(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2006/0228329 A1
`(43) Pub. Date:
`Oct. 12, 2006
`Brady et al.
`
`US 20060228329Al
`
`(54) HOMOGENEOUS PREPARATIONS OF IL-31
`
`Publication Classification
`
`(76)
`
`Inventors: Lowell J. Brady, Tacoma, WA (US);
`Thomas R. Bukowski, Seattle, WA
`(US); Chung-leung Chan, lssaquah,
`WA (US)
`
`Correspondence Address:
`ZymoGenetics, Inc.
`1201 Eastlake Avenue East
`Seattle, WA 98102 (US)
`
`(21) Appl. No.:
`
`11/344,451
`
`(22)
`
`Filed:
`
`Jan. 30, 2006
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/648,189, filed on Jan.
`28, 2005.
`
`(51)
`
`Int. Cl.
`(2006.01)
`C07K 14/52
`(2006.01)
`A61K 38/20
`(2006.01)
`A61K 39/395
`(2006.01)
`C07H 21/04
`(2006.01)
`C12P 21/02
`(2006.01)
`C12N 1/21
`(52) U.S.Cl.
`................. ..424/85.1;530/351;530/388.23;
`435/69.5; 435/320.1; 435/252.33;
`536/235; 424/145.1
`
`(57)
`
`ABSTRACT
`
`Homogeneous preparations of human and murine IL-31
`have been produced by mutating one or more of the cysteine
`residues in the polynucleotide sequences encoding the
`mature proteins. The cysteine mutant proteins can be shown
`to either bind to their cognate receptor or exhibit biological
`activity.
`
`APOTEX EX1005
`
`Page 1
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`APOTEX EX1005
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`

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`US 2006/0228329 A1
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`Oct. 12, 2006
`
`HOMOGENEOUS PREPARATIONS OF IL-31
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application claims the benefit of U.S. Provi-
`sional Application Ser. No. 60/648,189, filed Jan. 28, 2005,
`which is herein incorporated by reference.
`
`BACKGROUND OF THE INVENTION
`
`[0002] The increased availability and identification of
`genes from human and other genomes has led to an
`increased need for efficient expression and purification of
`recombinant proteins. The expression of proteins in bacteria
`is by far the most widely used approach for the production
`of cloned genes. For many reasons, expression in bacteria is
`preferred to expression in eukaryotic cells. For example,
`bacteria are much easier to grow than eukaryotic cells. More
`specifically,
`the availability of a wealth of sophisticated
`molecular genetic tools and thousands of mutants make E.
`coli, as an expression host, extremely useful for protein
`production. However,
`the high-level production of func-
`tional proteins in E. coli., especially those from eukaryotic
`sources has often been difficult.
`
`IL-31 is a recently discovered protein having the
`[0003]
`structure of a four-helical-bundle cytokine. This new cytok-
`ine is fully described in co-owned PCT application WO
`03/060090 and Dillon, et al., Naulre Immunol. 5:752-760,
`2004; both incorporated by reference herein. IL-31 is a
`ligand with high specificity for the receptor IL-31RA and at
`least one additional subunit comprising OncostatinM recep-
`tor beta. IL-31 was isolated from a cDNA library generated
`from activated human peripheral blood cells (hPBCs), which
`were selected for CD3. CD3 is a cell surface marker unique
`to cells of lymphoid origin, particularly T cells.
`
`[0004] Both the murine and human forms of IL-31 are
`known to have an odd number of cysteines. (PCT applica-
`tion WO 03/060090 and Dillon, et al., supra.) Expression of
`recombinant IL-31 can result in a heterologous mixture of
`proteins composed of intramolecular disulfide binding in
`multiple conformations. The separation of these forms can
`be difficult and laborious. It is therefore desirable to provide
`IL-31 molecules having a single intramolecular disulfide
`bonding pattern upon expression and methods for refolding
`and purifying these preparations to maintain homogeneity.
`Thus, the present invention provides for compositions and
`methods to produce homogeneous preparations of IL-31.
`
`[0005] Despite advances in the expression of recombinant
`proteins in bacterial hosts, there exists a need for improved
`methods for producing biologically active and purified
`recombinant IL-31 proteins in prokaryotic systems which
`result in higher yields for protein production. These and
`other aspects of the invention will become evident upon
`reference to the following detailed description. In addition,
`various references are identified below and are incorporated
`by reference in their entirety.
`
`[0006] The present invention provides such polypeptides
`for these and other uses that should be apparent to those
`skilled in the art from the teachings herein.
`
`SUMMARY OF THE INVENTION
`
`selected from the group consisting of SEQ ID NOs: 14, 15,
`16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.
`
`[0008] Within another aspect, the invention provides an
`expression vector comprising the following operably linked
`elements: a transcription promoter; a DNA segment encod-
`ing a polypeptide comprising an amino acid sequence
`selected from the group consisting of SEQ ID NOs: 14, 15,
`16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30; and
`a transcription terminator.
`
`the invention provides a
`[0009] Within another aspect,
`cultured cell into which has been introduced an expression
`vector comprising a DNA segment encoding a polypeptide
`comprising an amino acid sequence selected from the group
`consisting ofSEQ ID NOs:14,15,16,17,18,19,21,22,23,
`24, 25, 26, 27, 28, 29, and 30, wherein the cell expresses the
`polypeptide encoded by the DNA segment. Within an
`embodiment the cultured cell is a prokaryotic cell. Within
`another embodiment the cell is a gran1 negative cell. Within
`another embodiment
`the cell
`is E. coli. Within another
`embodiment, the E. coli cell is E. coli strain W3110.
`
`the invention provides a
`[0010] Within another aspect,
`process for producing a polypeptide comprising:
`
`culturing a cell into which has been introduced an
`[0011]
`expression vector comprising a DNA segment encoding a
`polypeptide comprising an amino acid sequence selected
`from the group consisting of SEQ ID NOs: 14, 15, 16, 17,
`18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, wherein the
`cell expresses the polypeptide encoded by the DNA seg-
`ment; and recovering the expressed polypeptide.
`
`[0012] Within another aspect, the invention provides an
`antibody or antibody fragment that specifically binds to a
`polypeptide comprising an amino acid sequence selected
`from the group consisting of SEQ ID NOs: 14, 15, 16, 17,
`18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. Within an
`embodiment the antibody is selected from the group con-
`sisting of a polyclonal antibody, a murine monoclonal anti-
`body, a humanized antibody derived from a murine mono-
`clonal
`antibody,
`an antibody fragment,
`neutralizing
`antibody, and a human monoclonal antibody. Within another
`embodiment the antibody fragment
`is selected from the
`group consisting of F(ab'), F(ab), Fab‘, Fab, Fv, scFv, and
`minimal recognition unit.
`
`[0013] Within another aspect is provided an anti-idiotype
`antibody comprising an anti-idiotype antibody that specifi-
`cally binds to the antibody.
`
`[0014] Within another aspect the invention provides an
`isolated polypeptide consisting of an amino acid sequence
`selected from the group consisting of SEQ ID NOs: 4, 15,
`16, 17, 18, 19, 21, 22, 23,24, 25, 26, 27,28, 29, and 30.
`
`[0015] Within another aspect is provided a formulation
`comprising:
`
`an isolated polypeptide selected from the group
`[0016]
`consisting of SEQ ID NOs: 4, 15, 16, 17, 18, 19, 21, 22, 23,
`24, 25, 26, 27, 28, 29, and 30; and
`
`a pharmaceutically acceptable vehicle. Within an
`[0017]
`embodiment, formulation is provide in a kit.
`
`[0007] Within one aspect, the invention provides an iso-
`lated polypeptide comprising an amino acid sequence
`
`[0018] Within another aspect, the polypeptide comprising
`an amino acid sequence selected from the group consisting
`
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`US 2006/0228329 A1
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`Oct. 12, 2006
`
`of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25,
`26, 27, 28, 29, and 30 is proinflamrnatory.
`
`[0019] Within another aspect the invention provides an
`isolated polypeptide comprising the amino acid sequence
`from residue 2 to residue 133 of SEQ ID NO: 23.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0020] Prior to setting forth the invention in detail, it may
`be helpful to the understanding thereof to define the follow-
`ing terms:
`
`[0021] The term “affinity tag” is used herein to denote a
`polypeptide segment
`that can be attached to a second
`polypeptide to provide for purification or detection of the
`second polypeptide or provide sites for attachment of the
`second polypeptide to a substrate. In principal, any peptide
`or protein for which an antibody or other specific binding
`agent is available can be used as an affinity tag. Affinity tags
`include a poly-histidine tract, protein A (Nilsson et al.,
`EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol.
`198:3, 1991), glutathione S transferase (Smith and Johnson,
`Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et
`al., Proc. Natl. Acad. Sci. USA 82:79524, 1985), substance
`P, FlagTM peptide (Hopp et al., Biotechnology 6:1204-10,
`1988),
`streptavidin binding peptide, or other antigenic
`epitope or binding domain. See,
`in general, Ford et al.,
`Protein Expression and Purification 2: 95-107, 1991. DNAs
`encoding affinity tags are available from commercial sup-
`pliers (e.g., Pharmacia Biotech, Piscataway, N.J.).
`
`[0022] The term “allelic variant” is used herein to denote
`any of two or more alternative forms of a gene occupying the
`same chromosomal locus. Allelic variation arises naturally
`through mutation, and may result in phenotypic polymor-
`phism within populations. Gene mutations can be silent (no
`change in the encoded polypeptide) or may encode polypep-
`tides having altered amino acid sequence. The term allelic
`variant is also used herein to denote a protein encoded by an
`allelic variant of a gene.
`
`[0023] The terms “amino-terminal” and “carboxyl-termi-
`nal” are used herein to denote positions within polypeptides.
`Where the context allows, these terms are used with refer-
`ence to a particular sequence or portion of a polypeptide to
`denote proximity or relative position. For example, a certain
`sequence positioned carboxyl-terminal
`to a
`reference
`sequence within a polypeptide is located proximal to the
`carboxyl terminus of the reference sequence, but is not
`necessarily at
`the carboxyl
`terminus of the complete
`polypeptide.
`
`[0024] The term “complement/anti-complement pair”
`denotes non-identical moieties that form a noncovalently
`associated, stable pair under appropriate conditions. For
`instance, biotin and avidin (or streptavidin) are prototypical
`members of a complement/anti-complement pair. Other
`exemplary
`complement/anti-complement pairs
`include
`receptor/ligand pairs,
`antibody/antigen (or hapten or
`epitope) pairs, sense/antisense polynucleotide pairs, and the
`like. Where subsequent dissociation of the complement/anti-
`complement pair is desirable, the complement/anti-comple-
`ment pair preferably has a binding afiinity of <109 M-1.
`
`mentary base sequence and reverse orientation as compared
`to a reference sequence. For example,
`the sequence 5'
`ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
`
`[0026] The term “contig” denotes a polynucleotide that
`has a contiguous stretch of identical or complementary
`sequence to another polynucleotide. Contiguous sequences
`are said to “overlap” a given stretch of polynucleotide
`sequence either in their entirety or along a partial stretch of
`the polynucleotide. For example, representative contigs to
`the polynucleotide sequence 5'-ATGGCTTAGCTT-3' are
`5'-TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5'.
`
`sequence”
`term “degenerate nucleotide
`[0027] The
`denotes a sequence of nucleotides that includes one or more
`degenerate codons (as compared to a reference polynucle-
`otide molecule that encodes a polypeptide). Degenerate
`codons contain different triplets of nucleotides, but encode
`the same amino acid residue (i.e., GAU and GAC triplets
`each encode Asp).
`
`[0028] The term “expression vector” is used to denote a
`DNA molecule, linear or circular, that comprises a segment
`encoding a polypeptide of interest operably linked to addi-
`tional segments that provide for its transcription. Such
`additional
`segments
`include promoter and terminator
`sequences, and may also include one or more origins of
`replication, one or more selectable markers, an enhancer, a
`polyadenylation signal, etc. Expression vectors are generally
`derived from plasmid or viral DNA, or may contain ele-
`ments of both.
`
`[0029] The term “isolated”, when applied to a polynucle-
`otide, denotes that the polynucleotide has been removed
`from its natural genetic milieu and is thus free of other
`extraneous or unwanted coding sequences, and is in a form
`suitable for use within genetically engineered protein pro-
`duction systems. Such isolated molecules are those that are
`separated from their natural environment and include cDNA
`and genomic clones. Isolated DNA molecules of the present
`invention are free of other genes with which they are
`ordinarily associated, but may include naturally occurring 5'
`and 3' untranslated regions such as promoters and termina-
`tors. The identification of associated regions will be evident
`to one of ordinary skill in the art (see for example, Dynan
`and Tijan, Nature 316:774-78, 1985).
`
`[0030] An “isolated” polypeptide or protein is a polypep-
`tide or protein that is found in a condition other than its
`native environment, such as apart from blood and animal
`tissue.
`In a preferred form,
`the isolated polypeptide is
`substantially free of other polypeptides, particularly other
`polypeptides of animal origin. It is preferred to provide the
`polypeptides in a highly purified form, i.e., greater than 95%
`pure, more preferably greater than 99% pure. When used in
`this context, the term “isolated” does not exclude the pres-
`ence of the same polypeptide in alternative physical forms,
`such as dimers or alternatively glycosylated or derivatized
`forms.
`
`[0031] The term “neoplastic”, when referring to cells,
`indicates cells undergoing new and abnormal proliferation,
`particularly in a tissue where in the proliferation is uncon-
`trolled and progressive, resulting in a neoplasm. The neo-
`plastic cells can be either malignant,
`i.e.,
`invasive-and
`metastatic, or benign.
`
`[0025] The term “complements of a polynucleotide mol-
`ecule” denotes a polynucleotide molecule having a comple-
`
`[0032] The term “operably linked”, when referring to
`DNA segments, indicates that the segments are arranged so
`
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`US 2006/0228329 A1
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`
`that they function in concert for their intended purposes,
`e.g.,
`transcription initiates in the promoter and proceeds
`through the coding segment to the terminator.
`
`[0033] The term “ortholog” denotes a polypeptide or pro-
`tein obtained from one species that is the functional coun-
`terpart of a polypeptide or protein from a different species.
`Sequence differences among orthologs are the result of
`speciation.
`
`“Paralogs” are distinct but structurally related pro-
`[0034]
`teins made by an organism. Paralogs are believed to arise
`through gene duplication. For example, or-globin, [3-globin,
`and myoglobin are paralogs of each other.
`
`[0035] A “polynucleotide” is a single- or double-stranded
`polymer of deoxyribonucleotide or ribonucleotide bases
`read from the 5' to the 3' end. Polynucleotides include RNA
`and DNA, and may be isolated from natural sources, syn-
`thesized in vitro, or prepared from a combination of natural
`and synthetic molecules. Sizes of polynucleotides are
`expressed as base pairs (abbreviated “bp”), nucleotides
`(“nt”), or kilobases (“kb”). Where the context allows, the
`latter two terms may describe polynucleotides that are
`single-stranded or double-stranded. When the term is
`applied to double-stranded molecules it is used to denote
`overall length and will be understood to be equivalent to the
`term “base pairs”. It will be recognized by those skilled in
`the art that the two strands of a double-stranded polynucle-
`otide may differ slightly in length and that the ends thereof
`may be staggered as a result of enzymatic cleavage; thus all
`nucleotides within a double-stranded polynucleotide mol-
`ecule may not be paired.
`
`[0036] A “polypeptide” is a polymer of amino acid resi-
`dues joined by peptide bonds, whether produced naturally or
`synthetically. Polypeptides of less than about 10 amino acid
`residues are commonly referred to as “peptides”.
`
`[0037] The term “promoter” is used herein for its art-
`recognized meaning to denote a portion of a gene containing
`DNA sequences that provide for the binding of RNA poly-
`merase and initiation of transcription. Promoter sequences
`are commonly, but not always, found in the 5' non-coding
`regions of genes.
`
`[0038] A “protein” is a macromolecule comprising one or
`more polypeptide chains. A protein may also comprise
`non-peptidic components, such as carbohydrate groups. Car-
`bohydrates and other non-peptidic substituents may be
`added to a protein by the cell
`in which the protein is
`produced, and will vary with the type of cell. Proteins are
`defined herein in terms of their amino acid backbone struc-
`
`tures; substituents such as carbohydrate groups are generally
`not specified, but may be present nonetheless.
`
`[0039] The term “receptor” denotes a cell-associated pro-
`tein that binds to a bioactive molecule (i.e., a ligand) and
`mediates the effect of the ligand on the cell. Membrane-
`bound receptors are characterized by a multi-peptide struc-
`ture comprising an extracellular ligand-binding domain and
`an intracellular effector domain that is typically involved in
`signal transduction. Binding of ligand to receptor results in
`a conformational change in the receptor that causes an
`interaction between the effector domain and other mol-
`
`ecule(s) in the cell. This interaction in turn leads to an
`alteration in the metabolism of the cell. Metabolic events
`
`that are linked to receptor-ligand interactions include gene
`
`dephosphorylation,
`phosphorylation,
`transcription,
`increases in cyclic AMP production, mobilization of cellular
`calcium, mobilization of membrane lipids, cell adhesion,
`hydrolysis of inositol lipids and hydrolysis of phospholipids.
`In general, receptors can be membrane bound, cytosolic or
`nuclear; monomeric (e.g.,
`thyroid stimulating hormone
`receptor, beta-adrenergic receptor) or multimeric (e.g.,
`PDGF receptor, growth hormone receptor, IL-3 receptor,
`GM-CSF receptor, G-CSF receptor, erythropoietin receptor
`and IL-6 receptor).
`
`[0040] The term “secretory signal sequence” denotes a
`DNA sequence that encodes a polypeptide (a “secretory
`peptide”) that, as a component of a larger polypeptide,
`directs the larger polypeptide through a secretory pathway of
`a cell in which it is synthesized. The larger polypeptide is
`commonly cleaved to remove the secretory peptide during
`transit through the secretory pathway.
`
`[0041] The term “splice variant” is used herein to denote
`alternative forms of RNA transcribed from a gene. Splice
`variation arises naturally through use of alternative splicing
`sites within a transcribed RNA molecule, or less commonly
`between separately transcribed RNA molecules, and may
`result in several mRNAs transcribed from the same gene.
`Splice variants may encode polypeptides having altered
`amino acid sequence. The term splice variant is also used
`herein to denote a protein encoded by a splice variant of an
`mRNA transcribed from a gene.
`
`[0042] Molecular weights and lengths of polymers deter-
`mined by imprecise analytical methods (e.g., gel electro-
`phoresis) will be understood to be approximate values.
`When such a value is expressed as “about” X or “approxi-
`mately” X, the stated value of X will be understood to be
`accurate to 110%.
`
`[0043] All references cited herein are incorporated by
`reference in their entirety.
`
`[0044] The present invention provides expression vectors
`and methods for producing recombinant IL-31 protein from
`a prokaryotic host and is based in part upon the discovery of
`compositions and methods to produce homogeneous prepa-
`rations of IL-31. IL-31 is a recently discovered protein
`having the structure of a four-helical-bundle cytokine. This
`cytokine was previously identified as IL-31 and is fully
`described in co-assigned U.S. patent application Ser. No.
`10/352,554, filed Jan. 21, 2003. See published U.S. Patent
`Application No. 2003-0224487, and PCT application WO
`03/060090, both herein incorporated by reference. See also,
`Dillon, et al., Naulre Immunol. 5:752-760, 2004. IL-31 is a
`ligand with high specificity for the receptor IL-31RA and at
`least one additional subunit comprising OncostatinM recep-
`tor beta (OSMRbeta). The native polynucleotide and
`polypeptide sequences for human IL-31 are shown in SEQ
`ID NOs:
`1 and 2, respectively. SEQ ID NO:3 shows the
`degenerate polynucleotide for the polypeptide having the
`amino acid sequence as shown in SEQ ID NO:2. The native
`polynucleotide and polypeptide sequences for mouse IL-31
`are shown in SEQ ID NOs: 4 and 5, respectively. SEQ ID
`NO:6 shows the degenerate polynucleotide for the polypep-
`tide having the amino acid sequence as shown in SEQ ID
`NO:5. The native polynucleotide and polypeptide sequences
`for human IL-31RA are shown in SEQ ID NOs: 7 and 8,
`respectively. The native polynucleotide and polypeptide
`sequences for mouse HL-31RA are shown in SEQ ID NOs:
`
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`US 2006/0228329 A1
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`
`respectively. The native polynucleotide and
`9 and 10,
`polypeptide sequences for human OSMRbeta are shown in
`SEQ ID NOs: 11 and 12, respectively.
`
`[0045] Both the murine and human forms of IL-31 are
`known to have an odd number of cysteines. (PCT applica-
`tion WO 03/060090 and Dillon, et al., supra.) Expression of
`recombinant IL-31 can result in a heterologous mixture of
`proteins composed of intramolecular disulfide binding in
`multiple conformations. The separation of these forms can
`be difficult and laborious. It is therefore desirable to provide
`IL-31 molecules having a single intramolecular disulfide
`bonding pattern upon expression and methods for refolding
`and purifying these preparations to maintain homogeneity.
`
`In particular, the expression vectors and methods of
`[0046]
`the present invention comprise an E. coli expression system.
`Using the expression vectors described herein significantly
`improved the yield of recombinant protein recovered from
`the bacteria.
`
`invention provides polynucleotide
`[0047] The present
`molecules, including DNA and RNA molecules, that encode
`Cysteine mutants of IL-31 that result in expression of a
`recombinant IL-31 preparation that is a homogeneous prepa-
`ration. For the purposes of this invention, a homogeneous
`preparation of IL-31 is a preparation which comprises at
`least 98% of a single intramolecular disulfide bonding
`pattern in the purified polypeptide. In other embodiments,
`the single disulfide conformation in a preparation of purified
`polypeptide is at 99% homogeneous.
`In general,
`these
`Cysteine mutants will maintain some biological activity of
`the wildtype IL-31, as described herein. For example, the
`molecules of the present invention can bind to the IL-31
`receptor with some specificity. Generally, a ligand binding to
`its cognate receptor is specific when the KD falls within the
`range of 100 nM to 100 pM. Specific binding in the range
`of 100 mM to 10 nM KD is low affinity binding. Specific
`binding in the range of 2.5 pM to 100 pM KD is high affinity
`binding. In another example, biological activity of IL-31
`Cysteine mutants is present when the molecules are capable
`of some level of activity associated with wildtype IL-31 as
`described in detail herein.
`
`[0048] When referring to native IL-31, the term shall mean
`IL-31 and zcytor17lig. When referring to IL-31RA, the term
`shall mean IL-31RA and zcytor17.
`
`[0049] The present invention also provides methods for
`recovering recombinant IL-31 protein from a prokaryotic
`host when the IL-31 protein is expressed by the host and
`found within the host cell as an unglycosylated, insoluble
`inclusion body. When the prokaryotic cell is lysed to isolate
`the inclusion bodies (also called refractile bodies),
`the
`inclusion bodies are aggregates of IL-31. Therefore,
`the
`inclusion bodies must be disassociated and dissolved to
`
`isolate the IL-31 protein, and generally this requires the use
`of a denaturing chaotropic solvent, resulting in recovering a
`polypeptide that must be refolded to have significant bio-
`logical activity. Once the IL-31 protein is refolded,
`the
`protein must be captured and purified. Thus, the present
`invention provides for methods for isolating insoluble IL-31
`protein from prokaryotic cells, dissolving the insoluble
`IL-31 protein material in a chaotropic solvent, diluting the
`chaotropic solvent in such a manner that the IL-31 protein is
`refolded and isolated. The present invention also includes
`methods for capturing the renatured IL-31 from the dilute
`
`refold bulfer using cation exchange chromatography, and
`purifying the refolded IL-31 protein using hydrophobic
`interaction chromatography. Further purification is achieved
`using anion exchange in binding assays using an IL-31
`receptor and the like.
`
`[0050] The present invention provides mutations in the
`IL-31 wildtype sequences as shown in SEQ ID NOs: 1, 2, 3,
`4, 5, and 6, that result in expression of single forms of the
`IL-31 molecule. Because the heterogeneity of forms is
`believed to be a result of multiple intramolecular disulfide
`bonding patterns,
`specific embodiments of the present
`invention includes mutations to the cysteine residues within
`the wildtype IL-31 sequences. The mature human IL-31
`polypeptide is shown in SEQ ID NO:13, with SEQ ID
`NO:49 showing the mature human IL-31 polypeptide with a
`start methionine. Molecules of the mature human IL-31
`
`polypeptide can have disulfide bonds between the cysteine
`residue at position 46 and position 107 of SEQ ID NO:13,
`between position 46 and 121 of SEQ ID NO:13, and
`between position 107 and 121 of SEQ ID NO: 13. Amutation
`of any of these three cysteines results in a mutant form of the
`human IL-31 protein that will only form one disulfide bond.
`Thus a mutation at postion 46 will result in a protein that
`forms a disulfide bond between position 107 and position
`121 of SEQ ID NO: 13; a mutation at position 107 will result
`in a protein that forms a disulfide bond between position 46
`and position 121 of SEQ ID N013; and a mutation at
`position 121 will result in a protein that forms a disulfide
`bond between position 46 and position 107 of SEQ ID
`NO:13. The cysteines in these positions can be mutated, for
`example, to a serine, alanine, threonine, valine, or aspar-
`agine. For example, a human IL-31 protein having a muta-
`tion from cysteine to serine at position 46 of SEQ ID NO: 13
`is shown in SEQ ID NO: 14; a human IL-31 protein having
`a mutation from cysteine to serine at position 107 of SEQ ID
`NO:13 is shown in SEQ ID NO:15; a human IL-31 protein
`having a mutation from cysteine to serine at position 121 of
`SEQ ID NO:13 is shown in SEQ ID NO:16.
`
`[0051] When human IL-31 is expressed in E. coli, an
`N-terminal or amino-terminal Methionine is present. SEQ
`ID NOs: 17-19, for example, show the nucleotide and amino
`acid residue sequences for IL-31 when the N-terminal Met
`is present in these mutants.
`
`[0052] Similar mutations can be made to the mouse IL-31
`polypeptide sequence. The mature mouse IL-31 polypeptide
`is shown in SEQ ID NO:20. Molecules of the mature murine
`IL-31 polypeptide can have disulfide bonds between the
`cysteine residue at position 44 and position 87 of SEQ ID
`NO:20, between position 44 and 107 of SEQ ID NO:20,
`between position 44 and 121 of SEQ ID NO:20; between
`position 44 and 133 of SEQ ID NO:20; between position 87
`and 107of SEQ ID NO:20; between position 87 and 121 of
`SEQ ID NO:20; between position 87 and 133 of SEQ ID
`NO:20; between position 107 and 121 of SEQ ID NO:20;
`between position 107 and 133 of SEQ ID N020; and
`between position 121 and 133 of SEQ ID NO:20. Amutation
`of any of these cysteines results in a mutant form of the
`mouse IL-31 protein. The cysteines in these positions can be
`mutated, for example, to a serine, alanine, threonine, valine,
`or asparagine. For example, a mouse IL-31 protein having a
`mutation from cysteine to serine at position 44 of SEQ ID
`NO:20 is shown in SEQ ID NO:21; a mouse IL-31 protein
`having a mutation from cysteine to serine at position 87 of
`
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`US 2006/0228329 A1
`
`Oct. 12, 2006
`
`SEQ ID NO:20 is shown in SEQ ID NO:22; a mouse IL-31
`protein having a mutation from cysteine to serine at position
`107 of SEQ ID NO:20 is shown in SEQ ID NO:23; a mouse
`IL-31 protein having a mutation from cysteine to serine at
`position 121 of SEQ ID NO:20 is shown in SEQ ID NO:24;
`and a mouse IL-31 protein having a mutation from cysteine
`to serine at position 133 of SEQ ID NO:20 is shown in SEQ
`ID NO:25.
`
`[0053] When mouse IL-31 is expressed in E. coli, an
`N-terminal or amino-terminal Methionine is present. SEQ
`ID NOs:26-30, for example, show the nucleotide and amino
`acid residue sequences for IL-31 when the N-terminal Met
`is present in these mutants. When the mouse IL-31 Cys
`mutants of the present invention were made in E. coli with
`serine at position 107 of SEQ ID NO: 20,
`the purified
`N-terminus was determined to begin at the phenylalanine
`(Phe) instead of the alanine. Thus, one embodiment of the
`invention is the polypeptide comprising or consisting of the
`amino acid sequence from position 2 (Phe) to position 133
`(Cys) of SEQ ID NO: 20, or of SEQ ID NOs: 21-30.
`
`[0054] The polynucleotide and polypeptide molecules to
`the present invention have a mutation at one or more of the
`cysteines present in the native IL-31 molecules, yet retain
`some biological activity as described herein. When referring
`to the cysteine mutants of IL-31, the term shall mean any of
`the mutated forms of IL-31 desribed above, and shall
`include, for example, any of SEQ ID NOs: 14-19 or 21-30,
`generally referred to as IL-31Cys mutants.
`
`[0055] A cell line that is dependent on the OSMRbeta and
`zcytor17 receptor linked pathway for survival and growth in
`the absence of other growth factors can be used to measure
`the activity of i the IL-31 Cys mutants described herein. The
`preferred growth factor-dependent cell line that can be used
`for transfection and expression of IL-31 receptor is BaF3
`(Palacios and Steinmetz, Cell 41: 727-734, 1985; Mathey-
`Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986). However,
`other growth factor-dependent cell lines, such as FDC-P1
`(Hapel et al., Blood 64: 786-790, 1984), and MO7e (Kiss et
`al., Leukemia 7: 235-240, 1993) are suitable for this pur-
`pose.
`
`[0056] One of ordinary skill in the art will appreciate that
`different species can exhibit “preferential codon usage.” In
`general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912,
`1980; Haas, et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson,
`et al., Gene 13:355-64, 1981; Grosjean and Fiers, Gene
`18:199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87,
`1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As used
`herein, the term “preferential codon usage” or “preferential
`codons” is a term of art referring to protein translation
`codons that are most frequently used in cells of a certain
`species, thus favoring one or a few representatives of the
`possible codons encoding each amino acid. For example, the
`amino acid Threonine (Thr) may be encoded by ACA, ACC,
`ACG, or ACT, but in mammalian cells ACC is the most
`commonly used codon; in other species, for example, insect
`cells, yeast, viruses or bacteria, different Thr codons may be
`preferential. Preferential codons for a particular species can
`be introduced into the polynucleotides of the present inven-
`tion by a variety of methods known in the art. Introduction
`of preferential codon sequences into recombinant DNA can,
`for example, enhance production of the protein by making
`protein translation more efficient within a particular cell type
`
`or species. Therefore, the degenerate codon sequence dis-
`closed in SEQ ID NO:3 serves as a template for optimizing
`expression of polynucleotides in various cell
`types and
`species commonly used in the art and disclosed herein.
`Sequences containing preferential codons can be tested and
`optimized for expression in various species, and tested for
`functionality as disclosed herein.
`
`[0057] As previously noted, the isolated polynucleotides
`of the present invention include DNA and RNA. Methods
`for preparing DNA and RNA are well known in the art. In
`general, RNA is isolated from a tissue or cell that produces
`large amounts of IL-31 RNA. Such tissues and cells are
`identified by Northern blotting (Thomas, Proc. Natl. Acad.
`Sci. USA 77:5201, 1980), or by screening conditioned
`medium from various cell types for activity on target cells or
`tissue. Once the activity or RNA producing cell or tissue is
`identified, total RNA can be prepared using guanidinium
`isothiocyanate extraction followed by isolation by centrifu-
`gation in a CsCl gradient (Chirgwin et al.,