WO2004020578A2 - Il-20 modifiés par glycosylation - Google Patents

Il-20 modifiés par glycosylation Download PDF

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WO2004020578A2
WO2004020578A2 PCT/US2003/023265 US0323265W WO2004020578A2 WO 2004020578 A2 WO2004020578 A2 WO 2004020578A2 US 0323265 W US0323265 W US 0323265W WO 2004020578 A2 WO2004020578 A2 WO 2004020578A2
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sequence
polypeptide
glycosylation
seq
modified
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PCT/US2003/023265
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WO2004020578A3 (fr
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Patrick Francis Grealish
Scott William Rowlinson
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Eli Lilly And Company
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Priority to EP03791607A priority Critical patent/EP1587904A2/fr
Priority to AU2003263812A priority patent/AU2003263812A1/en
Publication of WO2004020578A2 publication Critical patent/WO2004020578A2/fr
Publication of WO2004020578A3 publication Critical patent/WO2004020578A3/fr

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    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/91—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation

Definitions

  • the present invention relates to novel, glycosylation-modified IL-20 polypeptides that preferentially signal through one of the IL-20 receptor complexes and polynucleotides that identify and encode the polypeptides. Also provided are vector, host cells and recombinant methods for producing the same. The invention further provides therapeutic methods for using the polypeptides and polynucleotides of the invention.
  • Interleukin 20 is a member of the interleukin-10 (IL-10) protein family. Additional members of this protein family include, but are not limited to, interleukin 19 (IL-19) and interleukin 24 (IL-24). These proteins share limited primary sequence identity, some structural homology and receptor subunits (for review see Kotenko, S., Cvtokine & Growth Factor Reviews 13:223, 2002).
  • the IL-20 polynucleotide and polypeptide sequences are described in International Patent Publication Nos. WO 99/27103 and WO 00/12708.
  • the IL-19 polynucleotide and polypeptide sequence are described in U.S. Patent 5,985,614 and the IL-24 polynucleotide and polypeptide sequence are described in International Patent Publication No. WO
  • IL-10 plays major roles in inflammatory and immune responses (Moore, K. et al. Ann. Rev. Immunol. 19:683, 2001). Some of the other members of the IL-10 protein family have been shown to be involved in the regulation of inflammatory responses in various tissues.
  • IL-19 The function of IL-19 is unknown although its expression closely follows (temporally) the expression of IL-10 which may suggest a role for IL-19 as a feedback inhibitor of proinflammatory cytokine production, similar to EL- 10, or it may limit the anti-inflammatory action of IL-10 (Kotenko, S., Cvtokine & Growth Factor Reviews 13:223, 2002).
  • IL-24 has been shown to be associated with chromatin in cells undergoing mitosis and it has also been linked to the promotion of apoptosis in cancer cells (Su, Z., et al.. Proc. Nail. Acad. Sci. 95: 14400, 1998, U.S. Patents 6,355,622 and 5,710,137).
  • IL-20 has been linked to skin development and stimulation of platelet proliferation (International Patent Publication No. WO 99/27103), as well as activity resulting in an increased number of CFU-GEMM cells which are hematopoietic progenitor cells of red blood cells, platelets, granulocytes and monocytes (U.S. Patent Applications with serial numbers 60/272,242 filed February 28, 2001; 60/332,740 filed November 19, 2001 and 60/353,789 filed February 1, 2002) and anti-obesity activity (International Patent Application No. PCT/US02/00498).
  • Cytokines such as IL-20, IL-19 and IL-24, are secreted proteins that exert their actions by binding to specific cell-surface receptors that leads to signaling, i.e., activation of cytokine-specific signal transduction pathways.
  • a cytokine that signals through multiple, different receptor complexes may thereby activate multiple, different signal transduction pathways.
  • All IL-10-related cytokines signal through receptors belonging to the class II cytokine receptor family (Bazan, J., Proc. Natl. Acad. Sci.. 87:6934, 1990).
  • Rl and R2 The class II cytokine receptor subunits can be referred to as Rl and R2.
  • Rl The class II cytokine receptor subunits
  • the Rl subunit binds the ligand with high affinity, has a long intracellular domain which is associated with Jakl tyrosine kinase, becomes phosphorylated at tyrosine residues after ligand binding and then acts to recruit various signal transducing proteins to the receptor complex.
  • Rl defines the specificity of the signaling.
  • the R2 subunit acts to initiate signal transduction by bringing an additional tyrosine kinase to the receptor complex.
  • IL-20 does not follow the established receptor-binding paradigm and requires both receptor subunits for high affinity binding and does not detectably bind to either subunit expressed alone (Blumber, H. et al. Cell 104:9, 2001).
  • IL-20, IL-19 and IL-24 share some receptor subunits (Dumoutier, L, et al, J. Immunology 167:3545. 2001; Wang, M. et al, J. Biol. Chem. 277:7341. 2002).
  • the IL-19 receptor complex consists of IL20R1 and IL20R2 subunits (U.S. Patent No. 5,945,511 and International Patent Application No. PCT/US99/03735). Both IL-20 and IL-24 signal through two receptor complexes one of which is identical to the IL-19 receptor complex, the other consists of IL22R1 and IL20R2 subunits.
  • IL20R2 is common to both receptor complexes used by IL-20 and IL-24. Some studies have reported the presence of these receptor subunits in particular tissues, but none have shown which of the receptor subunits, if any, are expressed in sources that contain hematopoietic stem cells such as the bone marrow or the thyroid and would therefore be available for IL-20 to activate CFU-GEMM proliferation (summarized in Kotenko, S., Cvtokine & Growth Factor Reviews 13:223, 2002).
  • IL-20 The diverse biological activities of IL-20, that likely result from signaling through a particular receptor for a particular activity, have led to a need for glycosylation-modified IL-20 polypeptides, and polynucleotides that encode them, which preferentially signal through one of the IL-20 receptor complexes and thereby preserve a particular therapeutic utility while decreasing or eliminating another utility.
  • the present invention addresses the need for modified forms of IL-20 polypeptides capable of preferentially signaling through one IL-20 receptor complex, and related compositions and methods.
  • the present invention embodies a glycosylation-modified IL-20 polypeptide that preferentially signals through one of the IL-20 receptor complexes, preferably at a level at least 1.3, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 or 9.0 times the level it signals through a different IL-20 receptor complex.
  • the preferred IL-20 receptor complex comprises IL20R1 and IL20R2 subunits.
  • the invention embodies multiple forms of glycosylation-modified IL-20 polypeptides.
  • the glycosylation-modified IL-20 polypeptides of the invention encompass natural allelic variants.
  • the invention further contemplates a glycosylation-modified IL-20 polypeptide that preferentially signals through one of the IL-20 receptor complexes and comprises an amino acid sequence at least about 95%, even more preferably at least 96%, 97%, or 98% and most preferably at least 99% identical or homologous (i.e., amino acid sequence identity) to the sequence of amino acids 25 through 125, amino acids 1 through 125, amino acids 25 through the C- terminus, or amino acids 1 through the C-terminus, of SEQ ID NO: 5, 6, 7, 8, 14, 15, 16, 17, 18, 19, 20 or 21 in which the N-linked glycosylation consensus sequences [NX(T/S)] are such that N is an asparagine amino acid, X is any amino acid, preferably not a proline, and T
  • N-linked glycosylation sequences of the glycosylation-modified IL- 20 polypeptides of the invention may be shifted from their existing position, as shown in SEQ TD NOs: 5-9 and 14-21, in either direction, by 1, 2 or 3 amino acids.
  • polypeptides of the present invention may have the IL-20 signal sequence replaced with the signal sequence of a different protein.
  • the invention embodies glycosylation-modified IL-20 polypeptides that have one or more of the glycosylation modifications illustrated in SEQ ID NOs: 14-17.
  • a glycosylation-modified IL-20 polypeptide may have two N-linked glycosylation sites, one at amino acids 61-63 as shown in Gly4 (SEQ ID NO: 8 or 17) and one at amino acids 140-142 as shown in Glyl (SEQ ID NO: 5 or 14; see SEQ ID NOs: 18-21). It is noted that the N-linked glycosylation site for all polypeptides of the invention need not be that sequence specifically illustrated in SEQ ID NOs.
  • NXT/S N-linked glycosylation site
  • the invention embodies a fusion polypeptide comprising a first portion and a second portion joined by a peptide bond.
  • the first portion of the fusion polypeptide comprises amino acids 25 through 125, amino acids 1 through 125, amino acids 25 through the C-terminus, or amino acids 1 through the C-terminus, of a glycosylation- modified IL-20 polypeptide or variant thereof, with or without the signal sequence.
  • the second portion of the fusion polypeptide consists of another polypeptide including, but not limited to, an affinity tag.
  • the affinity tag is an immunoglobulin Fc polypeptide.
  • the affinity tag is FLAG and/or His6.
  • the signal sequence is provided by the second portion of the fusion polypeptide.
  • the first portion of the fusion polypeptide may be operably linked to the amino terminal or carboxy terminal end of the second portion.
  • the present invention further embodies glycosylation-modified IL-20 polypeptides comprising, or alternatively, consisting of, a polypeptide selected from the group consisting of about amino acids 25 through 125, amino acids 1 through 125, amino acids 25 through the C-terminus, and amino acids 1 through the C-terminus of SEQ ID NOs: 5, 6, 7, 8, 14, 15, 16, 17, 18, 19, 20 and 21.
  • the present invention embodies a nucleic acid molecule comprising a polynucleotide encoding a glycosylation-modified human IL-20 polypeptide that preferentially signals through one IL-20 receptor complex, preferably at a level at least 1.3, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, or 9.0 times the level it signals through a different IL-20 receptor complex.
  • the invention further embodies a nucleic acid molecule comprising a polynucleotide encoding a glycosylation-modified human IL-20 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 14, 15, 16, 17, 18, 19, 20 or 21.
  • the present invention further embodies a nucleic acid molecule comprising a polynucleotide encoding a glycosylation-modified human IL-20 having an amino acid sequence at least about 95%, even more preferably at least 96%, 97%, or 98% and most preferably at least 99% identical (i.e., amino acid sequence identity) to that shown in SEQ ID NOs: 5, 6, 7, 8, 14, 15, 16, 17, 18, 19, 20, or 21, in which the N-linked glycosylation consensus sequences [NX(T/S)] are such that the N is an asparagine amino acid, the X is any amino acid, preferably not a proline, and the T/S is either a threonine or a serine.
  • glycosylation-modified human IL-20 molecules encoded by the nucleic acid molecules of the invention may have a NX(T/S) sequence shifted in either direction by 1, 2 or 3 amino acids from where it exists in SEQ ID NOs: 5, 6, 7, 8, 14, 15, 16, 17, 18, 19, 20, or 21 and/or may optionally have the IL-20 signal sequence replaced with the signal sequence of a different protein.
  • the invention further embodies a nucleic acid molecule comprising a polynucleotide with the sequence shown in SEQ ID NOs: 10, 11, 12, or 13, with or without the sequence encoding the first 24 amino acids (signal sequence).
  • Those nucleic acid molecules not encoding the IL-20 signal sequence are operably linked to a nucleic acid molecule encoding a different signal sequence thereby enabling secretion of the polypeptide encoded by the nucleic acid molecule upon expression.
  • the present invention also embodies a recombinant vector, preferably an expression vector, that comprises a nucleic acid molecule of the present invention and a host cell, preferably a mammalian cell, comprising said recombinant vector.
  • the invention also provides a vector, preferably an expression vector, encoding a fusion polypeptide of the invention and a host cell, preferably a mammalian cell, transfected with such a vector to produce a fusion polypeptide of the invention.
  • a vector preferably an expression vector
  • a host cell preferably a mammalian cell
  • the invention also embodies methods of making such vectors and host cells of the invention and methods for using them for production glycosylation-modified IL-20 polypeptides.
  • the invention provides a method of modulating the physiology or development of a cell in vivo or in situ comprising introducing into such cell, or the environment of such cell, a therapeutically effective amount of a glycosylation-modified IL-20 polypeptide of the invention.
  • the present invention embodies use of a therapeutically effective amount of a glycosylation-modified IL-20 polypeptide of the invention in treating hematopoietic disorders, cancer, cardiovascular disorders, skin disorders such as psoriasis, inflammation and/or immune system disorders and pharmaceutical compositions comprising said polypeptide.
  • the invention further embodies a pharmaceutical composition
  • a pharmaceutical composition comprising, alternatively consisting of or consisting essentially of, a hematopoietic progenitor cell- stimulating amout, or alternatively a CFU-GEMM cell-stimulating amount of at least one glycosylation-modified IL-20 polypeptide and/or variant thereof and a pharmaceutically acceptable carrier, diluent or excipient.
  • Fig. 1 provides an alignment of the amino acid sequences of wild-type IL-20, IL- 19, IL-24 and the glycosylation-modified IL-20 named Glyl, Gly2, Gly3 and Gly4 with the N-linked glycosylation sites underlined.
  • amino acid is used herein in its broadest sense, and includes naturally occurring amino acids as well as non-naturally occurring amino acids, including amino acid analogs and derivatives. The latter includes molecules containing an amino acid moiety.
  • Reference herein to an amino acid includes naturally occurring proteogenic L- amino acids; D-amino acids; chemically modified amino acids, such as amino acid analogs and derivatives; naturally occurring non-proteogenic amino acids such as norleucine, ⁇ -alanine, ornithine, etc.; and chemically synthesized compounds having properties known in the art to be characteristic of amino acids.
  • glycoslation-modified IL-20 polypeptide refers to an amino acid moiety.
  • IL-20 polypeptide (SEQ ID NO: 1) with or without about the first (amino terminal) 24 amino acid signal sequence, said polypeptide modified to contain one or more N-linked glycosylation sites at amino acid positions 61-63, 97-99 and/or 140-142 or 1, 2, or 3 amino acids in either direction of these positions.
  • glycosylation- modified IL-20 polypeptide includes glycosylation-modified IL-20 variants as described herein. The sequence of exemplary glycosylation-modified IL-20 polypeptides is shown in SEQ ID NOs: 5-8, 14-21.
  • N-linked glycosylation site refers to a 3-amino acid sequence (or the nucleotides encoding said sequence) of NX(T/S) in which the N is an asparagine amino acid, the X is any amino acid, and the T/S is either a threonine or a serine amino acid.
  • glycosylation-modified IL-20 polynucleotide refers to a polynucleotide that encodes a glycosylation-modified IL-20 polypeptide or a glycosylation-modified IL-20 variant polypeptide.
  • the term further refers to a glycosylation-modified IL-20 variant polynucleotide as described herein.
  • glycosylation-modified IL-20 variant refers to a glycosylation-modified IL-20 polynucleotide or IL-20 polypeptide, e.g., as shown in SEQ ID NOs: 10-13 (polynucleotides), or 5-8 or 14-21 (polypeptides), that further comprises at least one of the various types of modifications contemplated herein.
  • the methods of the present invention contemplate alterations in a glycosylation-modified IL-20 polypeptides that may include one or more amino acid insertions, deletions, substitutions, truncations, fusions, shuffling of subunit sequences, and the like, either from natural mutations (e.g., allelic variants) or human manipulation, provided that the sequences produced by such modifications have substantially the same activity(ies) as the glycosylation-modified IL- 20 polypeptides (i.e., the parent molecule) without such additional modification(s). It is contemplated that such modification should result in a polypeptide no less than 95% homologous to the original glycosylation-modified IL-20 polypeptide sequence.
  • glycosylation-modified IL-20 variant as applied to a polypeptide, is intended to refer to a "functional" or “active” glycosylation-modified IL-20 polypeptide, having at least about 95% amino acid sequence identity with a glycosylation-modified IL- 20 polypeptide having the deduced amino acid sequences as shown in SEQ ID NOs: 5, 6, 7, 8, 14, 15, 16, 17, 18, 19, 20 or 21 , with or without the signal sequence, and in which the N-linked glycosylation site may be shifted 1, 2, or 3 amino acids in either direction.
  • glycosylation-modified IL-20 polypeptide variants include, for instance, glycosylation-modified IL-20 polypeptides wherein one or more amino acid residues are added, substituted or deleted, at the N- or C-terminus or within the sequence of SEQ ID NOs: 5, 6, 7, 8, 14, 15, 16, 17, 18, 19, 20 or 21.
  • a glycosylation-modified IL- 20 polypeptide variant will have at least about 95%, 96% or 97% amino acid sequence identity, preferably at least about 98% amino acid sequence identity, yet more preferably at least about 99% amino acid sequence identity with the amino acid sequence as shown in SEQ ID NOs: 5, 6, 7, 8, 14, 15, 16, 17, 18, 19, 20 or 21, with or without the signal peptide and in which the amino acids of an N-linked glycosylation site may be shifted 1, 2, or 3 amino acids in either direction.
  • Variants are contemplated to included natural allelic variants of IL-20, i.e., encoded by the various alleles present in a population of mammals, preferably humans.
  • Variants are also contemplated to include polypeptides comprising a fusion polypeptide in which the glycosylation-modified IL-20, or variant thereof, is operably linked to another polypeptide, e.g., an polypeptide encoding an affinity tag.
  • the second portion i.e., that portion that is not a glycosylation-modified IL-20 polypeptide or variant thereof
  • glycosylation-modified IL-20 variant polynucleotide refers to a polynucleotide that encodes a glycosylation-modified IL-20 variant polypeptide as described above or a polynucleotide that has at least about 95%, 96%, 97% polynucleotide sequence identity, preferably at least about 98% polynucleotide sequence identity, yet more preferably at least about 99% polynucleotide sequence identity with the polynucleotide sequence shown in SEQ ID NOs: 10, 1 1, 12, or 13, with or without the sequence encoding the first 24 amino acids (the signal sequence) and in which the nucleotides encoding an N-linked glycosylation site may be shifted 3, 6, or 9 nucleotides (the equivalent of 1, 2, or 3 amino acids) in either direction.
  • the terms “complementary” or “complementarity” are used in reference to nucleic acids (i.e., a sequence of nucleotides) related by the well-known base- pairing rules that A pairs with T and C pairs with G.
  • nucleic acids i.e., a sequence of nucleotides
  • the sequence 5'-A-G-T- 3' is complementary to the sequence 3'-T-C-A-5'.
  • Complementarity between two single- stranded molecules can be “partial,” in which only some of the nucleic acid bases are matched according to the base pairing rules.
  • DNA sequence polymo ⁇ hisms that lead to changes in the amino acid sequence of IL-20 may exist within a population (e.g., the human population). Such genetic polymo ⁇ hism in the IL-20 gene may exist among individuals within a population due to natural allelic variation.
  • An "allelic variant” is an alternate form of a polynucletide sequence that may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • a polynucleotide of the present invention can be composed of any RNA or DNA, which may be unmodified or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded regions.
  • a polynucleotide may contain one or more modified nucleotides.
  • “Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of such modifications can be made; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • Percent (%) amino acid sequence identity with respect to the glycosylation- modified IL-20 amino acid sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence (e.g., glycosylation-modified EL-20) that are identical with the amino acid residues in a reference polypeptide (e.g., wild type IL-20) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • a candidate sequence e.g., glycosylation-modified EL-20
  • a reference polypeptide e.g., wild type IL-20
  • Alignment for pu ⁇ oses of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN, ALIGN-2, Megalign (DNASTAR) or BLAST (e.g., Blast, Blast-2, WU-Blast-2) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the percent identity values used herein can be generated using WU-BLAST-2 (Altschul et al, Methods in Enzymology 266:460, 1996). Most of the WU-BLAST-2 search parameters are set to the default values.
  • a percent amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between the amino acid sequence of the polypeptide of interest and the comparison amino acid sequence of interest (i.e., the sequence against which the polypeptide of interest is being compared) as determined by WU-BLAST-2, by (b) the total number of amino acid residues of the polypeptide of interest, respectively.
  • Percent (%) nucleic acid sequence identity with respect to the polynucleotide sequences identified herein is defined as the percentage of nucleotides in a candidate sequence (e.g., glycosylation-modified IL-20) that are identical with the nucleotides in the reference sequence (e.g., wild type IL-20) after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for pu ⁇ oses of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN, Align-2, Megalign (DNASTAR), or BLAST (e.g., Blast, Blast-2) software.
  • a percent nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the polypeptide-encoding nucleic acid molecule of interest and the comparison nucleic acid molecule of interest (i.e., the sequence against which the polypeptide-encoding nucleic acid molecule of interest is being compared) as determined by WU-BLAST-2, by (b) the total number of nucleotides of the polypeptide-encoding nucleic acid molecule of interest.
  • mature protein or "mature polypeptide” as used herein refers to the form(s) of the protein as would be produced by expression in a mammalian cell.
  • proteins secreted by mammalian cells have a signal peptide (SP) sequence which is cleaved from the complete polypeptide to produce a "mature" form of the protein.
  • SP signal peptide
  • cleavage of a secreted protein is not uniform and may result in more than one species of mature protein.
  • the cleavage site of a secreted protein is determined by the primary amino acid sequence of the complete protein and generally cannot be predicted with complete accuracy.
  • a cleavage point may exist within the N-terminal domain between amino acid 10 and amino acid 35. More specifically the cleavage point is likely to exist after amino acid 15 but before amino acid 31. Cleavage sites sometimes vary from organism to organism and may even vary from molecule to molecule within a cell and cannot be predicted with absolute certainty. Optimally, cleavage sites for a secreted protein are determined experimentally by amino-terminal sequencing of the one or more species of mature proteins found within a purified preparation of the protein.
  • IL-20 (and glycosylation-modified forms of IL-20) has a signal sequence extending from amino acid residue 1 , a methionine, and including amino acid residue 24, a glycine, of SEQ ID NO: 1.
  • the mature IL-20 or mature form of any glycosylation- modified IL-20 extends from about amino acid residue 25, a leucine, to and including the C-terminal amino acid residue at position 176, a glutamic acid.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in the correct reading frame. However, enhancers do not have to be in the same reading frame.
  • vector refers to a nucleic acid compound used for introducing exogenous or endogenous DNA into host cells.
  • a vector comprises a nucleotide sequence that may encode one or more protein molecules. Plasmids, cosmids, viruses, and bacteriophages, in the natural state or which have undergone recombinant engineering, are examples of commonly used vectors.
  • the terms “treating”, “treatment” and “therapy” as used herein refer to curative therapy, prophylactic therapy, and preventive therapy.
  • An example of “preventive therapy” is the prevention or lessened targeted pathological condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • a “therapeutically-effective amount” is the minimal amount of active agent (e.g., a glycosylation-modified IL-20 polypeptide) necessary to impart therapeutic benefit to a mammal.
  • a "therapeutically-effective amount" to a mammal is such an amount that reduces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression, physiological conditions associated with or resistance to succumbing to the aforedescribed disorder.
  • inhibit refers to a decrease, whether partial or whole, in function or activity.
  • inhibition of signaling activity refers to any decrease in signaling activity that is being measured (directly or indirectly), including complete elimination of said activity.
  • Activity or “activity” or “functional” in the context of a glycosylation-modified
  • IL-20 polypeptide refers to retention of a biological function of the wild type IL-20 polypeptide including e.g., the ability to induce production of an antibody against an antigenic epitope possessed by the IL-20 polypeptide at levels near that of the wild type IL-20 polypeptide, or preferably the ability to signal through one of the IL-20 receptors.
  • Activity or “function” also refers to a biological function (either inhibitory or stimulatory) caused by a reference polypeptide (e.g., wild type IL-20).
  • Exemplary biological activities include, but are not limited to, the ability of such molecules to inhibit inflammation, to increase the presence of CFU-GEMM cells, to treat the following: psoriasis or other skin disorder, an immune system disorder, inflammation, obesity, a hematological disorder, a cancer and/or a cardiovascular disorder.
  • Glycosylation-modified IL-20 polypeptides of the invention are typically administered to a subject in "substantially pure” form.
  • the term “substantially pure” as used herein refers to a glycosylation-modified IL-20 polypeptide that is substantially free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated.
  • One skilled in the art can purify a glycosylation-modified IL-20 polypeptideusing standard techniques for protein purification.
  • Cells which are targeted by the methods of the present invention, such as, e.g., stem cells, hematopoietic stem cells or keratinocytes include isolated cells maintained in culture as well as cells within their natural context in vivo.
  • the present invention is based in part upon the discovery and synthesis of glycosylation- modified IL-20 polypeptides that preferentially signal through one of the multiple IL-20 receptor complexes, preferably at a level at least 1.3, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 or 9.0 times the level it signals through a different IL-20 receptor complex.
  • the glycosylation- modified IL-20 polypeptides may have altered receptor binding, not because of the particular glycosylation group per se, but rather, because there is a bulky group now attached to the IL-
  • Example 5 demonstrates glycosylation-modified IL-20 polypeptides that preferentially signal through the receptor complex comprising IL20R1 and IL20R2 subunits.
  • the present invention is further based upon the discovery of glycosylation-modified IL-20 polypeptide and their use in treating, preventing, and diagnosing hematopoietic disorders, obesity, cancer, cardiovascular disorders, skin disorders such as psoriasis, and/or immune system disorders such as autoimmune diseases and inflammatory disorders.
  • Glycosylation Modified IL-20 are examples of glycosylation-modified IL-20 polypeptide and their use in treating, preventing, and diagnosing hematopoietic disorders, obesity, cancer, cardiovascular disorders, skin disorders such as psoriasis, and/or immune system disorders such as autoimmune diseases and inflammatory disorders.
  • a type of covalent modification of the polypeptides included within the scope of this invention comprises altering the glycosylation pattern of the wild type IL-20 polypeptide.
  • "Altering the native glycosylation pattern” is intended for pu ⁇ oses herein to mean adding one or more N-linked glycosylation sites that are not present in the native sequences of IL-20. These sites are preferably added at a location equivalent to where a glycosylation site exists within IL-19 and or IL-24 or within 1, 2 or 3 amino acids of said site.
  • Addition of glycosylation sites to polypeptides may be accomplished by altering the amino acid sequence thereof.
  • the amino acid sequences may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids (NX(TJS)).
  • Another means of increasing the number of carbohydrate moieties on a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in International Patent Publication No. WO 87/05330 and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
  • the polypeptides of the present invention are glycosylation modified IL-20. These molecules have one or more N-linked glycosylation sites at amino acids 61-63, 97-99 and/orl40-142 (numbering based on full-length IL-20 protein sequence as shown in SEQ ID NO: 1). The location of the N-linked glycosylation site may vary by 1, 2 or 3 amino acids in the glycosylation-modified IL-20 and still maintain the desired property of preferentially signaling through one of the IL-20 receptor complexes.
  • the invention is contemplated to include those glycosylation-modified IL-20 polypeptides that have at least one N-linked glycosylation sites at amino acids 61-63 (site 1), 97-99 (site 2) and/orl40-142 (site 3), plus or minus 1 , 2, or 3 amino acids for each of the sites, (e.g., amino acid residues
  • polypeptides of the invention when inside the cell, may have a signal peptide sequence to enable protein transport within the cell; however, this signal peptide sequence is not present in the mature glycosylation-modified IL-20 polypeptide as it exists when outside the cell.
  • the signal peptide may be that as present in the wild type IL-20 polypeptide (amino acid residues 1 to about 24 of SEQ ID NO: 1) or it may be the signal peptide of another polypeptide operably linked to the mature sequence of IL-20 that begins at about amino acid 25 of SEQ ID NO: 1.
  • a signal peptide comprisesd of about 10-30 hydrophobic amino acids, targets the nascent protein from the ribosome to the endoplasmic reticulum (ER). Once localized to the ER, the proteins can be further directed to the Golgi apparatus within the cell. The Golgi distributes proteins to vesicles, lysosomes, the cell membrane, and other organelles. Proteins targeted to the ER by a signal sequence can be released from the cell into the extracellular space. This is the case for glycosylation-modified IL-20 polypeptides of the present invention. For example, vesicles containing proteins to be moved outside the cell can fuse with the cell membrane and release their contents into the extracellular space via a process called exocytosis.
  • Exocytosis can occur constitutively or after receipt of a triggering signal. In the latter case, the proteins are stored in secretory vesicles until exocytosis is triggered. Proteins that transit through this pathway are either released into the extracellular space or retained in the plasma membrane. The IL-20 polypeptide is released into extracellular space. In many instances the amino acids comprising the signal peptide are cleaved off the protein during transport or once its final destination has been reached. Specialized enzymes, termed signal peptidases, are responsible for the removal of the signal peptide sequences from proteins. These enzymes are activated once the signal peptide has directed the protein to the desired location.
  • a glycosylation-modified IL-20 polyeptide may be produced recombinantly, not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which e.g., may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the signal sequence may be a component of an expression vector, or it may be a part of the polypeptide-encoding DNA that is inserted into such a vector.
  • yeast secretion the signal sequence may be, e.g., the yeast invertase leader, aha factor leader (including Saccharomyces and Kluyveromyces cc-factor leaders, the latter described in U.S.
  • mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species as well as viral secretory leaders.
  • nucleic acid molecules comprising polynucleotides encoding the polypeptides. Exemplary polynucleotides are shown in SEQ ID NOs: 14-17 Also provided are polynucleotide variants that are homologous or substantially identical to SEQ ID NOs: 14-17.
  • the present invention encompasses variants of a polynucleotide sequence encoding a glycosylation-modified IL-20 polypeptide, or variant polypeptide thereof, disclosed in SEQ ID NOs: 10-13 and their complementary strands.
  • the present invention also encompasses variants of the glycosylation-modified IL-20 polypeptide sequences disclosed in SEQ ID NOs: 5-8 and 14-21.
  • variant refers to a polynucleotide or polypeptide differing from a polynucleotide sequence or a polypeptide sequence as shown in the SEQ ID NOs of the present invention (respectively), but retaining essential properties thereof such as a particular activity or function of interest.
  • a variant glycosylation-modified IL-20 polypeptide encompassed within the scope of the invention would preferentially signal through one IL-20 receptor complex at a level greater than through another IL-20 receptor complex, substantially similar to the non- variant form of the glycosylation-modified IL-20 polypeptide.
  • variants are closely similar overall in structural and/or sequence identity, and, in many regions, identical to a polynucleotide or polypeptide of the present invention.
  • variant is further described in the definitions herein.
  • the present invention encompasses nucleic acid molecules that comprise or alternatively consist of, a polynucleotide sequence that is at least 95%, 96%, 97%, 98%, or most preferably at least 99% identical to a polynucleotide comprising the sequence of SEQ ID NOs: 10-13 (or a strand complementary thereto); or a polynucleotide sequence encoding a polypeptide that is at least 95%, 96%, 97%, 98%, or most preferably at least 99% identical to that of SEQ ID NO: 5, 6, 7, 8, 14, 15, 16, 17, 18, 19, 20 or 21 in which the NX(T/S) sequence encoding the N-linked glycosylation site may be shifted 1, 2, or 3 amino acids in either direction and still fall within the scope of the invention.
  • the present invention is also directed to polypeptides that comprise, or alternatively consist of, an amino acid sequence that is at least: 95%, 96%, 97%, 98%, or 99% identical to a polypeptide sequence of SEQ ID NOs: 5, 6, 7, 8, 14, 15, 16, 17, 18, 19, 20 or 21 in which the NX(T/S) sequence encoding the N-linked glycosylation site may be shifted 1, 2, or 3 amino acids in either direction and still fall within the scope of the invention.
  • a polynucleotide sequence having at least some "percentage identity,” (e.g., 95%) to another polynucleotide sequence means that the sequence being compared (e.g., the test sequence or candidate sequence) may vary from another sequence (e.g. the reference sequence) by a certain number of nucleotide differences (e.g., a test sequence with 95% sequence identity to a reference sequence can have, on average, up to five point mutations per each 100 contiguous nucleotides of the referent sequence).
  • a test sequence to exhibit at least 95% identity to a reference sequence up to 5% of the nucleotides in the reference may differ, e.g., be deleted or substituted with another nucleotide, or a number of nucleotides (up to 5% of the total number of nucleotides in the reference sequence) may be inserted into the reference sequence.
  • determining if a particular nucleic acid molecule or polynucleotide sequence exhibits at least about: 95%, 96%, 97%, 98% or 99% identity to a polynucleotide sequence can be accomplished using known computer programs. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al, J Mol Biol 215:403. 1990.
  • polynucleotide variants containing alterations which produce silent substitutions (i.e., no change in amino acid encoded thereby), additions, or deletions, but do not alter the properties or activities of the encoded polypeptide.
  • a further indication that two nucleic acid sequences of polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described herein.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
  • a polypeptide exhibiting or having at least about, e.g., 95% "sequence identity" to another amino acid sequence may include, e.g., up to five amino acid alterations per each 100 amino acid (on average) stretch of the test amino acid sequence.
  • a first amino acid sequence that is at least 95% identical to a second amino acid sequence can have up to 5% of its total number of amino acid residues different from the second sequence, e.g., by insertion, deletion, or substitution of an amino acid residue.
  • Alterations in amino residues of a polypeptide sequence may occur, e.g., at the amino or carboxy terminal positions or anywhere between these terminal positions, interspersed either individually among residues in the sequence or in one or more contiguous sections, portions, or fragments within the sequence.
  • a reference polypeptide e.g., Glyl as shown in SEQ ID NO: 5
  • a fusion protein in which the reference polypeptide is joined to e.g., an affinity tag, (e.g., Glyl fused to FLIS tag as discussed in the Examples); it is not intended in the present invention that the portion of the fusion protein that does not encode a glycosylation-modified IL-20 (in this example, the FLIS tag) be included in the alignment or the percent identity analysis, but rather; only that portion of the fusion protein encoding a glycosylation-modified IL-20 should be included in the alignment to the reference polypeptide.
  • an affinity tag e.g., Glyl fused to FLIS tag
  • Variants encompassed by the present invention may contain alterations in the coding regions, non-coding regions, or both. Moreover, variants in which 1-2, 1-5, or 5-10 amino acids are substituted, deleted, or added in any combination are preferred. Even more preferably the variant glycosylation-modified IL-20 polypeptides preferentially signal through one of the IL-20 receptor complexes.
  • the invention encompasses polypeptide variants that show a biological activity of the reference glycosylation- modified IL-20 such as, e.g., ligand binding for an IL-20 receptor complex or antigenicity.
  • Such variants include, e.g., deletions, insertions, inversions, repeats, and substitutions selected so as to have little effect on activity using general rules known in the art.
  • teachings on making phenotypically silent amino acid substitutions are provided, e.g., by Bowie, et al, Science 247:1306, 1990.
  • a polypeptide of the invention in order of ever-increasing preference, it is highly preferable for a polypeptide of the invention to have an amino acid sequence that comprises an amino acid sequence of the present invention which contains zero or one, but not more than: 10, 9, 8, 7, 6, 5, 4, 3,
  • Recombinant expression vectors are typically self-replicating DNA or RNA constructs containing a desired gene to be expressed that is operably linked to a promoter and optionally other control elements that will be recognized in a suitable host cell.
  • the specific type of control elements necessary to effect expression depends on the host cell used and the level of expression desired.
  • Proteins of the invention can be expressed in mammalian cells, yeast, insect or other cells under the control of appropriate promoters and which are capable of glycosylating the protein at N-linked glycosylation consensus sites of NX(TJS).
  • Vectors as used herein, encompass plasmids, viruses, bacteriophage, integratable
  • Plasmids are the most commonly used form of vector, but many other forms of vectors that perform an equivalent function are also suitable for use.
  • Expression and cloning vectors will typically contain at least one selection gene.
  • Expression vectors further contain a promoter operably linked to the polypeptide-encoding nucleic acid sequence to direct mRNA synthesis.
  • a typical mammalian expression vector contains at least one promoter element that mediates initiation of transcription of mRNA, the polypeptide of interest's coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional optional elements include enhancer(s), a Kozak sequence and an intervening sequence (intron) flanked by donor and acceptor sites for RNA splicing.
  • a signal sequence also known as a leader sequence, prepro sequence or pre sequence is provided in the expression vector.
  • the signal sequence may be that of the IL-20 protein, or may be derived from another secreted protein (e.g., t-PA), or synthesized de novo.
  • the secretory signal sequence is joined to the DNA sequence encoding the glycosylation-modified IL-20 in the correct reading frame.
  • Signal sequences are commonly positioned 5' to the DNA sequence encoding the mature polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence.
  • Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Tranformation of insect cells and production of foreign polypeptides therein is disclosed in U.S. Patent No. 5,162,222.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al, J. Biosci. 11:47, 1987.
  • Insect cells can be infected with recombinant baculovirus (see Richardson, C. Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, (Humana Press, Totowa, NJ, 1995) or BAC-TO-BAC of Life Technologies.
  • Methods for introducing exogenous DNA into mammalian host cells or other cells are well known in the art and include calcium phosphate-mediated transfection, Wigler, et al, Cell 14:725, 1978; electroporation, Neumann et al, EMBO J.
  • nucleic acid encoding the polypeptide of interest is expressed in stable cell lines, cultured mammalian cells that contain the nucleic acid integrated into a host chromosome.
  • DHRF dihydrofolate reductase
  • GPT GPT neomycin
  • hygromycin allows the identification and isolation of the transfected cells.
  • the transfected gene can also be amplified to express large amounts of the encoded polypeptide.
  • the DHFR marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
  • Another useful selection marker is the enzyme glutamine synthase (Mu ⁇ hy, et al, Biochem. J. 227:277, 1991; Bebbington, et al, BioTechnology 10:169, 1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of polypeptides.
  • CHO cells are efficient at producing glycosylated polypeptides.
  • the description below relates primarily to production of a polypeptide of the invention by culturing cells transformed or transfected with a vector containing polypeptide- encoding nucleic acid. It is contemplated that alternative methods, well known in the art, may be used to prepare polypeptides. For instance, the sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al, Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J.
  • In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions. Various portions of a polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce a full-length .
  • Host cells are transfected or transformed with expression vectors described herein for polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the culture conditions such as media, temperature, pU and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al, supra.
  • Suitable host cells for cloning the nucleic acid in the vectors herein include prokaryote, yeast, or higher eukaryote cells, however for expression of glycosylation- modified IL-20, bacteria may not be used. Alternatively, PCR or other nucleic acid polymerase reactions, are suitable.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the polypeptide expressing vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.
  • Suitable host cells for the expression of glycosylated polypeptides of the invention are derived from multicellular organisms.
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sp, Spodoptera high5 as well as plant cells.
  • useful mammalian host cell lines include, e.g., CHO and COS cells.
  • Polypeptides of the invention may be recovered from culture medium or from host cell lysates and analyzed e.g., for their signaling ability as demonstrated in the Examples herein. It is preferred to purify the polypeptides of the invention to greater than about 80% purity, more preferably to at least 90% purity, even more preferably to at least 95% purity, and free of infectious and pyrogenic agents.
  • the glycosylation-modified IL-20 polypeptides of the present invention are not membrane-bound.
  • Cells employed in expression of polypeptides can be disrupted for protein purification by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • the following procedures are exemplary of suitable purification procedures: fractionation on an ion-exchange column; ethanol precipitation; reversed-phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G- 75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of polypeptides.
  • Various methods of protein purification may be employed and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182:83, 1990 and Scopes, Protein Purification: Principles and Practice.
  • the purification step(s) selected will depend, for example, on the nature of the production process used and the particular polypeptide produced. Many types of analyses can be performed with the polypeptides of the present invention to demonstrate their role in the development, pathogenesis, and treatment of hematopoietic disorders, cancer, cardiovascular disease, skin disorders and immune related disease. Certain analyses are exemplified in the Examples herein. Animal models can be used to further understand the role of the polypeptides of the invention.
  • Glycosylation-modified IL-20 polypeptides are useful for the prevention, diagnosis, and treatment of cancer, cardiovascular disorders and immune system disorders, skin disorder such as psoriasis, inflammation, obesity, and hematopoietic disorders.
  • glycosylation-modified IL-20 may be expressed and glycosylated in mammalian cells and secreted therefrom (Examples 2 and 3).
  • the data further demonstrate that glycosylation-modified IL-20, as exemplified by Glyl, Gly3 and Gly4, may preferentially signal through one of the multiple IL-20 receptor complexes (Example 5).
  • Particular cancers suitable for treatment with the polypeptides of the invention include, but are not limited to, acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocyticleukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia and chronic granulocyticleukemia.
  • Additional cancers suitable for treatment with the polypeptides of the invention inculde are not limited to, adenocarcinoma, lymphoma, melanoma, myeloma, Hamartoma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
  • Particular cardiovascular disorder suitable for treatment with the polypeptides of the invention include, but are not limited to, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, complications of cardiac transplantation, arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins
  • Particular immune system disorders suitable for treatment with the polypeptides of the invention include, but are not limited to, inflammatory disorders, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis- ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
  • hematopoietic disorders suitable for treatment with the polypeptides of the invention include, but are not limited to, various diseases arising from imbalances between degradation and reconstitution of blood cells or from generation of inappropriate numbers of certain types of blood cells and requiring enhancing or stimulating of hematopoiesis, erythropoiesis, leukopoiesis, thrombocytopoiesis, production of neutrophils, granulocytes, and/or platelets by stimulating the proliferation and/or differentiation of progenitors of such cells, as needed in various conditions and/or situations, including, but not limited to, the following:
  • abnormal platelet function such as Glanzmann's thrombasthenia, acute/chronic leukemia, myeloproliferative disorders, uremia, platelet storage pool disease, Non Willebrand disease, and postoperative cardiovascular dysfunction, and (d) other blood coagulation disorders such as afibrinogenemia or wounds of any origin.
  • CFU-GEMM stimulating amount when referring to a glycosylation-modified IL-20 polypeptide refers to an amount of glycosylation-modified IL- 20 that raises the baseline number of CFU-GEMM cells by at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 80% or greater over the number of CFU-GEMM cells present in a mammal, human, or patient in the absence of glycosylation-modified IL-20.
  • hematopoietic progenitor stimulating amount when referring to glycosylation- modified IL-20 refers to an amount of glycosylation-modified IL-20 that raises the baseline number of any hematopoietic progenitor cell type by at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 80% or greater over the number of that type of hematopoietic progenitor cells present in a mammal, human, or patient in the absence of glycosylation-modified IL-20.
  • the glycosylation-modified IL-20 polypeptide when the coding sequence for a glycosylation-modified IL-20 polypeptide encodes a protein which binds to another protein as is the case for extracellular glycosylation-modified IL-20 of the present invention, the glycosylation-modified IL-20 polypeptide can be used in assays to identify the other proteins or molecules involved in the binding interaction or subsequent signaling response. By such methods, inhibitors of the receptor/ligand binding interaction or signaling can be identified. Proteins involved in such binding interactions or signaling can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction or signaling. Also, the receptor polypeptide can be used to isolate correlative ligand(s).
  • the active agents of the present invention are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebral, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, intraoccular, intralesional, oral, topical, inhalation, pulmonary, and/or through sustained release.
  • intravenous administration as a bolus or by continuous infusion over a period of time
  • intramuscular, intraperitoneal, intracerebral, intracerobrospinal subcutaneous, intra-articular, intrasynovial, intrathecal, intraoccular, intralesional, oral, topical, inhalation, pulmonary, and/or through sustained release.
  • a polypeptide of the invention may be combined with other therapeutic regimens.
  • the appropriate dosage of an active agent will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic pu ⁇ oses, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician.
  • the agent is suitably administered to the patient at one time or over a series of treatments.
  • Dosages and desired drug concentration of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan.
  • An effective amount of at least one of the glycosylation-modified TL-20 in combination with a pharmaceutically acceptable sterile vehicle may be determined as described, for example, in Remingtons' Pharmaceutical Sciences; Drug Receptors and Receptor Theory, 18th ed., Mack Publishing Co., Easton, Pa. (1990). Animal experiments provide reliable guidance for the determination of effective does for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti and Chappell, "The Use of Interspecies Scaling in Toxicokinetics," in
  • dosages may be administered by one or more separate administrations or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is readily monitored by conventional techniques and assays.
  • compositions of the invention may further contain common carriers and excipients such as for example, corn-starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid.
  • common carriers and excipients such as for example, corn-starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid.
  • compositions to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Examples
  • Site directed mutagenesis was performed using a nucleic acid encoding a FLIS- tagged (FLAG + HIS6) wild-type IL-20 to generate four different IL-20 glycosylation mutants named Glyl, Gly2, Gly3 and Gly4 (Fig. 1).
  • the nucleic acid encoding IL-20 in a pGEM plasmid (Promega Co ⁇ .), was mutagenized using QUIKCHANGETM Site- Directed Mutagenesis Kit (Stratagene) using the method described by the manufacturer.
  • the sequence encoding the FLIS tag was present at the 3' end of the FLIS-tagged IL-20 gene to accommodate the purification of the protein when expressed; however, it is not necessary for function of the protein.
  • Glyl is IL-20 altered such that the lysine residue at position 141 in the wild type IL-20 protein is replaced with an asparagine, the lysine residue at position 142 in the wild type protein is replaced with an alanine, and the tyrosine residue at position 143 in the wild type protein is replaced with a threonine, thereby creating an N-linked glycosylation sequence (NAT) at the equivalent position where the second N-linked glycosylation sequence (NAT) exists in IL-19 (see Fig. 1).
  • NAT N-linked glycosylation sequence
  • Gly2 is IL-20 altered such that an asparagine-arginine pair of residues is inserted between the glutamine residue at position 99 and the threonine residue at position 100 of the wild type IL-20 protein, thereby creating an N-linked glycosylation sequence (NRT) at the equivalent position where an NRT glycosylation sequence is located in IL-24 and creating a protein that is two amino acids larger than the wild type IL-20 protein (Fig. 1).
  • NRT N-linked glycosylation sequence
  • Gly3 is IL-20 altered such that the tyrosine residue at position 98 and the glutamine at position 99 of the wild type IL-20 protein are replaced with an asparagine- arginine pair of residues, thereby creating an N-linked glycosylation sequence (NRT) at the equivalent position where an NRT glycosylation sequence is located in IL-24 and creating a protein that is identical in size to the wild type IL-20 (see Fig. 1).
  • NRT N-linked glycosylation sequence
  • Gly 4 is IL-20 altered such that the aspartic acid residue at position 61 in the wild type IL-20 protein is replaced with an asparagine, the isoleucine residue at position 62 in the wild type protein is replaced with a valine and the arginine residue at position 63 in the wild type protein is replaced with a threonine, thereby creating an N-linked glycosylation sequence (NNT) at the same location where the first ⁇ -linked glycosylation sequence ( ⁇ NT) exists in IL-19 (see Fig. 1).
  • NNT N-linked glycosylation sequence
  • IL-10 and the receptor subunits used by IL-20, with the symbol (*) indicating either a stop codon or additional sequence encoding a polypeptide (or stop signal or the amino acid sequence of fusion protein for the amino acid sequences) operably linked to the upstream sequence, e.g., an affinity tag useful for purification.
  • These sequences do not include the optional FLIS tag encoded by 5' gatatcgactacaaggatgacgacgacaagcacgtgcatcaccatcaccatcactag 3' (SEQ ID NO: 22).
  • the consensus N-linked glycosylation domain of NXT/S is underlined. N represents asparagine, X is any amino acid and T/S is either a threonine or a serine.
  • Glyl polynucleotide sequence (SEQ ID NO: 10) atgaaagcctctagtcttgccttcagccttctctctgctgcgtt atctcctatggact ccttccactggactgaagacactcaatttgggaagctgtgtga cgccacaaaccttcag gaaatacgaaatggattttctgagatacggggcagtgtgcaagccaaagatggaaacatt gacatcagaatcttaaggaggactgagtctttgcaagacacaaagcctgcgaatcgatgc tgcgccatttgctaagactctatctggacagggtattttaaaactaccagacc cctgaccattatactctccggggact ctg
  • Gly2 polynucleotide sequence (SEQ ID NO: 11) atgaaagcctctagtcttgccttcagccttctctctctgctgcgtttatctcctatggact ccttccactggactgaagacactcaatttgggaagctgtgtgatcgccacaaaccttcag gaaatacgaaatggattttctgagatacggggcagtgtgcaagccaaagatggaaacatt gacatcagaatcttaaggaggactgagtctttgcaagacacaaagcctgcgaatcgatgc tgcgccatttgctaagactctatctggacagggtattttaaaactaccagaat agaacccctgaccattatactctccccctg
  • Gly3 polynucleotide sequence (SEQ ID NO: 12) atgaaagcctctagtcttgccttcagccttctctctgctgcgtttatctcctatggact ccttccactggactgaagacactcaatttgggaagctgtgtgatcgccacaaaccttcag gaaatacgaaatggattttctgagatacggggcagtgtgcaagccaaagatggaacatt gacatcagaatcttaaggaggactgagtctttgcaagacacaaagcctgcgaatcgatgc tgcgccatttgctaagactctatctggacagggtattttaaaaacagaacccctccgga
  • Gly4 polynucleotide sequence (SEQ ID NO: 13) atgaaagcctctagtcttgccttcagccttctctctgctgcgtttatctcctatggact ccttccactggactgaagacactcaatttgggaagctgtgtgatcgccacaaaccttcag gaaatacgaaatggattttctgagatacggggcagtgtgcaagccaaagatggaacatt aacgtcacaatattaaggaggactgagtctttgcaagacacacaaagcctgcgaatcgatgc tgcgccattttgctaagactctaaaaactcta ctggacagggtattttaaaactaccagacc cctgaccattatactct
  • IL-20 polypeptide sequence (SEQ ID NO: 1 ) mkasslafsllsaafyllwtpstglktlnlgscviatnlqeirngfseirgsvqakdgni dirilrrteslqdtkpanrccllrhllrlyldrvfknyqtpdhytlrkisslansfltik kdlrlchahmtchcgeeamkkysqilshfeklepqaawkalgeldillqwmeete* IL-10 polypeptide sequence (SEQ ID NO: 4) mhssallcclvlltgvraspgqgtqsenscthfpgnnn lrdlrdafsrvktffqmkdqld nilIkeslledfkgylgcqalsemiqfyleevmpqaenqdp
  • Glyl polypeptide sequence (SEQ ID NO: 5) mkasslafsllsaafyllwtpstglktlnlgscviatnlqeirngfseirgsvqakdgni dirilrrteslqdtkpanrccllrhllrlyldrvfknyqtpdhytlrkisslansfItik kdlrlchahmtchcgeeamnatsqilshfeklepqaawkalgeldillqwmeete*
  • Gly2 polypeptide sequence (SEQ ID NO: 6) mkasslafsllsaafyllwtpstglktlnlgscviatnlqeirngfseirgsvqakdgni dirilrrteslqdtkpanrccllrhllrlyldrvfknyqnrtpdhytlrkisslansfIt ikkdlrlchahmtchcgeeamkkysqilshfeklepqaawkalgeldillqwmeete*
  • Gly3 polypeptide sequence (SEQ ID NO: 7) mkasslafsilsaafyllwtpstglktlnlgscviatnlqeirngfseirgsvqakdgni dirilrrteslqdtkpanrccllrhllrlyldrvfknrtpdhytlrkisslansfltikk dlrlchahmtchcgeeamkkysqilshfeklepqaawkalgeldillqwmeete*
  • Gly4 polypeptide sequence (SEQ ID NO: 8) mkasslafsllsaafyllwtpstglktlnlgscviatnlqeirngfseirgsvqakdgni nvtilrrteslqdtkpanrccllrhllrlyldrvfknyqtpdhytlrkisslansfItik kdlrlchahmtchcgeeamkkysqilshfeklepqaawkalgeldillqwmeete*
  • Generic Glyl polypeptide sequence (SEQ ID NO: 14) mkasslafsilsaafyllwtpstglktlnlgscviatnlqeirngfseirgsvqakdgni dirilrrteslqdtkpanrccllrhllrlyldrvfknyqtpdhytlrkisslansfItik kdlrlchahmtchcgeeamnx(t/s) sqilshfeklepqaawkalgeldillqwmeete * Generic Gly2 polypeptide sequence (SEQ ID NO: 15) mkasslafsllsaafyllwtpstglktlnlgscviatnlqeirngfseirgsvqakdgni dirilrrteslqdtkpanrccllrhllrlyldrvfkn
  • Generic Gly3 polypeptide sequence (SEQ ID NO: 16) mkasslafsllsaafyllwtpstglktlnlgscviatnlqeirngfseirgsvqakdgni dirilrrteslqdtkpanrccllrhllrlyldrvf nx (t/s) pdhytlrkisslansf1 tikkdlrlchahmtchcgeeamkkysqilshfeklepqaawkalgeldillqwmeete*
  • Generic Gly4 polypeptide sequence (SEQ ID NO: 17) mkasslafs11saafyllwtpstglktlnlgscviatnlqeirngfseirgsvqakdgni nx(t/s) ilrrteslqdtkpanrccllrhllrlyldrvfknyqtpdhytlrkisslansf
  • Glyl plus Gly4 polypeptide sequence (SEQ ID NO: 18) mkasslafsilsaafyllwtpstglktlnlgscviatnlqeirngfseirgsvqakdgni nx(t/s) ilrrteslqdtkpanrccllrhllrlyldrvfknyqtpdhytlrkisslansf ltikkdlrlchahmtchcgeeamnx (t/s) sqilshfeklepqaavvkalgeldillqwm eete*
  • Glyl plus Gly3 polypeptide sequence (SEQ ID NO: 19) mkasslaf sllsaafyllwtpstglktlnlgscviatnlqeirngf seirgsvqakdgni dirilrrteslqdtkpanrccllrhllrlyldrvfknx (t/s) pdhytlrkisslansf 1 tikkdlrlchahmtchcgeeamnx ( t/s) sqilshf eklepqaavvkalgeldillqwme ete*
  • Gly3 plus Gly4 polypeptide sequence (SEQ ID NO: 20) mkasslafsilsaafyllwtpstglktlnlgscviatnlqeirngfseirgsvqakdgni nx(t/s) ilrrteslqdtkpanrccllrhllrlyldrvf nx(t/s)pdhytlrkissla nsfltikkdlrlchahmtchcgeeamkkysqilshfeklepqaavvkalgeldillq me ete*
  • IL22R1 (SEQ ID NO: 2) mrt11tiItvgslaahapedpsdllqhvkfqssnfeniltwdsgpegtpdtvysieykty gerdwvakkgcqritrkscnltvetgnltelyyarvtavsaggrsatkmtdrfsslqhtt lkppdvtciskvrsiqmivhptptpiragdghrltledifhdlfyhlelqvnrtyqmhlg gkqreyeffgltpdteflgtimicvptwakesapymcrvktdrtwtysfsgafIfsmgf1 vavlcylsyryvtkppappnslnvqrvltfqplrfiqehvlipv
  • mutant polynucleotide sequences were verified and then transferred from the pGEM vector to an expression vector.
  • Any expression vector containing a promoter sequence functional in mammalian cells, yeast, or baculovirus could be used to express the glycosylated proteins of the present invention.
  • 293 EBNA cells (Invitrogen) were grown in a T75 cm 2 flask in DMEM/F12 supplemented with 10% (v/v) fetal bovine serum (FBS) 37°C. After PBS washing, the cells were trypsinized and resuspended to a concentration of 0.5 x 10 6 cells/ml.
  • FBS fetal bovine serum
  • HEK293EBNA cells adapted to suspension culture in animal protein-free medium (AFPM), are grown in spinner flasks.
  • Western blot analysis was performed on media from transiently transfected cells described in Example 2 to detect secreted IL-20 wild-type and glycosylation-mutant polypeptides. 100 ⁇ l of media was removed from the transfected cells and added to a tube containing 100 ⁇ l of 2X sample buffer (Novex, Tris-glycine SDS sample buffer). Samples were heated at 100°C, 3 min., and loaded onto a Tris-glycine SDS gel.
  • 2X sample buffer Novex, Tris-glycine SDS sample buffer
  • Electrophoresis was performed and the protein transferred onto 0.2 ⁇ m nitrocellulose.
  • Membranes were rinsed in PBS supplemented with 0.1 % v/v Tween-20 (PBST) prior to blocking for 16 hour at 4°C in PBS/5 % milk.
  • PBST 0.1 % v/v Tween-20
  • Membranes were incubated with the primary antibody that reacts to the FLAG affinity tag that was fused to the carboxy terminus of the IL-20 protein, anti-FLAG M2-HRP fused antibody (Sigma, St. Louis,
  • glycosylation of the Glyl -4 proteins was confirmed by observing an upward size shift of the protein band on the western blot along with the change of a previously tight band (i.e. wild-type IL-20) into a fuzzy band (i.e. the Glyl -4 mutants).
  • This fuzziness is a classic sign of protein glycosylation indicating a heterologously sized product due to the various numbers of sugar moieties added.
  • Glycosylation of Gly4 was further confirmed by mass spec analysis.
  • Cell culture media containing the polypeptide of interest is concentrated in an Amicon ProFlux M12 tangential filtration system using an Amicon S3Y10 UF membrane.
  • the concentrated media is passed over an immobilized metal-affinity chromatography column (Pharmacia) at a flow rate of 2 ml/min.
  • the column is washed with buffer A
  • This material is passed over a Superdex 75 (Pharmacia, 26/60) sizing column equilibrated with PBS, 0.5 M NaCl, pH 7.4, at a flow rate of 3 ml/min. Fractions containing the polypeptide of interest are analyzed by SDS-PAGE.
  • the Gly4 IL-20 glycosylation mutant shows preferential receptor specificity.
  • the resulting mutant acts more like IL-19 in that it signals better through the IL20R1/ IL20R2 receptor complex than through the
  • IL20R2/IL22R1 receptor complex Table 1 IL20R1/TL20R2 cell line
  • Transcriptional assays were conducted by transient transfection of 293 EBNA cell lines (Edge Biosystems) using a STAT3-luciferase reporter plasmid (Clontech). This assay works by expression of IL-20 or a mutant form of IL-20 which, if it binds to the IL- 20 receptor also being expressed in the cells, signals through the Stat3 pathway and upregulates expression of luciferase.
  • the cells were plated at -50-60% confluency 24 hours pre-transfection in poly-lysine coated 96 well plates with DMEM-F12/10% FBS (Gibco).
  • the DNA-liposome complexes were created in OptiMEM (Gibco) media using Lipofectamine 2000 (Gibco) according to manufacturer's instructions and applied to the cells in a 100 ⁇ l volume in the absence of serum.
  • Two subunits of the receptor (IL20R1 and IL20R2 or IL22R1 and IL20R2 were also co-transfected into the cells at equal DNA concentrations (lOng each) and the STAT-luciferase plasmid was present in the transfection solution at lOOng.
  • a DNA construct for a ligand protein (IL-20, IL-19, Glyl, Gly2, Gly3 or Gly 4) was added to the transfection solution at lng per well along with the other DNAs.
  • a transfection DNA solution would have four DNAs: (1) the STAT- luciferase plasmid, (2 and 3) two expression vectors, one for each of the two IL-20 receptor subunits to be studied, and (4) an expression vector capable of expressing a ligand protein of interest.
  • the ligand genes used for this experiment were FLIS-tagged. The stimulation was stopped on day three by aspirating the media and adding 75 ⁇ l of Glo lysis buffer (Promega). Cells were lysed at room temperature for five minutes before 70 ⁇ l of each sample was transferred to a white plate.
  • the plate had 70 ⁇ l of BRIGHT-GLOTM reagent added to each well and the two volumes were mixed by pipet and allowed to incubate about 5 minutes before being read on a Luminoskan luminometer. Samples were read at normal gain for 10 seconds/well. All data points are done in 4-8 replicates, the average is reported hereinbelow.
  • Table 4 demonstrates that the Glyl, Gly3 and Gly4 mutant IL-20s preferentially signal through the IL20R1/IL20R2 receptor complex as does IL- 19.
  • JX22R1/IL20R2 none 25.26 JX22R1/IL20R2 IL-20 284.63
  • the vector pC4 is one exemplary vector used for the expression of a polypeptide of interest in CHO cells.
  • Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).
  • the plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
  • CHO cells or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (aha minus MEM, Invitrogen) supplemented with methofrexate.
  • amplification of the DHFR genes in cells resistant to methofrexate (MTX) has been well documented (see, e.g., J. L.
  • Plasmid pC4 contains the LTR strong promoter of the Rous Sarcoma Virus (Cullen, et al, Molec. Cell. Biol. 5:438, 1985) plus a fragment isolated from the enhancer of the immediate early gene of human CMV (Boshart, et al, Cejl 41:521, 1985).
  • plasmid Downstream of the promoter are restriction enzyme cleavage sites that allow insertion of the genes. Downstream of these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene.
  • Other high efficiency promoters can also be used for expression, (e.g., human b-actin promoter, SV40 early or late promoters, or the LTR from other retroviruses).
  • Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the polypeptide of interest in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547, 1992).
  • polyadenylation signals e.g., from the human growth hormone or globin genes
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co- transfection with a gene expressing a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methofrexate.
  • Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for transfection.
  • Five micrograms of the expression plasmid containing the gene of interest is cotransfected with 0.5 ⁇ g of the plasmid pSV2-neo using e.g., lipofectin.
  • the plasmid pSV2neo contains a dominant selectable marker, the neomycin resistance gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in aha minus MEM supplemented with 1 ⁇ g/ml G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in aha minus MEM supplemented with 10, 25, or 50 ng ml of methofrexate plus 1 ⁇ g/ml G418.
  • hybridoma cloning plates Gibco-Fi Protected Eagle's Cell
  • clonal colonies are independently trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methofrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).
  • Clones growing at the highest concentrations of methofrexate are then independently transferred to a new well of a 6-well plate containing even higher concentrations of methofrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 mM methofrexate.
  • Expression of the desired gene product is analyzed, for instance, by SDS-PAGE, Western blot or by reverse phase HPLC analysis.
  • Polypeptides can be assayed for their ability to stimulate development of human megakaryocytes from CD34+ progenitor cells.
  • CD34+ selected cells are obtained from bone marrow as described (Hokom, M. et al , Molecular Biology of Haematopoiesis 3:15, 1994) and incubated in Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island, NY) with 2 mM Glutamine, 2-mercaptoethanol (10 "4 M), 1% bovine serum albumin, low density lipoprotein (40 ⁇ g/ml, Sigma); bovine pancreatic insulin (10 ⁇ g /ml), human transferrin (200 ⁇ g /ml), human recombinant thrombopoietin (50 ng/ml, R&D System); human recombinant stem cell factor (50 ng/ml, R&D Systems) and plus or minus isolated polypeptide of interest at various concentrations (typically about 200
  • CD34+ cells are plated at 3300 cells/ml final concentrations on 2 well chamber slides purchased from StemCell Technologies (Vancouver, Canada). Cells are incubated at 37°C for 12 days in humidified boxes in 5% C0 2 in air. The cells are then fixed directly to the culture wells with 1:3 methanol: acetone solution, and incubated with a monoclonal antibody, anti-GPIIb/IQa, (StemCell Technologies). The immune reaction is developed with biotin-conjugated goat-anti-mouse Ig G followed by avidin-alkaline phosphatase conjugate, identified by pink color, and counted with an inverted-phase microscope at 100X magnification.
  • CD34 + human bone marrow cells are plated in polypropylene V- bottomed 96 well plates at 10,000 cells/well with three wells/group.
  • Stem Cell Factor (SCF) is used at 10 ng/ml
  • interleukin-3 (IL-3) is used at 0.1 ng/ml
  • erythropoietin (EPO) is used at 1 U/ml and the polypeptide of interest is used at various concentrations, (typically about 400 ng/ml).
  • cytokine conditions are tested in TMDM 30% FBS + antibiotics: ( 1 ) SCF/IL-3/( ⁇ polypeptide of interest), (2) IL-3/EPO/( ⁇ polypeptide of interest); (3) SCF/IL-3/EPO/( ⁇ polypeptide of interest); (4) SCF/TL-3/macrophage colony stimulating factor; and (5) SCF/jX-3/EPO/TGF ⁇ .
  • Sample 1 SCF + IL-3 is a negative control with minimal growth expected in the absence of an additional factor (e.g., IL-20).
  • Sample 2 IL-3 + EPO is a negative control with minimal growth expected in the absence of an additional growth factor.
  • Sample 3 SCF + IL-3 + EPO is expected to produce strong erythroid growth and/or differentiation in the absence of an additional factor and also demonstrates the amount of erythroid growth and/or differentiation in excess of that observed when SCF + IL-3 or IL-3 + EPO are used in the absence of the third factor (plus or minus IL-20).
  • Sample 4 SCF + IL-3 + MCSF is used to demonstrate detectable monocytic growth and/or differentiation in comparison to using SCF + IL-3 in the absence of MCSF (plus or minus IL-20).
  • Sample 5 SCF + IL-3 + EPO + TGF ⁇ is used to demonstrate modulation of erythroid growth and/or differentiation in comparision to using SCF + IL-3 + EPO in the absence of TGF ⁇ (plus or minus IL-20).
  • Cultures are incubated at 37°C, 5% C0 2 , 95% humidity for 10 days with a breathable sealing membrane to prevent evaporation. Feeding occurs at days 4 and 7 by replacing 400 ⁇ l of the medium with fresh medium. At day 10 the cells are transferred to V-bottomed plates and stained for CD14 (FITC) and CD36 (PE) cell surface antigens. The cells are centrifuged and incubated 15 minutes at 4°C with 50 ⁇ g/ml human IgG (Sigma). Monocytes are CD14+, cells of the erythrocyte lineage are CD36+, cells that are negative for both CD 14 and CD36 are termed "undefined" and may subsequently differentiate into monocytes or erythrocytes.
  • FITC CD14
  • PE CD36
  • CFU-GEMM CD34 + cells are seeded into methylcellulose culture or Agar culture medium (Stem Cell Technologies) using standard procedures. Colony growth is stimulated with the following combinations of recombinant growth factors with and without polypeptide of interest (at about 200 ng/ml, or various test concentrations): (1) Epo (2 U/ml) plus SCF (50 ng/ml) and (2) Epo + SCF + IL-3 (10 ng/ml). All the commercially available cytokines are available from R&D Systems (Minneapolis, MN). After culture at 37°C for 2 weeks, the different types of colonies are counted from each dish under an inverted microscope. CFU-GEMM ultimately differentiate into red blood cells, granulocytes, monocytes, and platelets.
  • Example 8 Additional in vivo Testing in Normal Mice for Hematopietic Modulators a. Assay for Recovery of Blood Cells after Bone Marrow Transplantation.
  • Bone marrow (BM) is harvested by gentle flushing the hind limbs of normal 8- to 10-week-old Balb C mice (purchased from Harlan Sprague Dawley, Lndianapoils, IN) using RPMI medium containing 10% fetal calf serum.
  • donor mice are pretreated with 5-fluorouracil (5-FU) at 150-mg/kg-body weight intraperitoneally (IP) 3 days before harvesting BM for infusion.
  • IP intraperitoneally
  • 1 X 10 6 bone marrow cells are injected intravenously (IV) into lethally irradiated mice.
  • IL-20 (250 ⁇ g/kg body weight) is diluted in PBS and injected subcutaneously in 0.2-ml volume daily starting on the same day as irradiation and infusion of donor bone marrow cells. Control mice receive the same volume of PBS. Administration of polypeptides Glyl, Gly2, Gly3 or Gly4 occurs during days 0-17. Mice are weighed every 4 days during the post-transplantation period. Hematologic analysis of leukocyte cell counts and platelet counts are performed on orbit bleeds on a CDC HemavetTM machine. Blood smears are stained with Wright-Giemsa using standard methods and examined at 100X for differentiation analysis. Hematocrits are performed by spinning capillary tubes for 5 minutes in a Model MB Micro-Capillary Centrifuge.
  • mice Eight- to ten-week old BDF1 mice (Harlan Sprague Dawley) are administered carboplatin at 60-mg/kg body weights intraperitoneally (IP) 1 hour before sub-lethal irradiation (0.5 Gy total body irradiation for 20-22 mg mouse).
  • a polypeptide of interest e.g., Glyl, Gly2, Gly3 or Gly4 (with or without EPO or G-CSF) is injected subcutaneously in a 0.2 ml volume daily starting on the same day as irradiation.
  • Negative control mice receive the same volume of PBS as the treated mice. Polypeptide administration lasts for 17 days. The mice are analyzed at various times post-radiation.
  • mice are weighed every 2 to 4 days during the post-radiation period. Hematologic analysis of leukocyte cell counts and platelet counts are performed on orbit bleeds on a CDC Hemave TM machine. Blood smears are stained with Wright-Giemsa using standard methods and examine at 100X for differentiation analysis. Hematocrit measurements are performed by spinning capillary tubes for 5 minutes in a Model MB Micro-Capillary Centrifuge. Glyl, Gly2, Gly3 and/or Gly4 polypeptides may be useful in accelerating recovery of peripheral blood cell counts after chemotherapy and/or radiation therapy.
  • the exhypoxic polycythemic mouse bioassay may be used to quantify the inco ⁇ oration of 5' Fe (iron) into newly synthesized red blood cells as a measure of the increase in erythropoiesis in mice in response to an exogenously administered test sample.
  • the assay as described in WO/0024893, is a modification of the method of Cotes and Bangham (Nature 191:1065 (1961)).
  • test agent(s) may be administered by any of several routes of administration (e.g. i.v., s.c, i.p., or by minipump or cannula) and suitable test animals include normal mice as well as transgenic mice similar to those described in Example 9. Controls for non-specific effects for these treatments are done using vehicle with or without the active agent of similar composition in the same type animal monitoring the same parameters.
  • routes of administration e.g. i.v., s.c, i.p., or by minipump or cannula
  • suitable test animals include normal mice as well as transgenic mice similar to those described in Example 9. Controls for non-specific effects for these treatments are done using vehicle with or without the active agent of similar composition in the same type animal monitoring the same parameters.
  • Example 9 Transgenic animal development Transgenic mice with the gene encoding Glyl , Gly2, Gly3 or Gly4 or other variants thereof, are generated using established techniques (Hogan, B. et al. (1986) Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory, NY as modified by Fox and Solter (Mol. Cell. Biol. 8: 5470, 1988). Briefly, a DNA fragment encompassing the human apolipoprotein E (hApoE) gene promoter-5'hApoE untranslated region-polypeptide of interest (Glyl-4)/FLAG-hepatic control region (HCR) fusion gene is purified by gel electrophoresis and glass bead extraction.
  • hApoE human apolipoprotein E
  • HCR FLAG-hepatic control region
  • the purified DNA fragment is microinjected into the male pronuclei of newly fertilized one-cell-stage embryos (zygotes) of the FVB/N strain.
  • the embryos are cultured in vitro overnight to allow development to the two-cell-stage.
  • Two-cell embryos are then transplanted into the oviducts of pseudopregnant ICR strain mice to allow development to term.
  • a small piece of toe is removed from each animal and digested with proteinase K to release the nucleic acids.
  • a sample of the toe extract is subjected to PCR analysis using primers specific for the hApoE untranslated region to identify transgene-containing mice. Five founder transgenic mice are identified.
  • mice are analyzed for the number CFU-GEMM in the spleen.
  • Mouse bone marrow cells and splenocytes are isolated from Glyl, Gly2, Gly3 and Gly4 (or variants thereof) transgenic mice and age-matched wild type mice. Then, 1 X 10 5 mononuclear cells from the bone marrow or 1 X 10 6 mononuclear cells from the spleen of each mouse are cultured in methylcellulose (Stem Cell Technologies) in the presence of 0.1 mM hemin using standard protocol known in the art.
  • methylcellulose StemM hemin
  • Colony growth is stimulated with the following combinations of recombinant growth factors: Medium A: Epo (1 U/ml, R&D System) plus SCF (50 ng/ml, R&D System) and PWM-SCM (conditioned medium 5%, Stem Cell Technologies) or Medium B: Epo 1 U/ml. After culturing the cells at 37°C for seven days, the different types of colonies are counted from each dish under an inverted microscope. Group mean and SD are calculated. b. HCT Recovery in Transgenic Mice
  • mice Ten to twelve week-old transgenic mice and age matched, wild type mice are exposed to myelosuppresive therapy of 400 rads total body irradiation followed by a single intraperitoneal injection of 0.8 mg carboplatin as described by Kaushansky, et al. (J. Clin. Invest. 96:1883, 1996).
  • recombinant human Epo is injected 20 IU daily s.c. for 12 days. Blood counts are performed on 50 ⁇ l samples obtained by retro-orbital route, using a Hemavet 1500 hematology analyzer (CDC Technologies).

Abstract

L'invention concerne des polypeptides IL-20 modifiés par glycosylation qui effectuent des signalisations, de préférence, à travers l'un des multiples complexes du récepteur IL-20 et qui maintiennent au moins une activité associée à IL-20 de type sauvage tout en diminuant ou éliminant au moins une autre activité associée à IL-20 de type sauvage. Les sites consensus de glycosylation à liaison N sont modifiés en IL-20 au niveau d'un ou de plusieurs sites qui correspondent à un site consensus de glycosylation à liaison N qui existe dans IL-19 ou IL-24 de type sauvage. L'invention concerne également des polynucléotides codant pour les polypeptides selon l'invention, des vecteurs et des cellules hôtes comprenant un polynucléotide selon l'invention ainsi que des méthodes thérapeutiques permettant d'utiliser les polypeptides et les polynucléotides selon l'invention.
PCT/US2003/023265 2002-08-27 2003-08-14 Il-20 modifiés par glycosylation WO2004020578A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005014028A1 (fr) * 2003-08-08 2005-02-17 Novo Nordisk A/S Interleukine-20 dans le traitement et le diagnostic des etats associes a la neovascularisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BLUMBERG H. ET AL: 'Interleukin 20: Discovery, Receptor Identification and Role in Epidermal Function' CELL vol. 104, 12 January 2001, pages 9 - 19, XP002210052 *
PARRISH-NOVAK J. ET AL: 'Interleukin 19,20 and 24 Signal through Two Distinct Receptor Complexes' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 277, no. 49, 06 December 2002, pages 47517 - 47523, XP008047447 *
RICH B.E.: 'IL-20: A New Target for the Treatment of Inflammatory Skin Disease' EXPERT OPINION THERAPY TARGETS vol. 7, no. 2, 2003, pages 165 - 174, XP009038159 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005014028A1 (fr) * 2003-08-08 2005-02-17 Novo Nordisk A/S Interleukine-20 dans le traitement et le diagnostic des etats associes a la neovascularisation

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