Classifications

• • 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
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5412—IL-6
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to IL-6 antagonist peptides, isolatable from a peptide library through the two-hybrids system by their ability to bind to the intracellular domain of gpl30 and containing at least 5 amino acids.
• peptides comprise an amino acid sequence, which is selected from the group consisting of : SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ LD NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, as well as salts, functional derivatives, precursors and analogs thereof.
• Another object of the present invention is to provide the peptide in substantially purified form, in order to be suitable for use in pharmaceutical compositions, as active ingredient, in pathologies that require IL-6 activity inhibition.
• the Two Hybrid System is a method that uses transcriptional activity as a system to detect protein-protein interactions.
• a gene fusion is constructed to encode the DNA-binding domain of the yeast transcription factor GAL4 as a hybrid with any protein 'X' (usually a defined mammalian protein being the "bait” binding target).
• An additional gene fusion construct will encode the transcription activation domain of GAL4 fused to any protein 'Y' (usually a library of diverse proteins, the "fish”) (Fields et al., 1994). Whenever an X-Y interaction does occur, it will bring the activation domain close to sites on the DNA recognised by the GAL4 DNA-binding domain, therefore resulting in the expression of a flanking reporter gene regulated by these DNA sites.
• the reporter genes commonly used include: 1) lacZ, which produces blue colonies on plates or filters containing X-Gal; and 2) His3, a yeast gene involved in histidine biosynthesis, required for growth of host yeast cells.
• the receptor system for Interleukin-6 (D -6) is composed of two distinct receptor "subunits" designated gp80 and gpl30 (see Hirano et al., 1994).
• the IL-6 type cytokines elicit their signal through receptors that share the gpl30 protein.
• gpl30 homo- or heterodimerizes with the LIF and OSM receptor, thereby activating associated JAK tyrosine kinases.
• the JAKs phosphorylate the signal transducer (gpl30) and latent transcription factors of the ST AT family (Signal Transducer and Activator of Transcription), like ST ATI and STAT3 in the case of IL-6.
• STAT factors dimerize, translocate to the nucleus and bind to enhancer elements of IL-6 responsive genes (Lilttiken et al., 1993).
• JAK kinases Two functions can be ascribed to JAK kinases: 1) activation of STAT-mediated gene expression; 2) activation of STAT-independent mitogenic activity at least in some hematopoietic cells.
• Additional kinases are known to associate with to the intracellular portion of gpl30, such as Hck, Fes, Btk and Tec (Matsuda et al., 1995). However these interactions have not been elucidated at the molecular level. Moreover, Tanner et al. have demonstrated that the boxl domain of cytokine receptors is required but not sufficient for interaction with JAK kinases and have suggested that the boxl sequences cooperate with other cytoplasmic domain sequences to effect JAK kinase association (Tanner et al., 1995). Even the molecular counterpart on JAK kinases of boxl and box2 has not been defined.
• Synthetic peptides that inhibit IL-6 activity have been described in the International Patent Application WO 97/13781 (YEDA), which relates to peptides derived from the gp80 protein.
• gpl30-ICD human IL-6 receptor
• This THS screening should therefore identify candidate peptides which are able to directly interact with gpl30-ICD in a phosphorylation- independent manner.
• Phosphorylation independent-interactions with gpl30-ICD are known to occur in the transduction of the signal triggered by IL-6 type cytokines.
• gpl30-ICD interacting counterparts of this type include protein kinases of the JAK family (Darnell et al., 1994).
• the main object of the present invention is an IL-6 antagonist peptide, isolatable from a peptide library through the two-hybrids system by their ability to bind to the intracellular domain of gpl30 and containing at least 5 amino acids.
• such peptides contain up to 30 amino acids, more preferably 5-20, most preferably 8-16.
• the "bait" ("X') used in the THS screening is the intracellular domain (ICD) of the gpl30 protein. Such domain corresponds to the region from amino acid at position 642 to amino acid at position 918 (Yamasaki K. et al., 1988) of the IL6-R (gpl30).
• the "fish" in the THS screening is a library of random peptides.
• Such library can be any commercial library or can be produced "in-house” by known methods.
• such peptides comprise an amino acid sequence, which is selected from the group consisting of : SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, as well as salts, functional derivatives, precursors and analogs thereof.
• the peptide of the invention comprises the amino acid sequence of SEQ ID NO: 2.
• Analogs as used in the present application, means those peptides, in which one or more of the amino acids in the above sequences are changed without substantially affecting the IL-6 antagonist activity.
• preferred changes for analogs in accordance with the present invention are what are known as “conservative” substitutions.
• Conservative amino acid substitutions include amino acids replacements with synonymous amino acids within the same group, which have sufficiently similar physicochemical properties that substitution between members of the group will preserve the biological function of the molecule, Grantham, Science. Vol. 185, pp. 862-864 (1974).
• the synonymous amino acid groups are those defined in Table I. More preferably, the synonymous amino acid groups are those defined in Table II; and most preferably the synonymous amino acid groups are those defined in Table III. TABLE I Preferred Groups of Synonymous Amino Acids
• Lys Glu Gin, His, Arg, Lys
• salts herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the peptides of the invention or analogs thereof.
• Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like.
• Acid salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulphuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid.
• any such salts must have substantially similar activity to the peptides of the invention or its analogs.
• the definition "functional derivatives” as herein used refers to derivatives which can be prepared from the functional groups present on the lateral chains of the amino acid moieties or on the terminal N- or C- groups according to known methods and are comprised in the invention when they are pharmaceutically acceptable i.e. when they do not destroy the protein activity or do not impart toxicity to the pharmaceutical compositions containing them.
• Such derivatives include for example esters or aliphatic amides of the carboxyl-groups and N-acyl derivatives of free amino groups or O-acyl derivatives of free hydroxyl-groups and are formed with acyl-groups as for example alcanoyl- or aroyl-groups.
• the "precursors” are compounds which are converted into the peptides of the invention in the human or animal body.
• the "IL-6 antagonist activity” means ability to inhibit IL-6 activity by antagonizing the binding of HL-6 to its receptor and/or by interfering with the function of the receptor system which transduces, intracellularly, molecular signals leading to the gene activation in IL-6-dependent cells, such as, for example, myeloma cells. Therefore, such activity may be measured by any of the assays known in the art. Such assays include proliferation of murine plasmacytoma T1165 cells, growth inhibition of mouse Ml myeloid leukemia cells, or production of acute phase proteins from hepatoma cells.
• the peptides of the invention may be prepared by any well known procedure in the art, such as solid phase synthesis or liquid phase synthesis.
• solid phase synthesis for example, the amino acid corresponding to the C-terminus of the peptide to be synthesised is bound to a support which is insoluble in organic solvents, and by alternate repetition of reactions, one wherein amino acids with their ⁇ -amino groups and side chain functional groups protected with appropriate protective groups are condensed one by one in order from the C-terminus to the N-terminus, and one where the amino acids bound to the resin or the protective group of the ⁇ -amino groups of the peptides are released , the peptide chain is thus extended in this manner.
• Solid phase synthesis methods are largely classified by the tBoc method and the Fmoc method, depending on the type of protective group used.
• protective groups include tBoc (t-butoxycarbonyl), Cl-Z (2- chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloxycarbonyl), Bzl (benzyl), Fmoc (9- fluorenylmethoxycarbonyl), Mbh (4,4'-dimethoxydibenzhydryl), Mtr (4-methoxy-2,3,6- trimethylbenzenesulphonyl), Trt (trityl), Tos (tosyl), Z (benzyloxycarbonyl) and Cl 2 -Bzl (2,6-dichlorobenzyl) for the amino groups; NO 2 (nitro) and Pmc (2,2,5,7,8- pentamethylchromane-6-sulphonyl) for the guanidino groups); and tBu (t-butyl) for the hydroxyl groups).
• Such peptide cutting reaction may be carried with hydrogen fluoride or trifluoromethane sulfonic acid for the Boc method, and with TFA for the Fmoc method.
• the crude peptide thus obtained is then subjected to purification.
• Purification is carried out by any one of the methods known for this purpose, i.e. any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like.
• HPLC high performance liquid chromatography
• the elution can be carried using a water-acetonitrile-based solvent commonly employed for protein purification.
• Another object of the present invention is, therefore, to provide the peptide in substantially purified form, in order to be suitable for use in pharmaceutical compositions, as active ingredient, in pathologies that require D -6 activity inhibition.
• Pathologies in which the new peptides according to the invention are advantageously used for prophylactic, therapeutic or diagnostic uses include haematological diseases, immune system diseases, bone diseases, tumours and autoimmune diseases, as well as therapy for transplantation including solid organ and cellular transplants.
• CLL chronic lymphocytic leukaemia
• CD Castleman's disease
• osteoporosis osteoporosis
• psoriasis multiple sclerosis
• lupus erithematosus diabetes
• rheumatoid arthritis as well as anaemia and wasting in chronic diseases.
• An embodiment of the invention is the administration of a pharmacologically active amount of the peptide of the invention to subjects at risk of developing pathologies requiring IL-6 activity inhibition or to subjects already showing such pathologies.
• any route of administration compatible with the active principle can be used, but particularly preferred is the parenteral administration because it permits to have, in short times, systemic effects.
• the dose of peptide to be administered depends on the basis of the medical prescriptions according to age, weight and the individual response of the patient.
• the pharmaceutical composition for parenteral use can be prepared in injectable form comprising the active principle and a suitable vehicle.
• Vehicles for the parenteral administration are well known in the art and comprise, for example, water, saline solution and physiologic buffers.
• the vehicle can contain smaller amounts of excipients in order to maintain the solution stability and isotonicity.
• Figure 1 Vectors used in the screening for gpl30-binding peptides.
• the plasmid pASgpl30 encodes GAL4 DNA-binding domain (residues 1-147 a. a.) fused to gpl30- ICD; the plasmid, pGADGH encodes a library of random 16-mer peptides (NNK) ⁇ 6 fused to the GAL4 activation domain (residues 768-881 a.a.).
• Figure 2 Western blotting of yeast extracts.
• CG-1945 (lanes 1 and 2) was transformed with pAS2-l, coding for GAL4BD (lane 3) or with pASg ⁇ l30, coding for g ⁇ l30- GAL4BD (lane 4).
• Protein extracts were separated on a 15% acrylamide-SDS gel and analysed by a chemiluminescence detection system. Molecular weights are indicated (kilodaltons). The black arrow denotes gpl30-GAL4BD; the grey arrow denotes GAL4BD.
• Figure 3 Peptide library transformation.
• the yeast strain CG-1945 was transformed with 60 ⁇ g of library plasmids.
• the cells were grown on SD/-Trp/-Leu/-His/+ 10 mM 3 -AT agar medium at 30°C. After 4 days, some His + colonies appeared on the plates.
• In the first transformation we have isolated 20 His + clones; only 9 of them, numbered in the figure, were also lacZ + .
• Figure 4 Peptides.
• the combination of clone E and gpl30-ICD in yeast cells generated about 3 units/ml of LacZ activity. The value is obtained from three independent experiments.
• Figure 6 Relevant homologies. Sequence alignment of the peptides E (Ei) and C with several gpl30-associated tyrosine kinases. Identities are indicated as italics; basic amino acids are marked as (*); acidic amino acids are underlined.
• a 16-mer random peptides library has been screened with the Two-Hybrid System (THS).
• the yeast GAL4 activation domain (AD) coding sequence was ligated in frame to a random library of synthetic oligonucleotides which coding for the peptides.
• the vector used, pGADGH is a centromeric plasmid carrying the ADH1 promoter and the yeast Leu2 gene as a selectable marker (Fig. l).
• the random peptide library is estimated to contain about 10 7 independent clones (see Material and Methods).
• RT-PCR was performed in HepG2 cells, in order to isolate the intracellular portion of human gpl30.
• the corresponding cDNA was cloned in frame with the yeast GAL4 binding domain (BD) coding sequence in plasmid pAS2-l. This is a centromeric plasmid carrying the ADH1 promoter and the yeast TRP1 gene as a selectable marker.
• BD yeast GAL4 binding domain
• yeast strain CG-1945 which carries two reporter genes, lac Z and His3 (Estojak et al., 1995).
• the THS-selected plasmids coding for the peptide library have been back-transformed into the original screening strain, CG-1945, in the following conditions: 1) with no additional plasmid; 2) with a plasmid encoding the GAL4 DNA-binding domain alone (pAS2-l); 3) with the full "bait” plasmid or 4) with an unrelated fusion protein (like the human lamin- C fused to GAL4-BD) (Bartel et al., 1993).
• a true positive clone produces a positive signal only in the third condition listed above.
• AD/library plasmids have been selectively amplified in KColi using its LEU2 marker to complement the E. Coli leuB mutation of strain HB 101.
• plasmids have been used to back-transform yeast strain in order to eliminate false positives as described above: after these procedures, we selected nine positive clones (Fig. 4).
• the data obtained from the sequence analysis have indicated that (i) although all clones screened contain more than one (NNK)i6 oligonucleotide, only the first oligonucleotide sequence is expressed as a GAL4 AD/peptide fusion due to the in-frame stop codon at the end of every oligonucleotide; (ii) most of the isolated peptides have the expected full length of 16 amino acids; (ii) some of the peptides presented homologies with known gp 130-ICD interacting proteins.
• JH1 corresponds to the tyrosine kinase domain and JH2 corresponds to the putative serine-threonine kinase domain.
• the domains JH3 trough JH7 are non-catalytic domains and have no know function.
• Clone E shows a little region of homology with JAKl : this homology falls in the JH4 domain.
• the other clones show identities with other kinases like LTKR or with proteins like ANKl, whose functions include the attachment of integral membrane proteins to cytoskeletal elements.
• SH2 and Src homology 3 (SH3) domains are regions of 60-100 amino acids that interact with phosphorylated tyrosine residues or proline-rich regions, respectively.
• the boxl domain of gp 130 is an eight amino acid motif rich in proline residues. This domain should directly be involved in the interaction with the JAK kinases, also if it seems more probable that the boxl sequence plays a critical role in generating a secondary structure that is required for the interaction between JAK kinases and the cytokine receptors (Murakami et al., 1991).
• Another domain of gpl30 involved in protein-protein interactions is the consensus sequence YXPQ involved in STAT3 and STAT1 activation (Gerhartz et al., 1996).
• the PCR reactions contained 10 ⁇ l of cDNA of HepG2 cells, 50 pmoles of each primer (see below), 2.5 units of Stratagene Pfu polymerise, 0.2 mM of each of the four deoxynucleotide triphosphates, 10 ⁇ l of Pfu buffer, in a reaction volume of 100 ⁇ l, overlaid with 50 ⁇ l of mineral oil.
• Amplification was performed for 30 cycles with a temperature profile of 45 seconds at 94°C, 45 seconds at 60°C and 6 minutes at 72°C.
• PCR fragments were digested with appropriate restriction enzymes (£coRI/B ⁇ /wHI for hgpl30) overnight at 37°C.
• the digested PCR products were purified by low melting agarose gel electrophoresis and by Microcon 100 (Amicon). These fragments were ligated with the Rapid DNA ligation kit (Boehringer Mannheim) into both the pAS2-l and pGADGH vectors and transformed into Exoli Topi OF competent cells (Invitrogen).
• Upgpl30 5' CTG GAA TTC AAT AAG CGA GAC CTAATT AAA AAA CAC ATC TGGCCT AAT GTT C 3' (SEQ IDNO: 9)
• ACAGTC 3' (SEQ IDNO: 10)
• the human gpl30-ICD cDNA was PCR amplified using Upgpl30 and Logpl30.
• Primer 1 (GAL4 BD): 5' TCA TCG GAA GAG TAG 3' (SEQ ID NO: 11)
• HBIO hsdS20 ( b, nib), recA13, ara-14, pro A2, lacYl, galK2, rspL20 (Sm r ), xyl-
• LB agar (11) 10 g Bactotryptone 5 g Yeast extract 10 g NaCl 1.5% agar
• the Saccharomyces cerevisiae strain CG-1945 (Mat a, ura 3-52, his3-200, lys 2-801, ode 2-101, trp 1-901, leu2-3, 112, gal4-542, gal 80-538, cyh r 2, LYS::GAL1 UASs - GAL1 TATA -HIS3, URA3:: GAL4 ⁇ ⁇ w-CyCl T ⁇ T ⁇ -IOCZ) (Clontech Matchmaker) were used for all assays.
• CG-1945 carries two reporter genes under the control of different promoters: lacZ gene under the control of the CYCl promoter, which has its own upstream activation sequence replaced with GAL4 binding sites, and His3 gene under the control of the Gall promoter.
• Yeast cultures were grown at 30°C in either YPD medium (1% yeast extract, 2% peptone and 2% glucose) or SD minimal medium (0.5% yeast nitrogen base without amino acids, 2% glucose, and 1% desired amino acid dropout solution).
• Yeast Transformation and ⁇ -galactosidase assay Fusion genes were introduced into CG-1945 strain by the lithium acetate transformation procedure (16).
• interaction occurs between gpl30-ICD and a peptide the two GAL4 functional domains are tethered, resulting in the histidine expression.
• Yeast cells with interacting hybrid proteins can thus grow in a medium lacking of this amino acid.
• 3-AT 3-AT (3-aminol,2,4-triazole), a competitive inhibitor of the yeast HIS3 protein (imidazoleglycerol-phosphate dehydratase), is used to inhibit low levels of His3p leaky expressed in some reporter strains.
• Transformants were allowed to grow at 30°C, usually for 2-4 days, until colonies were large enough to assay for ⁇ -galactosidase activity. Transformants were replicated on sterile Whatman number 1 filter that had been layered on selective growth media. After colonies had grown, we perform two or more freeze/thaw cycles, placing the filter in liquid nitrogen and at room temperature for 0.5-1 minute.
• the filter was placed in 5 ml Z-buffer/X-gal solution in a clean 100-mm plate and incubated at 30°C typically from 30 minutes to 8 hours. The filter was dried and photographed to record the data.
• Mouse p53 protein and SV40 large T-antigen are known to interact in the THS.
• plasmids which encodes the GAL4 DNA-binding domain-murine p53 hybrid
• pTDl-1 which encodes the GAL4 activation domain- SV40 large T-antigen hybrid
• the culture was centrifuged at 14,000 rpm for 30 seconds: in the next step, we remove the supematants, wash the cells with a volume of Z-buffer and resuspend the pellet in 900 ⁇ l of Z buffer. Immediately, two or more freeze/thaw cycles were performed, placing the tubes in liquid nitrogen and in a 37°C water bath for 0.5-1 minute. Finally, we added 0.7 ml of ⁇ -mercaptoethanol-Zbuffer solution and 160 ⁇ l of ONPG (o-nitrophenyl ⁇ -galactopyranoside, Sigma) 4 mg/ml dissolved in Z buffer to each tubes: the tubes were incubated at 30°C until the yellow colour developed.
• ONPG o-nitrophenyl ⁇ -galactopyranoside
• JAK2 a third member of the JAK family of protein tyrosine kinases. Oncogene 7, 1347-1353; Hirano T. et al., (1994) Stem Cells, 12, 262-277;

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