this receptor system will be useful in inflammation, leukaemia
  • Antagonists of IL-2R ⁇ / ⁇ may be useful as immunosuppressants.
  • Antagonists of LIFR may be useful for the prevention of implantation of embryos in uteri.
  • Antagonist of IL-4/IL-13 will inhibit IgE production and may be useful in treating asthma and allergies.
  • Antagonist of IL-3 may be useful in treating allergy and follicular B cell lymphoma.
  • Antagonists of IL-4 may inhibit IgE production, and be useful for treatment of asthma and allergy.
  • Antagonists of IL-6R may be useful as an anti-inflammatory and may be used to inhibit mycloma growth.
  • Antagonists against IL-7 may be useful as an immunosuppressant.
  • Antagonists of the leptin receptor (OBR) may be useful in the treatment of cachexia, weight loss in conditions such as AIDS, cancer, and parasitic diseases.
  • the invention may also encompass agonists.
  • the residue identified clearly plays a pivotal role in the action of the system and whilst some molecules that interact with the F′-G′ loop of ⁇ c or corresponding residues in other receptors (such as shown in table 3) can be expected to have an antagonist effect it is equally possible that they have an agonist effect particularly in receptors that can be activated by homodimerization.
  • Agonists agents that bind to ⁇ c may be used to stimulate hemopoiesis, and to boost immune response against microorganisms.
  • Agonists agents that bind to LIFR may be useful in the suppression of embryonic stem cell differentiation.
  • Agonists agents that bind to IL-2R ⁇ may be used in immunostimulation.
  • Agonists agents that bind to IL-4R/IL-13 may have anti-tumour activity.
  • Agonists agents that bind IL-3R may be use in the in vivo and ex vivo expansion of early hemopoietic cells.
  • Agonists agents that bind to IL-4R may have useful anti-tumour activity.
  • Agonists agents that bind to IL-7R may have useful anti-tumour immunity.
  • Agonists agents that bind IL-11 may prove a useful adjunct to cancer therapy.
  • Agonists agents that bind to EPOR may be used to correct anemia of chronic renal failure, of chronic inflammatory diseases and of malignant diseases.
  • Agonists agents that bind to TPOR may be useful for correcting thrombocytopenia (such as may be associated with chronic inflammatory diseases, malignancies, chemo- and radiotherapy).
  • useful agonists are those for erythropoiedin and thrombopoietin to elevate erythrocyte and platelet numbers in blood following blood cell loss, chemotherapy, radiotherapy, immunosuppression or bone marrow transplantation.
  • Agonists of OBR may be used to induce weight loss, and in particular for obesity which is considered to be a contributing factor of hypertension, coronary heart disease and non insulin-dependent diabetes mellitus.
  • the molecules whether agonist or antagonist can be isolated on the basis of their ability to interact with the identified residue.
  • FIG. 1A Is a schematic representation of the extracellular domain of human ⁇ c showing two cytokine receptor modules (CRM1 & 2) (Goodall et al., 1993) and conserved features of the cytokine receptor superfamily (Bazan 1990).
  • FIG. 1B Represents the amino acid sequences corresponding to the putative F′-G′ loop in the membrane proximal CRM2 of ⁇ c (Hayashida et al., 1990) aligned with the F′-G′ loops of growth hormone receptor (GHR) (Leung et al., 1987) and prolactin receptor (PRLR) (Boutin et al., 1988).
  • GHR growth hormone receptor
  • PRLR prolactin receptor
  • conserveed structural motifs are boxed and previously identified residues that are involved in ligand (GH) contact (De Vos et al., 1992, and Somers et al., 1994) are shown in bold. Mutant forms of ⁇ c which were made to identify ligand binding determinants are shown with mutated residues in bold.
  • FIG. 2 are graphic representations showing that alanine substitution of residues 418-422 in ⁇ c abolishes high affinity GM-CSF and IL-3 binding.
  • Scatchard transformation of saturation binding experiments using 125 I-GM-CSF (left panel) and 125 I-IL-3 (right panel) were carried out on COS cells expressing both GM-CSFR and IL-3R ⁇ -chains together with wild type (o) or mutant 418 AAAAA 422 ( ⁇ ) ⁇ c .
  • the radio-iodinated ligand concentration range for both GM-CSF and IL-3 was 10 pM-10 nM and non-specific binding was determined in the presence of 1 ⁇ M unlabelled ligand.
  • the broken line indicates the high and low affinity binding components using wild type ⁇ c
  • the solid line represents the line of best fit for the 418 AAAAA 422 mutant ⁇ c as determined using the LIGAND program.
  • a representative experiment is shown and the K d values derived from these and several other experiments are shown in Table 2.
  • FIG. 3 Shows the effect of individual alanine substitution of residues Arg 418 , Thr 419 , Gly 420 , Tyr 421 and Asp 422 of human ⁇ c on high affinity GM-CSF and IL-3 binding.
  • Scatchard transformation of saturation binding studies with 125 I-GM-CSF (top panel) and 125 I-IL-3 (bottom panel) were carried out as described in FIG. 2 .
  • the broken line indicates the high and low affinity binding components using wild type ⁇ c (o), and the solid line represents the line of best fit for each mutant ⁇ c ( ⁇ ) as determined using the LIGAND program.
  • a representative experiment is shown and the K d values derived from these and similar studies are shown in Table 2.
  • FIG. 4 Shows the effect of re-introducing Tyr 421 into a poly-alanine substituted ⁇ c ( 418 AAAYA 422 ) on high affinity GM-CSF and IL-3 binding.
  • Scatchard transformation of saturation binding studies with 125 I-GM-CSF (left panel) and 125 I-IL-3 (right panel) were carried out as described in FIG. 2 .
  • the broken line indicates the high and low affinity binding components using wild type ⁇ c (o), and the solid line represents the line of best fit for the 418 AAAYA 422 mutant ⁇ c ( ⁇ ) as determined using the LIGAND program.
  • a representative experiment is shown and the K d values derived from these and similar studies are shown in Table 2.
  • FIG. 5A Is a representation of an autoradiogram showing that STAT5-like (STAT5-L) activity is induced by GM-CSF and IL-3 only in Jurkat cells expressing appropriate ⁇ -chains together with wild type (Wt) ⁇ c .
  • Nuclear extracts were prepared from transfected Jurkat cells stimulated for 1 hr with or without 300 nM cytokine and analysed by electrophoretic mobility shift assay using a STAT5 specific probe.
  • FIG. 5B Is a representation of an autoradiogram similar to that of FIG. 5A showing that the DNA binding protein induced by cytokine in receptor transfected Jurkat cells behaves like STAT5.
  • Nuclear extract prepared from GM-CSF-stimulated transfected Jurkat cells was subjected to electrophoretic mobility shift assay in the presence of 50-fold molar excess of competing non-radioactive oligonucleotides.
  • the oligonucleotides used for competition were: the ⁇ -casein promoter element ( ⁇ -cas), a mutant of the ⁇ -casein promoter element (mut ⁇ -cas), an AP-1 consensus binding site (AP-1) and the haemopoeitin receptor response element (HRRE).
  • FIG. 6A Is a graphical representation that Y421A ⁇ c activates STAT5-L with reduced potency in response to GM-CSF stimulation.
  • a dose-response study of cytokine-induced STAT5-L activity was carried out on Jurkat cells transfected with (x-chains and either wild type ⁇ c (o), or Y421A ( ⁇ ).
  • STAT5-L activity was determined by electrophoretic mobility shift assay using a STAT5 specific probe and subsequently quantified by phosphorimager analysis. The results are expressed as a percentage of maximal activity obtained after correction for background and protein concentration.
  • FIG. 6B Is a graphical representation that Y421A ⁇ c activates STAT5-L with reduced potency in response to IL-3 stimulation.
  • the experimentation and procedures are as in FIG. 6 A.
  • FIG. 7 A model of the special arrangement between the putative B′-C′ and F′-G′ loops of ⁇ c and the first ⁇ helix of GM-CSF, IL-3 or IL-5 based on the GH-GHR crystal structure. Only the membrane proximal domains of ⁇ c (CRM2) and the first a helix (helix A) of GM-CSF, IL-3 or IL-5 are shown, with Tyr 365 and Tyr 421 of ⁇ c and the conserved Glu or GM-CSF, IL-3 or IL-5 shown in space-filling representation.
  • CRM2 membrane proximal domains of ⁇ c
  • helix A helix of GM-CSF, IL-3 or IL-5
  • ⁇ c The region of ⁇ c we have targeted for mutagenesis is flanked by regions that show conservation throughout the cytokine receptor superfamily: the VRXR consensus described previously (Patthy 1990) where X most commonly represents an aliphatic residue, and the WSXWS motif (Cosman et al., 1990; Bazan 1990) (FIG. 1 ).
  • the WSXWS motif has long been recognized as a characteristic feature of this receptor family and several studies have been directed towards understanding its role in receptor function (Miyazaki et al., 1991; Yoshimura et al., 1992; Jo et al., 1992; Rozakis-Adcock and Kelly 1992; Baumgartner et al., 1994).
  • COS cells transfected with mutant penta-alanine ⁇ c ( 418 AAAAA 422 ) GM-CSFR and IL-3R ⁇ -chain exhibited cell-surface expression of all receptor chains comparable to wild type transfectants (Table 2) but exhibited no high affinity binding of GM-CSF or IL-3 ( FIG. 2 , Table 2). This indicates that substitution of the region 418-422 in ⁇ c has interfered with the ability of ⁇ c to support high affinity GM-CSF and IL-3 binding and suggests that residues in this region may be involved in ligand interaction.
  • Tyr 421 is Necessary and Sufficient for GM-CSF, IL-3 and IL-5 High Affinity Binding.
  • each residue in this region was substituted separately with alanine (FIG. 1 ).
  • the ability of individual mutants to mediate high affinity ligand binding was determined by carrying out radioligand saturation binding studies on COS cells expressing mutant ⁇ c together with GM-CSFR and IL-3R ⁇ -chains. Results showed that all alanine substitution mutants were cell surface expressed similarly to wild type ⁇ c (Table 2) and alanine substitution of residues Thr 419 , Glu 420 and Asn 422 had no affect on high affinity ligand binding ( FIG. 3 , Table 2).
  • STAT5 has been described in many haemopoietic cell lines including cells of the myeloid and lymphoid lineages (Mui et al., 1995; Azam et al., 1995; Gouilleux et al., 1995; Pallard et al., 1995).
  • GM-CSFR myeloid and lymphoid lineages
  • IL-3R IL-3R
  • IL-5R receptor ⁇ -chain together with wild type ⁇ c .
  • a cytokine inducible nuclear DNA binding protein was detected that retarded an oligonucleotide probe containing the STAT5 binding consensus sequence (TTC-N 3 -GAA) in an electrophoretic mobility shift assay.
  • mutant receptors to activate STAT5-L protein were determined by cotransfecting Jurkat cells with mutant ⁇ c and GM-CSFR or IL-3R ⁇ -chain cDNAs. Cell surface expression of receptor subunits was confirmed by flow cytometry using chain specific antibodies and the expression of mutant and wild type ⁇ c was found to be comparable (data not shown).
  • the transfectants were stimulated with either GM-CSF or IL-3 over a range of concentrations and nuclear extracts prepared.
  • the induction of STAT5-L protein was determined by electrophoretic mobility shift assay (EMSA) using the canonical STAT5 DNA binding site from the ⁇ -casein promoter as a probe.
  • ESA electrophoretic mobility shift assay
  • STAT5-L was activated in response to either GM-CSF or IL-3 in a dose-dependent fashion in the presence of the appropriate ⁇ -chain and wild type ⁇ c (FIG. 6 A & B).
  • Alanine substitution of Tyr 421 produced a ⁇ c that supported STAT5-L activation in response to GM-CSF with approximately 100-1000 fold reduction in potency (FIG. 6 A).
  • This reduction in GM-CSF responsiveness with this mutant ⁇ c indicates that Tyr 421 has a role in GM-CSF induced receptor activation.
  • Y421A ⁇ c had a greater effect on IL-3-induced STAT5-L activation producing only very low levels of detectable STAT5-L ( FIG. 6B ) and even at 3 ⁇ M IL-3 half maximal activation was not achieved. This suggests that the tyrosine residue is also crucial for the interaction of IL-3 with ⁇ c .
  • Monoclonal antibodies can be generated by immunizing with ⁇ c or with a fragment of ⁇ c containing the F′-G′ loop or with a peptide containing the F′-G′ sequence. After specificity controls demonstrate specific binding to the F′-G′ loop the antibodies may be selected for blocking GM-CSF, IL-3 and IL-5 high affinity binding and for blocking GM-CSF, IL-3 and IL-5 stimulation of function.
  • an appropriate monoclonal antibody has been identified and shown to block GM-CSF/IL-3/IL-5 high affinity binding and function, smaller fragments may be generated; e.g. F(ab) 2 , Fab and ultimately Fv.
  • F(ab) 2 fragment of fragment
  • Fab fragment antigen binding
  • Fab fragment fragments
  • Short peptides of similar sequences to the F′-G′ loop may be synthesized that block cytokine interaction with the F′-G′ loop of B c .
  • the reverse is also possible: short peptides of similar sequences to the helix A of GM-CSFIIL-31IL-5 (this is the region in the cytokines predicted to interact with the F′-G′ loop) may also block cytokine interaction.
  • a large pool of randomly synthesized oligonucleotides can be passed through an F′-G′ peptide immobilized on a solid matrix (Bock et al., 1992,—which reference is incorporated herein). Following washing, the strongly binding oligonucleotides remain and can then be eluted under different conditions (salt, pH etc). The sequence can then be determined by PCR and tested for inhibition of ⁇ c -mediated function on a real cell system.
  • gp130 and ⁇ c contain domains that are related to the fibronectin type III domain that is a characteristic feature of most cytokine receptors. gp130 has 6 of these domains, ⁇ c has four of these domains.
  • Each domain contains its own F-G loop, thus gp130 has 6 and ⁇ c has 4. Only one of these F-G loops is the important one as far as the present invention is concerned.
  • the important domain is characterised by the presence of i) a conserved pattern of Cys residues, ii) several Trp residues iii) a YXXRV/IR motif, and iv) a WSXWS motif (where X is any amino acid).
  • Tyr 421 is postulated to lie within the F-G loop of the fourth domain loop in ⁇ c (F′-G′).
  • the equivalent functionally important tyr in gp130 is postulated to lie within the F-G loop of the second domain.

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AU9289098A (en) * 1997-09-23 1999-04-12 Astra Aktiebolag Inhibitors of hematopoietins interleukin-3, interleukin-5 and granulocyte/macrophage colony stimulating factor and uses thereof
CA2235420A1 (fr) * 1998-06-17 1999-12-17 Paolo Renzi Oligonucleoside antisens pour traiter ou prevenir les maladies atopiques et la proliferation de cellules neoplastiques
AUPP525198A0 (en) * 1998-08-13 1998-09-03 Medvet Science Pty. Ltd. Monoclonal antibody inhibitor of GM-CSF, IL-3 and IL-5 and other cytokines and uses thereof
WO2000047620A1 (fr) 1999-02-08 2000-08-17 Medvet Science Pty Ltd Domaine de fixation à la cytokine
AU3885901A (en) 1999-09-21 2001-04-24 Emory University Methods and compositions for treating platelet-related disorders
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US7455836B2 (en) * 2000-05-08 2008-11-25 The University Of Melbourne Method of treatment and agents useful for same
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