Soluble zalpha11 cytokine receptors

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DESCRIPTION

Soluble zalfall cytokine receptors.

Hormones and polypeptide growth factors control the proliferation and differentiation of cells of multicellular organisms. These diffusible molecules allow cells to communicate with each other and work together to form cells and organs, and to repair damaged tissue. Examples of hormones and growth factors include steroid hormones (eg, estrogen, testosterone), parathyroid hormone, follicle-stimulating hormone, interleukins, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.

Hormones and growth factors influence cell metabolism by binding to receptors. Receptors can be integral membrane proteins that bind to signaling pathways within the cell, such as second messenger systems. Other classes of receptors are soluble molecules, such as transcription factors. Of particular interest are cytokine receptors, molecules that promote cell proliferation and / or differentiation. Examples of cytokines include erythropoietin (EPO), which stimulates the development of red blood cells; thrombopoietin (TPO), which stimulates the growth of cells of the megakaryocyte lineage; and granulocyte colony stimulating factor (G-CSF), which stimulates neutrophil development. These cytokines are useful for restoring normal red blood cell levels in patients suffering from anemia, thrombocytopenia, and neutropenia or receiving chemotherapy for cancer.

Parrish J. et al., American J. of Human Genetics, Vol. 65, No. 4, October 19, 1999, page A378 describe the identification and characterization of the zalpha11 cytokine receptor.

WO 00/17235 describes the zalpha11 cytokine receptor, and related compositions and methods.

WO 00/08152 describes nucleic acids encoding orphan mammalian receptor polypeptides, designated OCR-10 and OCR-10A, and related assay systems and methods.

EP 1 088 831 describes hemopoietin receptor proteins.

The demonstrated in vitro activities of these cytokines illustrate the enormous clinical potential for, and the need for, other cytokines, cytokine agonists, and cytokine antagonists or binding partners.

The present invention addresses these needs by providing an isolated polynucleotide encoding a heterodimeric or multimeric receptor complex comprising soluble receptor subunits, wherein the receptor complex comprises a sequence of amino acid residues that is at least 90% identical to the sequence. of amino acids shown in SEQ ID NO: 6, and a soluble receptor II, -2Ry polypeptide (SEQ ID NO: 4).

Within one aspect, the present invention provides an isolated polynucleotide encoding a soluble receptor polypeptide comprising an amino acid residue sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 6, and in the that the soluble receptor polypeptide encoded by the polynucleotide sequence binds to a ligand comprising a polypeptide of SEQ ID NO: 10 or SEQ ID NO: 47, or antagonizes the activity of the ligand. In one embodiment, the isolated polynucleotide is as described above, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a homodimeric receptor complex.

Within another aspect, the present invention provides an isolated polynucleotide encoding a soluble receptor polypeptide comprising an amino acid residue sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 6, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a heterodimeric or multimeric receptor complex. In one embodiment, the isolated polynucleotide is as described above, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a heterodimeric or multimeric receptor complex that further comprises a soluble Class I cytokine receptor.

In one embodiment, the isolated polynucleotide is as described above, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a heterodimeric or multimeric receptor complex that further comprises a soluble IL-2Ry receptor polypeptide (SEQ ID NO: 4 ) or a soluble IL-13a 'receptor polypeptide (SEQ ID NO: 82). In another embodiment, the isolated polynucleotide is as described above, wherein the polypeptide further comprises a WSXWS motif as shown in SEQ ID NO: 13.

Within another aspect, the present invention provides an isolated polynucleotide encoding a soluble receptor polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 6, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a heterodimeric or multimeric receptor complex. In one embodiment, the isolated polynucleotide is as described above, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a heterodimeric or multimeric receptor complex that further comprises a soluble IL-2Ry receptor polypeptide (SEQ ID NO: 4 ) or a soluble IL-13a 'receptor polypeptide (SEQ ID NO: 82). In another embodiment, the isolated polynucleotide is as described above, wherein the soluble receptor polypeptide is encoded by the polynucleotide shown in SEQ ID NO: 7. In another embodiment, the polynucleotide2

Isolated ES 2 279 809 T3 tide is as described above, wherein the soluble receptor polypeptide further comprises an affinity tag.

Within a second aspect, the present invention provides an expression vector comprising the following operably linked elements: (a) a transcription promoter; a first DNA segment encoding a soluble receptor polypeptide having an amino acid sequence as shown in SEQ ID NO: 6, and a transcription terminator; and (b) a second transcription promoter; a second DNA segment encoding a soluble receptor II, -2Ry polypeptide (SEQ ID NO: 4); and a transcription terminator; and wherein the first and second DNA segments are contained within a single expression vector.

Within a third aspect, the present invention provides a cultured cell comprising one or more expression vectors.

The present invention also provides one or more expression vectors comprising the following elements: (a) a transcription promoter; a first DNA segment encoding a soluble receptor polypeptide having an amino acid sequence as shown in SEQ ID NO: 6, and a transcription terminator, wherein said promoter, DNA segment, and terminator are operably linked; and (b) a second transcription promoter; a second DNA segment encoding a soluble IL-2Ry receptor polypeptide (SeQ ID NO: 4); and a transcription terminator, wherein said promoter, DNA segment, and terminator are operably linked; wherein said first and second DNA segments are contained within a single expression vector or are contained within independent expression vectors; and wherein the polypeptides expressed from said DNA segments associate to form a heterodimeric or multimeric receptor complex.

In one embodiment, the expression vector described above further comprises a secretory signal sequence operably linked to the first and second DNA segments.

Within a third aspect, the present invention provides a cultured cell comprising one or more expression vectors comprising the following elements: (a) a transcription promoter; a first DNA segment encoding a soluble receptor polypeptide having an amino acid sequence as shown in SEQ ID NO: 6, and a transcription terminator, wherein said promoter, DNA segment, and terminator are operably linked; and (b) a second transcription promoter; a second DNA segment encoding a soluble IL-2Ry receptor polypeptide (SEQ ID NO: 4); and a transcription terminator, wherein said promoter, DNA segment, and terminator are operably linked; wherein the first and second DNA segments are contained within a single expression vector or are contained within independent expression vectors; and wherein the cell expresses the polypeptides encoded by the DNA segments.

In one embodiment, the cultured cell comprising an expression vector is as described above, wherein the first and second DNA segments are located in independent expression vectors and are co-transfected into the cell, and the cell expresses the polypeptides encoded by the DNA segments. In another embodiment, the cultured cell comprising an expression vector is as described above, wherein the cell expresses a soluble heterodimeric or multimeric receptor polypeptide encoded by the DNA segments. In another embodiment, the cultured cell comprising an expression vector is as described above, wherein the cell secretes a soluble receptor polypeptide heterodimer or multimer complex that binds to a ligand comprising a polypeptide of SEQ ID NO. : 10 or SEQ ID NO: 47, or antagonizes the activity of the ligand.

Within another aspect, the present invention provides a DNA construct encoding a fusion protein comprising: a first DNA segment encoding a polypeptide having an amino acid residue sequence as shown in SEQ ID NO: 6; and at least one other DNA segment encoding a soluble Class I cytokine receptor polypeptide, wherein the first and the other DNA segments are linked in frames; and in which the first and the other DNA segments encode the fusion protein. In one embodiment, the DNA construct encodes a fusion protein as described above, wherein at least one other DNA segment encodes a soluble IL-2Ry receptor polypeptide (SEQ ID NO: 4) or a receptor polypeptide of Soluble IL-13a '(SEQ ID NO: 82).

Within another aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA construct encoding a fusion protein as described above; and a transcription terminator, wherein the promoter is operably linked to the DNA construct, and the DNA construct is operably linked to the transcription terminator.

Within another aspect, the present invention provides a cultured cell comprising an expression vector as described above, wherein the cell expresses a polypeptide encoded by the DNA construct.

Within another aspect, the present invention provides a method of producing a fusion protein comprising: culturing a cell as described above; and isolating the polypeptide produced by the cell.

Within another aspect, the present invention provides an isolated soluble receptor polypeptide comprising an amino acid residue sequence that is at least 90% identical to an amino acid sequence as

ES 2 279 809 T3 is shown in SEQ ID NO: 6, and wherein the soluble receptor polypeptide binds to a ligand comprising a polypeptide of SEQ ID NO: 10 or SEQ ID NO: 47, or antagonizes the activity of the ligand. In one embodiment, the isolated polypeptide is as described above, wherein the soluble receptor polypeptide forms a homodimeric receptor complex.

Within another aspect, the present invention provides an isolated polypeptide comprising an amino acid residue sequence that is at least 90% identical to an amino acid sequence as shown in SEQ ID NO: 6, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex. In one embodiment, the isolated polypeptide is as described above, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex that further comprises a soluble Class I cytokine receptor polypeptide. In another embodiment, the isolated polypeptide is as described above, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex that further comprises a soluble IL2Ry receptor polypeptide (SEQ ID NO: 4) or a receptor polypeptide. of soluble IL-13a '(SEQ ID nO: 82). In another embodiment, the isolated polypeptide is as described above, wherein the polypeptide further comprises a WSXWS motif as shown in SEQ ID NO: 13.

Within another aspect, the present invention provides an isolated soluble receptor polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 6, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex. In one embodiment, the isolated polypeptide is as described above, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex that further comprises a soluble Class I cytokine receptor polypeptide. In another embodiment, the isolated polypeptide is as described above, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex comprising a soluble IL-2Ry receptor polypeptide (SEQ ID NO: 4) or a polypeptide of soluble IL-13a 'receptor (SEQ ID NO: 82). In another embodiment, the isolated polypeptide is as described above, wherein the soluble receptor polypeptide further comprises an affinity tag, chemical moiety, toxin, or label.

Within another aspect, the present invention provides an isolated multimer or heterodimeric soluble receptor complex comprising soluble receptor subunits, wherein at least one of the soluble receptor subunits comprises a soluble receptor polypeptide comprising a sequence of amino acid residues. as shown in SEQ ID NO: 6. In one embodiment, the isolated multimer or heterodimeric soluble receptor complex described above further comprises a soluble Class I cytokine receptor polypeptide. In another embodiment, the isolated multimer or heterodimeric soluble receptor complex described above further comprises a soluble IL-2Ry receptor polypeptide (SEQ ID NO: 4) or a soluble IL-13a 'receptor polypeptide (SEQ ID NO: 82).

Within another aspect, the present invention provides a method of producing a soluble receptor polypeptide that forms a heterodimeric or multimeric complex comprising: culturing a cell as described above; and isolating the soluble receptor polypeptides produced by the cell.

Within another aspect, the present invention provides a method of producing an antibody to a soluble receptor polypeptide comprising: inoculating an animal with a soluble receptor polypeptide complex selected from the group consisting of: (a) a polypeptide comprising a complex homodimeric soluble receptor comprising SEQ ID NO: 6; (b) a polypeptide comprising a soluble receptor heterodimeric or multimeric receptor complex comprising SEQ ID NO: 6; (b) a polypeptide comprising a soluble receptor heterodimeric or multimeric receptor complex comprising SEQ ID NO: 6, and further comprising a soluble Class I cytokine receptor polypeptide; (c) a polypeptide comprising a soluble receptor heterodimeric or multimeric receptor complex comprising SEQ ID NO: 6, and further comprising a soluble IL-2Ry receptor polypeptide (SEQ ID NO: 4); (d) a polypeptide comprising a soluble receptor heterodimeric or multimeric receptor complex comprising SEQ ID NO: 6, and further comprising a soluble IL-13a 'receptor polypeptide (SEQ ID NO: 82): and wherein the polypeptide complex elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.

Within another aspect, the present invention provides an antibody produced by the method described above that specifically binds to a homodimeric, heterodimeric, or multimeric receptor complex comprising a soluble receptor polypeptide comprising SEQ ID NO: 6. In one embodiment, the antibody described above is a monoclonal antibody.

Within another aspect, the present invention provides an in vitro method for inhibiting a ligand comprising a polypeptide of SEQ ID NO: 10 or SEQ ID NO: 47, or antagonizing the proliferation induced by ligand activity of hematopoietic cells and progenitors. of hematopoietic cells comprising culturing bone marrow or peripheral blood cells with a composition comprising an amount of soluble receptor comprising SEQ ID NO: 6 sufficient to reduce proliferation of hematopoietic cells in bone marrow or peripheral blood cells compared to bone marrow or peripheral blood cells cultured in the absence of soluble receptor. In one embodiment the method is as described above, wherein the hematopoietic cells and hematopoietic progenitor cells are lymphoid cells. In another embodiment the method is as described above, wherein the lymphoid cells are NK cells or cytotoxic T cells.

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Within another aspect, the present invention provides the use of a soluble multimer or heterodimeric receptor complex comprising soluble receptor subunits, wherein at least one of the subunits comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 6, and wherein the receptor complex further comprises a soluble receptor II, -2Ry polypeptide (SEQ ID NO: 4), for the manufacture of a medicament to reduce the proliferation of neoplastic B or T cells in a mammal with a B or T cell neoplasm.

Within another aspect, the present invention provides the use of a soluble multimeric or heterodimeric receptor complex comprising soluble receptor subunits, wherein at least one of the subunits comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 6, and wherein the receptor complex further comprises a soluble IL-2Ry receptor polypeptide (SEQ ID NO: 4), in an acceptable pharmaceutical carrier, for the manufacture of a medicament for suppressing an immune response in a mammal exposed to an antigen or pathogen.

These and other aspects of the invention will become apparent with reference to the following detailed description of the invention.

Before beginning the invention in detail, it may be helpful for your understanding to define the following terms:

The term "affinity tag" is used herein to mean a polypeptide segment that can be incorporated into a second polypeptide to provide purification or detection of the second polypeptide or to provide sites for incorporation of the second polypeptide into a substrate. In principle, 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: 7952-4, 1985), substance P, Flag® peptide (Hopp et al. ., Biotechnology 6: 1204-10, 1988), streptavidin binding peptide or other antigen epitope or binding domain. See, generally, Ford et al., Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (eg, Pharmacia Biotech, Piscataway, NJ).

The terms "amino-terminal" and "carboxyl-terminal" as used herein indicate positions within polypeptides. Where context permits, these expressions are used with reference to a particular sequence or portion of a polypeptide to indicate proximity or relative position. For example, a certain sequence located carboxyl-terminal to a reference sequence within a polypeptide is located close to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the entire polypeptide.

The term "complement / anti-complement pair" indicates non-identical residues that form a stable, non-covalently associated pair under appropriate conditions. For example, biotin and avidin (or streptavidin) are prototypical members of a complement / anti-complement pair. Other examples of complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs, and the like. When subsequent dissociation of the complement / anti-complement pair is desirable, the complement / anti-complement pair preferably has a binding affinity <10⁹ M ¹.

The term "complements of a polynucleotide molecule" is a polynucleotide molecule that has a complementary base sequence and reverse orientation compared to a reference sequence. For example, the sequence 5 'ATGCACGGG 3' is complementary to 5 'CCCGTGCAT 3'.

The term "degenerate nucleotide sequence" indicates a nucleotide sequence that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different nucleotide triplets, but encode the same amino acid residue (ie, GAU and GAC triplets each encode Asp).

The term "expression vector" is used to denote a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest operably linked to additional 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 elements of both.

The term "isolated", when applied to a polynucleotide, indicates that the polynucleotide has been separated from its natural genetic environment and is thus free of other foreign or unwanted coding sequences, and is in a form suitable for use within systems of genetically engineered protein production. Such isolated molecules are those that are detached from their natural environment and include cDNA and genomic clones. The 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 terminators. The identification of associated regions will be apparent to one of ordinary skill in the art (see, for example, Dynan and Tijan, Nature 316: 77478, 1985).

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An "isolated" polypeptide or protein is a polypeptide or protein that is found in a state 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, ie greater than 95% purity, more preferably greater than 99% purity. When used in this context, the term "isolated" does not exclude the presence of the same polypeptde in alternative physical forms, such as dimers or alternatively glycosylated or derived forms.

The term "operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, for example, transcription is initiated at the promoter and proceeds through the segment of encoding up to the terminator.

The term "ortholog" indicates a polypeptide or protein derived from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences between orthologs are the result of specialization.

"Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, α-globin, / i-globin, and myoglobin are paralogues of each other.

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 can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Polynucleotide sizes are expressed as base pairs (abbreviated "bp"), nucleotides ("nt") or kilobases ("kb"). When the context allows, the last two terms can describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules, it is used to indicate full length and will be understood as equivalent to the expression "base pairs". It will be recognized by those skilled in the art that the two strands of a double stranded polynucleotide may differ slightly in length and that their ends may be staggered as a result of enzymatic cleavage; thus, all nucleotides within a double-stranded polynucleotide molecule may not be paired.

A "polypeptide" is a polymer of amino acid residues linked by peptide bonds, produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides."

"Probes and / or primers" as used herein can be RNA or DNA. The DNA can be cDNA or genomic DNA. Polynucleotide probes and primers are double-stranded DNA or RNA, generally synthetic oligonucleotides, but can be generated from cloned cDNA or genomic sequences or their complements. Analytical probes will be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used. PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt. Short polynucleotides can be used when the target of analysis is a small region of the gene. For gross gene analysis, a polynucleotide probe can comprise a complete exon or more. Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescent agent, paramagnetic particle, and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using methods that are well known in the art.

The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene that contains DNA sequences that provide for RNA polymerase binding and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5 'noncoding regions of genes.

A "protein" is a macromolecule that comprises one or more polypeptide chains. A protein can also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents can 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 structures; substituents such as carbohydrate groups are not generally specified, but may be present nonetheless.

The term "receptor" is used herein to denote a cell-associated protein, or polypeptide subunit of such a protein, that binds to a bioactive molecule (the "ligand") and mediates the effect of the ligand on the cell. Binding of the ligand to the receptor produces a conformational change in the receptor (and, in some cases, receptor multimerization, that is, association of identical or different receptor subunits) that causes interactions between the effector domain (s) ( es) and other molecule (s) of the cell. These interactions lead in turn to alterations in the cell's metabolism. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, cell proliferation, increases in cyclic AMP production, cellular calcium mobilization, membrane lipid mobilization, cell adhesion, lipid hydrolysis of inositol and hydrolysis of phospholipids. Cell surface cytokine receptors are characterized by a multi-domain structure as discussed in more detail below. These receptors are anchored in the cell membrane by a transmembrane domain characterized by a sequence of hydrophobic amino acid residues.

ES 2 279 809 T3 (typically around 21-25 residues), which are commonly flanked by positively charged residues (Lys or Arg). In general, the receptors can be membrane bound, cytosolic, or nuclear; monomers (eg, thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimers (eg, PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor , erythropoietin receptor and IL-6 receptor). The term "receptor polypeptide" is used to denote entire receptor polypeptide chains and portions thereof, including isolated functional domains (eg, ligand-binding domains).

A "secretory signal sequence" is 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 major peptide is commonly cleaved to separate the secretory peptide during transit through the secretory pathway.

A "soluble receptor" is a receptor polypeptide that is not bound to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides lacking transmembrane and cytoplasmic domains. Soluble receptors can comprise additional amino acid residues, such as affinity tags that provide purification of the polypeptide or provide sites for incorporation of the polypeptide into a substrate, or immunoglobulin constant region sequences. Many cell surface receptors have naturally-occurring, soluble counterparts that are produced by proteolysis. Soluble receptor polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.

The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splicing variation arises naturally through the use of alternative splice sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and can produce several transcribed mRNAs from the same gene. The splice variants can encode polypeptides that have altered amino acid sequence. The term "splice variant" is also used herein to denote a protein encoded by a splice variant of a mRNA transcribed from a gene.

It will be understood that the molecular weights and lengths of polymers determined by imprecise analytical methods (eg, gel electrophoresis) are approximate values. When such a value is expressed as "around" X or "approximately" X, the stated X value will be understood to have an accuracy of ± 10%.

The present invention is based in part on the discovery of a novel heterodimeric soluble receptor protein having the structure of a class I cytokine receptor. The heterodimeric soluble receptor includes at least one soluble zalpha11 receptor subunit, described in the application for Commonly owned US Patent No. 09 / 404,641. A second soluble receptor polypeptide included in the heterodimeric soluble receptor belongs to the receptor subfamily that includes the IL-2 receptor γ-common (IL-2Ry, or yc), the IL-2 receptor β subunit, and the receptor β-common (i.e., receptor β subunits of IL-3, IL-5, IL-13, IL-15, and GM-CSF), receptor subunits of IL-13a, IL-13a ', IL-15, and the like. The soluble human and mouse zalpha11 receptor (IL-21R) monomeric and homodimeric were shown to antagonize the activity of the natural ligand for the zalpha11 receptor, Zalpha11 Ligand (IL21) (Parris-Novak, J. et al., Nature 408: 57-63, 2000). The zalpha11 Ligand is described in commonly owned US Patent Application No. 09 / 522,217. According to the present invention, it was shown that a heterodimeric soluble zalpha11 receptor, exemplified by a preferred embodiment of a soluble zalpha11 receptor + heterodimeric soluble IL-2Ry receptor (zalpha11 / IL-2Ry), acts as an effective antagonist of the zalpha11 Ligand . As described in the present examples, the preferred zalpha11 / IL-2Rγ heterodimer was a more effective antagonist of zalpha11 Ligand activity, and thus a more superior antagonist than a zalpha11 homodimer or monomer.

Furthermore, soluble receptors comprising zalpha11 homodimers and monomers, as well as receptors comprising zalpha11 homodimers, heterodimers and multimers that are capable of intracellular signaling, are also considered by the present invention. Such receptors can comprise at least one extracellular domain of a zalpha11 receptor, and an intracellular domain of zalpha11 or another class I cytokine receptor. The additional heterodimeric or multimeric subunit may comprise the extracellular domain of IL-2Ry receptor (eg SEQ ID NO: 4), IL-13a receptor (also known as IL-13RA2; SEQ ID NO: 84), IL-13R (also known as IL13RA1; SEQ ID NO: 82, IL-15 (SEQ ID NO: 86), or other class I receptor, and an intracellular domain of zalpha11 or other class I cytokine receptor.

The nucleotide sequence of a representative zalpha11-encoding DNA is described in SEQ ID NO: 1 (from nucleotides 1 to 1614), and its deduced 538 amino acid sequence is described in SEQ ID NO: 2. In its entirety, the polypeptide of zalpha11 (SEQ ID NO: 2) represents a full-length polypeptide segment (residue 1 (Met) to residue 538 (Ser) of SEQ ID NO: 2). The domains and structural aspects of the zalpha11 polypeptide are described further below.

Analysis of the zalpha11 polypeptide encoded by the DNA sequence of SEQ ID NO: 1 revealed an open reading frame encoding 538 amino acids (SEQ ID NO: 2) comprising a secretory signal peptide of 19 amino acid residues (residue 1 (Met) to residue 19 (Gly) of SEQ ID NO: 2) and a mature polypeptide of 519 amino acids (residue 20 (Cys) to residue 538 (Ser) of SEQ ID NO: 2). In addition to the WSXWS motif (SEQ ID NO: 13)

ES 2 279 809 T3 corresponding to residues 214 to 218 of SEQ ID NO: 2, the receptor comprises a cytokine binding domain of approximately 200 amino acid residues (residues 20 (Cys) to 237 (His) of SEQ ID NO: 2); a linker domain (residues 120 (Pro) to 123 (Pro) of SEQ ID NO: 2); a penultimate chain region (residues 192 (Lys) to 202 (Ala) of SEQ ID NO: 2); a transmembrane domain (residues 238 (Leu) to 255 (Leu) of SeQ ID NO: 2); a complete intracellular signaling domain (residues 256 (Lys) to 538 (Ser) of SEQ ID NO: 2) containing a "Box I" signaling site (residues 267 (Ile) to 273 (Pro) of SEQ ID NO: 2) and a "Box II" signaling site (residues 301 (Leu) to 304 (Gly) of SEQ ID NO: 2). In addition there is a STAT3 (YXXQ) binding site located near the C terminus of residues 519 (Tyr) to 522 (Gln) of SEQ ID NO: 2. Those skilled in the art will recognize that these domain boundaries are approximate, and are based on alignments with known proteins and protein fold predictions. In addition to these domains, the receptor aspects conserved in the encoded receptor include (as shown in SEQ ID NO: 2) a conserved Trp residue at position 138 and a conserved Arg residue at position 201. In addition, zalpha11 contains typical conserved Cys residues of cytokine class I receptors, shown at residues 25, 35, 65 and 81 of SEQ ID NO: 2, and the corresponding regions of SEQ ID NO: 6 and SEQ ID NO: 69 described later. The corresponding polynucleotides encoding the zalpha11 polypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ ID NO: 1. The human zalpha11 soluble receptor polypeptide comprising residues 20 (Cys) to 237 (His) of SEQ ID NO: 2 is shown in SEQ ID NO: 6, and the corresponding polynucleotide sequence for the human zalpha11 soluble receptor polypeptide is shown in SEQ ID NO: 5.

SEQ ID NO: 3 is a polynucleotide sequence comprising a human IL-2Ry receptor fragment encoding a soluble 232 amino acid IL-2Ry receptor polypeptide (SEQ ID NO: 4). Those skilled in the art will recognize that these domain boundaries for the IL-2Ry receptor extracellular domain are approximate, and that other soluble IL-2Ry receptor polypeptides, such as those that include a sequence, are encompassed within the scope of the present invention. IL-2Ry receptor polypeptide secretory signal or additional IL-2Ry receptor polypeptide amino acids in the extracellular domain.

A variant form of the human zalpha11 polypeptide (WIPO Publication No. WO 00/27822, shown as SEQ ID NO: 3 and SEQ ID NO: 4 herein) was identified and is shown in the DNA sequence of SEQ ID NO: 64; and the corresponding polypeptide sequence is shown in SEQ ID NO: 65). This particular alternative zalpha11 receptor polypeptide contains 568 amino acids, and comprises a predicted secretory signal peptide of 20 amino acid residues (residue 1 (Met) to residue 20 (Gly) of SEQ ID NO: 65), and a mature polypeptide of 548 amino acids (residue 21 (Met) to residue 568 (Ser) of SEQ ID NO: 65). In addition to the WSXWS motif (SEQ ID NO: 13) corresponding to residues 244 to 248 of SEQ ID NO: 65, the receptor comprises a cytokine-binding domain of approximately 200 amino acid residues (residues 21 (Met) to 267 (His ) of SEQ NO: 65); no domain linker; a penultimate chain region (residues 222 (Lys) to 232 (Ala) of SEQ ID NO: 65); a transmembrane domain (residues 268 (Leu) to 285 (Leu) of SEQ ID NO: 65); a complete intracellular signaling domain (residues 268 (Lys) to 568 (Ser) of SEQ ID NO: 65) containing a "Box I" signaling site (residues 297 (Ile) to 303 (Pro) of SEQ ID NO: 65) and a "Cja II" signaling site (residues 331 (Leu) to 334 (Gly) of SEQ ID NO: 65). In addition, there is a STAT3 (YXXQ) binding site located near the C-terminus of residues 549 (Tyr) to 552 (Gln) of SEQ ID NO: 65. Those skilled in the art will recognize that these domain boundaries are approximate, and are based on alignments with known proteins and protein fold predictions. In addition to these domains, the receptor aspects conserved in the encoded receptor include (as shown in SEQ ID NO: 65) a conserved Trp residue at position 168, and a conserved Arg residue at position 231. Corresponding polynucleotides encoding the zalpha11 polypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ ID NO: 64. This particular human soluble zalpha11 receptor variant polypeptide, comprising residues 21 (Met) to 267 (His) of SEQ ID NO: 65 (SEQ ID NO: 69) and the corresponding polynucleotide sequence for this particular human soluble zalpha11 receptor polypeptide are shown in SEQ ID NO: 68. This variant form of the human zalpha11 receptor is included in the heterodimeric and multimeric zalpha11 receptor complexes of the present invention, described herein.

Furthermore, other variant forms of zalpha11 receptor are considered by the present invention, wherein the extracellular domain of the variant form described above (eg, 21 (Met) to 267 (His) of SEQ ID NO: 65, or SEQ ID NO: 69) comprises a domain linker comprising the PAPP amino acids (SEQ ID NO: 70) inserted between amino acids 161 (Ser) and 162 (Arg) of SEQ ID NO: 65, or the corresponding region of SEQ ID NO: 69. A preferred domain linker comprises an amino acid sequence preferably 4 to 14 amino acids in length, more preferably 14 amino acids in length, wherein any amino acid may be present next to the PAPP motif sequence (SEQ ID NO: 70). For example, a representative linker-containing variant zalpha11 soluble receptor is shown in SEQ ID NO: 71. In addition, other variant zalpha11 sequences may include, referring to SEQ ID NO: 65, a Gly at position 162 in place of an Arg, or the same ARG for substitution of Gly in the corresponding region of SEQ ID nO: 69 or SEQ ID NO: 71, or another variant of SEQ ID NO: 65 or SEQ ID NO: 69 that contains a domain linker as described above. Corresponding DNA sequences encoding such variants can be readily determined by one of skill in the art using the information present in Table 1 and Table 2.

The zalpha11 Ligand is a four helical bundle cytokine secreted in a "short helix" fashion. The polynucleotide sequence of the zalpha11 Ligand is shown in SEQ ID NO: 9 and the corresponding amino acid sequence is shown in SEQ ID NO: 10. The secretory signal sequence comprises amino acid residues 1 (Met) to 31 (Gly) , and the mature polypeptide comprises amino acid residues 32 (Gln) to 162 (Ser) (as

ES 2 279 809 T3 is shown in SEQ ID NO: 10). In general, cytokines are predicted to have a structure of four alpha helices, with the A, C and D helices being the most important in ligand-receptor interactions, and they are most highly conserved among family members. With reference to the amino acid sequence of the human zalpha11 Ligand shown in SEQ ID NO: 10, the alignment of the amino acid sequences of human zalpha11 Ligand, human IL-15, human IL-4 and human GM-CSF, is predicted that zalpha11 Ligand helix A is defined by amino acid residues 41-56; helix B by amino acid residues 69-84; helix C for amino acid residues 92-105; and helix D for amino acid residues 135-148; as shown in SEQ ID NO: 10. Structural analysis suggests that the A / B loop is long, the B / C loop is short, and the C / D loop is parallel long. Conserved cysteine ​​residues within Zaipha11 Ligand correspond to amino acid residues 71, 78, 122 and 125 of SEQ ID NO: 10. A consistent cysteine ​​placement is further confirmation of the four helical bundle structure. Also highly conserved in the family comprising IL-15, IL-2, IL-4, GM-CSF and zalpha11 ligand is the Glu-Phe-Leu sequence as shown in SEQ ID NO: 10 at residues 136-138 .

Further analysis of Zalpha11 Ligand based on multiple alignments of known cytokines predicts that amino acid residues 44, 47 and 135 (as shown in SEQ ID nO: 10) play an important role in the binding of Zalpha11 Ligand to its cognate receptor. Based on a comparison between human and mouse zalpha11 Ligand sequences, well conserved residues were found in the regions predicted to encode alpha A and D helices. The corresponding polynucleotides encoding the zalpha11 Ligand regions, domains, motifs, residues, and polypeptide sequences described herein are as shown in SEQ ID NO: 9. The mouse zalpha11 Ligand is shown in SEQ ID NO: 46, and the sequence of the corresponding polypeptide in SEQ ID NO: 47.

The activity of the molecules of the present invention can be measured using a variety of assays that measure proliferation and / or binding to cells expressing the zalpha11 receptor. Changes in zalpha11 Ligand-dependent cells are of particular interest. A suitable cell line was designed to be zalpha11 Ligand dependent comprising an IL-3 dependent BaF3 cell line (Palacios and Steinmetz, Cell 41: 727-734, 1985; MatheyPrevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986). Furthermore, other suitable cell lines to be designed to be zalpha11 Ligand dependent include FDC-P1 (Hapel et al., Blood 64: 786-790, 1984); and MO7e (Kiss et al., Leukemia 7: 235-240, 1993). Growth factor-dependent cell lines can be established according to published methods (eg, Greenberger et al., Leukemia Res. 8: 363-375, 1984; Dexter et al., In Baum et al. Reds., Experimental Hematology Today, 8th Ann. Mtg. Int. Soc. Exp. Hematol. 1979, 145-156, 1980).

The zalpha11 Ligand stimulates the proliferation, activation, differentiation and / or induction or inhibition of specialized cellular function of cells involved in hematopoiesis homeostasis and immune function. In particular, zalpha11 Ligand polypeptides stimulate proliferation, activation, differentiation, induction or inhibition of specialized cellular functions of cells of the hematopoietic lineages, including, but not limited to, T cells, B cells, NK cells, dendritic cells, monocytes and macrophages; as well as epithelial cells. Hematopoietic cell proliferation and / or differentiation can be measured in vitro using cultured cells or in vivo by administering zalpha11 Ligand to the appropriate animal model. Assays that measure cell proliferation or differentiation are well known in the art and are described herein. For example, assays that measure proliferation include assays such as neutral red dye chemosensitivity (Cavanaugh et al., Investigational New Drugs 8: 347-354, 1990), incorporation of radiolabeled nucleotides (Cook et al., Analytical Biochem. 179: 1-7, 1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82: 169-179, 1985, and the use of salts tetrazolium (Mosmann, J. Immunol. Methods 65: 55-63, 1983; Alley et al., Cancer Res. 48: 589-601, 1988; Marshall et al., Growth Reg. 5: 69-84, 1995; and Scudiero et al., Cancer Res. 48: 4827-4833, 1988. Assays that measure differentiation include, for example, measurement of cell surface markers associated with phase-specific expression of a tissue, enzyme activity, activity functional or morphological changes (Watt, FASEB, 5: 281-284, 1991; Francis, Differentiation 57: 63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989). Conversely, these assays can be used in a competition to assess the antagonist or zalpha11 Ligand binding activity of the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry receptors of the present invention. Furthermore, soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry receptors of the present invention can be used as antagonist or Ligand binding agent to modulate the immune system and hematopoietic activities of the zalpha11 Ligand.

Zalpha11 Ligand was isolated from tissue known to have important immune function and containing cells that play a role in the immune system. The zalpha1 Ligand is expressed in selected, activated CD3 + peripheral blood cells, and it has been shown that the expression of zalpha11 Ligand increases after activation of T cells. In addition, the results of experiments described in the Patent Application of the USA Common Property No. 09 / 522,217, and the present Examples section, demonstrate that the zalpha11 Ligand has an effect on the growth / expansion and / or differentiated status of NK cells or NK progenitors. Additional evidence demonstrates that the zalpha11 Ligand affects the proliferation and / or differentiation of T cells and B cells in vivo. Factors that stimulate the proliferation of hematopoietic progenitors and activate mature cells are generally known. NK cells are sensitive to IL-2 alone, but proliferation and activation generally require additional growth factors. For example, it has been shown that IL-7 and Link Factor (c-team ligand) were required for colony formation of NK progenitors. IL-15 + IL-2 in combination with IL-7 and Link Factor was more effective (Mrózek et al., Blood 87: 2632-2640, 1996). In addition, unidentified cytokines may be required for proliferation of specific subsets of NK cells and / or NK progenitors (Robertson et al., Blood 76: 2451-2438, 1990). A composition comprising zalpha11 Ligand and IL-15 stimulates NK progenitors and NK cells, with the

ES 2 279 809 T3 evidence that this composition is more effective than the factors and combinations of factors previously described. Such compositions may further comprise equipment ligand or stem cell factor. Thus, soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry receptors of the present invention can be used as antagonist or Ligand binding agent to decrease the activity of zalpha11 Ligand in NK cells.

Furthermore, the tissue distribution of a receptor for a given cytokine provides a strong indication of the potential sites of action of that cytokine. Northern analysis of the zalpha11 receptor revealed transcripts in human spleen, thymus, lymphoid node, bone marrow, and peripheral blood leukocytes. Specific cell types were identified as expressing zalpha11 receptors, and strong signals were seen in a mixed lymphocyte reaction (MLR) and in Burkitt's Raji lymphoma. The two monocytic cell lines, THP-1 (Tsuchiya et al., Int. J. Cancer 26: 171-176, 1980) and U937 (Sundstrom et al., Int. J. Cancer 17: 565-577, 1976), they were negative. The zalpha11 receptor is expressed at relatively high levels in MLR, in which peripheral blood mononuclear cells (PBMNC) from two individuals mix, causing mutual activation. Detection of high levels of transcript in the MLR but not in resting T or B cell populations suggests that zalpha11 receptor expression may be induced in one or more cell types during activation. Activation of isolated populations of T and B cells can be achieved artificially by stimulating cells with PMA and ionomycin. When sorted cells were subjected to these activation conditions, zalpha11 receptor transcript levels increased in both cell types, supporting a role for this receptor and zalpha11 Ligand in immune responses, especially in autocrine and paracrine T and B cell expansions. during activation. The zalpha11 ligand may also play a role in the expansion of more primitive progenitors involved in lymphopoiesis. Thus, soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry receptors of the present invention can be used as antagonist or Ligand binding agent to modulate the lymphopoietic activities of the zalpha11 Ligand.

The zalpha11 receptor was found to be present at low levels in resting T and B cells, and upregulated during activation in both cell types. Interestingly, B cells also downregulate the message more rapidly than T cells, suggesting that signal amplitude and signal turn-off time are important for proper regulation of B cell responses.

Furthermore, a large proportion of intestinal lamina propria cells show positive hybridization signals with the zalpha11 receptor. This tissue consists of a mixed population of lymphoid cells, including activated CD4 + T cells and activated B cells. Immune dysfunction, particularly chronic activation of the mucosal immune response, plays an important role in the etiology of Crohn's disease and inflammatory bowel disease (IBD); abnormal response to and / or pro-inflammatory cytokine production is also a suspected factor (Braegger et al., Annals Allergy 72: 135-141, 1994; Sartor RB Am. J. Gastroenterol. 92: 5S-11S, 1997 ). Zalpha11 Ligand in concert with IL-15 expands bone marrow progenitor NK cells and increases NK cell effector function. The zalpha11 Ligand also co-stimulates mature B cells stimulated with anti-CD40 antibodies, but inhibits the proliferation of B cells to signals through IgM. The zalpha11 Ligand increases T cell proliferation in concert with a signal through the T cell receptor, and overexpression in transgenic mice leads to lymphopenia and an expansion of monocytes and granulocytes. These pleiotropic effects of the zalpha11 Ligand suggest that molecules that antagonize or bind to the zalpha11 Ligand, such as the molecules of the present invention, may provide therapeutic utility for a wide range of diseases arising from defects in the immune system, including (although but not limited to) systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), myasthenia gravis, Crohn's disease, IBD, and diabetes. It is important to note that these diseases are the result of a complex network of immune dysfunction (SLE, for example, is the manifestation of defects in T and B cells), and that immune cells depend by interaction with others to obtain an effective immune response . Thus, the zalpha11 Ligand (or a Ligand antagonist, such as a molecule of the present invention) that can be used to manipulate more than one type of immune cell is an attractive therapeutic candidate for intervention in multiple phases of disease.

Similarly, the tissue distribution of the mRNA corresponding to the IL-2Ry receptor cDNA shows expression in hematopoietic and lymphoid cells, including CD4 + T cells, CD8 + T cells, CD20 + B cells, CD56 + NK cells, CD14 + monocytes, as well as granulocytes. The IL-2Ry receptor cDNA is not generally found in other cell types, including epithelial cells and fibroblast cells. The expression pattern of this receptor is correlated with the activities of the zalpha11 Ligand and the location of the zalpha11 receptor. Furthermore, antibodies to the IL-2Ry receptor diminish or erode the effect of the zalpha11 Ligand on B cells and BaF3 / zalpha11 receptor cells, demonstrating that the zalpha11 receptor and the IL-2Ry receptor can heterodimerize in vivo and in vitro.

The zalpha11 Ligand promotes the expansion of bone marrow NK cell populations and regulates the proliferation of mature T and B cells in response to activating stimuli. The zalpha11 ligand acts through a receptor complex that includes at least one zalpha11 receptor subunit and the y subunit._c of IL2R, even though the zalpha11 receptor cytoplasmic domain is capable of signal transducing in a heterodimeric configuration (Commonly Proprietary US Patent Application No. 09 / 522,217). IL4Ra is also capable of signaling as a homodimer (Kammer, W. et al., J. Biol. Chem. 271: 23634-23637, 1996), although the true functional IL4 receptor complex is a heterodimer of IL4Ra / y_c. Signaling at BaF3 / zalpha11 receptor could have resulted from interactions of the human zalpha11 receptor with endogenous mouse yc, and the present Examples show that antibodies to the yc subunit decrease zalpha11 Ligand signaling in these cells.

ES 2 279 809 T3

Furthermore, the IL2 receptor has been studied in detail and is composed of an α-β-γ „heterotrimer. The β and y subunits_c are both essential for signal transduction and are members of the hematopoietin receptor superfamily (Cosman, D., Cytokine 5: 95-106, 1993), while the α subunit appears to be primarily involved in high affinity binding conversion and it is structurally distinct from the hematopoietin receptor family. It has been shown that the y subunit_c participates in forming receptors for IL4, IL7, IL9, and IL15, in addition to IL2 (for a review, see Sugamura, K. et al., Ann. Rev. Immunol. 14: 179-205, 1996), and has been shown than null mutations in the y gene_c cause severe X-linked combined immunodeficiency (X-SCID) (Noguchi, M. et al., Cell 73: 147-157, 1993).

Zalpha11 Ligand antagonism of IL4-induced and anti-IgM B cell proliferation (commonly owned US Patent Application No. 09 / 522,217, and examples thereof) may be due to competition for Y_c; however, it is clear that IL4 can signal through a receptor independent of y_c (IL4Ra + IL13Ra ') (Murata, T. et al., Blood 91: 3884-3891, 1998). B cells from human SCID patients proliferate normally in response to anti-IgM and IL4 (Matthews, DJ et al., Blood 85: 38-42, 1995), and IL4 responsiveness in normal human B cells is associated with with IL13 responsiveness and IL13R levels (Ford, D. et al., J. Immunol. 163: 3185-3193, 1999). Similarly, the zalpha11 Ligand can signal through a heterodimer, heterotrimer, or multimer complex that includes the zalpha11 receptor and a non-y subunit._c. As such, the present invention considers soluble zalpha11 receptor heterodimeric antagonists and zalpha11 Ligand binding agents that do not include the subunit and_c, but include an additional Class I cytokine subunit, eg, IL13Ra 'and the like.

The soluble receptors of the present invention are useful as antagonists of the zalpha11 Ligand cytokine. Such antagonistic effects can be achieved by direct neutralization or binding of the zalpha11 Ligand. In addition to antagonistic uses, the soluble receptors of the present invention can bind to the zalpha11 Ligand and act as carrier proteins for the zalpha11 Ligand cytokine, in order to transport the Ligand to different tissues, organs and cells within the body. As such, the soluble receptors of the present invention can be fused or coupled to molecules, polypeptides, or chemical moieties that direct the soluble receptor-Ligand complex to a specific site, such as a tissue, specific immune cell, or tumor. Thus, the soluble receptors of the present invention can be used to specifically target the action of the zalpha11 Ligand. See Cosman, D. Cytokine 5: 95-106, 1993; and Fernandez-Botran, R. Exp. Opin. Invest. Drugs 9: 497-513, 2000.

Furthermore, the soluble receptors of the present invention can be used to stabilize the zalpha11 Ligand, to increase the bioavailability, therapeutic longevity and / or the efficacy of the Ligand by stabilizing the degradation or clearance Ligand, or by targeting the ligand to a place of action within the body. For example, naturally occurring soluble IL-6 / IL-6R complex stabilizes IL-6 and can signal through the gp130 receptor. See Cosman, D. supra and Fernandez-Botran, R. supra.

For example, the zalpha11 Ligand will be useful in treating tumor genesis, and would therefore be useful in treating cancer. The zalpha11 Ligand inhibits the IL-4-stimulated proliferation of normal B cells stimulated by anti-IgM and a similar effect is observed in B-cell tumor lines, suggesting that there may be therapeutic benefit to treating patients with the Zalpha11 Ligand with the in order to induce the B cell tumor cells to a less proliferative state. The ligand could be administered in combination with other agents already in use, including conventional chemotherapeutic agents as well as immune modulators such as interferon alpha. Alpha / beta interferons have been shown to be effective in treating some leukemias and animal disease models, and the growth inhibitory effects of interferon-alpha and zalpha11 Ligand are additive to at least one cell lineage derived from B cell tumors. Furthermore, stabilization of the zalpha11 Ligand or the ability to target the Ligand to specific sites of action with the soluble receptors of the present invention would be desirable in this therapeutic endeavor.

The present invention provides a method of reducing the proliferation of neoplastic B or T cells which comprises administering to a mammal with a B or T cell neoplasm an amount of a zalpha11 Ligand antagonist composition, such as the soluble receptors herein. invention, sufficient to reduce the proliferation of neoplastic B or T cells. In other embodiments, the composition may comprise at least one other cytokine selected from the group consisting of IL-2, IL-15, IL-4, GM-CSF, Flt3 ligand, or stem cell factor. Furthermore, the zalpha11 Ligand antagonist may be a toxic fusion. Similarly, the soluble receptor-toxic fusions and soluble receptors of the present invention can be used to reduce the proliferation of lymphoid and hematopoietic neoplasms that overexpress or grow in response to zalpha11 Ligand. Furthermore, the indirect effects of the soluble receptors of the present invention can modulate the function of NK cells induced by zalpha11 Ligand, and thereby indirectly increase the killing of tumor cells.

The present invention provides polynucleotide molecules, including DNA and RNA molecules that encode the heterodimeric zalpha11 receptor polypeptides described herein. Those skilled in the art will recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible between these polynucleotide molecules. SEQ ID NO: 7 is a degenerate DNA sequence that encompasses all DNAs encoding the soluble zalpha11 receptor polypeptide of SEQ ID NO: 6. SEQ ID NO: 66 is a degenerate DNA sequence that encompasses all DNAs that encode the soluble zalpha11 receptor polypeptide of SEQ ID NO: 69. SEQ ID NO: 8 is a degenerate DNA sequence that encompasses all DNAs that encode the IL-2Ry polypeptide

Soluble human ES 2 279 809 T3 of SEQ ID NO: 4. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 7, SEQ ID NO: 66 and SEQ ID NO: 8 also provides all RNA sequences that encode SEQ ID NO: 6, SEQ ID NO: 69 and SEQ ID NO: 4 respectively substituting U for T. Thus, polynucleotides encoding zalpha11 polypeptide comprising nucleotide 1 to nucleotide 654 of SEQ ID NO: 7 or comprising nucleotide 1 to nucleotide 741 of SEQ ID NO: 66, polynucleotides encoding IL polypeptide, are considered by the present invention Soluble human -2Ry comprising nucleotide 1 to nucleotide 696 of SEQ ID NO: 8, and their RNA equivalents. Table 1 outlines the one letter codes used within SEQ ID NO: 7, SEQ ID NO: 66 and SEQ ID NO: 8 to indicate degenerate nucleotide positions. "Resolutions" are the nucleotides indicated by a letter of the code. "Complement" indicates the code for the complementary nucleotide (s). For example, the code Y indicates C or T, and its complement R indicates A or G, with A being complementary to T and G being complementary to C.

TABLE 1

<td>Nucleotide</td><td>Resolution</td><td>Complement</td><td>Resolution</td>

<td>TO</td><td>TO</td><td>T</td><td>T</td>

<td>C</td><td>C</td><td>G</td><td>G</td>

<td>G</td><td>G</td><td>C</td><td>C</td>

<td>T</td><td>T</td><td>TO</td><td>TO</td>

<td>R</td><td>A | G</td><td>Y</td><td>C | T</td>

<td>Y</td><td>C | T</td><td>R</td><td>A | G</td>

<td>M</td><td>A | C</td><td>K</td><td>G | T</td>

<td>K</td><td>G | T</td><td>M</td><td>A | C</td>

<td>s</td><td>C | G</td><td>S</td><td>C | G</td>

<td>w</td><td>A | T</td><td>w</td><td>A | T</td>

<td>H</td><td>A | C | T</td><td>D</td><td>A | G | T</td>

<td>B</td><td>C | G | T</td><td>V</td><td>A | C | G</td>

<td>V</td><td>A | C | G</td><td>B</td><td>C | G | T</td>

<td>D</td><td>A | G | T</td><td>H</td><td>A | C | T</td>

<td>N</td><td>A | C | G | T</td><td>N</td><td>A | C | G | T</td>

The degenerate codons used in SEQ ID NO: 7, SEQ ID NO: 66 and SEQ ID NO: 8 encompass all possible codons for a given amino acid, they are indicated in Table 2.

ES 2 279 809 T3

TABLE 2

<td>Amino acid</td><td>Code of one letter</td><td>Codons</td><td>Codon degenerate</td>

<td>Cys</td><td>C</td><td>TGC TGT</td><td>TGY</td>

<td>To be</td><td>S</td><td>AGC AGT TCA TCC TCG TCT</td><td>WSN</td>

<td>Thr</td><td>T</td><td>ACA ACC ACG ACT</td><td>ACN</td>

<td>Pro</td><td>P</td><td>CCA CCC CCG CCT</td><td>CCN</td>

<td>To</td><td>TO</td><td>GCA GCC GCG GCT</td><td>GCN</td>

<td>Gly</td><td>G</td><td>GGA GGC GGG GGT</td><td>GGN</td>

<td>Asn</td><td>N</td><td>AAC AAT</td><td>AAY</td>

<td>Asp</td><td>D</td><td>GAC GAT</td><td>Gay</td>

<td>Glu</td><td>AND</td><td>GAA GAG</td><td>GAR</td>

<td>Gln</td><td>Q</td><td>CAA CAG</td><td>CAR</td>

<td>His</td><td>H</td><td>CAC CAT</td><td>CAY</td>

<td>Arg</td><td>R</td><td>AGA AGG CGA CGC CGG CGT</td><td>MGN</td>

<td>Lys</td><td>K</td><td>AAA AAG</td><td>AAR</td>

<td>Met</td><td>M</td><td>ATG</td><td>ATG</td>

<td>lie</td><td>I</td><td>ATA ATC ATT</td><td>ATH</td>

<td>Leu</td><td>L</td><td>CTA CTC CTG CTT TTA TTG</td><td>YTN</td>

<td>Val</td><td>V</td><td>GTA GTC GTG GTT</td><td>GTN</td>

<td>Phe</td><td>F</td><td>TTC TTT</td><td>TTY</td>

<td>Tyr</td><td>Y</td><td>TAC TAT</td><td>TAY</td>

<td>Trp</td><td>W</td><td>TGG</td><td>TGG</td>

<td>Ter</td><td> -</td><td>TAA TAG TGA</td><td>TRR</td>

<td>Asn | Asp</td><td>B</td><td></td><td>Ray</td>

<td>Glu | Gln</td><td>Z</td><td></td><td>HE</td>

<td>Any</td><td>X</td><td></td><td>NNN</td>

One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, code for arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, code for serine (AGY). There is a similar relationship between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 69, or SEQ ID NO: 4. The functionality of variant sequences can be easily tested as described herein.

One of ordinary skill in the art will also appreciate that different species may exhibit "preferential codon usage." In general, see Grantham et al., Nuc. Acids Res. 8: 1893-912, 1980; Haas et al. Curr. Biol. 6: 31513

ES 2 279 809 T3

24, 1996; Wain-Hobson et al., Gene 13: 335-64, 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 an expression of the art that refers to protein translation codons that are used more frequently in cells of a certain species, thus favoring one or a few representative of the possible codons that code for each amino acid (see Table 2). For example, the amino acid Threonine (Thr) can be encoded by ACA, ACC, ACG or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, eg, insect, yeast, virus or bacterial cells, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of preferential codon sequences into recombinant DNA can, for example, increase protein production by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence described in SEQ ID NO: 7, SEQ ID NO: 66 and SEQ ID NO: 8 serves as a template to optimize the expression of polynucleotides and polypeptides in various types and species of cells commonly used in the art. and described here. Sequences containing preferential codons for expression in various species can be tested and optimized, and their functionality tested as described herein.

Within preferred embodiments of the invention, isolated polynucleotides hybridize to similarly sized regions of SEQ ID NO: 5, SEQ ID NO: 68 or SEQ ID NO: 3, or a sequence complementary to them, under stringent conditions. In general, stringent conditions are selected to be approximately 5 ° C lower than the thermal melting point (T_m) for the specific sequence with a defined ionic strength and pH. The t_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Numerous equations for calculating Tm are known in the art, and are specific for DNA, RNA, and DNA-RNA hybrids, and probe sequences of varying length (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al. (reds.) Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (reds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26: 227 (1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, CA), as well as places on the Internet, are tools available to analyze a given sequence and calculate Tm. based on criteria defined by the user. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences (e.g.,&gt; 50 base pairs) is performed at temperatures of about 2025 ° C below the T_m calculated. For smaller probes (eg &lt;50 base pairs) hybridization is typically performed at Tm or 5-10 ° C below. This allows the maximum hybridization ratio for DNA-DNA and DNA-RNA hybrids. Higher degrees of stringency can be achieved at lower temperatures with the addition of formamide, which reduces the Tm of the hybrid by approximately 1 ° C for every 1% formamide in the buffer solution. Suitable stringent hybridization conditions are equivalent to an incubation of approximately 5 hrs overnight at approximately 42 ° C in a solution comprising: approximately 40-50% formamide, up to approximately 6X SSC, approximately 5X Denhardt's solution, from zero to about 10% dextran sulfate and about 10-20 µg / ml denatured commercially available carrier DNA. Generally, such stringent conditions include temperatures of 20-70 ° C and a hybridization buffer containing up to 6X SSC and 0-50% formamide; hybridization is then followed by washing the filters in up to about 2X SSC. For example, a suitable wash stringency is equivalent to 0.1X SSC to 2X SSC, 0.1% SDS, between 55 ° C and 65 ° C. Different degrees of rigidity can be used during hybridization and washing to achieve maximum specific binding to the target sequence. Typically, washes following hybridization are performed with increasing degrees of stringency to separate unhybridized polynucleotide probes from hybridized complexes. Hybridization and stringent washing conditions depend on the length of the probe, reflected in the T_m, hybridization and wash solutions used, and are routinely determined empirically by one of ordinary skill in the art.

As previously indicated, 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 zalpha11 receptor RNA or the RNA for the heterodimeric component of the receptor, such as IL-2Ry, or another class of cytokine receptor class I. Such tissues and cells they are identified by Northern spots (Thomas, Proc. Natl. Acad. Sci. USA 77: 5201, 1980), and include PBLs, spleen, thymus, and lymphoid tissues, Raji cells, human erythroleukemia cell lines (e.g., TF-1), acute monocytic leukemia cell lines, other lymphoid and hematopoietic cell lines, and similar, for the zalpha11 receptor. RNA for a heterodimeric component of the receptor, such as IL-2Ry, or another class I cytokine receptor can be isolated from lymphoid cells, such as those described above, and other cells and tissues as is known in the art for these receptors. total RNA is prepared using guanidinium isothiocyanate extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18: 52-94, 1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972). Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. Alternatively, genomic DNA can be isolated. Polynucleotides encoding zalpha11 polypeptides are then identified and isolated by, for example, hybridization or polymerase chain reaction (PCR) (Mullis, US Patent No. 4,683,202).

The polynucleotides of the present invention can also be synthesized using DNA synthesis machines. Currently the method of choice is the phosphoramidite method. If synthesized double-stranded DNA is required

ES 2 279 809 T3 chemically for an application such as the synthesis of a gene or gene fragment, each complementary strand is done separately. The production of short polynucleotides (60 to 80 bp) is technically simple and can be achieved by synthesizing the complementary strands and then reassembling them. However, to produce longer polynucleotides (&gt; 300 bp), special strategies are usually employed, because the coupling efficiency of each cycle during chemical DNA synthesis is rarely 100%. To overcome this problem, synthetic (double-stranded) genes are assembled in modular fashion from single-stranded fragments that are 20 to 100 nucleotides in length.

An alternative way to prepare a full-length gene is to synthesize a specified set of overlapping oligonucleotides (40 to 100 nucleotides). After reassociating the 3 'and 5' short overlapping complementary regions (6 to 10 nucleotides), large gaps still remain, but the short base pair regions are of sufficient length and sufficiently stable to hold the structure together. The gaps are filled and the DNA duplex is completed by enzymatic DNA synthesis by E. coli DNA polymerase I. After completion of the enzymatic synthesis, the sections are closed with T4 DNA ligase. Double stranded constructs are sequentially linked together to form the complete gene sequence which is verified by DNA sequence analysis. See Glick and Pasternak, Molecular Biotechnology, Principles &amp; Applications of Recombinant DNA, (ASM Press, Washington, DC 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al., Proc. Ntl. Acad. Sci. USA 87: 633-7, 1990. In addition, other sequences containing signals for appropriate initiation and termination of transcription and translation are generally added.

The present invention further provides equivalent polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to, mammals, birds, amphibians, reptiles, fish, insects, and other vertebrate and invertebrate species. Of particular interest are soluble heterodimeric receptor complexes that combine soluble zalpha11 receptor and soluble human IL-2Ry or other soluble Class I cytokine receptor polypeptides from other mammalian species, including mouse, porcine, ovine, bovine, canine, feline, polypeptides. equine and other primate. Known and unknown orthologs of soluble human zalpha11 receptor and soluble human IL-2Ry or other soluble Class I cytokine receptors can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type, such as lymphoid cells, that expresses zalpha11 receptor, human IL-2Ry, or other Class I cytokine receptors. In addition, suitable sources of mRNA can be identified by probing Northern spots with probes designed from the sequences described herein. A library is then prepared from mRNA from a positive tissue or cell line. A cDNA encoding zalpha11 can then be isolated by a variety of methods, such as probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the described sequences. A cDNA can also be cloned using PCR (Mullis, supra), using primers designed from the representative human zalpha11 sequence, or or the soluble human IL-2Ry sequence, described herein. Within a further method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with a zalpha11 polypeptide antibody. Similar techniques can also be applied to the isolation of genomic clones.

Cytokine receptor subunits are characterized by a multi-domain structure comprising an extracellular domain, a transmembrane domain that anchors the polypeptide on the cell membrane, and an intracellular domain. The extracellular domain of the zalpha11 receptor is a ligand-binding domain, which binds Ligand of zalpha11, and the intracellular domain is an effector domain involved in signal transduction, although the ligand-binding and effector functions may reside in separate subunits of a multimer receiver. The ligand binding domain may itself be a multi-domain structure. Multimeric receptors include homodimers (e.g., αα and ββ isoforms of PDGF receptor, erythropoietin receptor, MPL, and G-CSF receptor), heterodimers whose subunits each have ligand-binding and effector domains (e.g., αβ isoform PDGF receptor), and multimers that have component subunits with disparate functions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-13, IL -15 and GM-CSF). Some receptor subunits are common to a plurality of receptors. For example, the AIC2B subunit, which cannot bind ligand by itself but includes an intracellular signal transduction domain, is a component of IL-3 and GM-CSF receptors. Many cytokine receptors can be placed in one of four related families on the basis of structure and function. Hematopoietic receptors, for example, are characterized by the presence of a domain containing conserved cysteine ​​residues and the WSXWS motif (SEQ ID NO: 13). The structure of the cytokine receptor has been examined by Urdal, Ann. Reports Med. Chem. 26: 221-228, 1991; and Cosman, Cytokine 5: 95-106, 1993. Under selective pressure for organisms to acquire new biological functions, new members of the receptor family are likely to emerge from gene duplication of existing receptors leading to the existence of multi-gene families. Family members thus contain traces of the ancestral gene, and these characteristic features can be exploited in the isolation and identification of additional family members. Thus, the cytokine receptor superfamily is subdivided into several families, eg, the immunoglobulin family (including receptors for CSF-1, MGF, IL-1, and PDGF); the hematopoietin family (including IL-2 receptor β subunit, GM-CSF receptor α subunit, GM-cSf receptor β subunit; and G-CSF, EpO, IL-3, IL-4 receptors, IL-5, IL-6, IL-7, IL-9, IL13 and IL-15); the TNF receptor family (including TNF (p80) receptors, TNF (p60), CD27, CD30, CD40, Fas, and NGF receptor).

Sequence analysis of the zalpha11 receptor suggests that it is a member of the same receptor subfamily as the IL-2 receptor β subunit, IL-4 and IL-9 receptors. Certain receptors in this subfamily (eg, EPO-R or MPL) associate to form homodimers that transduce a signal. Other members of the subfamily (for

Example ES 2 279 809 T3, IL-6, IL-11 and LIF receptors) combine with a second subunit (termed a β subunit ) to bind ligand and transduce a signal. Specific β subunits associate with a plurality of specific cytokine receptor subunits. For example, the β gp130 subunit (Hibi et al., Cell 63: 1149-1157, 1990) associates with receptor subunits specific for IL-6, IL-11 and LIF (Gearing et al., EMBO J. 10: 2839-2848, 1991; Gearing et al., US Patent No. 5,284,755). Oncostatin M binds to a LIF receptor heterodimer and gp130. CNTF binds to trimeric receptors comprising CNTF receptor, LIF receptor, and gp130 subunits. Furthermore, IL-4 and IL-13 elicit responses through IL-4 and IL-13 receptors by acting on various functional heterodimeric receptor complexes, for example, with and without the y subunit._c, and such heterodimeric receptor complexes can affect whether cytokines act on hematopoietic or non-hematopoietic cells (Andersson, A. et al., Eur. J. Immunol. 27: 1762-1768, 1997; Murata T. et al., Blood 10 : 3884-3891, 1998). Furthermore, the binding affinity of IL-4 at its receptor is increased when the subunit and_c of the IL4R complex is replaced by an IL-13Ra 'subunit (Murata, T. et al., supra). Thus, the soluble receptors of the present invention include zalpha11 receptor homodimers; and heterodimers having a zalpha11 receptor component, such as soluble zalpha11 / IL-2Ry or soluble zalpha11 receptor heterodimerized with another soluble Class I cytokine receptor, such as IL-13Ra (SEQ ID NO: 84), IL-13Ra '( SEQ ID NO: 82) or an IL-15 receptor subunit (SEQ ID NO: 86).

For example, suitable soluble Class I cytokine receptors that can heterodimerize with a soluble zalpha11 receptor component (eg, SEQ ID NO: 6), include a soluble receptor for IL-13Ra as shown in SEQ ID NO: 84, IL-13Ra 'as shown in SEQ ID NO: 82 or IL-15 as shown in SEQ ID NO: 86. In addition, functional sub-fragments, such as minimal cytokine-binding fragments, of these soluble receptors can be used. Class I cytokine. Such functional fragments include 1 to 322, 7 to 322 and 105 to 322 of SEQ ID NO: 82, 1 to 317, 10 to 317 and 105 to 317 of SEQ ID NO: 84, and 1 to 173 of SEQ ID NO: 86 The corresponding polynucleotide sequences are as shown in SEQ ID NO: 81, SEQ ID NO: 83 and SEQ ID NO: 85 respectively. It is well within the level of one skilled in the art to define which sequences of a known class I cytokine sequence comprise the extracellular cytokine binding domain devoid of a transmembrane domain and intracellular domain.

A polynucleotide sequence for the human zalpha11 receptor mouse ortholog has been identified and is shown in SEQ ID NO: 11, and the corresponding amino acid sequence is shown in SEQ ID NO: 12. Analysis of the mouse zalpha11 polypeptide encoded by the DNA sequence of SEQ ID NO: 11 revealed an open reading frame encoding 529 amino acids (SEQ ID NO: 12) comprising a predicted secretory signal peptide of 19 amino acid residues (residue 1 (Met) to residue 19 (Ser) of SEQ ID NO: 12), and a 510 amino acid mature polypeptide (residue 20 (Cys) to residue 529 (Ser) of SeQ ID NO: 2). In addition to the WSXWS motif (SEQ ID NO: 13) corresponding to residues 214 to 218 of SEQ ID NO: 12, the receptor comprises a cytokine-binding domain of approximately 200 amino acid residues (residues 20 (Cys) to 237 (His ) of SEQ ID NO: 12); a linker domain (residues 120 (Pro) to 123 (Pro) of SEQ ID NO: 12); a penultimate chain region (residues 192 (Lys) to 202 (Ala) of SEQ ID NO: 12); a transmembrane domain (residues 238 (Met) to 254 (Leu) of SEQ ID NO: 12); a complete intracellular signaling domain (residues 255 (Lys) to 529 (Ser) of SEQ ID NO: 12) containing a "Box I" signaling site (residues 266 (Ile) to 273 (Pro) of SEQ ID NO: 12) and a "Box II" flag site (residues 301 (Ile) to 304 (Val) of SEQ ID NO: 2). A comparison of the human and mouse amino acid sequences reveals that the human and orthologous polypeptides contain structural aspects described above. The mature sequence for mouse zalpha11 begins at Cys₂₀ (as shown in SEQ ID NO: 12), which corresponds to Cys₂₀ (as shown in SEQ ID NO: 2) in the human sequence. There is approximately 69% identity between the mouse and human zalpha11 sequences on the extracellular cytokine binding domain corresponding to residues 20 (Cys) to 237 (His) of SEQ ID NO: 2 (SEQ ID NO: 6 ) and residues 20 (Cys) to 237 (His) of SEQ ID NO: 12. The above percentage identities were determined using a FASTA program with ktup = 1, gap opening penalty = 12, gap extension penalty = 2, and substitution matrix = BLOSUM62, setting other parameters of the FASTA program as default. The corresponding polynucleotides encoding the mouse zalpha11 polypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ ID NO: 11.

The present invention also provides a heterodimeric soluble receptor, wherein the soluble zalpha11 receptor polypeptide isolated therein is substantially similar to the polypeptides of SEQ ID NO: 6 and their orthologs. Furthermore, in a preferred embodiment, the present invention also provides a heterodimeric soluble receptor, wherein the soluble IL-2Ry receptor polypeptide isolated therein is substantially similar to the polypeptides of SEQ ID NO: 4 and their orthologs. The term "substantially similar" is used herein to indicate polypeptides that have at least 70%, more preferably at least 80%, sequence identity with the sequences shown in SEQ ID NO: 6 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and even more preferably 95% or more identical to SEQ ID NO: 6 or SEQ ID NO: 4 or their orthologs. Percent sequence identity is determined by standard methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992. Briefly, two amino acid sequences are aligned to optimize alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) As shown in Table 3 (amino acids are indicated by standard one letter codes). The percent identity is then calculated as:

ES 2 279 809 T3

Total number of identical pairs x 100

[length of the longest sequence plus the number of gaps introduced in the longest sequence in order to align the two sequences]

TABLE 3

<td> 0 »1</td><td> 1</td><td> 0 0-3</td><td> 5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>B -1</td><td> 0</td><td> 0 2-4</td><td> 2</td><td> 5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>α or</td><td> -2</td><td> 0 -2 -3</td><td> -2</td><td> -2 6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>K -3</td><td> 0</td><td> 1 -1 -3</td><td> 0</td><td>0 -2 S</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>I -1</td><td> -3</td><td>--¾ ai"</td><td> -3</td><td> -3 -4 -3</td><td> 4</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>L -1</td><td> •2</td><td>-3 -4 -X</td><td> -2</td><td> -3 -4 -3</td><td></td><td> 4</td><td></td><td></td><td></td><td></td><td></td>

<td>K -1</td><td> 2</td><td> 0 -1 -3</td><td> 1</td><td> 1 -2 -1</td><td> -3</td><td> -2 5</td><td></td><td></td><td></td><td></td><td></td>

<td>Μ -1</td><td> -1</td><td> -2 -3 -1</td><td> 0</td><td> -2 -3 -2</td><td> 1</td><td> 2 -1</td><td>S</td><td></td><td></td><td></td><td></td>

<td>F -2</td><td> -3</td><td> -3 -3 -2</td><td> -3</td><td>-3 -3 -X</td><td> 0</td><td> 0 -3</td><td> 0</td><td> 6</td><td></td><td></td><td></td>

<td>P -1</td><td> -2</td><td> •2 -1 -3</td><td> -1</td><td>-i -a -2</td><td> -3</td><td> -3 -1</td><td> -2</td><td> -4 7</td><td></td><td></td><td></td>

<td>S 1</td><td> -1</td><td> 10-1</td><td> 0</td><td> 0 0-1</td><td> **</td><td> -2 .0</td><td> -1</td><td>-2 Ί</td><td> 4</td><td></td><td></td>

<td>T 0</td><td> -1</td><td> 0 -1 -1</td><td>• X</td><td>«U 4b“ 45</td><td> -1</td><td> -1 -1</td><td> -1</td><td> -2 -1</td><td> 1</td><td> 3</td><td></td>

<td>W -3</td><td> -3</td><td> -4 -4 -2</td><td> -2</td><td></td><td> -3</td><td></td><td> -1</td><td>X -4</td><td> -3</td><td> -2</td><td> 11</td>

<td>Y -2</td><td> -2</td><td> -2 -3 -2</td><td>-X</td><td> -2 -3 2</td><td> -1</td><td> -1 -2</td><td> -1</td><td> 3 -3</td><td> -2</td><td>'"« Fi</td><td> 2</td>

<td>V 0</td><td> -3</td><td> -3 -3 -1</td><td> -2</td><td> -2 -3 -3</td><td> 3</td><td> 1 -2</td><td> 1</td><td> -1 -2</td><td> -2</td><td> 0</td><td> -3</td>

The identity of polynucleotide molecule sequences is determined by similar methods using a relationship as described above.

Those skilled in the art appreciate that there are many established algorithms available for aligning two amino acid sequences. The Pearson and Lipman "FASTA" search for similarity algorithm is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence described herein and the amino acid sequence of a putative zsig57 variant. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat'lAcad. Sci. USA 85: 2444 (1988), and by Pearson, Meth. Enzymol. 183: 63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the sequence in question (for example, SEQ ID NO: 6) and a test sequence, which have the highest density of identities (if the variable ktup is 1) or pairs of identities (if ktup = 2), without considering conservative amino acid substitutions, insertions or deletions. The ten regions with the highest density of identities are re-scored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "fixed" to include only those residues that contribute to the score. highest. If there are multiple regions with scores greater than the “cut-off” value (calculated using a predetermined formula based on the length of the sequence and the value of ktup), the initial arranged regions are examined to determine whether the regions can be joined to form a approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48: 444 (1970); Sellers, SIAM J. Appl. Math. 26: 787 (1974)), which allows amino acid insertions and deletions. The preferred program parameters for FASTA analysis are: ktup = 1, gap opening penalty = 10 and gap extension penalty = 1, and substitution matrix = BLOSUM62, other parameters being set as default. These FASTA program parameters can be entered into a FASTA program by modifying the scoring matrix file ("SMATRIX") as explained in Appendix 2 of Pearson, Meth. Enzymol. 183: 63 (1990).

ES 2 279 809 T3

FASTA can also be used to determine nucleic acid molecule sequence identity using a relationship as described above. For nucleotide sequence comparisons, the ktup value can range from one to six, preferably three to six, more preferably three, with other program parameters set as the default.

The BLOSUM62 Table (Table 3) is an amino acid substitution matrix derived from approximately 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 related protein clusters (Henikoff and Henikoff, Proc. Nat 'l. Acad. Sci. USA 89: 10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that can be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely on chemical properties (as discussed below), the term "conservative amino acid substitutions" preferably refers to a substitution represented by a BLOSUM62 value greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 ( eg 1, 2 or 3), while the more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (eg 2 or 3).

Variant zalpha11 polypeptides or substantially homologous zalpha11 polypeptides are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably small in nature, that is, conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. The present invention thus includes soluble zalpha11 receptor polypeptides of about 190 to about 245 amino acid residues comprising a sequence that is at least 80%, preferably at least 90%, and more preferably 95% or more identical to the corresponding region of SEQ. ID NO: 6. Polypeptides comprising affinity tags may further comprise a proteolytic cleavage site between the zalpha11 polypeptide and the affinity tag. Suitable sites include thrombin cleavage sites and factor Xa cleavage sites.

TABLE 4

Conservative Basic Amino Acid Substitutions: Arginine Lysine Histidine

Acids: glutamic acid aspartic acid

Polar: glutamine asparagine

Hydrophobes: Leucine Isoleucine Valine

Aromatics: phenylalanine tryptophan tyrosine

Small: glycine alanine serine threonine methionine

ES 2 279 809 T3

The present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions. For example, a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as zalpha11 / IL-2Ry, can be prepared as a fusion to a dimerizing protein as described in US Patent Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include immunoglobulin constant region domains, eg, IgGy1, and the human κ light chain. Immunoglobulin soluble zalpha11 receptor or immunoglobulin soluble zalpha11 heterodimeric polypeptide, such as zalpha11 / IL-2R and immunoglobulin soluble fusions, can be expressed in cells genetically engineered to produce a variety of multimeric zalpha11 receptor analogs. Helper domains can be fused to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, to target specific cells, tissues, or macromolecules (eg, collagen or cells expressing the zalpha11 Ligand). A zalpha11 polypeptide can be fused to two or more residues, such as an affinity tag for purification and a target binding domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See Tuan et al., Connective Tissue Research 34: 1-9, 1996.

The proteins of the present invention may also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine , pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine and 4-fluorophenylalanine. Various methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system comprising an extract of E. coli S30 and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113: 2722, 1991; Ellman et al., Methods Enzymol. 202: 301, 1991; Chung et al., Science 259: 806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90: 10145-9, (1993). In a second method, translation is performed in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271: 1991-8, 1996). Within a third method, E. coli in the absence of a naturally occurring amino acid to be substituted (e.g. phenylalanine) and in the presence of desired non-naturally occurring amino acid (s) (e.g. 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine or 4 -fluorophenylalanine). The non-naturally occurring amino acid is incorporated into protein in place of its natural partner. See Koide et al., Biochem. 33: 7470-7476, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modifications. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

Zalpha11 amino acid residues can be replaced by a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and non-natural amino acids.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88: 4498-502, 1991). In the latter technique, single alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are assayed for biological activity (eg, ligand binding and signal transduction) as described below to identify amino acid residues. that are critical to the activity of the molecule. See also Hilton et al., J. Biol. Chem. 271: 4699-4708, 1996. Ligand-receptor, protein-protein or other biological interaction sites can also be determined by physical analysis of the structure, determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity marking, in conjunction with amino acid mutation of the contact site. putative. See, for example, de Vos et al., Science 255: 306-312, 1992; Smith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. The identities of essential amino acids can also be inferred from analyzes of homologies with related receptors.

The determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be defined. Within these regions, specific residues can be determined that will be more or less tolerant to change and maintain the total tertiary structure of the molecule. Methods for analyzing a sequence structure include, but are not limited to, multiple sequence alignment with high amino acid or nucleotide identity and computer analysis using available software (eg, the Insight II® viewer and homology modeling tools; MSI, San Diego, CA), secondary structure propensities, binary models, complementary packaging, and masked polar interactions (Barton, Current Opin. Struct. Biol. 5: 372-376, 1995; and Cordes et al., Current Opin. Struct. Biol. 6: 3-10, 1996). In general, when designing modifications of molecules or identifying specific fragments, the determination of the structure will be accompanied by evaluation of the activity of the modified molecules.

Changes are made to the amino acid sequences in soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, to minimize disruption of a higher order structure essential for biological activity. For example, when the soluble zalpha11 receptor or zalpha11 heterodimeric polypeptide

ES 2 279 809 T3 soluble, such as soluble zalpha11 / IL-2Ry, comprises one or more helices, changes will be made in amino acid residues so as not to break the geometry of the helix and other components of the molecule in which changes in conformation decrease some critical function, eg, binding of the molecule to the zalpha11 Ligand or antagonism of the zalpha11 Ligand activity. The effects of amino acid sequence changes can be predicted, for example, by computer modeling as described above or determined by crystal structure analysis (see, for example, Lapthorn et al., Nat. Struct. Biol. 2 : 266-268, 1995). Other methods that are well known in the art compare the folding of a variant protein with a standard molecule (eg, the native protein). For example, a comparison of the cysteine ​​pattern in variant and standard molecules can be made. Mass spectrometry and chemical modification using reduction and alkylation provide methods for determining cysteine ​​residues that are associated with disulfide bonds or are free from such associations (Bean et al., Anal. Biochem. 201: 216-226, 1992; Gray , Protein Sci. 2: 1732-1748, 1993; and Patterson et al., Anal. Chem. 66: 3727-3732, 1994). It is generally believed that if a modified molecule does not have the same disulfide bond pattern as the standard molecule, folding would be affected. Another well known and accepted method of measuring fold is circular dichrosism (CD). Measurement and comparison of CD spectra generated by a modified molecule and a standard molecule are routine (Johnson, Proteins 7: 205-214, 1990). Crystallography is another well-known method for analyzing fold and structure. Nuclear magnetic resonance (NMR), digestive peptide representation and epitope representation are also known methods for analyzing fold and structural similarities between proteins and polypeptides (Schaanan et al., Science 257: 961-964, 1992).

A Hopp / Woods hydrophilicity profile of the zalpha11 protein sequence can be generated as shown in SEQ ID NO: 6 (Hopp et al., Proc. Natl. Acad. Sci. 78: 3824-3828, 1981; Hopp, J. Immun. Meth. 88: 1-18, 1986, and Triquier et al., Protein Engineering 11: 153-169, 1998). The profile is based on a sliding six-residue window. Masked G, S and T residues and exposed H, Y and W residues were ignored. For example, in the soluble zalpha11 receptor, hydrophilic regions include amino acid residues 55 to 60 of SEQ ID NO: 2, amino acid residues 56 to 61 of SEQ ID NO: 2, amino acid residues 139 to 144 of SEQ ID NO: 2, and amino acid residues 227 to 232 of SEQ ID NO: 2. The corresponding hydrophilic regions in reference to SEQ ID NO: 6 can be made with reference to the above amino acid residues of SEQ ID NO: 2.

Those skilled in the art will recognize that hydrophilicity or hydrophobicity will be considered when designing modifications to the amino acid sequence of a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, so as not to disrupt the total structural and biological profile. Of particular interest for substitution are hydrophobic residues selected from the group consisting of Val, Leu and Ile or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp. For example, substitution tolerant residues could include residues such as shown in SEQ ID NO: 6, SeQ ID NO: 69 and SEQ ID NO: 4. However, cysteine ​​residues could be relatively intolerant of substitution.

The essential amino acid identities can also be inferred from sequence similarity analysis between members of the Class I cytokine receptor family with soluble zalpha11 receptor or soluble IL-2Ry receptor. Using methods such as the "FASTA" analysis described previously, regions of high similarity are identified within a protein family and used to analyze amino acid sequence for conserved regions. An alternative method of identifying a variant extracellular domain zalpha11 polynucleotide based on structure is to determine whether a nucleic acid molecule encoding a potential variant zalpha11 polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 68, as discussed above. Similarly, soluble class I cytokine receptor variants contained within a zalpha11 heterodimeric polypeptide, such as the soluble zalpha11 / IL-2Ry soluble IL-2Ry receptor component, can be identified as described above.

Other methods for identifying essential amino acids in the polypeptides of the present invention are procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081, 1989; Bass et al., Proc Natl. Acad. Sci. USA 88: 4498, 1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering", in Proteins: Analysis and Design, Angeletti (red.) Pages 259-311 (Academic Press, Inc . 1998)). In the latter technique, single alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are assayed for biological activity as described below to identify amino acid residues that are critical to the activity of the molecule. See also Holton et al., J. Biol. Chem. 271: 4699 (1996).

The present invention also includes soluble zalpha11 receptor polypeptide functional fragments or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry polypeptides and nucleic acid molecules encoding such functional fragments. A soluble zalpha11 receptor polypeptide or "functional" soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry polypeptide, or fragment thereof defined herein is characterized by its ability to bind an anti-zalpha11 antibody, or a Ligand of zalpha11 (soluble or immobilized), or to antagonize Ligand activity of zalpha11 in, for example, a biological or binding assay. As previously described herein, the zalpha11 receptor is characterized by a class I cytokine receptor structure. Thus, the present invention further provides fusion proteins that encompass: (a) homodimeric or multimeric polypeptide molecules that comprise a described extracellular domain. here; and (b) functional fragments that comprise one or more of these domains. The other polypeptide portion of the fusion protein may be contributed by another class I cytokine receptor, e.g., IL-2Ry, IL-2 receptor β subunit, and the common β-receptor (i.e., receptor β subunits IL3, IL5, and GM-CSF), IL-13a, IL-13a 'or IL20 receptor subunits

ES 2 279 809 T3

15, or by a non-native and / or unrelated secretory signal peptide that facilitates the secretion of the soluble fusion protein.

Routine suppression analyzes of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule encoding a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry. As an illustration, DNA molecules having the nucleotide sequence of SEQ ID NO: 1 or fragments thereof can be digested with Bal31 nuclease to obtain a series of nested deletions. These DNA fragments can be inserted into expression vectors in the appropriate reading frame, and the expressed polypeptides are isolated and tested for antagonism of zalpha11 Ligand or zalpha11 Ligand binding biological activity; or the ability to bind soluble anti-zalpha11 receptor or anti-soluble zalpha11 heterodimeric polypeptide antibodies; or the ligand binding capacity of zalpha11. An alternative to exonuclease digestion is to use oligonucleotide directed mutagenesis to introduce deletions or stop codons to specify the production of a desired polypeptide fragment. Alternatively, particular fragments of a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide polynucleotide can be synthesized using the polymerase chain reaction.

Standard methods for identifying functional domains, such as Ligand binding domains, are routine for those skilled in the art. For example, studies on truncation in either or both terms of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66: 507 (1995). In addition, standard techniques for functional analysis of proteins are described, for example, by Treuter et al., Molec. Eng. Genet. 240: 113 (1993); Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon”, in Biological Interferon Systems, Proceedings of ISIR-TNO Meeting on Interferon Systems, Cantel (red.), Pages 65-72 (Nijhoff 1987); Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation 1, Boyinton et al. (reds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol. Chem. 270: 29270 (1995); Fukunaga et al., J. Biol. Chem. 270: 25291 (1995), Yamaguchi et al., Biochem. Pharmacol. 50: 1295 (1995); and Meisel et al., PlantMolec. Biol. 30: 1 (1996).

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those described by Reidhaar-Olson and Sauer (Science 241: 53-57, 1988) or Bowie and Sauer (Proc. Natl. Acad. Scie. USA) 86: 2152-2156, 1989). Briefly, these authors describe methods for randomizing two or more positions in a polypeptide, selecting a functional polypeptide, and then determining the sequence of the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (eg, Lowman et al., Biochem 30: 10832-10837, 1991; Ladner et al., US Pat. No. 5,223,409; Huse, WIPO Publication WO 92/062045) and region-directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988).

Soluble zalpha11 receptor DNA variants or soluble zalpha11 heterodimeric polypeptide and polypeptide sequences can be generated by DNA mixing as described by Stemmer, Nature 370: 389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91: 10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by homologous recombination in vitro by random fragmentation of a parent DNA followed by reassembly using PCR, producing randomly introduced point mutations. This technique can be modified using a family of parent DNAs, such as allelic variants of DNAs from different species, to introduce additional variability into the process. Selection or examination for the desired activity, followed by additional iterations of mutagenesis and testing provides rapid sequence "evolution" by selecting for desirable mutations while simultaneously selecting against deleterious changes.

Mutagenesis methods as described herein can be combined with high throughput automated screening methods to detect binding or zalpha11 Ligand antagonist activity in cloned, mutagenized soluble zalpha11 receptor host cells or soluble zalpha11 heterodimeric receptor polypeptides. Preferred assays in this regard include cell proliferation assays and biosensor-based ligand binding assays, which are described below in the Examples. Mutagenized DNA molecules encoding active receptors or portions thereof (eg, ligand-binding fragments and the like) can be recovered from host cells, and the sequence rapidly determined using modern equipment. These methods allow rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

The present invention thus provides a series of new hybrid molecules in which a segment comprising one or more of the soluble zalpha11 receptor domains is fused to another soluble receptor polypeptide. Fusion is preferably done by splicing at the DNA level to allow expression of chimeric molecules in recombinant production systems. The resulting molecules are then tested for properties such as improved solubility, improved stability, extended clearance half-life, improved secretion and expression levels, and pharmacodynamics. Such hybrid molecules may further comprise additional amino acid residues (eg, a polypeptide linker) between the component proteins or polypeptides.

Using the methods discussed herein, one of ordinary skill in the art can identify and / or prepare a variety of fragments or variants of polypeptides of SEQ ID NO: 6 that retain antagonist or zalpha11 Ligand binding activity. For example, a soluble zalpha11 receptor can be made by preparing a variety of polypeptides that are substantially homologous to the cytokine binding domain (residues 20 (Cys) to 237 (His) of SEQ ID NO:

ES 2 279 809 T3 (SEQ ID NO: 6), or a subsequence thereof that binds to Ligand of zalpha11 or allelic variants or species orthologs thereof) and retain ligand-binding activity of wild-type zalpha11 protein . Such polypeptides can include additional amino acids from, for example, part or all of the signal peptide sequence, transmembrane and intracellular domains. Such polypeptides can also include additional polypeptide segments as generally described herein such as tags, affinity tags, and the like. Similarly, one of ordinary skill in the art can identify and / or prepare a variety of polypeptide fragments or variants of SEQ ID NO: 4, or other soluble class I cytokine receptors that form zalpha11 receptor heterodimers.

For any soluble zalpha11 receptor polypeptide, including variants and fusion or protein polypeptides, one of ordinary skill in the art can easily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.

The soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides of the present invention, including full-length soluble receptor polypeptides, biologically active fragments, and fusion polypeptides can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include cultured bacteria, fungal cells, and higher eukaryotic cells. Eukaryotic cells are preferred, particularly cultured cells of multicellular organisms. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are described as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^to ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., reds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a zalpha11 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcriptional promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that, within certain systems, selectable markers can be provided in separate vectors, and replication of exogenous DNA can be provided by integration. in the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors, and other elements is a matter of routine design within the level of ordinary skill in the art. Many of these items are described in the literature and are available from commercial suppliers.

To direct a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence) is provided in the expression vector. , prepro sequence or pre sequence). The secretory signal sequence can be that of the zalpha11 receptor described here, IL-2Ry (amino acid 1 (Met) to 22 (Gly) of SEQ ID NO: 18), or it can be derived from another secreted protein (for example, t- PA) or synthesized de novo. The secretory signal sequence is operably linked to the zalpha11 DNA sequence, that is, the two sequences are linked in the correct reading frame and positioned to direct the newly synthesized polypeptide to the secretory pathway of the host cell. Secretory signal sequences are commonly located 5 'to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be located elsewhere in the DNA sequence of interest (see, for example, Welch et al. ., US Patent No. 5,037,743; Holland et al., US Patent No. 5,143,830).

Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52 : 456, 1973), electroporation (Neumann et al., EMBO J. J_: 841-845, 1982), DEAE-dextran-mediated transfection (Ausubel et al., Ibid), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15: 73, 1993; Ciccarone et al., Focus 15: 80, 1993) and viral vectors (Miller and Rosman, BiuoTechniques 7: 980-90, 1989, Wang and Finer, Nature Med. 2: 714-716, 1996). The production of recombinant polypeptides in cultured mammalian cells is described, for example, by Levinson et al., US Pat. No. 4,713,339; Hagen et al., US Patent No. 4,784,950; Palmiter et al., US Patent No. 4,579,821; and Ringold, US Patent No. 4,656,134. Suitable cultured mammalian cells include the cell lines COS-1 (ATCC No. CRL 1650), COS7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen Virol. 36: 59.72, 1977) and Chinese hamster ovary (eg CHO-K1; ATcc No. CCL 61). Additional suitable cell lines are known in the art and are available from public repositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus (CMV). See, for example, US Pat. No. 4,956,288. Other suitable promoters include those for metallothionein genes (US Pat. Nos. 4,579,821 and 4,601,978) and the major adenovirus late promoter.

Drug screening is generally used to select cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants." Cells that have been cultured in the presence of the selective agent and are capable of passing the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is done in the presence of a neomycin-like drug, such as G-418 or the like. They can

ES 2 279 809 T3 also used selection systems to increase the level of expression of the gene of interest, a procedure referred to as "multiplication". Multiplication is accomplished by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of the selective agent to select for cells that produce high levels of the introduced gene products. A preferred multiplicable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (eg hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, MHC Class I, placental alkaline phosphatase, can be used to separate transfected cells from non-transfected cells by means such as sorting. FACS, flow cytometry, or magnetic bead separation technology.

Other higher eukaryotic cells, including plant cells, insect cells, and bird cells, can also be used as hosts. The use of Agrobacterium rhizogenes as a vector to express genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11: 47-58 (1987). The transformation of insect cells and the production of foreign polypeptides in them is described by Guarino et al., US Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See King, LA and Possee, RD, The Baculovirus Expression System: A Laboratory Guide, London, Chapman &amp; Hall, O'Reilly, DR et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994 ; and Richardson, CD, red., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. A second method for producing recombinant zalpha11 baculovirus uses a transposon-based system described by Luckow (Luckow, VA et al., J Virol 67: 4566-79, 1993) . This system, which uses transfer vectors, is sold in the Bac-to-Bac® kit (Life Technologies, Rockville, MD). This system uses a transfer vector, pFastBac1^® (Life Technologies) containing a Tn7 transposon to move DNA encoding zalpha11 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." See Hill-Perkins, MS and Possee, RD, J Gen Virol 71: 971-6, 1990; Bonning, BC et al., J Gen Virol 75: 1551-6, 1994; and Chazenbalk, GD and Rapoport, B., J Biol Chem 270: 1543-9, 1995. In addition, transfer vectors may include a DNA-framed fusion encoding an epitope tag at the C or N termini of the expressed zalpha11 polypeptide, eg, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82: 79524, 1985). Using a method known in the art, it is transformed into E. coli a transfer vector containing zalpha11, and bacmids containing a disrupted lacZ gene, indicative of recombinant baculovirus, are examined. Bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, eg, Sf9 cells. Recombinant virus that expresses zalpha11 is then produced. Recombinant viral strains are produced by methods commonly used in the art.

The recombinant virus is used to infect host cells, typically a cell line derived from the late military caterpillar, Spodoptera frugiperda. See, generally, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, DC, 1994. Suitable cell control lineage is High FiveO® (Invitrogen) derived from Trichoplusia ni (US Pat. No. 5,300,435). Commercially available serum-free medium is used to grow and maintain cells. Suitable media are Sf900 II^® (Life Technologies) or ESF 921® (Expression Systems) for Sf9 cells; and Ex-cellO405® (JRH Biosciences, Lenexa, KS) or Express FiveO® (Life Technologies) for T ni cells. The procedures used are generally described in available laboratory manuals (King, LA and Possee, RD, ibid .; O'Reilly, DR et al., Ibid .; Richardson, CD, ibid.). Subsequent purification of the zalpha11 polypeptide from the supernatant can be accomplished using methods described herein.

Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are described, for example, by Kawasaki, US Patent No. 4,599,311; Kawasaki et al., US Pat. No. 4,931,373; Brake, US Patent No. 4,870,008; Welch et al., US Patent No. 5,037,743; and Murray et al., US Patent No. 4,845,075. Transformed cells are selected for phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (eg, leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system described by Kawasaki et al. (US Patent No. 4,931,373), which allows transformed cells to be selected for growth in glucose-containing media. Preferred promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, eg, Kawasaki, US Pat. No. 4,599,311; Kingsman et al., US Pat. No. 4,615,974; and Bitter, US Patent No. 4,977,092) and alcohol dehydrogenase genes. See also US Patent Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts are known in the art, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii, and Candida maltosa. See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459-3465, 1986 and Cregg, US Patent No. 4,882,279. Aspergillus cells can be used according to the methods of McKnight et al., US Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are described by Sumino et al., US Patent No. 5,162,228. Methods for transforming Neurospora are described by Lambowitz, US Patent No. 4,486,533.

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The use of Pichia methanolica as a host for the production of recombinant proteins is described in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536 and WO 98/02565. DNA molecules of use for transforming P. methanolica will commonly be prepared as circular double-stranded plasmids that are preferably linearized prior to transformation. For polypeptide production in P. methanolica, it is preferred that the promoter and terminator on the plasmid be that of a P. methanolica, such as an alcohol utilization gene from P. methanolica (AUG1 or AUG2). Other useful promoters include those for the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a Pichia methanolica ADE2 gene, encoding phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. For large scale industrial procedures, where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells defective in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica by electroporation using an exponentially decreasing pulsed electric field, having a field strength of 2.5 to 4.5 kv / cm, preferably around 3.75 kv / cm, and a time constant (t) of 1 to 40 milliseconds, more preferably around 20 milliseconds.

Also useful within the present invention are prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus, and other genera. Methods for transforming these hosts and expressing foreign DNA sequences cloned into them are well known in the art (see, for example, Sambrook et al., Ibid.). When a zalpha11 polypeptide is expressed in bacteria such as E. coli, the polypeptide can be conserved in the cytoplasm, typically as insoluble granules, or it can be directed to the periplasmic space by a bacterial secretion sequence. In the first case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recover the protein, thereby obviating the need for denaturation and folding.

The transformed or transfected host cells are cultured according to standard procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media are known in the art, including defined media and complex media, and generally include a carbon source, a nitrogen source, essential amino acids, vitamins, and minerals. The media can also contain components such as growth factors or serum, as required. The growth medium will generally select for cells that contain the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient that is complemented by the selectable marker carried in the expression vector or co-transfected into the host cell. Cells of P. methanolica in a medium comprising suitable carbon, nitrogen and trace nutrient sources at a temperature of approximately 25 ° C to 35 ° C. Liquid cultures with sufficient aeration are provided by conventional means, such as shaking small flasks or bubbling fermenters. A preferred culture medium for P methanolica is YEPD (2% D-glucose, 2% Bacto® Peptone (Difco Laboratories, Detroit, MI), 1% Bacto® Yeast Extract (Difco Laboratories), 0.004% Adenine and 0.006% L-leucine).

Mammalian cells of suitable use for testing antagonist activity of the new soluble receptors of the present invention express a zalpha11 receptor or receptor fusion capable of signaling and transducing a receptor-mediated signal of the zalpha11 Ligand. Such cells include cells that express a β subunit, such as gp130, IL-2Ry, and cells that co-express receptors (Gearing et al., EMBO J. 10: 2839-2848, 1991); Gearing et al., US Pat. No. 5,284,755). In this regard it is generally preferred to employ a cell that responds to other cytokines that bind to receptors of the same subfamily, such as IL-6 or LIF, because such cells contain the required signal transduction pathway (s) ( s). Preferred cells of this type include the human TF-1 cell line (ATCC number CRL-2003) and the DA-1 cell line (Branch et al., Blood 69: 1782, 1987; Broudy et al., Blood 75: 1622 -1626.1990). Alternatively, suitable host cells can be designed to produce a β subunit or other cellular component necessary for the desired cellular response. For example, the mouse cell line BaF3 (Palacios and Steinmetz, Cell 41: 727-734, 1985; Mathey-Provot et al., Mol. Cell. Biol. 6: 4133-4135, 1986) has been used to produce a Cell line that responds to the zalpha11 Ligand (see Examples). Other such lineages include a small hamster kidney (BHK) cell line, or the CTLL-2 (ATCC TIB-214) cell line can be transfected to express a II, -2Ry subunit in addition to the zalpha11 receptor. It is generally preferred to use a host cell and receptor (s) from the same species, although this method allows designing cell lines to express multiple receptor subunits from any species, thereby overcoming potential limitations arising from species specificity. Alternatively, species homologs of the human receptor cDNA can be cloned and used within cell lines of the same species, such as a mouse cDNA in the BaF3 cell line. Thus, cell lines that are dependent on a hematopoietic growth factor, such as IL-3, can be designed to become dependent on the zalpha11 ligand. Such cells can be used as described herein in the presence of zalpha11 Ligand to assess the antagonist activity of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry on signaling and proliferative activity of zalpha11 Ligand.

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Cells expressing soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, are used within screening assays. A variety of suitable assays are known in the art. These assays are based on the detection of a biological response in the target cell to the zalpha11 Ligand in the presence or absence of the soluble receptors of the present invention. Such an assay is a cell proliferation assay. Cells are cultured in the presence or absence of zalpha11 Ligand with or without the addition of other cytokines or proliferative agents and cell proliferation is detected, for example, by measuring the incorporation of tritiated thymidine or by colorimetric assay based on the metabolic breakdown of Alamar Blue® ( AccuMed, Chicago, IL) or 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (MTT) bromide (Mosman, J. Immunol. Meth. 65: 55-63,1983). An alternative assay format uses cells that are further engineered to express a reporter gene. The reporter gene is linked to a promoter element that responds to the receptor-linked pathway, and the assay detects the activation of reporter gene transcription. DNA response elements may include, but are not limited to, Cyclic AMP Response Elements (CRE), Hormone Response Elements (HRE), Insulin Response Element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87: 5273-7, 1990) and serum response elements (SRE) (Shaw et al., Cell 56: 563-72, 1989). Cyclic AMP response elements are discussed in Roestler et al., J. Biol. Chem. 263 (19): 9063-6; 1998 and Habener, Molec. Endocrinol. 4 (8): 1087-94, 1990. Hormone response elements are discussed in Beato, Cell 56: 335-44; 1989. A preferred promoter element in this regard is a serum responsive element or SRE (see, eg, Shaw et al., Cell 56: 563-572, 1989). One such preferred reporter gel is a luciferase gene (de Wet et al., Mol. Cell. Biol. 7: 725, 1987). Luciferase gene expression is detected by luminescence using methods known in the art (eg, Baumgartner et al., J. Biol. Chem. 269: 1909429101, 1994; Schenborn and Goiffin, Promega Notes 41: 11, 1993). Luciferase assay kits are commercially available from, for example, Promega Corp., Madison, WI. Target cell lines of this type can be used to screen chemical libraries, cell-conditioned culture media, mushroom broths, soil samples, water samples, and the like for antagonistic activity. Such cells can be used as described herein in the presence of zalpha11 Ligand in a competitive inhibition-type assay to assess the antagonist activity of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry in signaling activity and Zalpha11 Ligand proliferative.

T and B cell proliferation assay methods can also be used to assess the antagonist activity of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry on signaling and proliferative activity of zalpha11 Ligand in the presence of other cytokines, eg, IL-15, Flt3, and the like. Such assays are described herein in the examples, and are known in the art. Briefly, using flow cytometry, subsets of mature or immature T cells or B cells are isolated based on the presence or absence of various cell surface molecules (e.g., CD4, CD8, CD19, CD3, CD40, CD28, etc. .). Cells can be selected before or after exposure to Zalpha11 Ligand, depending on the type of cell studied and the effect on them of Zalpha11 Ligand. The soluble receptors or antibodies of the present invention can be added in a range of concentrations to assess antagonist or binding activity on the Ligand in the T-cell or B-cell proliferation assay. Such assays are well known in the art and are described. here.

In addition, a secretion trap method employing soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides can be used to isolate transfected cells expressing zalpha11 Ligand. See for method Aldrich et al., Cell 87: 1161-1169, 1996. A cDNA expression library prepared from a known or suspected source of ligand is transfected into COS-7 cells. The cDNA library vector generally has an SV40 origin for multiplying in COS-7 cells and a CMV promoter for high expression. The transfected COS-7 cells are grown in a monolayer and then fixed and permeabilized. The soluble zalpha11 receptor or biotin-labeled or soluble zalpha11 heterodimeric receptor polypeptides, described herein, are then contacted with the layer of cells and allowed to bind to cells of the monolayer that express a complementary anti-molecule, ie. say, a Ligand of zalpha11. A cell that expresses a ligand will thus bind with receptor molecules. An anti-tag antibody (anti-Ig for Ig fusions, M2 or anti-FLAG for FLAG-tagged fusions, streptavidin, and the like) that is conjugated to horseradish peroxidase (HRP) is used to visualize these cells to which are has bound soluble zalpha11 receptor or biotin-labeled or soluble zalpha11 heterodimeric receptor polypeptides. HRP catalyzes the deposition of a tyramide reagent, eg, tyramide-FITC. A commercially available kit (eg, Renaissance TSA-Direct® Kit; NEN Life Science Products, Boston, MA) can be used for this detection. Cells expressing zalpha11 receptor ligand will be identified under fluorescence microscopy as green cells, and harvested for subsequent cloning of the ligand using procedures for plasmid rescue as outlined in Aldrich et al., Supra, followed by subsequent rounds of the trap assay. secretion until single clones are identified.

In addition, histological and immunohistochemical methods employing soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides can be used to identify cells and tissue cells expressing zalpha11 Ligand. Such methods are known in the art and are described herein.

Additional assays to detect soluble zalpha11 receptor antagonist or binding activity or soluble zalpha11 heterodimeric receptor polypeptides provided by the present invention include the use of hybrid receptor polypeptides. These hybrid polypeptides belong to two general classes. Within the first class, the intracellular domain of zalpha11, comprising approximately residues 256 (Lys) to 528 (Ser) of SEQ ID NO: 2, is linked to the ligand-binding domain of a second receptor. It is preferred that the second receptor is a hematopoietic cytokine receptor, such as an mpl receptor (Souyri et al., Cell 63: 1137-1147, 1990). The hybrid receptor will further comprise a transmembrane domain, which can be derived from any receptor. A construc25

ES 2 279 809 T3 DNA encoding the hybrid receptor is then inserted into a host cell. Cells expressing the hybrid receptor are then cultured in the presence of a ligand for the binding domain and a response is assayed. This system provides a means to analyze zalpha11-mediated signal transduction while using readily available ligands. This system can also be used to determine whether particular cell lines are capable of responding to signals transduced by zalpha11. A second class of hybrid receptor polypeptides comprises the extracellular (ligand binding) domain of zalpha11 (approximately residues 20 (Cys) to 237 (His) of SEQ ID NO: 2) with a cytoplasmic domain of a second receptor, preferably a cytokine receptor, and a transmembrane domain. The transmembrane domain can be derived from any receptor. Such hybrid receptors are expressed in cells known to be capable of responding to signals transduced by the receptor comprising the extracellular domain, such as in the presence of the zalpha11 Ligand. Addition of soluble zalpha11 receptor or soluble zalpha11 receptor polypeptides , in the presence of zalpha11 Ligand, is used to assess the soluble zalpha11 receptor antagonist or binding activity of zalpha11 ligand or zalpha11 heterodimeric receptor polypeptides. soluble for the zalpha11 Ligand.

The tissue specificity and biological activities of zalpha11 Ligand expression suggest a role in early development of NK cells and thymocytes, expansion of mature B cells, stimulation of the general immune response, and regulation of the immune response. These processes involve stimulation of cell proliferation and differentiation in response to the binding of the zalpha11 Ligand to its cognate receptor, which comprises at least one zalpha11 receptor subunit. In view of the observed biological activity for this Ligand, antagonists have enormous potential in in vitro and in vivo applications. As antagonists of the zalpha11 ligand, soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides may find utility in suppressing the immune system, such as in the treatment of autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, diabetes mellitus, disease inflammatory bowel, Crohn's disease, and the like. Immune suppression can also be used to reduce rejection of tissue or organ transplants and grafts and to treat B-cell malignancies, T-cell specific leukemias or lymphomas, by inhibiting the proliferation of the affected cell type.

Soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides can also be used within diagnostic systems for the detection of circulating levels of zalpha11 Ligand. Within a related embodiment, antibodies or other agents that specifically bind to soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides can be used to detect circulating receptor polypeptides. Elevated or lowered levels of Ligand or receptor polypeptides can be indicative of disease states, including cancer. Soluble receptor polypeptides can contribute to disease processes and can be a surrogate marker for an underlying disease. For example, elevated levels of soluble IL-2 receptor in human serum have been associated with a wide variety of inflammatory and neoplastic conditions, such as myocardial infarction, asthma, myasthenia gravis, rheumatoid arthritis, acute T-cell leukemia, lymphomas of B cells, chronic lymphocytic leukemia, colon cancer, breast cancer, and ovarian cancer (Heaney et al., Blood 87: 847-857, 1996).

A zalpha11 receptor ligand binding polypeptide, such as soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, can be prepared by expressing a truncated DNA encoding the zalpha11 cytokine binding domain (approximately from residue 20 (Cys) to residue 237 (His) of the human receptor (SEQ ID NO: 2) (SEQ ID NO: 6) or the corresponding region of a non-human receptor (eg, SEQ ID NO: 12). A soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, can be prepared by co-expressing a truncated DNA encoding the zalpha11 cytokine-binding domain (SEQ ID NO: 6) and the truncated DNA encoding the extracellular domain. from another class I cytokine receptor, such as IL-2Ry (SEQ ID NO: 4). It is preferred to prepare the extracellular domains of the soluble zalpha11 homodimer or heterodimer in a form essentially free of transmembrane and intracellular polypeptide segments. Furthermore, ligand-binding polypeptide fragments within the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide (eg, soluble zalpha11 / IL-2Ry), or cytokine-binding domain, described above, may also serve as soluble zalpha11 receptors. for the uses described here. To direct the export of a receptor polypeptide from the host cell, the receptor DNA is linked to a second DNA segment that encodes a secretory peptide, such as a secretory peptide of t-PA, a secretory peptide of another cytokine receptor , another secreted molecule, or a zalpha11 receptor secretory peptide. To facilitate purification of the secreted receptor polypeptide, a C-terminal extension, such as a poly-histidine tag, substance P, Flag® peptide (Hopp et al., Bio / Technology 6: 1204-1210, 1988; available from Eastman Kodak Co., New Haven, CT), a Glu-glu tag (SEQ ID NO: 14), or other polypeptide or protein for which an antibody or other specific binding agent is available.

In an alternative method, an extracellular domain of the receptor can be expressed as a fusion with immunoglobulin heavy chain constant regions, typically an Fc fragment, which contains two constant region domains and lacks the variable region. Such fusions are typically secreted as multimeric molecules in which the Fc portions are disulfide linked to each other and two receptor polypeptides are placed in close proximity to each other. Fusions of this type can be used to affinity purify cognate ligand from solution, as an in vitro assay tool, to block signals in vitro by specifically titering Ligand, and as antagonists in vivo by administering them parenterally to bind circulating ligand and separate them from the circulation. To purify the ligand, a zalpha11-Ig chimera (eg, Zalpha11-Fc4 described herein) is added to a sample containing the ligand (eg, culture media or tissue extracts with conditioned cells) under conditions that facilitate binding. receptor-ligand (typically almost physiological temperature, pH and ionic strength). The complex

ES 2 279 809 T3 chimera-ligand is then separated by mixing using protein A, immobilized on a solid support (eg insoluble resin beads). The ligand is then eluted using standard chemical techniques, such as with a salt or pH gradient. Alternatively, the chimera itself can be attached to a solid support, binding and eluting as before. The collected fractions can be refracted to the desired level of purity.

Furthermore, soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11 / IL-2Ry, can be used as a "ligand sink", ie antagonist, to bind ligand in vivo or in vitro in therapeutic applications. or others in which the presence of the ligand is not desired. For example, in cancers expressing large amounts of bioactive zalpha11 Ligand, soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11 / IL-2Ry, can be used as a direct antagonist of the ligand in vivo, and can help reduce progression and symptoms associated with the disease, and can be used in conjunction with other therapies (for example, chemotherapy) to increase the effect of therapy in reducing progression and symptoms and preventing relapse. In addition, soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11 / IL-2Ry, can be used to slow the progression of cancers that overexpress zalpha11 receptors, binding to ligand in vivo that would otherwise increase proliferation of those cancers.

Furthermore, soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11 / IL-2Ry, can be used in vivo or in diagnostic applications to detect zalpha11 Ligand-expressing cancers in vivo or in tissue samples. For example, soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11 / IL-2Ry, can be conjugated to a radiolabel or fluorescent label as described herein, and used to detect the presence of the zalpha11 Ligand in a tissue sample using an in vitro ligand-receptor binding assay, or a fluorescent imaging assay. In addition, a soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble, radiolabeled zalpha11 / IL-2Ry could be administered in vivo to detect Ligand-expressing solid tumors via a radio-imaging method known in the art. technique.

It is preferred to purify the polypeptides of the present invention to&gt; 80% purity, more preferably&gt; 90% purity, even more preferably&gt; 95% purity, and a pharmaceutically pure state, which is greater than 99% pure with with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious agents and pyrogens. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.

Soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11 / IL-2Ry (or zalpha11 fusion or chimeric polypeptides), expressed recombinants can be purified using fractionation and / or conventional purification methods and means. They can be used for fractionation of samples, ammonium sulfate precipitation and acid or chaotrope extraction. Examples of purification steps can include hydroxyapatite, size exclusion, FPLC, and reverse phase high performance liquid chromatography. Suitable chromatographic media include derived dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. Derivatives of PEI, DEAE, QAE and Q are preferred. Examples of chromatographic media include media derived with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl-butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia), and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins, and the like, which are insoluble under the conditions in which they are to be used. These supports can be modified with reactive groups that allow the incorporation of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate residues. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. The selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles &amp; Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated by exploiting their biochemical, structural, and biological properties. For example, immobilized metal ion adsorption chromatography (IMAC) can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first loaded with divalent metal ions to form a chelate (Sulkowski, Tends in Biochem. 3: 1-7, 1985). Histidine-rich proteins will be adsorbed on this matrix with different affinities, depending on the metal ion used, and will be eluted by competitive elution, lowering the pH, or the use of strong chelating agents. Other purification methods include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher (red.), Academic Press, San Diego, 1990, pp. 529-39). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (eg, maltose-binding protein, an immunoglobulin domain) can be constructed to facilitate purification. In addition, zalpha11 Ligand affinity columns can be used to purify soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11 / IL-2Ry. Such affinity chromatography methods are well known in the art.

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Furthermore, using methods described in the art, polypeptide fusions, or hybrid zalpha11 proteins, are constructed using regions or domains of the zalpha11 of the invention in combination with those of other proteins of the human cytokine receptor family, or heterologous proteins ( Sambrook et al., Ibid., Altschul et al., Ibid., Picard, Cur. Opin. Biology, 5: 511-5, 1994, and references therein). These methods make it possible to determine the biological importance of major domains or regions in a polypeptide of interest. Such hybrids can alter reaction kinetics, bind, narrow, or expand substrate specificity, or alter the tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.

Soluble receptor fusion proteins or polypeptides can be prepared by methods known to those of skill in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding one or more components of the fusion protein in the appropriate reading frame can be generated using known techniques, and expressed by the methods described herein. For example, part or all of a domain (s) that confer (s) a biological function can be exchanged between zalpha11 of the present invention with the functionally equivalent domain (s) of another member of the cytokine family. Such domains include, but are not limited to, the extracellular cytokine binding domain, ligand and residue binding domain, transmembrane domain, as described herein. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known family proteins, depending on the fusion constructed. In addition, such fusion proteins can exhibit other properties as described herein.

Standard molecular cloning and biological techniques can be used to swap the equivalent domains between the zalpha11 polypeptide and the polypeptides to which it is fused. In general, a segment of DNA that encodes a domain of interest, for example, a domain of zalpha11 described herein, is operably linked framed with at least one other segment of DNA that encodes an additional polypeptide (for example, a domain or region of another cytokine receptor, such as IL-2Ry receptor) and inserted into an appropriate expression vector, as described herein. Generally, DNA constructs are made such that the various DNA segments encoding corresponding regions of a polypeptide are operably linked framed to make a single construct encoding the entire fusion protein, or a functional portion thereof. For example, a DNA construct would encode from the N-terminus to the C-terminus for a fusion protein comprising a signal polypeptide followed by a cytokine binding domain. Such fusion proteins can be expressed, isolated, and assayed for activity as described herein.

Soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as zalpha11 / IL-2Ry polypeptides, or fragments thereof, can also be prepared by chemical synthesis. Such polypeptides can be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.

The polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85: 2149, 1963; and Kaiser et al., Anal. Biochem. 34: 595, 1970. After the complete synthesis of the desired peptide on a solid support, the peptide-resin is with a reagent that cleaves the polypeptide from the resin and removes a good part of the protecting groups from side chains. . Such methods are well established in the art.

The activity of molecules of the present invention can be measured using a variety of assays that measure cell differentiation and proliferation. Such assays are well known in the art and are described herein.

The proteins of the present invention are useful, for example, in the treatment of lymphoid, immune, hematopoietic, inflammatory and similar disorders, and can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model. For example, host cells expressing a soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11 / IL-2Ry, can be embedded in an alginate environment and injected (implanted) into recipient animals. Alginate-poly-L-lysine microencapsulation, permsolective membrane encapsulation, and diffusion chambers are a means of entrapment of transfected mammalian cells or primary mammalian cells. These types of non-immunogenic "encapsulations" allow the diffusion of proteins and other macromolecules secreted or released by the captured cells to the recipient animal. Most importantly, the capsules hide and protect the embedded foreign cells from the immune response of the recipient animal. Such encapsulations can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells). Alginate fibers provide a simple and rapid means of generating embedded cells.

The materials necessary to generate the alginate fibers are known in the art. In an example procedure, prepare 3% alginate H₂Or sterile water, and it is sterile filtered. Immediately before preparing the alginate fibers, the alginate solution is filtered again. A suspension of cells of approximately 50% (containing about 5 x 10⁵ at approximately 5 x 10⁷ cells / ml) is mixed with 3% alginate solution. One ml of the alginate / cell suspension is extruded into a CaCl solution₂ sterile filtered 100 mM over a time period of ~ 15 min, forming a "fiber". The extracted fiber is then transferred to a CaCl solution₂fifty mM and then to a 25 mM CaCl2 solution. The fiber is then rinsed with deionized water before coating the fiber by incubating in a 0.01% solution of poly-L-lysine. Finally, the fiber is rinsed with Ringer's Solution

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Lactated and withdrawn from solution in a syringe barrel (without needle). A large gauge needle is then attached to the syringe, and the fiber is injected intraperitoneally into a recipient in a minimal volume of Lactated Ringer's Solution.

Adenoviral and other viral systems, such as vaccinia viruses, can be used to express and produce the proteins of the present invention. For example, using adenovirus vectors in which portions of the adenovirus genome have been deleted, inserts are incorporated into viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential E1 gene has been deleted from the viral vector, and the virus will not replicate unless the E1 gene is provided by the host cell (human 293 cell line is an example). If the adenoviral delivery system has an E1 gene deletion, the virus cannot replicate in human cells, but will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Furthermore, by deleting the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated. The generation of so-called "contentless" adenoviruses in which all viral genes are deleted is particularly advantageous for insertion of large heterologous DNA inserts. For a review, see Yeh, P. and Perricaudet, M., FASEB J. 11: 615-623, 1997.

The adenovirus system can be used for in vitro protein production. By growing non-293 cells infected with adenovirus under conditions in which the cells do not divide rapidly, the cells can produce proteins for extended periods of time. For example, BHK cells are grown to confluence in cell factories, and exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, allowing the infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (see Garnier et al., Cytotechnol. 15: 145-55, 1994) . With either method, an expressed and secreted heterologous protein can be isolated repeatedly from culture supernatant, lysate, or cell membrane fractions depending on the disposition of the expressed protein in the cell. Within the method of producing infected 293 cells, non-secreted proteins can also be efficiently obtained.

Soluble zalpha11 receptor antagonists or soluble zalpha11 receptor polypeptides, such as soluble zalpha11 / IL-2Ry, can be used in vitro in an assay to measure a decrease in stimulus of colony formation by zalpha11 Ligand from marrow cultures. isolated primary bone. Such assays are described herein and are well known in the art.

Zalpha11 Ligand antagonists and binding agents are also useful as research reagents to characterize sites of ligand-receptor interaction. Inhibitors of zalpha11 Ligand activity (zalpha11 Ligand antagonists) include soluble anti-zalpha11 receptor or anti-soluble zalpha11 heterodimeric receptor polypeptide antibodies, such as soluble anti-zalpha11 / IL-2Ry antibodies and soluble zalpha11 receptor or polypeptides. soluble zalpha11 heterodimeric receptors, such as soluble zalpha11 / IL-2Ry receptors, as well as other peptide and non-peptide agents (including ribozymes).

A soluble zalpha11 receptor ligand binding polypeptide or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11 / IL-2Ry of the present invention, can also be used to purify zalpha11 Ligand. The polypeptide is immobilized on a solid support, such as agarose beads, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or similar materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting medium will generally be configured in the form of a column, and ligand-containing fluids are passed through the column one or more times to allow the ligand to bind to the receptor polypeptide. The ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl), or pH to break ligand-receptor binding.

An assay system using a ligand-binding receptor (or an antibody, a member of a complement / anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (for example, BIAcore^®, Pharmacia Biosensor, Piscataway, NJ; or SELDI technology^®, Ciphergen, Inc. Palo Alto, CA). Such a receptor, antibody, member of a complement / anti-complement pair, or fragment is immobilized on the surface of a receptor chip. The use of this instrument is described by Karlsson, J. Immunol. Methods 145: 229-240, 1991 and Cunningham and Wells, J. Mol. Biol. 234: 554-63, 1993. A receptor, antibody, member or fragment is covalently incorporated, using amine or sulfhydryl chemistry, into dextran fibers that are incorporated into gold film within the flowing cell. A test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement / anti-complement pair is present in the sample, it will bind to the receptor, antibody, or immobilized member, respectively, causing a change in the refractive index of the medium, which is detected as a change. from the resonance of the surface plasmon of the gold film. This system allows the determination of ratios on and off, from which the binding affinity can be calculated, and the binding stoichiometry can be assessed.

Ligand binding receptor polypeptides, such as those of the present invention, can also be used within other assay systems known in the art. Such systems include Scatchard analysis to determine29

ES 2 279 809 T3 nar binding affinity (see Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949) and calorimetric assays Cunningham et al., Science 253, 545-48, 1991; Cunningham et al., Science 245, 821-25, 1991).

Soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry polypeptides, can also be used to prepare antibodies that bind to epitopes, peptides or polypeptides contained within the antigen. The zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry polypeptides or a fragment thereof, serves as an antigen (immunogen) to inoculate an animal and elicit an immune response. One skilled in the art would recognize that epitope-bearing antigenic polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a soluble zalpha11 receptor or heterodimeric zalpha11 polypeptide, such as soluble zalpha11 / IL-2Ry polypeptides (eg, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 4). Included are polypeptides that comprise a major portion of a soluble zalpha11 receptor or heterodimeric zalpha11 polypeptide, such as soluble zalpha11 / IL-2R polypeptides, ie, 30 to 100 residues up to the full length of the amino acid sequence. Immunogenic antigens or epitopes may also include incorporated tags, adjuvants, and carriers, as described herein. Suitable antigens include the zalpha11 polypeptide encoded by SEQ ID NO: 2 from amino acid number 20 (Cys) to amino acid number 237 (His) (SEQ ID NO: 6), or a contiguous AA fragment of amino acids 9 to 218. . Preferred peptides for use as antigen are the cytokine binding domain, described herein, and hydrophilic peptides of zalpha11 such as predicted by one skilled in the art from a hydrophobicity plot, determined for example from a hydrophilicity profile of Hopp / Woods based on a six residue sliding window, ignoring masked G, S and T residues and exposed H, Y and W residues. For example, zalpha11 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: (1) amino acid number 51 (Trp) to amino acid number 61 (Glu) of SEQ ID NO: 2; (2) from amino acid number 136 (Ile) to amino acid number 143 (Glu) of SEQ ID NO: 2; (3) from amino acid number 187 (Pro) to amino acid number 195 (Ser) of SEQ ID NO: 2; and (4) from amino acid number 223 (Phe) to amino acid number 232 (Glu) of SEQ ID NO: 2. The corresponding hydrophilic regions with reference to SEQ ID NO: 6 can be made with reference to the above amino acid residues of SEQ ID NO: 2. Furthermore, epitope-bearing polypeptide antigens are suitable antigens as predicted by a Jameson-Wolf plot, for example, using a DNASTAR Protean program (DNASTAR, Inc., Madisol, WI). In addition, conserved motifs, and variable regions between conserved zalpha11 soluble receptor motifs, are suitable antigens. Suitable antigens also include the zalpha11 polypeptides described above in combination with another extracellular cytokine class I domain, such as those that form soluble zalpha11 heterodimeric polypeptides, such as soluble zalpha11 / IL2Ry. In addition, corresponding regions of the soluble mouse zalpha11 receptor polypeptide (residues 20 (Cys) to 237 (His) SEQ ID NO: 12) can be used to generate antibodies against the soluble mouse zalpha11 receptor. In addition, antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan et al. (reds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, JGR, Red., Monoclonal Hybridoma Antibodies; Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982.

As would be apparent to one of ordinary skill in the art, polyclonal antibodies can be generated by incoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a soluble zalpha11 receptor or heterodimeric polypeptide. of soluble zalpha11, such as a soluble zalpha11 / IL-2Ry polypeptide or a fragment thereof. The immunogenicity of a zalpha11 polypeptide can be increased by the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Useful polypeptides for immunization also include fusion polypeptides, such as fusions of zalpha11 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The immunogenic polypeptide can be a full-length molecule or a portion thereof. If the portion of the polypeptide is "hapten-like", such portion may advantageously be linked or linked to a macromolecular vehicle (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization. .

As used herein, the term "antibodies" includes polyclonal antibodies, affinity purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F (ab ') proteolytic fragments.₂and Fab. Intact antibodies or genetically engineered fragments are also included, such as chimeric antibodies, Fv fragments, single chain antibodies, and the like, as well as synthetic antigen-binding peptides and polypeptides. Non-human antibodies can be humanized by grafting non-human CDRs into human frames and constant regions, or incorporating the entire non-human variable domains (optionally "covering" them with a human-like surface by substitution of exposed residues, in which the result is an antibody "plated"). In some cases, humanized antibodies can conserve non-human residues within human variable region framework domains to enhance appropriate binding characteristics. By humanizing antibodies, the biological half-life can be increased, and the potential for adverse immune reactions upon administration to humans is reduced.

Antibodies are considered to bind specifically if they: 1) exhibit a threshold level of binding activity and 2) do not significantly cross-react with related polypeptide molecules. A threshold level of binding is determined whether soluble anti-zalpha11 receptor or anti-soluble zalpha11 heterodimeric polypeptide antibodies, such as soluble anti-zalpha11 / IL-2Ry, bind here to a soluble zalpha11 receptor or zalpha11 heterodimeric polypeptide.

ES 2 279 809 T3 soluble, such as soluble zalpha11 / IL-2R polypeptide, peptide or epitope with an affinity at least 10 times greater than the binding affinity to control polypeptide (soluble non-zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble anti-zalpha11 / IL-2Ry). It is preferred that the antibodies have a binding affinity (K_to) from 10⁶ M ¹ or higher, preferably 10⁷ M ¹ or greater, more preferably 10⁸ M ¹ or higher, and even more preferably 10⁹ M ¹ or older. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).

Whether soluble anti-zalpha11 receptor or soluble anti-zalpha11 heterodimeric polypeptide antibodies, such as soluble anti-zalpha11 / IL-2Ry do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody that detects soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry polypeptide, but not known related polypeptides using standard Western blot analysis (Ausubel et al., ibid.). Examples of known related polypeptides are those described in the prior art, such as known orthologs and paralogs, and similar known members of a protein family. Screening can also be done using soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, non-human, and soluble zalpha11 receptor mutant polypeptides or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry. . In addition, antibodies "against" known related polypeptides can be screened to isolate a population that specifically binds to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry polypeptides. For example, antibodies produced to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, are adsorbed on related polypeptides adhered to an insoluble matrix; Antibodies specific for soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL2Ry, will flow through the matrix under the appropriate buffer conditions. The test allows the isolation of polyclonal and monoclonal antibodies that do not cross-react with known closely related polypeptides (Antibidies: A Laboratory Manual, Harlow and Lane (reds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan et al. . (reds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). The examination and isolation of specific antibodies is well known in the art. See Fundamental Immunology, Paul (reds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principles and Practice, Goding, JW (reds.), Academic Press. Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984. Soluble anti-zalpha11 receptor or anti-soluble zalpha11 heterodimeric polypeptide antibodies, such as soluble anti-zalpha11 / IL-2R, can be detected by a number of methods in the art, and are described below. .

A variety of assays known to those of skill in the art can be used to detect antibodies that bind to soluble zalpha11 receptor proteins or polypeptides or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry. Test examples are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Reds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), Western blot or spot assay, competition or inhibition assay, and sandwich assay. In addition, the binding of antibodies to soluble zalpha11 receptor protein or polypeptide or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, wild type versus mutant, can be examined.

Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL2Ry protein or peptide, and selection of libraries displaying antibodies on phage or similar vectors. (for example, by using soluble zalpha11 receptor protein or peptide or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, immobilized or marked). Genes encoding polypeptides having potential binding domains for soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2R polypeptide, can be obtained by examining libraries of random peptides displayed on phage (phage display) or in bacteria. , such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as by random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen peptides that interact with a known target which may be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Methods for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent No. 5,223,409; Ladner et al., US Patent No. 4,946,778; Ladner et al., US Patent No. 5,403,484 and Ladner et al., US Patent No. 5,571,698) and random peptide display libraries are commercially available and kits to screen such libraries, for example, from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using soluble zalpha11 receptor sequences or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2R and described herein to identify soluble zalpha11 receptor binding proteins or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry. These "binding polypeptides" that interact with soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry polypeptides, can be used to label cells; to isolate homologous polypeptides by affinity purification; they can be conjugated directly or indirectly to drugs, toxins, radonuclides, and the like. These binding polypeptides can also be used in analytical methods such as to screen expression libraries and neutralize activity, eg, to block ligand-receptor interaction, or viral binding to a receptor.

ES 2 279 809 T3

The binding polypeptides can also be used for diagnostic assays to determine circulating levels of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry polypeptides; to detect or quantify soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Rγ polypeptides, as markers of underlying disease or pathology. These binding polypeptides may also act as zalpha11 receptor or heterodimeric zalpha11 polypeptide "antagonists", such as zalpha11 / IL-2RY, to block the binding of zalpha11 receptor or zalpha11 heterodimeric polypeptide, such as zalpha11 / IL-2RY polypeptide. , and in vitro and in vivo signal transduction. Again these soluble anti-zalpha11 receptor binding polypeptides or soluble anti-zalpha11 heterodimeric polypeptide, such as soluble anti-zalpha11 / IL-2RY, would be useful to inhibit zalpha11 Ligand activity, as well as receptor activity or binding. protein. Antibodies produced to the heterodimeric or multimeric combinations of the present invention are preferred embodiments because they may act more specifically against the zalpha11 Ligand, or more effectively than antibodies raised to only one subunit. In addition, the antagonistic and binding activity of the antibodies of the present invention can be tested in the proliferation of zalpha11 Ligand and other biological assays described herein.

Antibodies to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL2Ry, can be used to label cells expressing zalpha11 receptor or zalpha11 heterodimeric polypeptides, such as zalpha11 / IL-2RY; to isolate soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2RY polypeptide by affinity purification; for diagnostic assays to determine circulating levels of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2RY polypeptides; to detect or quantify soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2RY, as a marker of underlying disease or pathology; in analytical methods using FACS; to examine expression libraries; to generate anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zalpha11 receptor or heterodimeric zalpha11 polypeptide, such as zalpha11 / IL-2RY, or zalpha11 Ligand activity in vitro and in vivo. Suitable direct labels or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles, and the like; indirect labels or tags may feature the use of biotin-avidin or other complement / anti-complement pairs as intermediates. The present antibodies can also be conjugated directly or indirectly to drugs, toxins, radionuclides, and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Furthermore, antibodies to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2RY or fragments thereof can be used in vitro to detect soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as zalpha11 / IL-2RY. soluble, denatured or undenatured, or fragments thereof in assays, eg, Western blots or other assays known in the art.

Antibodies to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL2Ry, are useful for labeling cells expressing the corresponding receptors and assaying their expression levels, for affinity purification, within diagnostic assays to determine levels. circulating soluble receptor polypeptides, analytical methods employing fluorescence-activated cell sorting. Furthermore, divalent antibodies and anti-idiotypic antibodies can be used as agonists to mimic the effect of the zalpha11 Ligand.

The present antibodies can also be conjugated directly or indirectly to drugs, toxins, radionuclides, and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For example, antibodies or binding polypeptides that recognize soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2RY polypeptides, of the present invention can be used to identify or treat tissues or organs that express an anti- corresponding complementary (ie, a zalpha11 receptor or zalpha11 heterodimeric receptor, such as zalpha11 / IL-2RY). More specifically, soluble anti-zalpha11 receptor or soluble anti-zalpha11 heterodimeric polypeptide antibodies, such as soluble anti-zalpha11 / IL-2RY; or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the zalpha11 receptor or zalpha11 heterodimeric receptor, such as zalpha11 / IL-2RY receptor molecules. Suitable detectable molecules can be directly or indirectly incorporated into soluble zalpha11 receptor-binding polypeptides or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL2Ry ("binding polypeptides," including binding peptides described above), antibodies, or bioactive fragments. or portions of them. Suitable detectable molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles, and the like. Suitable cytotoxic molecules can be incorporated directly or indirectly into the polypeptide or antibody, and include bacterial or plant toxins (eg, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, and the like), as well as therapeutic radionuclides such as iodine-131 , rhenium-188 or yttrium-90 (incorporated directly into the polypeptide or antibody, or indirectly incorporated via a chelating moiety, for example). The binding polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For indirect incorporation of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated to one member of a complementary / anti-complementary pair, wherein the other member is bound to the binding polypeptide or antibody portion. For these purposes, biotin / streptavidin is an example of a complementary / anti-complementary pair.

In another embodiment, polypeptide-binding toxin fusion proteins or antibody-toxin fusion proteins can be used for inhibition or ablation of target cells or tissues (eg, to treat cancer cells or tissues).

ES 2 279 809 T3

Alternatively, if the binding polypeptide has multiple functional domains (i.e., an activation domain or a ligand-binding domain, plus a target binding domain), a fusion protein that includes only the binding domain may be suitable. targeting to direct a detectable molecule, a cytotoxic molecule, or a complementary molecule to a cell or tissue type of interest. In those cases where the fusion protein that includes only a single domain includes a complementary molecule, the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule. Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell / tissue specific delivery of generic anti-complementary-detectable / cytotoxic molecule conjugates.

In another embodiment, soluble zalpha11 receptor binding polypeptide or soluble zalpha11 heterodimeric polypeptide fusion proteins, such as soluble zalpha11 / IL-2Ry-cytokine or antibody-cytokine, may be used to enhance in vivo killing of target tissues (e.g. , blood, lymphoid, colon and bone marrow cancers), if the soluble anti-xzalpha11 receptor or cytokine-binding polypeptide antibody or soluble anti-zalpha11 heterodimeric polypeptide antibody, such as soluble anti-zalpha11 / IL-2Ry, it targets the hyperproliferative cell (see, generally, Hornick et al., Blood 89: 4437-47, 1997). The fusion proteins described allow the targeting of a cytokine to a desired site of action, thereby providing a local high cytokine concentration. Zalpha11 anti-homodimer and heterodimer antibodies target an undesirable cell or tissue (ie, tumor or leukemia) and the fused cytokine mediates enhanced target cell lysis by effector cells. Suitable cytokines for this purpose include interleukin 2 and granulocyte macrophage colony stimulating factor (GM-CSF), for example.

Alternatively, soluble zalpha11 receptor binding polypeptide or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, or the antibody fusion proteins described herein can be used to enhance in vivo killing of target tissues by directly stimulating an apoptotic pathway. modulated by the zalpha11 receptor, causing the cell death of hyperproliferative cells expressing the zalpha11 receptor or a heterodimeric zalpha11 receptor, such as soluble zalpha11 / IL-2Ry receptor.

Four-helical bundle cytokines that bind to cytokine receptors as well as other proteins produced by activated lymphocytes play an important biological role in cell differentiation, activation, recruitment, and homeostasis of cells throughout the body. Therapeutic utility includes treatment of diseases that require immune regulation including autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, and diabetes. Zalpha11 Ligand antagonists, including soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, may be important in regulating inflammation, and would therefore be useful in the treatment of rheumatoid arthritis, asthma, ulcerative colitis, inflammatory bowel disease, Crohn's disease, and sepsis. There may be a role for zalpha11 Ligand antagonists, including soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, in mediating tumorgenesis, and would therefore be useful in the treatment of cancer. Zalpha11 Ligand antagonists, including soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, may be a therapeutic potential to suppress the immune system that would be important in reducing graft rejection. Soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, may have utility in preventing graft versus host disease.

Alternatively, antagonists of zalpha11 Ligand, including soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2R receptors, in conjunction with other cytokines may allow selective activation, enhancement or selective suppression of the immune system in conjunction with Ligand of zalpha11 on other cytokines that would be important to stimulate immunity to infectious diseases, treat immunocompromised patients, such as an HIV + patient or to improve vaccines. In particular, zalpha11 antagonists, including soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, could prevent expansion of a subset of the immune system involving zalpha11 Ligand (e.g., NK cells and mature B cells), while allowing the expansion of progenitors induced by other cytokines (eg, T cells), and would provide therapeutic value in the treatment of viral infection and other infection. For example, with Dengue virus infection, which causes Dengue hemorrhagic fever / Dengue shock syndrome (DHF / DSS), severe DHF / DSS is believed to occur as a result of “immune surge,” that is, increased replication. virus in the presence of pre-existing antibodies against another serotype. In the second infection with a different Dengue virus serotype, the immune system produces antibodies against the first virus that cross-react but do not neutralize the virus, and potentially aid its entry into macrophages. Thus, suppression of the antibody immune response, or B-cell response, during a second or third Dengue infection can help the immune system to react appropriately in the second infection to neutralize the virus by suppressing the "boosting" antibodies from the infection by the first serotype, thereby avoiding severe DHF / DSS. For a review, see White, DO and Fenner FJ (Reds.) Medical Virology, 3^to Ed., Academic Press, Orlando Fl, 1986, pages 479-508). Similarly, suppression of maternal antibody responses to fetal antigens by soluble receptors of the present invention can help prevent birth defects and miscarriage. Furthermore, in such applications the soluble receptors of the present invention can be used in conjunction with other cytokines to suppress some activities of the immune system (for example, proliferation of B cells, using soluble receptors) but allowing others to increase, for example, in the presence of other cytokines described herein and known in the art.

ES 2 279 809 T3

The antibody or bioactive binding polypeptide conjugates described herein may be delivered orally, intravenously, intraarterially, or intraductally, or may be introduced locally at the intended site of action. For pharmaceutical use, the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2R receptor polypeptides, of the present invention are formulated for parenteral delivery, particularly intravenous or subcutaneous, according to conventional methods. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, pharmaceutical formulations will include a soluble zalpha11 receptor polypeptide in combination with a pharmaceutically acceptable carrier, such as saline, buffered saline, 5% dextrose in water, or the like. The formulations may additionally include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent loss of protein from vial surfaces, etc. Formulation methods are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, Red., Mack Publishing Co., Easton, pA, 19^to ed., 1995. Therapeutic doses will generally be in the range of 0.1 to 100 jug / kg of patient weight per day, preferably 0.5-20 mg / kg per day, the exact dose being determined by the physician according to standards. accepted, taking into account the nature and severity of the condition to be treated, the characteristics of the patient, etc. Determination of dosage is within the level of ordinary skill in the art. Proteins can be given for acute treatment, for a week or less, often over a period of one to three days, or they can be used for chronic treatment, for several months or years. In general, a therapeutically effective amount of soluble zalpha11 receptor polypeptide is an amount sufficient to produce a clinically significant effect.

The invention is further illustrated by the following non-limiting examples.

Examples Example 1

Construction of expression vector expressing full-length zalpha11

The complete zalpha11 receptor was isolated from a plasmid containing zalpha11 receptor cDNA (SEQ ID NO: 1) using PCR with primers ZC19,905 (SEQ ID NO: 19) and ZC19,906 (SEQ ID NO: 20). The reaction conditions were as follows: 95 ° C for 1 min; 35 cycles at 95 ° C for 1 min, 55 ° C for 1 min, 72 ° C for 2 min; followed by 72 ° C in 10 min; and then a soak at 10 ° C. The PCR product was run on a 1% low melting point agarose gel (Boerhinger Mannheim) and zalpha11 cDNA of approximately 1.5 kb was isolated using a Qiaquick® gel extraction kit (Qiagen) according to manufacturer's instructions.

The purified zalpha11 cDNA was digested with BamHI (Boerhinger Mannheim) and EcoRI (BRL) according to the manufacturer's instructions. The entire digest was run on a 1% low melting agarose gel (Boerhinger Mannheim) and the split zalpha11 fragment was purified using the Qiaquick® gel extraction set (Qiagen) according to the manufacturer's instructions. The resulting cleaved zalpha11 fragment was inserted into an expression vector as described below.

The pZP-5N receptor expression vector was digested with BamHI (Boerhinger Mannheim) and EcoRI (BRL) according to the manufacturer's instructions, and gel purified as described above. This vector fragment was combined with the BamHI and EcoRI cleaved zalpha11 fragment isolated earlier in a ligation reaction using T4 Ligase (BRL). The ligation was incubated at 15 ° C overnight. A sample of the ligation was electroporated into E. DH10B electroMAX® electrocompetent coli coli (25 μ 200, 200 Ω, 2.3 v). Transformants were plated on LB + ampicillin plates and single colonies were examined by PCR for zalpha11 sequence using ZC19,905 (SEQ ID NO: 19) and ZC19,906 (SEQ ID NO: 20) using the conditions of PCR described above. Confirmation of the zalpha11 sequence was made by sequence analysis. The insert was approximately 1.6 kb, and was full length.

Example 2

Zalpha11-based proliferation in BAF3 assay using Alamar Blue

BaF3 cells expressing the full-length zalpha11 receptor were constructed using the zalpha11 expression vector described in Example 1. BaF3 cells expressing the zalpha11 receptor mRNA were designated BaF3 / zalpha11. These cells provide an assay system for detecting zalpha11 Ligand activity as described below in numerous Examples. Conversely, these cells also provide an assay system for detecting antagonist or inhibitory activity of zalpha11 Ligand by the soluble receptors and antibodies of the present invention.

A. Construction of BaF3 cells expressing human zalpha11 receptor

BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell line derived from mouse bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol . 6: 4133-4135, 1986), was maintained in complete medium (RPMI medium (JRH Bioscience Inc., Lenexa, KS) supplemented with 10% heat-inactivated fetal calf serum, 2 ng / ml IL-3 of mouse (mIL-3) (R&amp;D, Minneapolis, MN), 2 mM L-glutaMax-1® (Gibco BRL),

ES 2 279 809 T3 1 mM sodium pyruvate (Gibco BRL) and PSN antibiotics (GIBCO BRL)). Prior to electroporation, pZP-5N / zalpha11 plasmid DNA (Example 1) was prepared and purified using a Qiagen Maxi Prep kit (Qiagen) according to the manufacturer's instructions. BaF3 cells for electroporation were washed once in RPMI medium and then resuspended in RPMI medium with a cell density of 10⁷ cells / ml. One ml of the resuspended BaF3 cells was mixed with 30 pg of plasmid DNA from pZP-5N / zalpha11 and transferred to separate disposable electroporation chambers (GIBCO BRL). After a 15 min incubation at room temperature, the cells were given two serial shakes (800 IFad / 300 v; 1180 IFad / 300 v) given by an electrporation apparatus (CELL-PORATOR®, GIBCO BRL). After a 5 minute recovery time, the electroporated cells were transferred to 50 ml of complete medium and placed in an incubator for 15-24 hours (37 ° C, 5% CO₂). The cells were then centrifuged and resuspended in 50 ml of complete medium containing Geneticin® selection (Gibco) (500 pg / ml G418) in a T-162 flask to isolate the G418 resistant cluster. The signaling ability was tested as described after pools of the transfected BaF3 cells, hereinafter referred to as BaF3 / zalpha11 cells.

B. Assaying the signaling ability of BaF3 / zalpha11 cells using an Alamar Blue proliferation assay

BaF3 / zalpha11 cells were centrifuged and washed in complete medium, described above, but without mIL-3 (hereinafter referred to as "mIL-3-free medium"). The cells were centrifuged and washed 3 times to ensure separation of mIL-3. The cells were then counted on a hemacytometer. Cells were plated in a 96-well format at 5,000 cells per well in a volume of 100 µl per well using the mIL-3-free medium.

BaF3 / zalpha11 cell proliferation was assessed using conditioned medium from cells expressing zalpha11 Ligand diluted with medium free of mIL-3 to concentrations of 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375%; or purified zalpha11 ligand (US patent application Ser. Common Property No. 09 / 522,217) diluted with mIL-3-free medium to concentrations of 500 ng / ml, 250 ng / ml, 125 ng / ml, 62 ng / ml, 30 ng / ml, 15 ng / ml, 7.5 ng / ml, 3.75 ng / ml, 1.8 ng / ml, 0.9 ng / ml, 0.5 ng / ml, and 0.25 ng / ml. 100 µl of the diluted mTPO was added to the BaF3 / zalpha11 cells. The total assay volume is 200 µl. Negative controls were run in parallel using only medium free of mIL-3. Assay plates were incubated at 37 ° C and 5% CO2 for 3 days, at which time Alamar Blue (Accumed, Chicago, IL) was added at 20 µl / well. The Alamar Blue gives a fluorometric reading based on the metabolic activity of cells, and is thus a direct measurement of cell proliferation compared to a negative control. The plates were incubated again at 37 ° C and 5% CO2 for 24 hours. Plates were read on the Fmax plate reader^® (Molecular Devices Sunnyvale, CA) using the SoftMax program^® Pro, with wavelengths of 544 (Excitation) and 590 (Emission). The results confirmed the signaling capacity of the zalpha11 receptor, because the zalpha11 Ligand significantly induced proliferation over background levels.

Example 3

Zalpha11 Ligand Screening Using BaF3 / zalpha11 Cells Using an Alamar Blue Proliferation Assay

A. Activation of Primary Monkey Splenocytes to Test for the Presence of Zalpha11 Ligand

Monkey splenocytes were stimulated in vitro to produce conditioned medium to test for the presence of zalpha11 Ligand activity as described below. Spleens were obtained from 8-year-old female M. nesestrian monkeys. The spleens were disrupted to produce a single cell suspension. Mononuclear cells were isolated by Ficoll-Paque density gradient^® PLUS (Pharmacia Biotech, Uppsala, Sweden). Mononuclear cells were seeded at 2 x 10⁶ cells / ml in RPMI-1640 medium supplemented with 10% FBS and activated with 5 ng / ml of phorbol 12-myristate-13-acetate (PMA) (Calbiochem, San Diego, CA) and 0.5 mg / ml by Ionomycin^® (Calbiochem) for 48 h. The supernatant from stimulated monkey spleen cells was used to assay for BaF3 / zalpha11 cell proliferation as described below.

B. Zalpha11 Ligand Screening Using BaF3 / zalpha11 Cells Using an Alamar Blue Proliferation Assay

BaF3 / zalpha11 cells were centrifuged and washed in medium free of mIL-3. The cells were centrifuged and washed 3 times to ensure separation of mIL-3. The cells were then counted on a hemacytometer. Cells were plated in a 96-well format at 5,000 cells per well in a volume of 100 µl per well using the mIL-3-free medium.

BaF3 / zalpha11 cell proliferation was assessed using activated monkey spleen conditioned medium (see Example 3A). The conditioned medium was diluted with mIL-3-free medium to concentrations of 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75%, and 0.375%. 100 µl of the diluted conditioned medium was added to the BaF3 / zalpha11 cells. The total assay volume is 200 µl. Assay plates were incubated at 37 ° C and 5% CO₂ for 3 days, at which time Alamar Blue (Accumed, Chicago, IL) was added at 20 µl / well. The plates were incubated again at 37 ° C and 5% CO2 for 24 hours. Plates were read on the Fmax plate reader^® (Molecular Devices) as described above (Example 2).

ES 2 279 809 T3

The results confirmed the proliferative response of the BaF3 / zalpha11 cells to a factor present in the activated monkey spleen conditioned medium. The response, as measured, was approximately 4 times above background at the 50% concentration. Non-transfected BaF3 cells did not proliferate in response to this factor, showing that this factor is specific for the zalpha11 receptor.

C. Human primary source used to isolate Zalpha Ligand

100 ml blood draws were taken from each of six donors. Blood was drawn using 10X 10 ml vacutainer tubes containing heparin. Blood from six donors (600 ml) was pooled, diluted 1: 1 in PBS and separated using a Ficoll-Paque® PLUS (Pharmacia Biotech). The production of isolated primary human cells after separation in the ficoll gradient was 1.2 x 10⁹ cells.

Cells were suspended in 9.6 ml of MACS buffer (PBS, 0.5% EDTA, 2 mM EDTA). 1.6 ml of cell suspension were removed and 0.4 ml of CD3 microbeads (Miltenyi Biotec, Auburn, CA) were added. The mixture was incubated for 15 min at 4 ° C. These CD3 bead-labeled cells were washed with 30 ml of MACS buffer and then resuspended in 2 ml of MACS buffer.

A VS + column (Miltenyi) was prepared according to the manufacturer's instructions. The VS + column was then placed in a VarioMACS magnetic field.^® (Miltenyi). The column was equilibrated with 5 ml of MACS buffer. The isolated primary human cells were then applied to the column. CD3 negative cells were allowed to pass. The column was rinsed with 9 ml (3 x 3 ml) of MACS buffer. The column was then detached from the magnet and placed on a 15 ml falcon tube. The CD3 + cells were eluted by adding 5 ml of MACS buffer to the column and the bound cells were removed using the piston provided by the manufacturer. The incubation of the cells with the CD3 magnetic beads, the washes and the VS + column steps (incubation by elution) above were repeated five more times. The CD3 + fractions resulting from the six column separations were pooled. The production of human cells selected with CD3 + was 3 x 10⁸ total cells.

A sample of the CD3 + selected human cells was separated for staining and sorting on a fluorescent antibody cell sorter (FACS) to assess their purity. Human CD3 + selected cells were 91% CD3 + cells.

Human CD3 + selected cells were activated by incubating in RPMI + 5% FBS + 10 ng / ml PMA and 0.5 jug / ml Ionomycin (Calbiochem) for 13 hours at 37 ° C. Zalpha11 Ligand activity was assayed in the supernatant of these activated CD3 + selected human cells as described below. In addition, activated CD3 + selected human cells were used to prepare a cDNA library, as described in US patent application Ser. common property No. 09 / 522,217.

D. ZalphaIII Ligand Assay in CD3 + Selected Human Cell Supernatant Using BaF3 / zalpha11 Cells and an Alamar Blue Proliferation Assay

BaF3 / zalpha11 cells were centrifuged and washed in medium free of mIL-3. Cells were centrifuged and washed 3 times to ensure separation of mIL-3. The cells were then counted on a hemacytometer. Cells were plated in a 96-well format at 5,000 cells per well in a volume of 100 µl per well using the mIL-3-free medium.

The proliferation of BaF3 / zalpha11 cells was assessed using conditioned medium of selected human cells with activated CD3 + (see Example 5C) diluted with medium free of mIL-3 to concentrations of 50%, 25%, 12.5%, 6.25 %, 3.125%, 1.5%, 0.75% and 0.375%. 100 µl of the diluted conditioned medium was added to the BaF3 / zalpha11 cells. The total assay volume is 200 µl. Assay plates were incubated and tested as described in Example 5B.

The results confirmed the proliferative response of the BaF3 / zalpha11 cells to a factor present in the conditioned medium of human cells selected with CD3 +. The response, as measured, was approximately 10-fold over background at the 50% concentration. Non-transfected BaF3 cells did not proliferate in response to this factor, showing that this factor is specific for the zalpha11 receptor. Furthermore, the soluble zalpha11 receptor blocked this proliferative activity in BaF3 / zalpha11 cells (see Example 16).

Example 4

Construction of mammalian expression vectors expressing soluble zalphaÍÍ receptors: zalfa11CEE, zalfa11CFLG, zalfa11CHIS and zalfaÍ1-Fc4

A. Construction of a zalfaÍÍMAMMAL EXPRESSION VECTOR CONTAINING ZALFAÍCEE, ZalfaÍCFLG and zalfaÍÍCHIS

An expression vector was prepared for the expression of the soluble, extracellular domain of the zalpha11 polypeptide, pC4zalfa11CEE, in which the construct is designed to express a zalpha11 polypeptide consisting of the methio36

ES 2 279 809 Truncated predicted initiation T3 nin adjacent to the predicted transmembrane domain, and with a Glu-Glu C-terminal tag (SEQ ID NO: 14).

A 700 bp PCR generated zalpha11 DNA fragment was created using ZC19,931 (SEQ ID NO: 21) and ZC19,932 (SEQ NO: 22) as PCR primers to add Asp718 and BamHI restriction sites. A plasmid containing the zalpha11 receptor cDNA (SEQ ID NO: 1) was used as a template. The PCR amplification of the zalpha11 fragment was carried out as follows: Twenty-five cycles at 94 ° C for 0.5 minutes; five cycles at 94 ° C for 10 seconds; 50 ° C for 30 seconds; 68 ° C for 45 seconds, followed by a stop at 4 ° C. The reaction was purified by chloroform / phenol extraction and isopropanol precipitation, and digested with Asp718 and BamHI (Gibco BRL) according to the manufacturer's method. A band of the predicted size, 700 bp, was visualized by 1% agarose gel electrophoresis, the DNA was removed and purified using a QiaexII® purification system (Qiagen) according to the manufacturer's instructions.

The removed DNA was subcloned into plasmid pC4EE that had been cut with BamHI and Asp718. The pC4zalpha11CEE expression vector uses the native zalpha11 signal peptide and incorporates the Glu-Glu tag (SEQ ID NO: 14) at the C-terminus of the polynucleotide sequence encoding zalpha11 polypeptide. Plasmid pC4EE is a mammalian expression vector containing an expression cassette having the mouse metallothionein-1 promoter, multiple restriction sites for insertion of coding sequences, a stop codon, and a growth hormone terminator. human. The plasmid also has an origin of replication from E. coli, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene, and the SV40 terminator.

Approximately 30 ng of the restriction-digested zalpha11 insert and approximately 12 ng of the digested vector were ligated overnight at 16 ° C. One microliter of each ligation reaction was independently electroporated into DH10B competent cells (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instruction and plated on LB plates containing 50 mg / ml ampicillin, and incubated overnight. . Colonies were examined by restriction analysis of DNA prepared from 2 ml of single colony liquid cultures. The insert sequence of positive clones was verified by sequence analysis. Large scale plasmid preparation was made using a QIAGEN® Maxi prep kit (Qiagen) according to the manufacturer's instructions.

The same procedure was used to prepare soluble zalpha11 receptors with a C-terminal his tag, composed of 6 His residues in a row; and a C-terminal flag tag (SEQ ID NO: 23), zalfa11CFLAG. To construct these constructions, the above vector has the HIS or FLAG tag^® instead of the glu-glu tag (SEQ ID NO: 14).

B. Zalpha11-Fc4 Soluble Zalpha11 Receptor Mammalian Expression Construction

An expression plasmid containing all or part of a polynucleotide encoding zalpha11 was constructed by homologous recombination. A zalpha11 cDNA fragment was isolated using PCR, which includes the zalpha11 receptor extracellular domain polynucleotide sequence. The two primers used in the production of the zalpha11 fragment were: (1) The primers for PCR each include from the 5 'to the 3' end: 40 bp of the flanking sequence of the vector (5 'of the insert) and 17 bp corresponding to the 5 'end of the extracellular domain of zalpha11 (SEQ ID NO: 24); and (2) 40 bp from the 5 'end of the Fc4 polynucleotide sequence (SEQ ID NO: 25) and 17 bp corresponding to the 3' end of the extracellular domain of zalpha11 (SEQ ID NO: 26). The Fc4 fragment for fusion with zalpha11 was generated by PCR in a similar manner. The two primers used in the production of the Fc4 fragment were: (1) a 5 'primer consisting of 40 bp of sequence from the 3' end of the extracellular domain of zalpha11 and 17 bp from the 5 'end of Fc4 (SEQ ID NO: 27 ); and (2) a 3 'primer consisting of 40 bp of vector sequence (3' of the insert) and 17 bp of the 3 'end of Fc4 (SEQ ID NO: 28).

The PCR multiplication of each of the reactions described above was carried out as follows: one cycle at 94 ° C for 2 minutes; twenty-five cycles at 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute; one cycle at 72 ° C for 5 minutes; followed by a stop at 4 ° C. Ten µ of the 100 µ of the PCR reaction were run on a 0.8% LMP agarose gel (Seaplaque GTG) with 1 x TBE buffer for analysis. The remaining 90 µl of the PCR reaction were precipitated with the addition of 5 µl of 1 M NaCl and 250 µl of absolute ethanol. The expression vector used was derived from plasmid pCZR199 (deposited in the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, designated No. 98668). and cut with SmaI (BRL). The expression vector was derived from plasmid pCZR199, and is a mammalian expression vector containing an expression cassette having the CMV immediate early promoter, a consensus intron of the variable region of an immunoglobulin heavy chain site of mouse, multiple restriction sites for insertion of coding sequences, a stop codon and a human growth hormone terminator. The expression vector also has an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene, and the SV40 terminator. The expression vector used was constructed from pCZR199 by substituting the metallothionein promoter for the CMV immediate early promoter.

One hundred microliters of competent yeast cells (S. cerevisiae) were combined with 10 µg containing approximately 1 µg of each of the zalpha11 and Fc4 inserts and 100 ng of SmaI-digested expression vector (BRL), and transferred to a 0.2 cm electroporation cuvette. Yeast / DNA mixtures were electropulsed at 0.75

ES 2 279 809 T3 kv (5 kv / cm), “infinite” ohms, 25 μΙ^;. 600 µl 1.2 M sorbitol was added to each cuvette and the yeast was plated in two 300 µl aliquots on two URA-D plates and incubated at 30 ° C.

After approximately 48 hours, single plate Ura + yeast transformants were resuspended in 1 ml H2O and briefly centrifuged to globulize yeast cells. The cell bead was resuspended in 1 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundred microliters of the lysis mixture was added to an Eppendorf tube containing 300 μl of acid washed glass beads and 200 μl of phenol-chloroform, vortexed for 1 minute intervals two or three times, followed by centrifugation 5 minutes in an Eppendorf centrifuge at full speed. Three hundred microliters of the aqueous phase were transferred to a new tube, and the DNA was precipitated with 600 µl of ethanol (EtOH), followed by centrifugation for 10 minutes at 4 ° C. The DNA bead was resuspended in 100 µl H2O.

Transformation of electrocompetent E. coli cells (DH10B, GibcoBRL) is done with 0.5-2 ml of yeast DNA prep and 40 µl of DH10B cells. The cells were electropulsed at 2.0 kv, 25 mF, and 400 ohms. After electroporation, 1 ml of SOC (2% tryptone Bacto^® (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, MgCl₂ 10 mM, MgSO₄ 10 mM, 20 mM glucose) in 250 µl aliquots on four LB AMP plates (LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg / L ampicillin).

Individual clones harboring the correct expression construct for zalpha11-Fc4 were identified by restriction digesting to verify the presence of the zalpha11-Fc4 insert and confirm that the various DNA sequences had been correctly linked to each other. The insert of positive clones was subjected to sequence analysis. Plasmid DNA is isolated on a larger scale using the Qiagen Maxi kit (Qiagen) according to the manufacturer's instructions.

Example 5

Transfection and expression of soluble zalpha11 receptor polypeptides

BHK 570 cells (ATCC No. CRL-10314), step 27, were plated at 1.2 x 10⁶ cells / well (6-well plate) in 800 µl of serum-free (SF) DMEM medium (DMEM, Gibco / BRL high glucose) (Gibco BRL, Gaithersburg, MD). Cells were transfected with expression plasmids containing zalpha11CEE, zalpha11CFLG or zalpha11CHIS described above (see Example 4) using Lipofectin^® (Gibco BRL), in serum-free DMEM (SF). Three micrograms of zalpha11CEE, zalpha11CFLG or zalpha11CHIS were each diluted separately in 1.5 ml tubes to a total final volume of 100 µl DMEM SF. The Lipofectin® mixture was incubated at room temperature for 30-45 minutes, then the DNA mixture was added and allowed to incubate for approximately 10-15 minutes at room temperature.

The entire DNA: Lipofectin® mixture was added to the plated cells and distributed evenly over them. Cells were incubated at 37 ° C for approximately five hours and then transferred to separate 150 mm MAXI plates in a final volume of 30 ml DMEM / 5% fetal calf serum (FBS) (Hyclone, Logan, UT) . Plates were incubated at 37 ° C and 5% CO₂, overnight and the DNA: Lipofectin® mixture was replaced by selection means (5% FBS / DMEM with 1 µM methotrexate (MTX)) the next day.

Approximately 10-12 days post-transfection, the plates were washed with 10 ml of DMEM SF. The wash medium was aspirated and replaced with 7.25 ml of serum-free DMEM. Sterile Teflon meshes (Spectrum Medical Industries, Los Angeles, CA) pre-soaked in DMEM SF were then placed on the clone cell colonies. A sterile nitrocellulose filter pre-soaked in DMEM SF was then placed on the mesh. Orientation marks on the nitrocellulose were transferred to the culture plate. The plates were then incubated for 5-6 hours in an incubator at 37 ° C and with 5% CO.₂.

After incubation, the filters / meshes were removed, and the media was aspirated and replaced with 5% FBS / DMEM with 1 µM MTX. The filters were then blocked in 10% skimmed milk powder / Western A buffer (Western A: 50 mM Tris pH 7.4, 5 mM EDTA, 0.05% NP-40, 150 mM NaCl and 0.25% gelatin) for 15 minutes at room temperature on a rotary shaker. Filters were then incubated with anti-Glu-Glu, antiFLAG® or anti-HIS-HRP antibody conjugates, respectively, in 2.5% skim milk powder / Western A buffer for one hour at room temperature on a rotary shaker. Filters were then washed three times at room temperature with Western A for 5-10 minutes per wash. Filters were developed with ultra ECL reagent (Amersham Corp., Arlington Heights, IL) according to the manufacturer's instructions and viewed on the Lumi-Imager (Roche Corp.).

Positive expressing clonal colonies were mechanically plated into 12-well plates in 1 ml of 5% FCS / DMEM with 5 µM MTX, and then grown to confluence. Expression levels in conditioned medium samples were then assayed by SDS-PAGE and Western analysis. The three highest expressing clones were taken for each construct; two of the three were frozen as support and one was expanded for mycoplasma testing and large-scale factory seeding.

ES 2 279 809 T3

B. Expression of the soluble zalpha11 receptor zalpha11-Fc4 in mammals

BHK 570 cells (ATCC #: CRL-10314) were plated in 10 cm tissue culture plates and allowed to grow to approximately 50 to 70% confluency overnight at 37 ° C and 5% CO.₂, in DMEM / FBS medium (DMEM, Gibco / BRL high glucose content) (Gibco BRL, Gaithersburg, MD), 5% fetal calf serum (Hyclone, Logan, UT), 1 mM L-glutamine (JRH Biosciences, Lenexa, KS), 1 mM sodium pyruvate (Gibco BRL)). The cells were then transfected with the plasmid containing zalpha11-Fc4 (see Example 9), using Lipofectamine® (Gibco BRL), in serum-free medium (SF) formulation (DMEM, 10 mg / ml transferrin, 5 mg / ml of insulin, 2 mg / ml of fetuin, 1% of L-glutamine and 1% of sodium pyruvate). The plasmid containing zalpha11-Fc4 was diluted in 15 ml tubes to a final total volume of 640 ml with SF medium. 35 ml of Lipofectamine were mixed^® (Gibco BRL) with 605 ml of SF medium. The Lipofectamine Mix^® was added to the DNA mix and allowed to incubate for approximately 30 minutes at room temperature. Five milliliters of SF medium were added to the DNA mixture Lipofectamine®. The cells were rinsed once with 5 ml of SF medium, aspirated and the DNA-Lipofectamine® mixture was added. Cells were incubated at 37 ° C for five hours and then 6.4 ml of DMEM medium / 10% FBS, 1% PSN was added to each plate. Cells were incubated at 37 ° C overnight and the DNA-Lipofectamine® mixture was replaced with fresh 5% FBS / DMEM medium the next day. On day 2 post-transfection, cells were divided in selection medium (DMEM / FBS medium from above with addition of 1 mM methotrexate (Sigma Chemical Co., St. Louis, Mo.)) in 150 mm plates at 1: 10, 1:20 and 1:50. The medium in the cells was replaced by fresh selection medium on day 5 post-transfection. Approximately 10 days post-transfection, two 150 mm culture plates of methotrexate resistant colonies from each transfection were trypsinized, and the cells were pooled and plated in a T-162 flask and transferred to large scale culture.

Example 6

Purification of soluble zalpha11 receptors from BHK 570 cells

A. Purification of zalpha11CEE polypeptide from BHK 570

Unless otherwise indicated, all operations were carried out at 4 ° C. The following procedure was used to purify zalpha11 polypeptide containing C-terminal GluGlu (EE) tags. Thirty liters of cell factory conditioned medium was concentrated to 1.6 liters with an Amicon S10Y3 spiral cartridge in a ProFlux A30. A protease inhibitor solution was added to the 1.6 liters of cell factory conditioned medium concentrates from transfected BHK 570 cells (Example 5) to final concentrations of 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis, MO), 0.003 mM Leupeptin (Boehringer-Mannheim, Indianapolis, IN), 0.001 mM Pepstatin (Boehringer-Mannheim), and 0.4 mM Pefabloc (Boehringer-Mannheim). Samples were separated for analysis and most of the volume was frozen at -80 ° C until purification started. Total target protein concentrations of the concentrated cell factory conditioned medium were determined by SDS-PAGE and Western blot analysis with the conjugated anti-EE HRP antibody.

A 100 ml column of anti-EE G-Sepharose (prepared as described below) was poured into a Waters AP-5 5 cm x 10 cm glass column. The column was flow packed and equilibrated on a BioCad Sprint (PerSeptive BioSystems, Framingham, MA) with Phosphate Buffered Saline (PBS) pH 7.4. The concentrated cell factory conditioned medium was thawed, sterile filtered through 0.2 microns, the pH adjusted to 7.4, and then loaded onto the column overnight at a flow rate of 1 ml / minute. . The column was washed with 10 column volumes (CVs) of phosphate buffered saline (PBS, pH 7.4) and then flushed with 200 ml of PBS (pH 6.0) containing 0.5 mg / ml of peptide EE (Anaspec, San Jose, CA) at a rate of 5 ml / minute. The peptide EE used has the sequence EYMPME (SeQ ID NO: 14). The column was washed for 10 CVs with PBS and then eluted with 5 CVs of 0.2 M glycine, pH 3.0. The H from the column eluted with glycine was adjusted to 7.0 with 2 CVs of 5X PBS and then equilibrated in PBS (pH 7.4). 5 ml fractions were collected during full elution chromatography and absorbance was checked at 280 and 215 nm; The pass-through and wash groupings were also saved and analyzed. The target protein in the EE polypeptide elution peak fractions was analyzed by SDS-PAGE Silver staining and Western blots with the conjugated anti-EE antibody HRP. Elution fractions of polypeptide of interest were pooled and concentrated from 60 ml to 5.0 ml using a 10,000 Dalton molecular weight cut-off membrane centrifugal concentrator (Millipore, Bedford, MA) according to the manufacturer's instructions.

To separate zalpha11CEE from other co-purification proteins, the concentrated polypeptide choice pooled fractions were subjected to POROS HQ-50 (strong anion exchange resin from PerSeptive BioSystems, Framingham, MA) at pH 8.0. A 1.0 x 6.0 cm column was poured and flow packed into a BioCad Sprint. The column was charged with counter ions and then equilibrated in 20 mM TRIS pH 8.0 (Tris) Hydroxymethyl Aminomethane)). The sample was diluted 1:13 (to reduce the ionic strength of PBS) and then loaded onto the Poros HQ column at 5 ml / minute. The column was washed for 10 CVs with 20 mM Tris pH 8.0 and then eluted with a 40 CV gradient of 20 mM Tris / 1 M sodium chloride (NaCl) at a rate of 10 ml / minute. 1.5 ml fractions were collected during the full chromatography and the absorbance was checked at 280 and 215 nm. Elution peak fractions were analyzed by SDS-PAGE Silver staining. The fractions of interest were pooled and concentrated to 1.5-2 ml using a 10,000 Dalton molecular weight cut-off membrane centrifugal concentrator (Millipore, Bedford, MA) according to the manufacturer's instructions.

ES 2 279 809 T3

To separate zalpha11CEE polypeptide from free EE peptide and any contaminating co-purification proteins, pooled concentrated fractions were chromatographed on a 1.5 x 90 cm Sephadex S200 column (Pharmacia, Piscataway, NJ) equilibrated and loaded in PBS with a flow rate of 1.0 ml / min using a BioCad Sprint. 1.5 ml fractions were collected throughout the entire chromatography and the absorbance was checked at 280 and 215 nm. Peak fractions were characterized by SDS-PAGE Silver staining, and only the purest fractions were pooled. This material represented purified zalpha11CEE polypeptide.

This purified material was finally subjected to a 4 ml ActiClean Etox column (Sterogene) to remove any remaining endotoxins. The sample was passed over the PBS equilibrated gravity column four times and the column was then washed with a single volume of 3 ml of PBS, which was pooled with the "clean" sample. The material was then sterile filtered through 0.2 microns and stored at -80 ° C until aliquoted.

On Coomassie Blue and Silver stained SDSPAGE Western blot gels, the zalpha11CEE polypeptide had a major band with an apparent molecular weight of 50,000 Daltons. The mobility of this band was the same in reducing and non-reducing gels.

The protein concentration of the purified material was performed by BCA analysis (Pierce, Rockford, IL) and the protein was aliquoted and stored at -80 ° C according to standard procedures. In IEF gels (isoelectric focusing) the protein passes with a PI lower than 4.5. The zalpha11CEE polypeptide concentration was 1.0 mg / ml.

Purified zalpha11CEE polypeptide was prepared for injection into rabbits and sent to R&amp;R Research and Development (Stanwood, WA) for antibody production. Rabbits were injected to produce anti-Huzalfa11-CEE BHK serum (Example 10 below).

To prepare anti-EE Sepharose, a 100 ml bed volume of Protein G-Sepharose (Pharmacia, Piscataway, NJ) was washed 3 times with 100 ml of PBS containing 0.02% sodium azide using a filter unit of 0.45 micron Nalgene 500 mL. The gel was washed with 6.0 volumes of 200 mM triethanolamine, pH 8.2 (TEA, Sigma, St. Louis, MO), and an equal volume of EE antibody solution containing 900 mg of antibody was added. After an overnight incubation at 4 ° C, unbound antibody was removed by washing the resin with 5 volumes of 200 mM TEA as described above. The resin was resuspended in 2 volumes of TEA, transferred to a suitable container and dimethyl pimilimidate-2HCl (Pierce, Rockford, IL) dissolved in TEA was added to a final concentration of 36 mg / ml protein G-Sepharose gel. . The gel was rocked at room temperature for 45 min and the liquid was separated using the filter unit as described above. Non-specific sites on the gel were then blocked by incubating for 10 min, at room temperature, with 5 volumes of 20 mM ethanolamine in 200 mM TEA. The gel was then washed with 5 volumes of PBS containing 0.02% sodium azide and stored in this solution at 4 ° C.

B. Purification of zalpha11CFLAG polypeptide from BHK 570

Unless otherwise indicated, all operations were carried out at 4 ° C. The following procedure was used to purify zalpha11 polypeptide containing C-terminal FLAG® (FLG) tags (Sigma-Aldrich Co.). Thirty liters of cell factory conditioned medium was concentrated to 1.7 liters with an Amicon S10Y3 spiral cartridge in a ProFlux A30. A protease inhibitor solution was added to 1.7 liters of concentrated cell factory conditioned medium from transfected BHK 570 cells (see Example 5) to final concentrations of 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis, MO), 0.03 mM Leupeptin (Boehringer-Mannheim, Indianapolis, IN), 0.001 mM Pepstatin (Boehringer-Mannheim), and 0.4 mM Pefabloc (Boehringer-Mannheim). Samples were separated for analysis and most of the volume was frozen at -80 ° C until purification started. Total target protein concentrations of cell factory conditioned medium were determined by SDS-PAGE and Western blot analysis with anti-FLAG® (Kodak) HRP conjugated antibody. A 125 ml column of anti-FLAG® M2-Agarose affinity gel (Sigma-Aldrich Co.) was poured into a Waters AP-5 5 cm x 10 cm glass column. The column was flow packed and equilibrated on a BioCad Sprint (PerSeptive BioSystems, Framingham, MA) with Phosphate Buffered Saline (PBS) pH 7.4. The concentrated cell factory conditioned medium was thawed, sterile filtered through 0.2 microns, the pH adjusted to 7.4, and then loaded onto the column overnight at a flow rate of 1 ml / minute. . The column was washed with 10 column volumes (CVs) of phosphate buffered saline (PBS, pH 7.4) and then flushed with 250 ml of PBS (pH 6.0) containing 0.5 mg / ml of FLAG peptide^® (Sigma-Aldrich Co.) at a rate of 5 ml / minute. The FLAG® peptide used has the sequence DYKDDDDK (SEQ ID NO: 23). The column was washed for 10 CVs with PBS and then eluted with 5 CVs of 0.2M glycine, pH 3.0. The H from the column eluted with glycine was adjusted to 7.0 with 2 CVs of 5X PBS and then equilibrated in PBS (pH 7.4). Five ml fractions were collected during full elution chromatography and absorbance was checked at 280 and 215 nm; The passage through and the wash groupings were also saved and analyzed. The target protein was analyzed in the FLAG polypeptide elution peak fractions^® by SDS-PAGE Silver staining and Western blots with the conjugated anti-FLAG HRP antibody. Elution fractions of polypeptide of interest were pooled and concentrated from 80 ml to 12 ml using a 10,000 Dalton molecular weight cut-off membrane centrifugal concentrator (Millipore, Bedford, MA) according to the manufacturer's instructions.

To separate zalpha11CFLG from other co-purification proteins, polypeptide elution pooled fractions were subjected to POROS HQ-50 (strong anion exchange resin from PerSeptive BioSystems, Framingham,

ES 2 279 809 T3

MA) at pH 8.0. A 1.0 x 6.0 cm column was poured and flow packed into a BioCad Sprint. The column was charged with counter ions and then equilibrated in 20 mM TRIS pH 8.0 (Tris) Hydroxymethyl Aminomethane)). The sample was diluted 1:13 (to reduce the ionic strength of PBS) and then loaded onto the Poros HQ-50 column at 5 ml / minute. The column was washed for 10 column volumes (CVs) with 20 mM Tris pH 8.0 and then eluted with a 40 CV gradient of 20 mM Tris / 1 M sodium chloride (NaCl) at a rate of 10 ml / minute. 1.5 ml fractions were collected during the full chromatography and the absorbance was checked at 280 and 215 nm. Elution peak fractions were analyzed by SDS-PAGE Silver staining. The fractions of interest were pooled and concentrated to 1.5-2 ml using a 10,000 Dalton molecular weight cut-off membrane centrifugal concentrator (Millipore, Bedford, MA) according to the manufacturer's instructions.

To separate zalpha11CFLG polypeptide from free FLAG® peptide and any contaminating co-purification proteins, the pooled concentrated fractions were chromatographed on a 1.5 x 90 cm Sephacrfyl S200 column (Pharmacia, Piscataway, NJ) equilibrated and loaded in PBS at a flow rate of 1.0 ml / min using a BioCad Sprint. 1.5 ml fractions were collected throughout the entire chromatography and the absorbance was checked at 280 and 215 nm. Peak fractions were characterized by SDS-PAGE Silver staining, and only the purest fractions were pooled. This material represented purified zalpha11CFLG polypeptide.

This purified material was finally subjected to a 4 ml ActiClean Etox column (Sterogene) to remove any remaining endotoxins. The sample was passed over the PBS equilibrated gravity column four times and the column was then washed with a single volume of 3 ml of PBS, which was pooled with the "clean" sample. The material was then sterile filtered through 0.2 microns and stored at -80 ° C until aliquoted.

On Coomassie Blue and Silver stained SDSPAGE Western blot gels, the zalpha11CFLG polypeptide had a major band with an apparent molecular weight of 50,000 Daltons. The mobility of this band was the same in reducing and non-reducing gels.

The protein concentration of the purified material was performed by BCA analysis (Pierce, Rockford, IL) and the protein was aliquoted and stored at -80 ° C according to standard procedures. In IEF gels (isoelectric focusing) the protein passes with a PI lower than 4.5. The zalpha11CEE polypeptide concentration was 1.0 mg / ml.

C. Purification of zalpha1q1-Fc4 polypeptide from transfected BHK 570 cells

Unless otherwise indicated, all operations were carried out at 4 ° C. The following procedure was used to purify zalpha11 polypeptide containing C-terminal fusion to human IgG / Fc (zalpha11-Fc4; Examples 4 and 5). 12,000 ml of conditioned medium from zalpha11-Fc4 transfected BHK 570 cells (Example 5) were filtered through a 0.2 mm sterilizing filter and then supplemented with a solution of protease inhibitors, to final concentrations of 0.001 mM leupeptin. (Boehringer-Mannheim, Indianapolis, IN), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim). It was filled with a protein sepharose G (6 ml bed volume, Pharmacia Biotech) and washed with 500 ml of PBS (Gibco / BRL). The supplemented conditioned medium was passed over the column at a flow rate of 10 ml / minute, followed by washing with 1000 ml of PBS (BRL / Gibco). Zalpha11-Fc4 was eluted from the column with 0.1 M glycine pH 3.5 and 2 ml fractions were directly collected in 0.2 ml of 2 M Tris pH 8.0, to adjust the final pH to 7.0 in the fractions.

The eluted fractions were characterized by SDS-PAGE and Western blots with anti-human Fc antibodies (Amersham). Western blot analysis of reducing SDS-PAGE gels revealed an immunoreactive protein of 80,000 kDa in fractions 2-10. Silver stained SDS-PAGE gels also revealed an 80,000 kDa zalpha11: Fc polypeptide in fractions 2-10. Fractions 2-10 were pooled.

The protein concentration of the pooled fractions was performed by BCA analysis (Pierce, Rockford, IL) and the material was aliquoted and stored at -80 ° C according to our standard procedures. The concentration of the pooled fractions was 0.26 mg / ml.

Example 7

Assay using soluble zalpha11CEE, zalpha11CFLG and zalpha11Fc4 soluble receptor zalpha11 receptors in competitive inhibition assay

BaF3 / zalpha11 cells were centrifuged and washed in medium free of mIL-3. Cells were centrifuged and washed 3 times to ensure separation of mIL-3. The cells were then counted on a hemacytometer. Cells were plated in a 96-well format at 5,000 cells per well in a volume of 100 µl per well using the mIL-3-free medium.

Both means of monkey spleen cell activation and the selected CD3 + cells, described in Example 3, were added in separate experiments at concentrations of 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375%, with or without soluble zalpha11 receptors (CEE, C-tag and Fc4 constructions; see Example 6) at a rate of 10 pg / ml. The total assay volume was 200 µl.

ES 2 279 809 T3

Assay plates were incubated at 37 ° C and 5% CO₂ for 3 days, at which time Alamar Blue (Accumed) was added at 20 µl / well. The plates were incubated again at 37 ° C and 5% CO₂ for 24 hours. Plates were read on the Fmax plate reader^® (Molecular Devices) as described above (Example 2). The results demonstrated complete inhibition of cell growth of each of the different zalpha11 soluble receptor constructs with 10 µg / ml, confirming that the factor in each sample was specific for the zalpha11 receptor.

Titration curves were also made, diluting the soluble receptors, using the assay indicated above. Both soluble zalpha11 receptors zalpha11CEE and zalpha11CFLG were able to completely inhibit growth with as low as 20 ng / ml. The soluble zalpha11 receptor zalpha11-Fc4 was only as effective at 1.5 µg / ml.

Example 8

Expression of soluble human zalpha11 receptor in E. coli

A. Construction of expression vector pCZR225 expressing huzalfa11 / MBP-6H fusion polypeptide

An expression plasmid containing a polynucleotide encoding a soluble human zalpha11 receptor C-terminally fused to maltose binding protein (MBP) was constructed by homologous recombination. The polynucleotide sequence for the zalpha11 soluble receptor-MBP fusion polypeptide is shown in SEQ ID NO: 29, with the corresponding protein sequence shown in SEQ ID NO: 30. The fusion polypeptide, designated huzalpha11 / MBP-6H, in Example 9, contains a portion of MBP (amino acid 1 (Met) to amino acid 388 (Ser) of SEQ ID NO: 30) fused to the soluble human zalpha11 receptor (amino acid 389 (Cys) to amino acid 606 (His) of SEQ ID NO: 30). A human zalpha11 cDNA fragment (SEQ ID NO: 31) was isolated using PCR. Two primers were used in the production of the human zalpha11 fragment in a PCR reaction: (1) primer ZC20,187 (SEQ ID NO: 32), which contains 40 bp of the vector framework sequence and 25 bp corresponding to the amino terminus of the human zalpha11, and (2) primer ZC20,185 (SEQ ID NO: 33), containing 40 bp from the 3 'end corresponding to the flanking vector sequence and 25 bp corresponding to the carboxyl terminus of human zalpha11. The PCR reaction conditions were as follows: 25 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute; followed by soaking at 4 ° C, performed in duplicate. Two µl of the 100 µl PCR reaction were run on a 1.0% agarose gel with 1 x TBE buffer for analysis, and the expected approximately 660 bp fragment was seen. The remaining 90 µl of PCR reaction was combined with the second PCR tube precipitated with 400 µl of absolute ethanol. The precipitated DNA was used to recombine into the SmaI cut receptor vector pTAP98 to produce the construct encoding the MBP-zalpha11 fusion, as described below.

Plasmid pTAP98 was derived from plasmids pRS316 and pMAL-c2. Plasmid pRS316 is a Saccharomyces cerevisiae shuttle vector (Hieter P. and Sikorski, R., Genetics 122: 19-27, 1989). pMAL-C2 (NEB) is an E. coli expression plasmid. It carries the tac promoter that drives MalE (gene encoding MBP) followed by a His tag, a thrombin cleavage site, a cloning site, and the rrnB terminator. The vector pTAP98 was constructed using yeast homologous recombination. 100 ng of EcoRI cut pMAL-c2 were recombined with 1 µg of Pvu1 cut pRS316, 1 µg of linker and 1 µg of Sca1 / EcoRI cut pRS316. The linker consisted of the oligos ZC19,372 (SEQ ID NO: 34) (100 pmol): ZC19,351 (SEQ ID NO: 35) (1 pmol): ZC19,352 (SEQ ID NO: 36) (1 pmol) and ZC19,371 (SEQ ID NO: 37) (100 pmol) combined in a PCR reaction. The PCR reaction conditions were as follows: 10 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 30 seconds; followed by soaking at 4 ° C. The PCR products were concentrated by precipitation with 100% ethanol.

One hundred microliters of competent yeast cells (S. cerevisiae) were combined with 10 μl of a mixture containing approximately 1 μg of the above human zalpha11 receptor PCR product and 100 ng of SmaI-digested vector pTAP98, and transferred to a cuvette of 0.2 cm electroporation. The yeast / DNA mixture was electropulsed at 0.75 kv, infinite ohms, 25 μΕ 600 μl of 1.2 M sorbitol was added to each cuvette and the yeast was then plated in two 300 μl aliquots on two URA plates D and incubated at 30 ° C.

After approximately 48 hours, single plate Ura + yeast transformants were resuspended in 1 ml H2O and briefly centrifuged to globulize yeast cells. The cell bead was resuspended in 1 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundred microliters of the lysis mixture was added to an Eppendorf tube containing 300 μl of acid washed glass beads and 200 μl of phenol-chloroform and vortexed for 1 minute intervals two or three times, followed by centrifugation. 5 minutes in an Eppendorf centrifuge at full speed. Three hundred microliters of the aqueous phase were transferred to a new tube, and the DNA was precipitated with 600 µl of ethanol (EtOH), followed by centrifugation for 10 minutes at 4 ° C. The DNA bead was resuspended in 100 μl of H₂OR.

Transformation of electrocompetent E. coli cells (MC1061, Casadaban et al. J. Mol. Biol. 138, 179207) was done with 1 µl of yeast DNA prep and 40 µl of MC1061 cells. Cells were electropulsed at 2.0 kv, 25 µF, and 400 ohms. After electroporation, 0.6 ml of SOC (2% Bacto® tryptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl , MgCl₂ 10 mM, MgSO₄ 10 mM, 20 mM glucose) in an aliquot in 100 mg / L plates of MM / CA + AMP (Pryor and Leiting, Protein Expression and Purification 10: 309-319, 1997).

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Cells harboring the correct expression construct for the human zalpha11 receptor were identified by expression. Cells were grown in MM / CA with 100 jug / ml ampicillin for two hours, shaking, at 37 ° C. 1 ml of the culture was induced with 1 mM IPTG. 2-4 hours later the 250 μ \ of each culture were mixed with 250 μ. \ Of acid-washed glass beads and 250 μ \ of Thorner's buffer with 5% // ME and stain (8 M urea, Tris 100 mM pH 7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Samples were vortexed for one minute and heated at 65 ° C for 10 minutes. 20 µ per lane was loaded onto a 4% -12% V PAGE gel (NOVEX). Gels were run in 1XMES buffer. Positive clones were named pCZR225 and subjected to sequence analysis. The polynucleotide sequence of the MBP-zalpha11 fusion is shown in SEQ ID NO: 50.

B. Bacterial expression of human huzalpha11 / MBP-6H fusion polypeptide

One microliter of sequence determination DNA was used to transform strain BL21. Cells were electropulsed at 2.0 kv, 25 µF, and 400 ohms. After electroporation, 0.6 ml of MM / CA with 100 mg / l of ampicillin.

Cells were grown in MM / CA with 100 mg / l ampicillin for two hours, shaking, at 37 ° C. 1 ml of the culture was induced with 1 mM IPTG. The 250 μ \ of each culture were mixed 2-4 hours later with 250 μ \ acid-washed glass beads and 250 μ \ Thorner's buffer with 5% // ME and stain (8 M urea, Tris 100 mM pH 7.0, 0.10% glycerol, 2 mM EDTA, 5% SDS). Samples were vortexed for one minute and heated at 65 ° C for 10 minutes. 20 µ per lane was loaded onto 4% -12% PAGE gel (NOVEX). The gels were run in 1XMES buffer. Positive clones were used to develop protein purification of the huzalpha11 / MBP-6H fusion protein (Example 9 below).

Example 9

Purification of soluble huzalfa11 / MBP-6H receptor from E. coli fermentation

Unless otherwise indicated, all operations were carried out at 4 ° C. The following procedure was used to purify soluble huzalpha11 / MBP-6H receptor polypeptide. Cells of E. coli containing the pCZR225 construct and expressing soluble huzalpha11 / MBP-6H receptor (Example 8) in SuperBroth II (12 g / l casein, 24 g / l yeast extract, 11.4 g / l dipotassium phosphate, 1.7 g / L monopotassium phosphate; Becton Dickenson, Cockeysville, MD) and frozen in 0.5% glycerol. Twenty grams of the cells frozen in SuperBroth II + glycerol were used to purify the protein. The frozen cells were thawed and diluted 1:10 in a protease inhibitor solution (extraction buffer) prior to lysing the cells and releasing the soluble huzalpha11 / MBP-6H receptor protein. The diluted cells contained final concentrations of 20 mM Tris (JT Baker, Philipsburg, NJ), 100 mM sodium chloride (NaCl, Mallinkrodt, Paris, KY), 0.5 mM phenylmethylsulfonyl fluoride (PmSf, Sigma Chemical Co., St. Louis, MO), 2 jug / ml of leupeptin (Fluka, Switzerland) and 2 jug / ml of aprotinin (Sigma). A French press cell breaking system (Constant Systems Ltd., Warwick, UK) with a temperature of -7 to -10 ° C and 2,100 kg / cm was used to lyse the cells.². Breakage of lysed cells was checked by A readings₆₀₀ before and after the French press. The lysed cells were centrifuged at 18,000 G for 45 minutes to remove broken cell debris, and the supernatant was used to purify the protein. Total target protein concentrations of the supernatant were determined by BCA protein assay (Pierce, Rockford, IL), according to the manufacturer's instructions.

A 25 ml column of Talon metal affinity resin (Clontech, Palo Alto, CA) (prepared as described below) was poured into a Bio-Rad glass column, 2.5 cm diameter x 10 cm height . The column was packed and gravity equilibrated with 10 column volumes (CVs) of Talon equilibration buffer (20 mM Tris, 100 mM NaCl, pH 8.0). The supernatant was batch loaded onto Talon metal affinity resin and shaken overnight. The resin was poured back onto the column and washed with 10 CVs of Talon equilibration buffer by gravity and then eluted by gravity with 140 ml of elution buffer (Talon equilibration buffer + 200 mM imidazole-Fluka Chemical). The talon column was cleaned with 5 CVs of 20 mM 2- (N-morpholino) ethanesulfonic acid pH 5.0 (MES, Sigma) and 5 CVs of H₂Or distilled, and then stored in 20% ethanol / 0.1% sodium azide. Fourteen ml fractions were collected during full elution chromatography and fractions with absorbance at 280 and 320 nm and BCA protein assay were read; The pass through and wash pools were also saved and analyzed. Elution fractions of protein of interest were pooled and loaded directly onto amylose resin (New England Biolabs, Beverly, MA).

To obtain more pure huzalpha11 / MBP-6H polypeptide, the talon affinity elution pooled fractions were subjected to amylose resin (22 ml) at pH 7.4. A 2.5 cm diameter x 10 cm high Bio-Rad column was poured, packed and equilibrated in 10 CVs of 20 mM amylose-Tris equilibration buffer (JT Baker), 100 mM NaCl (Mallinkrodt), pMsf 1 mM (Sigma), 10 mM beta-mercaptoethanol (BME, ICN Biomedicals Inc., Aurora, OH) pH 7.4. The sample was loaded by gravity flow rate of 0.5 ml / min. The column was washed for 10 CVs with amylose equilibration buffer and then eluted with ~ 2 CVs of 10 mM amylose + maltose equilibration buffer (Fluka Biochemical, Switzerland) by gravity. 5 ml fractions were collected during the full chromatography and the absorbance at 280 and 320 nm was read. The amylose column was regenerated with 1 CV of distilled H2O, 5 CVs of 0.1% SDS (w / v) (Sigma), 5 CVs of H₂Or distilled and then 5 CVS of amylose equilibration buffer.

The fractions of interest were pooled and dialyzed on a Slide-A-Lyzer (Pierce) with 4 x 4 L of PBS pH 7.4 (Sigma) to remove low molecular weight contaminants, buffer exchange and desalting. After the

ES 2 279 809 T3 changes from PBS, the harvested material represented the purified huzalpha11 / MBP-6H polypeptide. Purified huzalpha11 / MBP-6H polypeptide was analyzed by Coomassie staining of SDS-PAGE and Western blot analysis with conjugated rabbit anti-HRP antibody (Rockland, Gilbertsville, PA). The concentration of the huzalfa11 / MBP-6H polypeptide was 1.92 mg / ml determined by BCA analysis.

Purified huzalpha11 / MBP-6H polypeptide was prepared for injection into rabbits and sent to R&amp;R Research and Development (Stanwood, WA) for antibody production. Rabbits were injected to produce anti-huzalfa11 / MBP-6H antiserum (Example 10 below).

Example 10

Soluble zalpha11 receptor polyclonal antibodies

Polyclonal antibodies were prepared by immunizing two female New Zealand white rabbits with the purified huzalpha11 / MBP-6H polypeptide (Example 9), or the purified recombinant zalpha11CEE soluble receptor (Example 6A). The corresponding polyclonal antibodies were named rabbit anti-huzalfa11 / MBP-6H and rabbit anti-huzalfa11-CEE-BHK, respectively. Each rabbit was given an initial intraperitoneal (IP) injection of 200 mg of purifuged protein in complete Freund's adjuvant (Pierce, Rockford, IL), followed by booster IP injections of 100 mg of purified protein in incomplete Freund's adjuvant every third. weeks. Seven to ten days after the administration of the third booster injection, the animals were bled and the serum was collected. The rabbits were then boosted and bled every three weeks.

Polyclonal antibodies specific for zalpha11 were affinity purified from rabbit serum using a CNBr-SEPHAROSE 4B protein column (Pharmacia LKB), which was prepared using 10 mg of the purified huzalpha11 / MBP-6H polypeptide (Example 9) by gram of CNBr-SEPHAROSE, followed by 20X dialysis in PBS overnight. Antibodies specific for zalpha11 were characterized by an ELISA titer check using 1 mg / ml of the appropriate protein antigen as an antibody target. The lower limit of detection (LLD) of the affinity purified anti-huzalfa11 / MBP-6H antibody is a dilution of 500 pg / ml. The LLD of the affinity purified anti-huzalfa11-CEE-BHK antibody is a dilution of 50 pg / ml.

Example 11

Identification of cells expressing zalpha11 receptor using RT-PCR

Specific human cell types were isolated and examined for zalpha11 expression by RT-PCR. Fresh human tonsil B cells were isolated by mechanical disruption through 100 µm nylon cell filters (Falcon®; Bectin Dickenson, Franklin Lakes, NY). B cell suspensions were enriched for CD19 + B cells by positive selection with a VerioMACS VS + magnetic column and CD19 microbeads (Miltenyi Biotec, Auburn, CA) according to manufacturer's instructions. T cells and monocytes were isolated from apheresis human blood samples, CD3 + T cells were purified by VarioMACS positive selection of CD3 microbeads, and monocytes were purified by VarioMACS negative selection columns (Miltenyi) according to manufacturer's instructions. Samples from each population were stained and analyzed by fluorescent antibody cell sorting analysis (FACS) (Bectin Dickinson, San Jose, CA) to determine the percent enrichment and the resulting yields. CD19 + B cells were approximately 96% purified, CD3 + T cells were approximately 95% purified, and monocytes were approximately 96% purified.

RNA was prepared using a standard method in the art from the three cell types that were resting or activated. RNA from resting cells was isolated directly from the above column preparations. CD19 + and CD3 + cells were activated by culturing at 500,000 cells / ml in RPMI + 10% FBS containing 5 ng / ml of PMA (Calbiochem, La Jolla, CA) and 0.5 jug / ml of ionomycin (Calbiochem) for 4 and 24 hours. Monocytes were activated by culturing in RPMI + 10% FBS containing 10 ng / ml LPS (Sigma, St. Louis, MO) and 10 ng / ml rhIFN-γ (R&amp;D, Minneapolis, MN) for 24 hours. Cells were harvested and washed in PBS. RNA was prepared from cell beads using the RNeasy Midiprep kit^® (Qiagen, Valencia, CA) according to the manufacturer's instructions and first strand cDNA synthesis was generated with the Superscript II® kit (GIBCO BRL, Grand Island, NY) according to the manufacturer's method.

Oligos ZC19907 (SEQ ID NO: 38) and ZC19908 (SEQ ID NO: 39) were used in a PCR reaction to screen the samples described above for a 1.2 kb fragment corresponding to the zalpha11 message. The PCR multiplication was performed with Taq polymerase (BRL Grand Island NY), and the following conditions: 35 cycles of 95 ° C for 1 min, 60 ° C for 1 min, 72 ° C for 30 s; 1 cycle at 72 ° C for 10 min; and soak at 4 ° C. 10 µl of each 50 µl reaction volume was run on a 2% agarose IXTAE gel to identify the resulting products. The PCR products were scored (-) no product, (+) visible band, (++) increased band presence, and (+++) the most predominant band, with the results shown in Table 5 below.

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TABLE 5

<td>CDNA source</td><td>Activation</td><td>PCR product</td>

<td>CD19 + cells</td><td>0 h at rest 4 hours activated 24 hours activated</td><td> + ++ +++</td>

<td>CD3 + cells</td><td>0 h at rest 4 hours activated 24 hours activated</td><td> ++</td>

<td>monocytes</td><td>0 h at rest 24 hours activated</td><td> -</td>

The results indicated that the zalpha11 message is present in resting human CD19 + B cells and increases with mitogenic activation. It also appears to be expressed by human CD3 + T cells only after 4 hours of activation. There was no apparent message in resting or activated human monocytes.

Example 12

Immunohistochemistry of zalfa11

A. Cell and tissue preparations

Positive controls consisted of zalpha11 receptor transfected BaF3 cells (Example 2) and lymphoid tissues known to express zalpha11 receptor, including mouse lymph node, spleen and thymus received from HSD (Harlan Sprague Dawley, Indianapolis, IN), lymphoid node and monkey spleen received from Regional Primate Research Center (University of Washington, Seattle, WA), human lymphoid node and spleen received from CHTN (Cleveland, OH). Negative controls performed on each sample included: (1) untransfected BaF3 cells, (2) mouse and human brain and liver tissue known to be non-expressing zalpha11 receptor, (3) antibody dilution buffer staining (Ventann Bioteck Systems, Tucson, AZ) in the absence of primary antibody and (4) using soluble zaqlpha11 receptor protein in competition experiments.

Other cell samples were examined. Unstimulated and stimulated HL60 cells were tested. HL60 cells are a promyelocytic cell lineage, which can differentiate into myeloid or granulocyte lineages with different reagents. Stimulated HL60 samples were prepared as follows: (1) HL60 cells were treated with 10 ng / ml phorbolmyristate-phorbol acetate (PMA) (Sigma, St. Louis, MO) for 48 hours to differentiate into monocyte lineage cells; and (2) HL60 cells treated with 1.25% DMSO (Sigma) for 4 days to differentiate into neutrophil-like cells. In addition, human polymorphonuclear cells (PMN), human granulocytes, human peripheral blood lymphocytes (PBL), and fresh human blood human monocytes (prepared internally using routine methods in the art) were examined. The cells and tissues described above were fixed overnight in 10% NBF (Surgipath, Richmond, IL) and embedded in X-tra paraplast (Oxford Scientific, St. Louis, MO) and sectioned at 5 μιη with a micrometer. Reichart-Jung 2050 (Leica Instruments GmbH, Nussloch, Germany).

B. Immunohistochemistry

Tissue slices were deparaffinized, hydrated to buffer (water) and subjected to HIER treatment with steam in Antigen Retrieval Citra buffer (BioGenex, San Roman, CA) for 20 minutes. 5% normal goat serum (Vector, Burlingame, CA) was used to block non-specific binding for 10 minutes. Immunocytochemical screening analyzes were performed using polyclonal antibodies to soluble zalpha11 receptor protein (rabbit anti-huzalfa11-MBP-6H and rabbit anti-huzalfa11-CEE-BHK; Example 10) as main antibodies, with dilutions of 1: 200 and 1: 400 respectively. Biotin-conjugated goat anti-rabbit IgG (Vector; Cat. No. BA-1000, 1.5 mg / ml) was used as secondary antibody at 1: 200 dilution. In separate samples, protein competition was performed using additional soluble zalpha11CEE receptor protein (in excess of 10X) (Example 6A) for the main antibody to pre-block immunoreaction of the main antibody. This competition was used as a control for the specificity of the rabbit polyclonal antibody for zalpha11. Detection was performed on the Ventana ChemMate 500 instrument using a ChemMate DAB kit (labeled streptavidin-biotin kit with application of a streptavidin-horseradish peroxidase conjugate, and DAB substrate) according to the manufacturer's instructions and using the stain of manufacturer's hematoxylin contrast for 30 seconds (Ventana Biotek Systems, Tucson, AZ).

High expression of zalpha11 was observed in PMA activated HL60 cells. Low level of expression was observed in PBL and HL60 cells without stimulation. A subset of cells from the spleen, thymus and lymph node of the mouse showed positive staining. Human and mouse lymph node and spleen, and HL60 cells with DM45 stimulation

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SO showed minimal or no coloration. The signal seen in cells and tissues was mostly competed using the soluble zalpha11 receptor protein in excess. Negative control brain and liver tissues did not show staining.

Example 13

Identification of peripheral blood mononuclear cells (PBMNC's) expressing zalphaII receptor using polyclonal rabbit antisera for soluble zalphaII receptor

200 ml of fresh heparinized blood was obtained from a normal donor. Blood was diluted 1: 1 in PBS, and separated using a Ficoll-Paque PLUS gradient (Pharmacia Biotech, Uppsala, Sweden) and the lymphocyte interface was collected. Cells were washed 2X in PBS and resuspended in RPMI medium + 5% FBS with a concentration of 2 x 10⁶ cells / ml.

In order to determine if zalpha11 receptor expression is affected by the activation state of lymphocyte cells, that is, between resting and activated cells, several stimulation conditions were used: 1) unstimulated, that is, medium alone (RPMI medium + 5% FBS); stimulated with 10 ng / ml PMA + 0.5 jug / ml ionomycin (both from Calbiochem); and 3) activation with PHA (phytohemagglutinin-P, Difco / VWR). Cells were incubated at 37 ° C for 17 hours and then harvested for staining to detect zalpha11 receptor expression.

An indirect staining method was used. Briefly, human lymphocyte cells were suspended in staining buffer (PBS + 0.02% NaN₃ + 1% BSA 2% normal human serum) and plated at 2 x 10⁵ cells at 50 joules / well in a 96-well plate. Antibodies to the soluble receptor zalpha11CEE (Example 15) were used to determine whether they co-stained with a marker (CD14) on B cells (CD19), T cells (CD3), or mo nocytes on the isolated human lymphocytes. A rabbit polyclonal serum for soluble zalpha11 receptor (anti-huzalpha11-CEE-Rb BHK) (Example 10) was used as antibody at 10 jug / ml to identify zalpha11 in lymphocytes. A secondary antibody, goat anti-rabbit Ig-FITC (Biosource, Camarillo, CA) was used to visualize the binding of Rb anti-huzalpha11-CEE-BHK antibody to zalpha11 receptors. Other antibodies were used simultaneously to stain T cells (CD3-PE; PharMingen, San Diego, CA), B cells (CD19-PE) (PharMingen), and monocytes (CD14-PE) (PharMingen) in order to identify co-staining. of the anti-zalpha11 receptor antibody in these cell types. Various controls were used to determine non-specific binding and background levels of staining: (1) an irrelevant polyclonal rabbit serum was used as a non-specific control; and (2) a secondary antibody alone was used to determine background binding of that reagent. Purified zalpha11CEE soluble receptor (Example 6) in approximately 10-fold excess was used as a competitive inhibitor to verify the specificity of rabbit anti-huzalpha11CEE-BHK antibody for zalpha11 soluble receptor.

After plating the cells and adding the primary and co-staining antibodies, the cells were incubated on ice for 30 minutes, washed 2X with staining buffer, and stained with the secondary antibody, rabbit anti-Ig-FITC , from goat (Biosource), for 30 minutes on ice. Cells were washed 2X with staining buffer and resuspended at 200 µl per well in staining buffer containing 7AAD viability dye at a final concentration of approximately 1 jug / ml (Sigma, St. Louis, MO). Samples were read on the FACS-Caliber (Becton-Dickinson, San Jose, CA) and viable cells were analyzed.

The rabbit polyclonal for zalpha11 receptor stained resting B cells. The signal in resting B cells was brighter than the signal achieved using the irrelevant rabbit serum, and the signal decreased to a greater extent in B cells than in T cells with the addition of excess soluble receptor zalpha11-CEE. This experiment was repeated using separate B and T cells, and the results were very similar. Again, the staining with the rabbit polyclonal anti-huzalfa11-CEE-BHK antibody to the zalpha11 receptor was higher in resting B cells.

Example 14

ZalphaI receptor expression in various tissues using real-time quantitative RT / PCR

A. Primers and probes for quantitative RT-PCR

Quantitative real-time RT-PCR has been previously described using the ABI PRISM 7700 sequence detection system (PE Applied Biosystems, Inc., Foster City, CA) (see Heid, CA et al., Genome Research 6: 986-994 , 1996; Gibson, UEM et al., Genome Research 6: 995-1001, 1996; Sundaresan, S. et al., Endocrinology 139: 47564764, 1998). This method incorporates the use of a gene specific probe containing reporter and quench fluorescent dyes. When the probe is intact, the emission of the reporter dye is negated due to the close proximity of the quench dye. During PCR extension using additional gene-specific forward and reverse primers, the probe is cleaved by 5 'nuclease activity of Taq polymerase which releases the reporter dye from the probe resulting in increased fluorescent emission.

The primers and probes used for real-time quantitative RT-PCR analysis of zalpha11 receptor expression were designed using Primer Express® primer design software (PE Applied Biosystems, Foster City, CA). The human zalpha11 receptor primers were designed by extending an intron-exon junction to eliminate genomic DNA cloning. The forward primer, ZC22,277 (SEQ ID NO: 40) and the reverse primer, ZC22,276

ES 2 279 809 T3 (SEQ ID NO: 41) were used in a PCR reaction (post) with a concentration of approximately 300 nM to synthesize a 143 bp product. The corresponding zalpha11 TaqMan® probe, designated ZG31 (SEQ ID NO: 42) was synthesized and labeled by PE Applied Biosystems. The ZG31 probe was labeled at the 5 'end with a reporter fluorescent dye (6-carboxy-fluorescein) (FAM) (Applied Biosystems) and at the 3' end with a quencher fluorescent dye (6-carboxy-tetramethyl-rhodamine) ( TAMRA) (PE Applied Biosystems).

As a control to test the integrity and quality of the RNA samples tested, the rRNA of all RNA samples (later) was examined using a fixed primer and probe ordered from PE Applied Biosystems (Cat. No. 4304483). The kit contains rRNA forward primer (SEQ ID NO: 43) and rRNA reverse primer (SEQ ID NO: 44), rRNA TaqMan® probe (SEQ ID NO: 45). The rRNA probe was labeled at the 5 'end with a VIC reporter fluorescent dye (PE Applied Biosystems) and at the 3' end with the quencher fluorescent dye TAMRA (PE Applied Biosystems). The rRNA results also serve as an internal control and allow normalization of the zalpha11 mRNA expression results seen in the test samples.

RNA samples of CD3, CD19, and monocyte cell types were prepared as described in Example 11 above. Control RNA was prepared using the RNeasy Miniprep® kit (Qiagen, Valencia, CA) according to manufacturer's instructions, starting at approximately 10 million BaF3 cells expressing human zalpha11 receptor (Example 2A).

B. Real-time quantitative RT-PCR

Relative levels of zalpha11 mRNA were determined by analyzing total RNA samples using the one-step RT-PCR method (PE Applied Biosystems). Total RNA was isolated from BaF3 cells expressing human zalpha11 receptor by standard methods and used to generate a standard curve used for quantitation. The curve consisted of 10-fold serial dilutions ranging from 2.5-2.5 x 10^-4 ng / pl for the rRNA examination and 250-0.25 ng / pl for the zalpha11 examination, each point of the standard curve being analyzed in triplicate. Total RNA samples from cells were also analyzed in triplicate for human zalpha11 receptor transcript levels and for endogenous control rRNA levels. In a total volume of 25 µl, each RNA sample was subjected to a one-step RT-PCR reaction containing: approximately 25 ng of total RNA in buffer A (50 mM KCl, 10 mM Tris-HCl); the internal standard dye, carboxy-x-rhodamine (ROX); appropriate primers (approximately 50 nM rRNA primers (SEQ ID NO: 43 and SEQ ID NO: 44) for the rRNA samples; and primers ZC22,277 (SEQ ID NO: 40) and ZC22,276 (SEQ ID NO: 41 ) about 300 nM for zalpha11 samples); the appropriate probe (rRNA TaqMan® probe (SEQ ID NO: 45) about 50 nM for rRNA samples, ZG31 probe (SEQ ID NO: 42) about 100 nM for zalpha11 samples); MgCl₂ 5.5 mM; each 300 µM d-CTP, d-ATP and d-GTP and 600 µ / M d-UTP; MuLV reverse transcriptase (0.25 U / pl); AmpliTaq® Gold DNA polymerase (0.025 U / pl) (PE Applied Biosystems); and RNase inhibitor (0.4 U / pl) (PE Applied Biosystems). The thermal cycling conditions of PCR were as follows: an initial reverse transcription (RT) step of one cycle at 48 ° C for 30 minutes; followed by an activation step with AmpliTaq Gold® (PE Applied Biosystems) of one cycle at 95 ° C for 10 minutes; followed by 40 multiplication cycles at 95 ° C for 15 seconds and at 60 ° C for 1 minute.

Relative levels of zalpha11 RNA were determined using the standard curve method as described by the manufacturer, PE Biosystems (User Bulletin No. 2: ABI Prism 7700 Sequence Detection System, Relative Quantitation of Gene Expression, Feb 11 December 1997). RRNA measurements were used to normalize zalpha11 levels and the resting CD3 + RNA sample was used as a calibrator. The resting CD3 calibrator was arbitrarily chosen and given a value of 1.00. The rest of the samples were compared relative to the calibrator. The data is shown in the following Table 6.

TABLE 6

<td>Sample</td><td>Resting</td><td>4-hour stimulation</td><td>24-hour stimulation</td>

<td>CD3</td><td> 1,00</td><td> 15,27</td><td> 16,70</td>

<td>CD19</td><td> 20,14</td><td> 65,08</td><td> 25,42</td>

<td>Monocytes</td><td> 0,05</td><td>no data</td><td> 0,26</td>

There was a 15-fold increase in zalpha11 receptor expression on CD3 + at 4 and 24 hours. Resting CD19 had a 20-fold increase in receptor expression relative to resting CD3 +. There was a 3-fold increase with a 4-hour stimulation that returned to resting levels within 24 hours. Monocytes showed no detectable zalpha11 receptor expression in this assay.

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C. Purified human T, NK, and B cells as primary source used to assess human zalpha11 receptor expression

Whole blood (150 ml) was collected from a healthy human donor and mixed 1: 1 with PBS in 50 ml conical tubes. Thirty ml of diluted blood was then boosted with 15 ml of Ficoll Paque Plus (Amersham Pharmacia Biotech, Uppsala, Sweden). These gradients were centrifuged 30 min at 500 g and allowed to stop without streaking. RBC depleted cells from the interface (PBMC) were harvested and washed 3 times with PBS. The production of isolated human PBMC was 200 x 10⁶ before the selection described later.

The PBMCs were suspended in 1.5 ml of MACS buffer (PBS, 0.5% EDTA, 2 mM EDTA) and set aside 3 x 10⁶ for control RNA and for flow cytometric analysis. The 0.25 ml of anti-human CD8 microbeads (Miltenyi Biotec) were added and the mixture was incubated for 15 min at 4 degrees C. These cells labeled with CD8 beads were washed with 30 ml of MACS buffer and then resuspended in 2 ml MACS buffer.

A VS + column was prepared according to the manufacturer's instructions. The VS + column was then placed in a VarioMACS magnetic field (Miltenyi). The column was equilibrated with 5 ml of MACS buffer. The isolated primary mouse cells were then applied to the column. CD8 negative cells were allowed to pass through. The column was rinsed with 9 ml (3 x 3 ml) of MACS buffer. The column was then removed from the magnetic field and placed on a 15 ml falcon tube. The CD8 + cells were eluted by adding 5 ml of MACS buffer to the column and the bound cells were removed using the piston provided by the manufacturer. The production of selected human peripheral CD8 + T cells was 51 x 10⁶ total cells. CD8 negative flow-through cells were collected, counted, stained with anti-human CD4 coated beads and then incubated and passed over a new VS + column at the same concentrations described above. The production of selected human peripheral CD4 + T cells was 42 x 10⁶ total cells.

A sample of each of the selected CD8 + and CD4 + human T cells was separated for staining and sorting on a fluorescence activated cell sorter (FACS) to assess its purity. A PE-conjugated anti-human CD4 antibody, an anti-human CD8-FITC Ab, and an anti-human CD19-CyChrome Ab (all from Pharmingen) were used to stain the selected CD8 + and CD4 + cells. The cells selected for CD8 in this first experiment were 80% CD8 +, and the cells selected for CD4 were 85% CD4 +. In 2 subsequent experiments (Example 14B), the purified CD8 + cells were 84% and 81% pure, and the CD4 + cells were 85% and 97% pure, respectively. In one experiment, non-binding (flow-through) cells were stained with anti-human CD19 coated beads (Miltenyi) and passed over a third column of magnetic beads to isolate CD19 + B cells (these were 92% purity).

Selected human CD8 +, CD4 + and CD19 + cells were activated by incubating 0.5 x 10⁶ cells / ml in RPMI + 5% human ultraserum (Gemini Bioproducts, Calabasas, CA) + 10 ng / ml PMA and 0.5 jug / ml ionomycin (Calbiochem) for 4, 16 or 24 hours at 37 ° C. T cells (2.5 x 10⁶/ well) were alternately stimulated in pre-coated 24-well plates overnight with 0.5 jug / ml of plate-bound anti-CD3 mAb UCHT1 (PharMingen) with or without soluble anti-CD28 mAb at a rate of 5 jug / ml. At each time point, cells were harvested, globulized, washed once with PBS, and globulized again. The supernatant was separated and the beads were snap frozen in an ice / ethanol bath, and then stored at -80 ° C for RNA preparation at a later date.

In a separate experiment, human NK cells were enriched from Ficolled PBMC by negative selection using the Human NK Enrichment System (consisting of antibodies to CD3, CD4, CD14, CD19, CD66b, and glycophorin A) from Stem Cell Technologies (Vancouver , BC, Canada). Cell beads were prepared from freshly isolated NK cells from 2 different donors, or from NK cells cultured 24 hours in medium alone or in medium supplemented with 20 ng / ml IL-15. RNA from a human NK cell line from a malignant non-Hodgkin's lymphoma and designated NK-92 (ATCC No. CRL-2407) was also tested. As positive controls, RNA was isolated from human B cell lines CESS (ATCC No. TIB-190), IM-9 (ATCC No. CCL-159) and HS-Sultan (CRL-1484).

Real-time PCR was performed on these selected human NK, CD8 +, CD4 + and CD19 + cells as described above to assess human zalpha11 receptor expression. Relative levels of zalpha11 receptor RNA were determined by analysis of total RNA samples using the one-step RT-PCR method (PE Applied Biosystems). RNA from BaF3 cells expressing human zalpha11 receptor was used to generate appropriate control for standard curves for the real-time PCR described in Example 14C above. The results of the experiments that analyze the expression of the zalpha11 Ligand and the zalpha receptor in stimulated and unstimulated cells are as described in Example 14D-E below.

D. Human zalpha11 receptor and ligand expression on CD4 +, CD8 + and CD19 + cells

The first experiment used RT-PCR, described above, to assess zalpha11 receptor expression in unstimulated and anti-CD3 stimulated CD4 + and CD8 + samples at the 0 h time points (unstimulated ("resting") cells) and at 4 h, 15.5 h, and 24 h after stimulation. The resting CD4 + sample was arbitrarily chosen as the calibrator and given a value of 1.00. There was approximately a 4-fold increase in receptor expression in unstimulated CD4 + cells from 4 h to 24 h of culture and an approximately 8-fold increase during the same time period in stimulated anti-CD3 CD4 + cells. CD8 + cells showed a

ES 2 279 809 T3 7-fold increase in zalpha11 receptor expression that peaked at 4 h and decreased with time. With anti-CD3 stimulation, CD8 + cells had a constant 8-fold increase in receptor expression.

The second experiment used RT-PCR to assess zalpha11 receptor expression in CD4 + and CD8 + samples stimulated anti-CD3, stimulated with PMA + ionomycin, and unstimulated at the time points of 0 h and 3.5 h, 16 h and 24 h. after activation. The resting CD8 + sample was arbitrarily chosen as the calibrator and given a value of 1.00. The resting CD4 + and CD8 + cells did not have significant amounts of receptor expression. Expression was approximately 3-fold higher in PMA + ionomycin-stimulated CD4 + samples at 3.5 hrs, 16 hrs, and 24 hrs after challenge. Expression in activated anti-CD3 CD4 + cells peaked 10-fold above background levels at 3.5 h after challenge, and then returned to 4-fold background levels at 16 h after challenge . CD8 + cells showed a 4-fold increase in expression at 3.5 h after PMA + ionomycin stimulation, with expression decreasing at subsequent time points. As in the first experiment, the anti-CD3 stimulated CD8 + cells again exhibited an 8-fold induction of receptor expression on the background.

The final experiment used RT-PCR to assess zalpha11 receptor expression in CD4 + and CD8 + samples stimulated anti-CD3 and anti-CD3 / anti-CD28 and not stimulated at the time points of 0 h, and at 2 h, 4 h. 16 h after stimulation. Receptor expression was also examined in the same time intervals of CD19 + cells activated with PMA + ionomycin. The resting CD4 + sample was arbitrarily chosen as the calibrator and given a value of 1.00. Anti-CD3 stimulated CD4 + cells at 2 hr had only a 4-fold receptor induction, compared to the 10-fold induction seen at 3.5 hr in the previous experiment. The combination of anti-CD3 and anti-CD8 increased the expression 8-fold over the background. 16 hr anti-CD3 / anti-CD28 stimulated CD8 + cells had very low receptor expression levels, as seen in CD8 + cells in previous experiments (above). CD19 + cells stimulated with PMA + ionomycin had the most significant receptor expression with a 19-fold increase at 2 hr, but expression levels decreased back to those of resting cells at 16 hr.

A certain amount of variation was expected between blood draws (ie, multiple samples at different times from the same patient and between multiple patients). Therefore, data trends within each study or a single blood sample were analyzed, and the above three experiments were compared to obtain an overall conclusion. The trend from the real-time PCR experiments described above is that of all cell types tested, CD19 + B cells activated with PMA + ionomycin expressed the highest levels of zalpha11 receptor RNA. CD4 + and CD8 + cells can also be stimulated to express receptor, but at lower levels than in B cells.

E. Expression of human zalpha11 receptor in human NK cells

Real-time PCR was also performed on human NK cells, purified as described above in Example 14C. Sample NK-92 was arbitrarily chosen as the calibrator and given a value of 1.00. There was approximately a 4.5-fold increase in receptor expression in CESS positive control cells, a 1.5-fold increase in IM-9 cells, and no increase in HS-Sultan cells (0.9-fold relative to to NK-92). NK cells, fresh or cultured overnight with or without IL-15, expressed very similar levels of the zalpha11 receptor as NK-92 (with varying values ​​0.9-1.2 times different relative to NK-92).

Example 15

Identification of cells expressing zalpha11 receptor using in situ hybridization

Specific human tissues were isolated and their expression of zalpha11 was examined by in situ hybridization. The various human tissues prepared, sectioned and subjected to in situ hybridization included thymus, spleen, tonsil, lymphoid node and lung. Tissues were fixed in 10% buffered formalin and paraffin blocked using standard techniques. Tissues were sectioned at 4 to 8 microns. Tissues were prepared using a standard method ("Development of non-isotopic in situ hybridization" at http://dir.niehs.nih.gov//diríep/ish.html). Briefly, tissue sections were deparaffinized with HistoClear (National Diagnostics, Atlanta, GA) and then dehydrated with ethanol. They were then digested with Proteinase K (50 pg / ml) (Boehringer Diagnostics, Indianapolis, IN) at 37 ° C for 2 to 20 minutes. This step was followed with acetylation and rehydration of the tissues.

Two PCR-generated in situ probes were designed against the human zalpha11 sequence. Two groups of oligos were designed to generate probes for separate regions of the zalpha11 cDNA: (1) oligos ZC23,684 (SEQ ID NO: 60) and ZC23,656 (SEQ ID NO: 61) were used to generate a probe of 413 bp for zalpha11; and (2) oligos ZC23,685 (SEQIDNO: 62) and ZC23,657 (SeQIDNO: 63) were used to generate a 430 bp probe for zalpha11. The second probe is 1500 bp 3 'from the first zalpha11 probe. The antisense oligo from each group also contained the working sequence for the T7 RNA polymerase promoter to allow easy transcription of antisense RNA probes from these PCR products. The PCR reaction conditions were as follows: 30 cycles at 94 ° C for 30 s, 60 ° C for 1 min, 72 ° C for 1.5 min. The PCR products were purified by Qiagen spin columns followed by phenol / chloroform extraction and ethanol precipitation. The probes were then labeled with digoxigenin (Boehringer) or biotin (Boehringer) using an in vitro transcription system (Promega, Madison, WI) according to the manufacturer's instructions.

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In situ hybridization was performed with a digoxigenin or biotin-labeled zalpha11 probe (above). The probe was added to the slides at a concentration of 1 to 5 pmol / ml for 12 to 16 hours at 55-60 ° C. The slides were then washed in 2X SSC and 0.1X SSC at 50 ° C. Signals were multiplied using tyramide signal multiplication (TSA) (TSA, indirect set in situ; NEN) and visualized with Vector Red substrate set (Vector Lab) according to manufacturer's instructions. The slides were then stained-counted with hematoxylin (Vector Laboratories, Burlingame, CA).

A signal was seen in the thymus, tonsil, lung, and lymph node. The positive staining cells appeared to be lymphocytes.

Example 16

Secretion Trap Assay

A secretion trap assay was used to identify the cDNA for the zalpha11 Ligand. Positive DNA pools obtained from the expression cloning endeavor were described in commonly owned US Patent Application No. 09 / 522,217.

Conditioned medium of DNA clones transfected into BHK cells in 96-well format, using BaF3 / zalpha11 cells described in Example 2, was put into the proliferation assay. Several DNA pools gave positive activities that were repeated and neutralized with soluble zalpha11 receptors. (Example 6). A positive DNA pool was transfected into COS cells in 12-well format, using the Lipofectamine® method described below.

A secretion trap assay was then performed using soluble zalpha11 receptors (Glu-Glu C-terminal tagged with or without biotination; Flag C-terminal tagging; or soluble Fc4 zalpha11 receptor fusions) (Example 6) to test direct binding between the positive cluster zalpha11 Ligand and soluble zalpha11 receptors (see below). The result was positive, allowing the detection and isolation of clones expressing the zalpha11 Ligand. Plates were shaken at 37 ° C for 24 hours, and DNA minipreps (QiaPrep® 96 Turbo Miniprep Kit; Qiagen) were then prepared in 96-well format using a TomTech Quadra 9600. Plasmid DNA was then pooled in the row format and columns, was transfected into COS cells and secretion trap positive clusters were then determined as described below.

COS cell transfections

COS cell transfection was performed as follows: 3 μl of pooled DNA and 5 μl of Lipofectamine® are mixed in 92 μl of serum-free DMEM medium (55 mg of sodium pyruvate, 146 mg of L-glutamine, 5 mg of transferrin ,

2.5 mg of insulin, 1 µg of selenium and 5 mg of fetuin in 500 ml of DMEM), are incubated at room temperature for 30 minutes and then 400 µl of serum-free DMEM medium are added. This 500 μl mix is ​​added to

1.5 x 10⁵ COS cells / well placed in 12-well tissue culture plate and incubated for 5 hours at 37 ° C. Add 500 µl of 20% FBS DMEM medium (100 ml of FBS, 55 mg of sodium pyruvate and 146 mg of L-glutamine in 500 ml of DMEM) and incubate overnight.

Secretion Trap Assay

The secretion trap was performed as follows: cell medium was rinsed with PBS and then fixed for 15 minutes with 1.8% formaldehyde in PBS. The cells were then washed with TNT (0.1 M Tris-HCl, 0.15 M NaCl, and 0.05% Tween-20 in H₂O), and permeated with 0.1% Triton-X in PBS for 15 minutes and washed again with TNT. Cells were blocked for 1 hour with TNB (0.1 M Tris-HCl, 0.15 M NaCl and 0.5% Blocking Reagent (NEN Renaissance TSA-Direct Kit) in H2O), and washed again with TNT. If biotinylated protein was used, cells were blocked for 15 minute incubations with avidin and then biotin (Vector Labs), washing each other with TNT. Depending on which soluble receptor was used, cells were incubated for 1 hour with: (A) 1-3 µg / ml zalpha11-soluble receptor zalpha11 Fc4 fusion protein (Example 6); (B) 3 µg / ml of C-terminal FLAG-tagged zalpha11 soluble receptor, zalpha11CFLG (Example 6); (C) 3 µg / ml of soluble C-terminal GluGlu-tagged zalpha11 receptor, zalpha11CEE (Example 6); or (D) 3 µg / ml of soluble zalpha11 biotinylated receptor zalpha11CEE (Example 6) on TNB. The cells were then washed with TNT. Depending on which soluble receptor was used, cells were incubated for another hour with: (A) 1: 200 diluted goat anti-human Ig-HRP (specific for Fc); (B) M2-HRP diluted 1: 1000; (C) anti-GluGlu-HRP antibody diluted 1: 1000; or (D) streptavidin-HRP diluted 1: 300 (NEN set) in TNB. Cells were washed again with TNT.

Positive binding was detected with fluorescein tyramide reagent diluted 1:50 in dilution buffer (NEN kit) and incubated for 4-6 minutes, and washed with TNT. Cells were preserved with Vectashield Mounting Media (Vector Labs Burlingame, CA) diluted 1: 5 in TNT. Cells were visualized using a FITC filter under a fluorescent microscope.

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Example 17

Mouse Zalpha11 Ligand Binds Human Zalpha11 Soluble Receptor in Secretion Trap Assay

A plasmid containing DNA encoding the mouse zalpha11 Ligand (SEQ ID NO: 47) was transfected into COS cells, and the soluble human zalpha11 receptor zalpha11-Fc4 (Example 6C) to transfected COS cells was assayed for binding. secretion trap assay (Example 16). The assay confirmed that the mouse zalpha11 Ligand binds to the soluble human zalpha11 receptor.

COS cell transfection was performed according to Example 16 using 0.7 µg of the plasmid in 3 µΕ The secretion trap was performed according to Example 16 using 1 µg / ml of soluble zalpha11 Fc4 receptor fusion protein (Example 6C) in TNB , and goat anti-human Ig-HRP diluted 1: 200 (specific for Fc) in TNB for detectable antibody. Positive binding of soluble human zalpha11 receptor to fixed cells prepared with fluorescein tyramide reagent was detected, preserved and visualized according to Example 16. The positive result indicated that mouse zalpha11 Ligand binds soluble human zalpha11 receptor.

Example 18

Mouse zalpha11 ligand activates human zalpha11 receptor in BaF3 assay using Alamar Blue

BaF / zalpha11 cells were centrifuged, washed and plated in medium free of mIL-3 as described in Example 2. Proliferation of BaF3 / zalpha11 cells was assessed using serum-free conditioned medium of BHK cells expressing Mouse zalpha11 ligand (SEQ ID NO: 47). The conditioned medium was diluted with mIL-3 free medium at concentrations of: 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, or .75% and 0.375%. The proliferation assay was performed as in Example 2. The results confirmed the proliferative response of BaF3 / zalpha11 cells to mouse zalpha11 Ligand. The response, as measured, was approximately 5 times above background at the 50% concentration.

Example 19

Zalpha11 Ligand Activates Human Zalpha11 Receptor in Luciferase Assay

A. Construction of BaF3 / KZ134 / zalpha11 cell lineage

Plasmid KZ134 was constructed with complementary oligonucleotides ZC12,749 (SEQ ID NO: 48) and ZC12,748 (SEQ ID NO: 49) containing 4-gene STAT transcription factor binding elements. A modified c-fos Sis inducible element (m67SIE or hSIE) (Sadowski, H. et al., Science 261: 1739-1744, 1993), the p21 SIE1 of the p21 WAF1 gene (Chin, Y. et al., Science 272: 719-722, 1996), the mammary gland response element of the β-casein gene (Schmitt-Ney, M. et al., Mol. Cell Biol. 11: 3745-3755, 1991) and an inducible STAT element of the Fcg RI gene (Seidel, H. et al., Proc. Natl. Acad. Sci. 92: 3041-3045, 1995). These oligonucleotides contain compatible Asp718-XhoI ends and were ligated, using standard methods, into a receptor firefly luciderase reporter vector with a c-fos promoter (Poulsen, LK et al., J. Biol. Chem. 273: 6229-6232 , 1998) digested with the same enzymes and containing a selectable marker for neomycin. Plasmid KZ134 was used to stably transfect BaF3 cells, using standard transfection and selection methods, to make the BaF3 / KZ134 cell line.

A stable BaF3 / KZ134 reporter cell line, expressing the full-length zalpha11 receptor, was constructed according to Example 1, using approximately 30 µg of the zalpha11 expression vector. Clones were diluted, plated, and selected using standard techniques. Clones were examined by luciferase assay (see Example 19B below) using human zalpha11 Ligand conditioned medium as inducer. Clones with the highest luciferase response (by STAT luciferase) and the lowest background were selected. A stable transfectant cell line was selected. The cell line was named BaF3 / KZ134 / zalpha11.

B. Human and mouse zalpha11 ligand activates human zalpha11 receptor in BaF3 / KZ134 / zalpha11 luciderase assay

BaF3 / KZ134 / zalpha11 cells were centrifuged and washed in medium free of mIL-3. The cells were centrifuged and washed 3 times to ensure separation of mIL-3. The cells were then counted on a hemacytometer. Cells were plated in a 96-well format at approximately 30,000 cells per well in a volume of 100 µl per well using the mIL-3 free medium. The same procedure was used for non-transfected BaF3 / KZ134 cells for use as a control in the subsequent assay.

STAT activation of BaF3 / KZ134 / zalpha11 cells was assessed using conditioned medium from (1) BHK570 cells transfected with an expression vector encoding human zalpha11 Ligand (SEQ ID NO: 19) or (2) transfected BHK570 cells. with an expression vector encoding the mouse zalpha11 Ligand (SEQ ID NO: 47) or (3) medium free of mIL-3 to measure the control response of medium only. The conditioned medium was diluted with RPMI medium free of mIL-3 to concentrations of 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375%. 100 µl of the diluted conditioned medium was added to the BaF3 / KZ134 / zalpha11 cells. The test using the medium

ES 2 279 809 T3 conditioning was done in parallel on non-transfected BaF3 / KZ134 cells as control. The total assay volume was 200 µl. Assay plates were incubated at 37 ° C and 5% CO₂ for 24 hours, at which time the cells were globulized by centrifugation at 2,000 rpm for 10 minutes, the medium was aspirated and 25 µl of lysis buffer (Promega) was added. After 10 minutes at room temperature, the activation of the STAT reporter construct was measured on the plates by reading them on a luminometer (Labsystems Luminoskan, model RS) with 40 μ. \ Additions of luciferase assay substrate (Promega) in an integration five seconds.

The results confirmed the response of the STAT reporter from BaF3 / KZ134 / zalpha11 cells to the human zalpha11 Ligand. The response, as measured, was approximately 50 times over the control with only medium at the 50% concentration. Activation of STAT in response to human zalpha11 Ligand was absent in untransfected BaF3 / KZ134 control cells, showing that the response is mediated through the zalpha11 receptor.

The results also confirmed the STAT reporter response of BaF3 / KZ134 / zalpha11 cells to the mouse zalpha11 Ligand. The response, as measured, was approximately 40 times over the control with only medium at the 50% concentration. Furthermore, activation of STAT in response to mouse zalpha11 Ligand was evident (approximately 5-fold) in untransfected BaF / KZ134 control cells, suggesting that mouse BaF3 cells may have endogenous mouse receptor.

Example 20

Mouse zalpha11 ligand is active in mouse bone marrow assay

A. Isolation of non-adherent low-density marrow cells

Fresh mouse femur (marrow) aspirates were obtained from 6-10 week old male Balb / C or C57BL / 6 mice. The marrow was then washed with RPMI + 10% FBS (JRH, Leneka KS; Hyclone, Logan UT) and suspended in RPMI + 10% FBS as a whole marrow cell suspension. The whole marrow cell suspension was then subjected to a density gradient (Nycoprep, 1077, Animal; Gibco BRL) to enrich for low density, mostly mononuclear cells, as follows: the whole marrow cell suspension (approximately 8 ml ) was carefully pipetted on top of approximately 5 ml of Nycoprep gradient solution in a 15 ml conical tube, and then centrifuged at 600 xg for 20 minutes. The interface layer, containing the low-density mononuclear cells, was then separated, washed with RPMI + 10% excess FBS, and globulized by centrifugation at 400 xg for 5-10 minutes. This bead was resuspended in RPMI + 10% FBS and plated in a T-75 flask at a rate of approximately 10⁶ cells / ml, and incubated at 37 ° C and 5% CO₂ for about 2 hours. The resulting cells in suspension were low density non-adherent marrow cells (NALD).

B. 96-Well Assay

NALD mouse marrow cells were plated at 25,000 to 45,000 cells / well in 96-well tissue culture plates in RPMI + 10% FBS + 1 ng / ml mouse stem cell factor (mSCF ) (R&amp;D Systems, Minneapolis, MN) plus 5% conditioned medium from one of the following: (1) BHK 570 cells expressing mouse zalpha11 Ligand (SEQ ID NO: 47), (2) BHK 570 cells expressing Human zalpha11 ligand (SEQ ID NO: 10) or (3) control BHK 570 cells that contain vector and do not express any Ligand. These cells were then subjected to a variety of cytokine treatments to test marrow hematopoietic cell expansion or differentiation. To assay, plated NA LD mouse marrow cells were subjected to human interleukin-15 (hIL-15) (R&amp;D Systems) or one of a panel of other cytokines (R&amp;D Systems). Serial dilution of hIL-15 or the other cytokines was tested, with 2-fold serial dilution from a concentration of about 50 ng / ml to about 6025 ng / ml. After 8 to 12 days, the proliferation of cells from the 96-well assays was scored by Alamar Blue Assay as described in Example 2.

C. 96-Well NA LD Mouse Marrow Assay Results

The conditioned medium of BHK cells expressing mouse and human zalpha11 Ligand acted in synergy with hIL-15 to promote the expansion of a hematopoietic cell population in mouse NA LD marrow. This expansion of hematopoietic cells was not shown with control BHK plus IL15 conditioned medium. The hematopoietic cell population expanded by mouse zalpha11 Ligand with hIL-15 and hematopoietic cells expanded by human zalpha11 Ligand with hIL-15 were further propagated in cell culture. These hematopoietic cells were stained with a phycoerythrin-labeled anti-Pan NK cell antibody (Pharmingen) and subjected to flow cytometric analysis, which showed that the expanded cells stained positively for this natural killer (NK) cell marker. .

The same 96-well assay was performed using fresh human marrow cells purchased from Poietic Technologies, Gaithersburg, MD. Again, in conjunction with IL-15, mouse and human zalpha11 Ligand expanded a population of hematopoietic cells that were positively stained by the NK cell marker using the antibody described above.

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The soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2Ry, can be used in this assay to measure the binding, antagonistic or inhibitory effects of the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as zalpha11 / IL-2Ry soluble, on the zalpha11 Ligand.

Example 21

ZalphaII-MBP receptor purification

Unless otherwise indicated, all operations were carried out at 4 ° C. The following procedure was used to purify soluble receptor zalpha11-human (or mouse) MBP fusions from E. coli (Example 8). Paste of E. coli frozen pre-centrifuged and diluted in 2 liters of buffer B (0.02 M TRIS (EM Science); 0.2 M NaCl (Mallincrodt); 0.01 M 2-mercaptoethanol (EM Science); pH 8.0 ; with 5 mg / l pepstatin A (Boehringer Mannheim); 5 mg / l aprotinin (Boehringer Mannheim); and 1 mg / l PMSF (Fluka)) plus 1-2 ml of an anti-foaming reagent AF289 ( Sigma). The mixture was made in a pre-cooled French press cell breaker (Constant Systems LTD) with 1,400-2,100 kg / cm².

The lysate was then centrifuged at 18,000 xg for 45 minutes at 4 ° C and the supernatant was retained. A 200 ml suspension of Amylose resin (New England BioLabs), pre-equilibrated in buffer A (0.02 M TRIS (EM Science), 0.2 M NaCl (Mallincrodt), 0-2-mercaptoethanol) was added to the lysate supernatant. 0.01M (EM Science); pH 8.0) and incubated overnight in 21 roller bottles to allow maximum uptake of the MBP fusion protein batch. The resin was washed in batch column format for&gt; 5 column volumes with buffer A, and the batch was then eluted with buffer C (buffer A with 0.02M maltose (Sigma)). Crude fractions were collected and checked by absorbance at 280 nm.

The eluted protein was analyzed by SDS NuPAGE (NOVEX) Coomassie (Sigma) staining. The sample and protein block were stored at -80 ° C.

Example 22

Activity of expanded cells of human and mouse zalphaÍÍ Ligand and mature mouse NK cells in NK cell cytotoxicity assays

A. NK cell assay

NK cell mediated target cytolysis was examined by a⁵¹ Standard Cr. Target cells (K562 cells (ATCC No. CCL-243) in human assays and YAC-1 cells (ATCC No. TIB-160) in mouse assays) lack expression of major histocompatibility complex (MHC) molecules, making them susceptible to NK cell-mediated lysis. A negative control target cell line in mouse assays is MHC + EL4 thymoma (ATCC No. TIB-39). K562, EL4 and YAC-1 cells were grown in RP10 medium (RPMI 1640 standard (Gibco / BRL, Grand Island, NY) supplemented with 10% FBS (Hyclone, Logan, UT) as well as 4 mM glutamine (Gibco / BRL ), 100 IU / ml penicillin + 100 MCG / ml streptomycin (Gibco / BRL), 50 pM β-mercaptoethanol (Gibco / BRL) and 10 mM HEPES buffer (Gibco / BRL). On the day of the assay, 1 were harvested -2 x 10⁶ cells were targeted and resuspended at 2.5-5 x 10⁶ cells / ml in RP10 medium. 50-100 µl of⁵¹5 mCi / ml sodium chromate (NEN, Boston, MA) and incubated for 1 hour at 37 ° C, then washed twice with 12 ml of PBS and resuspended in 2 ml of RP10 medium. After counting the cells in a hemacytometer, the target cells were diluted to 0.5-1 x 10⁵ cells / ml and 100 µl (0.5-1 x 10⁴ cells) with effector cells as described below.

In human trials, effector cells were prepared from selected and expanded human CD34 + BM cells that were harvested, washed, counted, mixed in various concentrations with target cells labeled with ⁵¹Cr in 96-well round bottom plates, and incubated for 4 hours at 37 ° C. After co-incubating effector cells and labeled target cells, half of the supernatant was collected from each well and counted in a gamma counter for 1 min / sample. The specific release percentage of⁵¹ Cr was calculated from the formula 100 x (XY) / (ZY), where X is release of ⁵¹ Cr in the presence of effector cells, Y is the spontaneous release in the absence of effectors and Z is the release of ⁵¹Total Cr of target cells incubated with 0.5% Triton X-100. Data were represented as% specific lysis versus effector to target ratio in each well.

B. Activity of expanded cells of human zalphaIII Ligand

Isolated CD34 + human HPCs were cultured with flt3 +/- zalpha11 Ligand and flt3 + IL-15 +/- zalpha11 Ligand, cells were harvested on day 15 to assess their ability to lyse MHC cells^- K562 in a release trial of ⁵¹Cr standard as described above and to analyze its surface phenotype by flow cytometry. As expected from previous reports (Mrozek, E et al., Blood 87: 2632-2640, 1996; and Yu, H et al., Blood 92: 3647-3657, 1998), the simultaneous addition of IL-15 and flt3L induced the result of a small population of CD56 + cells. Interestingly, although BM cells grown simultaneously with zalpha11 Ligand and flt3L did not expand significantly, there was a significant increase in total cell number in cultures containing a combination of flt3L, zalpha11 Ligand, and IL-15.

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For an assessment of the surface phenotype of these human BM cultures, small aliquots of the cells were stained for 3-color flow cytometric analysis with anti-CD3-FITC, anti-CD56-PE, and anti-CD16-CyChrome mAbs (all from PharMingen , San Diego, CA) and analyzed on a FACSCalibur using CellQuest software (Becton Dickinson, Mountain View, CA). This flow cytometric analysis confirmed that the cells produced from these cultures were differentiated NK cells, because they were large and granular and expressed CD56 and CD16, and were CD3.^- (Lanier, LL Annu. Rev. Immunol. 16: 359-393, 1998). Furthermore, these cells had a significantly higher effector function than cells grown with IL-15 and flt3. More specifically, cells grown on all three cytokines lysed more than 40% of K562 targets at an effector-to-target ratio (E: T) of 1.5, while cells grown on IL-15 + flt3L lysed less than 5 % of objectives in an E: T of 2. These data demonstrate that, in combination with IL-15, the zalpha11 Ligand (commonly owned US Patent Application No. 09 / 522,217) stimulates the differentiation of NK cells from CD34 + BM cells.

C. Mouse Zalpha11 Ligand Expanded Cell Activity

To test the effects of mouse zalpha11 Ligand (commonly owned US Patent Application No. 09 / 522,217) on mouse hematopoietic progenitor cells, purified Lineage-negative (Lin-) bone marrow cells were expanded. of C57Bl / 6 mice in flt3 + IL-15 +/- zalpha11 ligand. On day 6 of culture, cells ("effector") were harvested and counted, and then resuspended in 0.4 ml of RP10 medium (Example 22A). Two aliquots (0.15 ml each) of each expanded sample with or without zalpha11 Ligand (Example 22A) were serially diluted 3 times in duplicate in 96-well round bottom plates, for a total of 6 wells of 100 μl each. The remaining 100 µl of cells were stained for NK cell surface markers with FITC-anti-2B4 and PEanti-DX5 mAbs (PharMingen) and analyzed by flow cytometry. Each group of cells exposed to flt3 + IL-15 with or without the presence of mouse zalpha11 Ligand had similar fractions of 2B4 + DX5 + cells, ranging from 65 to 75% positive for both NK markers.

For the NK lysis assay, target cells (YAC-1 and EL4) were labeled with ⁵¹ Cr as described above. After counting the target cells in a hemacytometer, the target cells were diluted to 0.5-1 x 10⁵ cells / ml and 100 μl of YAC-1 or EL4 (0.5-1 x 10⁴ cells) with 100 μl of effector cells and incubated for 4 hours at 37 ° C. Specific lysis for each well was determined as described above.

Cells grown in the presence of flt3 + IL-15 + zalpha11 Ligand were found to exhibit increased lytic activity (about 2-fold) against YAC-1 targets (but did not kill the control MHC + EL4 cell line). With an effector-to-target ratio (E: T) of 5, NK cells generated in the presence of the 3 cytokines (zalpha11 ligand + flt3 + IL-15) lysed 12% of YAC-1 cells, while NK cells expanded with flt3 + IL-15 lysed 6% of the YAC-1 targets. Subsequent experiments confirmed this trend.

In a second method to determine the biological activity of zalpha11 Ligand on mouse NK cells, immature CD4 CD8 ("double negative", DN) thymocytes were isolated using routine methods and cultured with IL-15 + flt3 + IL -7 or IL-15 + flt3 + IL-2, with or without zalpha11 Ligand. On day 6 of culture, cells were harvested and YAC-1 and EL4 cells were assayed for NK lytic activity as described above. Cells grown in the presence of zalpha11 Ligand were found to have the highest lytic activity in this assay, with increased lytic activity over cells grown in the presence of the other cytokines. Specifically, DN thymocytes grown with IL-15 + flt3 + IL-7 killed 18% of YAC-1 cells with 24 E: T, while cells grown in the presence of IL-15 + flt3 + IL-7 more Ligand of zalpha11 destroyed 48% of the targets with the same E: T. DN thymocytes grown in IL-15 + flt3 + IL-2 destroyed 15% of YAC-1 targets with an E: T of 6, while cells grown with these 3 cytokines and Zalpha11 Ligand destroyed 35% of YAC-1 cells with an E: T of 9. Flow cytometry was performed on cultured cells one day prior to the NK lysis assay. As was true for bone marrow cultures, despite the proliferative effect of the zalpha11 Ligand (the number of cells increases approximately 2-fold when zalpha11 Ligand is added), the fraction of DX5 cells did not significantly increase.⁺ (17-20% of total cells in cultures with IL-7, and 35-46% of the total in cultures with IL-2). These data suggest that the zalpha11 Ligand, in combination with IL-15 and flt3, increases the lytic activity of NK cells generated from mouse bone marrow or thymus.

D. Mouse zalpha11 Ligand Activity on Mature Mouse NK Cells

In order to test the effects of mouse zalpha11 Ligand on mature NK cells, spleens were isolated from four 5 week old C57Bl / 6 mice (Jackson Laboratories, Bar Harbor, ME) and kneaded with glass slides of frosted termination to create a suspension of cells. Red blood cells were separated by hypotonic lysis as follows: cells were globulized and the supernatant was removed by aspiration. The bead was gently vortexed, and then 900 µl of sterile water was added while shaking, followed rapidly (less than 5 s later) by 100 µl of 10X HBSS (Gibco / BRL). The cells were then resuspended in 10 ml of 1X HBSS and debris was separated by passing the cells over a nylon mesh coated cell filter (Falcon). These RBC depleted spleen cells were globulized and then resuspended in MACS buffer (PBS + 1% BSA + 2 mM EDTA) and counted. 300 x 10 were colored⁶ cells with anti-DX5 coated magnetic beads (Miltenyi Biotec) and positively selected DX5 cells⁺ NK on a MACS VS + separation column, according to the manufacturer's instructions, leading to recovery of 8.4 x 10⁶ DX5 + and 251 x 10 cells⁶ DX5 cells^-. Each of these groups of cells was cultured in 24-well plates (0.67 x 10⁶ cells / well, 2 wells per treatment condition) in RP10 medium (Example 22A) alone or with 1) 30 ng / ml mouse zalpha11 Ligand

ES 2 279 809 T3 (Commonly owned US Patent Application No. 09 / 522,217), 2) 30 ng / ml recombinant mouse IL-2 (R&amp;D Systems, Inc., Minneapolis, MN), 3) 30 ng / ml recombinant human IL-15 (R&amp;D), 4) 30 ng / ml each of mouse zalpha11 Ligand and hIL-15 or 5) 30 ng / ml each of mIL-2 and hIL- fifteen. Cells were harvested after 21 hours, washed and resuspended in RP10 medium and counted. The cells were then tested for the ability to lyse YAC-1 or EL4 target cells labeled with ⁵¹ Cr, as described in Example 22A.

Overall, there was little NK activity from the DX5 groups^- (not NK cells), but DX5 cells^- cultured with zalpha11 Ligand and hIL-15 lysed 25% of YAC-1 target cells with an E: T of 82. As a comparison, DX5 cells^- cultured with hIL-15 alone lysed 14% of the YAC-1 targets with an E: T of 110. This suggests that the zalpha11 Ligand and IL-15 act together on the residual NK NK1.1 + cells in this preparation of cells. As for the DX5 + cell preparation, treatment with mouse zalpha11 Ligand alone did not significantly increase its effector function (its lysis of YAC-1 cells was similar to the untreated group). As expected, IL-2 and IL-15 significantly enhanced NK activity. The highest level of lysis, however, was detected in the group treated with zalpha11 Ligand and hIL-15 (65% lysis of YAC-1 cells with an E: T of 3.3, compared to 45% lysis with an E: T of 4 for the hIL-15 treatment group). Taken together, these results suggest that although zalpha11 Ligand alone may not increase NK cell lysis activity, it increases NK lysis activity of mature NK cells when administered with IL-15.

Soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2RY, can be used in this assay to measure binding, antagonistic, or inhibitory effects of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as zalpha11 / IL-2RY soluble, on the zalpha11 Ligand.

Example 23

Proliferation of zalpha11 Ligand from human and mouse T cells in a T cell proliferation assay

A. Proliferation of mouse zalpha11 Ligand from mouse T cells

T cells from C57Bl / 16 mice (Jackson Laboratories, Bar Harbor, ME) were isolated from splenocytes and pooled lymphocytes from axillary, brachial, inguinal, cervical and mesenteric lymph nodes (LNs). Spleens were kneaded with frosted end glass slides to create a cell suspension. The LNs were torn with forceps and passed through a cell sieve to remove debris. Pooled splenocytes and LN cells were separated into CD8 + and CD4 + subsets using two successive MACS magnetic separation columns, according to manufacturer's instructions (Miltenyi Biotec, Auburn, CA). Whole thymocytes were collected from the same mice.

Cells were cultured at 3 x 10⁵ cells / well (thymocytes) or 10⁵ cells / well (mature T cells) with increasing concentrations of purified mouse zalpha11 Ligand (0-30 ng / ml) (commonly owned US Patent Application No. 09 / 522,217) in flat-bottom plates of 96 wells pre-coated overnight at 4 ° C with various concentrations of anti-CD3 mAb 2C11 (PharMingen) for 3 days at 37 ° C. The anti-CD3 antibody served to activate mouse T cells through the T cell receptor. Each well was pulsed with 1 // Ci of³H-thymidine on day 2 and the plates were harvested and counted 16 hours later to assess proliferation.

When zalpha11 Ligand was tested in T cell proliferation assays, it was found to co-stimulate anti-CD3 activated mouse thymocytes, leading to accelerated production of CD8 + CD4 cells (most thymocytes cultured with anti-CD3 + Zalpha11 ligands were CD8 + CD4^- on day 3 of culture, whereas cells cultured with anti-CD3 alone did not significantly deviate to this phenotype until day 5). No significant levels of thymocyte proliferation to zalpha11 Ligand were observed in the absence of anti-CD3.

Interestingly, when mature peripheral mouse T cells were tested for their ability to respond to anti-CD3 zalpha11 + Ligand, it was found that only CD8 +, but not the CD4 + subgroup, responded in a dose-dependent manner to Ligand. by zalfa11. Weak but reproducible proliferation of CD8 + cells (but not CD4 + cells) was also observed in response to zalpha11 Ligand alone. Interestingly, this was not observed for human T cells (see Example 22B below).

B. Proliferation of human zalpha11 Ligand from human T cells

Human CD4 + and CD8 + T cells were isolated from PBMC as described in Example 14. Cells were cultured at a rate of approximately 10⁵ cells / well with increasing concentrations of purified human zalpha11 Ligand (0-50 ng / ml) (commonly owned US Patent Application No. 09 / 522,217) in pre-coated 96-well flat-bottom plates overnight at 4 ° C with various concentrations of anti-human CD3 mAb UCHT1 (PharMingen) for 3 days at 37 ° C. Each well was pulsed with 1 // Ci of³H-thymidine on day 2 and the plates were harvested and counted 16 hours later. In contrast to the results with mouse T cells, preliminary data suggest that human zalpha11 Ligand co-stimulates human CD4 + T cells but not CD8 +, in a dose-dependent manner.

Soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11 / IL-2RY, can be used in this assay to measure binding, antagonistic, or inhibitory effects of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as zalpha11 / IL-2RY soluble, on the zalpha11 Ligand.

ES 2 279 809 T3

Example 24

Human zalpha11 receptor monoclonal antibodies

Zalpha11 receptor monoclonal antibodies were prepared by immunizing 5 male BalbC mice (Harlan Sprague Dawley, Indianapolis, IN) with the purified recombinant protein huzalfa11-CEE-BHK (Example 6). Each mouse was given an initial intraperitoneal (IP) injection of 20 mg of purified protein in complete Freund's adjuvant (Pierce, Rockford, IL) followed by booster IP injections of 10 mg of purified protein in incomplete Freund's adjuvant every two weeks. . Seven to ten days after administering the third booster injection, the animals were bled and serum was collected.

Serum samples from mice produced for huzalpha11-CEE-BHK were characterized by an ELISA titer check using purified recombinant CHO huzalpha11-Fc protein (Example 10C) as an antibody target. A mouse serum sample had titer for the specific antibody target at a dilution of 1: 1,000,000 (1: 1E6). Four mouse serum samples were titer for the specific antibody target at a dilution of 1: 100,000 (1: 1E5).

Splenocytes were harvested from all 4 high-titer mice and fused to mouse SP2 / 0 myeloma cells using PEG 1500 (Boerhinger Mannheim, UK) in two separate fusion procedures using a 4: 1 fusion ratio of splenocytes to cells of myeloma (Antibodies: A Laboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Press). After 10 days of post-fusion growth, hybridomas producing specific antibodies were identified by ELISA using purified recombinant BHK human zalpha11-Fc4 protein as antibody target (Example 6C) and by FACS using Baf3 cells expressing the huzalpha11 sequence ( Example 2) as an antibody target. The 4 hybridomas that resulted positive by both methods were cloned three times by limiting dilution. The antibodies were named: 249.28.2.1.2.2; 247.10.2.15.4.6; 249.19.2.2.3.5; and 249.15.2.4.2.7.

Example 25

Dose-response study of purified recombinant human zalpha11 receptor protein in normal mice

A. Summary

Normal nine week old female C57Bl / 6 mice (Harlan Sprague Dawley, Indianapolis, IN) were treated by intraperitoneal injection once daily for seven days with one of three dose levels of purified recombinant human zalpha11-Fc4 soluble receptor (Example 6C) (5, 50 or 250 pg / mouse / day) or PBS vehicle plus 250 pg per dose of BSA. Body weights were checked every other day. On day seven, the five mice from the highest dose group and five from the vehicle control group were sacrificed. Blood, bone marrow, and tissues were harvested and analyzed. The remaining mice were sacrificed and harvested the next day. Potential disturbances in lymphoid tissues, as well as general physiological and toxicological parameters, were examined.

There was no clinical evidence of toxicity. Liver, kidney, spleen, thymus, and brain were weighed, and there were no differences between the treatment groups in organ weights. No histological changes were found in the examined tissues.

B. Preparation of dosing solutions

Purified recombinant human zalpha11-FC4 receptor fusion protein (zalpha11-FC4) (Example 6C) was diluted in sterile phosphate buffered saline (PBS) (GibcoBRL, Grand Island, NY) in concentrations to deliver 5, 50, or 250 micrograms of protein in 0.1 ml of PBS vehicle. Bovine serum albumin (BSA) (Sigma, St. Louis, MO) in PBS to make a dose of 250 pg per 0.1 ml, and then filtered through a 0.2 pm syringe tip filter for the vehicle control treatment. Solutions for daily dosing were made on Day 0, aliquoted and frozen in a -20 ° C freezer frosted for use. On the day of administration, the appropriate aliquots were thawed and 0.1 ml of solution was injected intraperitoneally at approximately mid-morning each day for seven days.

C. Study design

The mice were nine weeks old at the start of the study. Each zalpha11-FC4 treatment group consisted of five mice; the control group had 10 mice. The highest dose mice and half of the control mice were sacrificed the day after the last of the seven treatments (Day 7). The two remaining control and lowest dose groups were sacrificed the next day (Day 8).

The body weights of the mice were recorded every other day during the treatment. There was no difference in weight gain between the treatment groups during the treatment week.

At sacrifice, tissues were harvested to assess lymphocyte populations by FACS analysis, including bone marrow, thymus, and spleen. Flow cytometric analysis of lymphoid organs and bone marrow was performed with the FACSCalibur (Becton Dickinson, Mansfield, MA). Tissues harvested for histological examination for signs of protein toxicity included: spleen, thymus, liver, kidney, adrenal gland, mesenteric lymph node, duodenum,

ES 2 279 809 T3 pancreas, jejunum, sternum, uterus, ovaries, urinary bladder and gallbladder, salivary gland, heart and lungs. All tissues fixed for histology were kept at 4 ° C overnight in 10% normal buffered saline (Surgipath, Richmond, IL). The following day the NBF was replaced by 70% ethanol and the tissues were returned to 4 ° C until treatment for histology.

Tissues were treated and stained for H&amp;B analysis on site and then shipped to contract pathologist David Fairchild. Blood was collected for whole blood cell counts and serum chemistry profiles. CBC's were made in-house with the Cell Dyn 3500 hematology analyzer (Abbott Diagnostics, Abbott Park, IL). The serum was kept frozen in a frosted -20 ° C freezer until submission to the Phoenix Central Laboratory (Everett, WA) for complete serum chemistry panels. To compare myeloid: erythroid ratios between the 250 μg dose groups of zalfa11R and BSA, an aliquot of bone marrow from one femur was applied to CytoSpin plates (CYTOSPIN 3 CYTOCENTRIFUGE and CYTO SLIDES, Shandon, Pittsburg, PA). Bone marrow slides were tested at Phoenix Central Laboratories.

D. Study results

There were no obvious clinical indications of physiological effects or toxicity of the rh-zalpha11R-FC4 fusion protein at the doses tested (250 µg / day or less). Body weights remained normal for the duration of the treatments. Red blood cell and platelet counts were normal. There were two mice in the 250 μg zalpha11-FC4 dose group whose differential WBC count revealed a possible elevation in the percentage of monocytes, although the other three mice in the group had monocyte percentages equivalent to the mean of the control mice. The difference in monocyte counts from differential white blood cells is not considered a significant finding. There were no other differences in the whole blood counts. Bone marrow cytology did not reveal a change in the myeloid and erythroid progenitor populations, and all cell types present appeared normal. All standard serum chemistry parameters were in normal ranges. There were no differences between the treatment groups in thymus, spleen, kidney, liver, or brain weights. Histological evaluation of the following tissues showed no evidence of abnormalities: thymus, spleen, liver, kidney, adrenal capsule, duodenum, pancreas, jejunum, cecum, colon, mesenteric lymphoid nodes, uterus, ovaries, salivary gland, heart, trachea, lung and brain. The absence of physiological effects in normal mice indicates that the soluble zalpha11 receptor has low toxicity in vivo, which is desirable for a therapeutic agent.

Example 26

Zalpha11 Ligand-dependent proliferation of stimulated anti-CD40 or anti-IgM B cells

A. Purification of human B cells

One vial containing 1 x 10⁸ Apheresis human peripheral blood mononuclear cells (PBMCs), frozen, rapidly thawed in a 37 ° C bath and resuspended in 25 ml of B cell medium (RPMI 1640 medium (JRH Biosciences, Lenexa, KS), 10 % Heat Inactivated Fetal Calf Serum, 5% L-Glutamine, 5% Penicillin / Streptomycin) (Gibco BRL)) in a 50 ml tube (Falcon VWR, Seattle, WA). Cells were tested for viability using Trypan Blue (Gibco BRL). Ten milliliters of Ficoll / Hypaque Plus (Pharmacia LKB Biotechnology Inc., Piscataway, Nj) were layered under the cell suspension and centrifuged for 30 minutes at 1800 rpm and allowed to stop without braking. The interface was then detached and transferred to a fresh 50 ml Falcon tube, brought to a final volume of 40 ml with PBS and centrifuged for 10 minutes at 1200 rpm with the brake on. The viability of the isolated cells was tested using Trypan Blue. Alternatively, freshly drawn human blood was diluted 1: 1 with PBS (Gibco BRL) and layered on Ficoll / Hypaque Plus (Pharmacia), centrifuged and washed as before. Cells isolated from fresh or frozen sources gave equivalent results.

B cells were purified from Ficoll-floated peripheral blood cells from normal (above) human donors with anti-CD19 magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. The purity of the resulting preparations was verified by flow cytometric analysis with anti-CD22 FITC Ab (Pharmingen, San Diego, CA). B cell preparations were typically&gt; 90% pure.

B. Mouse B cell purification

A suspension of mouse splenocytes was prepared by tearing spleens from adult C57Bl / 6 mice (Charles River Laboratories, Wilmington, MA) with curved needles in B cell medium. RBCs were removed by hypotonic lysis. CD43 positive cells were separated with CD43 magnetic beads (Miltenyi Biotec) according to the manufacturer's instructions. The purity of the resulting preparations was checked by flow cytometric analysis with Ab antiCD45R FITC (Pharmingen). B cell preparations were typically&gt; 90% pure.

C. Proliferation of Anti-CD40 Stimulated B Cells in the Presence of Human or Mouse Zalpha11 Ligand

B cells from human or mouse source were resuspended with a final concentration of 1 x 10⁶ cells / ml in B cell medium and plated at 100 μl / well in a 96-well U-bottom plate (Falcon, VWR) containing various stimulation conditions to bring the final volume to 200 μl / well. well. For anti-CD40 stimulation, human cultures were supplemented with 1 μg / ml of anti-human CD40 (Genzyme,

ES 2 279 809 T3

Cambridge, MA) and mouse cultures were supplemented with 1 jug / ml of mouse anti-CD40 (Serotec, UK). Human or mouse zalpha11 Ligand (commonly owned US Patent Application No. 09 / 522,217) was added at varying dilutions of 1 pg / ml-100 ng / ml as appropriate. The specificity of the effect of zalpha11 Ligand was confirmed by inhibition of zalpha11 Ligand with 25 mg / ml soluble human zalpha11CEE (Example 6A). All treatments were carried out in triplicate. The cells were then incubated at 37 ° C in a humidified incubator for 120 hours (human) or 72 hours (mouse). Sixteen hours before harvesting, 1 juCi of³H-thymidine (Amersham, Piscataway, NJ) to assess whether B cells had proliferated. Cells were harvested in a 96-well filter plate (UniFilter GF / C, Packard, Meriden, CT) using a cell harvester (Packard ) and were collected according to the manufacturer's instructions. The plates were dried at 55 ° C for 20-30 minutes and the bottom of the wells were closed with an opaque plate sealer. 0.25 ml of scintillation fluid (Microsint-O, Packard) was added to each well and the plate was read using a TopCount Microplate scintillation counter (Packard).

Incubation with zalpha11 Ligand at concentrations of 3 ng / ml or more increased soluble anti-CD40-induced proliferation in a dose-dependent manner in both mouse and human B cells by as much as 30-fold. Mouse and human B cells also responded equally to their respective zalpha11 Ligand. In both species, the stimulation was specific for the zalpha11 ligand, because it was reversed by the presence of the soluble zalpha11 receptor in the culture.

D. Proliferation of stimulated anti-IgM B cells in the presence of human or mouse zalpha11 Ligand

B cells from human or mouse source as described above (parts A and B) were plated as described above (part C). For anti-human cell IgM stimulation, plates were pre-coated overnight with 10 mg / ml Abs anti-human IgM F (ab ')₂ (Southern Biotech Associates, Birmingham, Alabama) and washed with sterile medium immediately prior to use. The cultures were supplemented with 0-10 ng / ml hu rIL-4 (R&amp;D Systems, Minneapolis, MN). For anti-IgM stimulation of mouse cells, soluble anti-IgM (Biosource, Camarillo, CA) was added to the cultures at a rate of 10 mg / ml. For each of the foregoing anti-IgM / IL4 conditions, human or mouse zalpha11 Ligand was added at varying dilutions from 1 pg / ml to 100 ng / ml as described above. The specificity of the zalpha11 Ligand effect was confirmed by inhibition with soluble human zalpha11 receptor as described above (Part C). All treatments were carried out in triplicate. Cells were incubated, labeled with³H-thymidine, harvested and analyzed as described in part C above.

Incubation with zalpha11 Ligand at concentrations of 0.3 ng / ml or more inhibited proliferation induced by insoluble anti-IgM (mouse) or anti-IgM and IL-4 (human) in a dose-dependent manner. This inhibition was specific for zalpha11 Ligand, because it was reversed by the presence of soluble zalpha11 receptor in the culture.

E. Proliferation of anti-CD40 B cells requires IL-2 receptor gamma

Mouse B cells were purified and stimulated with anti-CD40 monoclonal antibody as described in Example 26B and C above. Mouse zalpha11 Ligand-induced co-stimulation was completely blocked by the addition of monoclonal anti-IL2 receptor gamma (IL-2Ry) antibodies that block IL-2y utilization. Antibodies 3E12 and TUG / m2 (PharMingen, San Diego, CA) were included in the proliferation assay at 50 jug / ml. These results demonstrate that IL-2Ry in B cells is physiologically involved with ligand stimulation of zalpha11 of B cells. Furthermore, these results provide indirect functional support in vivo to find that IL-2Ry heterodimerizes with the zalpha11 receptor in vitro (Example 27 below).

F. The effects of zalpha11 Ligand on B cells are inhibited by soluble zalpha11 receptor constructs

Mouse B cells were purified and stimulated with anti-CD40 monoclonal antibody or anti-IgM antibodies as described in Example 26C and D above. The mouse zalpha11 Ligand-induced effect was completely blocked by the addition of purified hu-zalpha11R: IL-2Ry heterodimeric soluble receptor (Example 28) or a mu-zalpha11-Fc heterodimeric soluble receptor (Example 6C). Again, these results provide additional functional support to find that IL-2Ry heterodimerizes with the zalpha11 receptor (Example 27 below), and acts as an antagonist to the effect of zalpha11 Ligand on B cells.

Example 27

Human zalpha11 receptor heterodimerized with IL-2 gamma receptor

A. Assay using conditioned medium from transfected BHK-570 cells expressing human zalpha11 Ligand

Soluble human zalpha11 receptor zalpha11CFLAG (Example 6B) or gp130 (Hibi, M. et al., Cell 63: 1149-1157, 1990) were biotined by reaction with a five-fold molar excess of sulfo-NHS-LC-biotin (Pierce , Inc., Rockford, IL) according to the manufacturer's method. Soluble zalpha11 receptor and Il-2-y receptor (sIL-2Ry) (R&amp;D Systems, Minneapolis, MN) were labeled with a five-fold molar excess of Ru-BPY-NHS (Igen, Inc., Gaithersburg, MD) according to the manufacturer's method. The biotinated and Ru-BPY-NHS-labeled forms of the soluble zalpha11 receptor are

ES 2 279 809 T3 named respectively Bio-zalpha11 and Ru-zalpha11 receptor; the biotinated and Ru-BPY-NHS-labeled forms of soluble IL-2Ry were named Bio-IL2Ry and Ru-IL2Ry, respectively.

For characterization of the initial receptor binding of human zalpha11 Ligand, conditioned medium from transfected BHK-570 cells expressing human zalpha11 Ligand or control medium from non-transfected BHK-570 cells were used to determine if the zalpha11 Ligand could mediate in zalpha11 receptor homodimerization and whether it could mediate zalpha11 receptor heterodimerization with IL-2Ry or gp130. To do this, 50 μl of conditioned medium, from control cells or conditioned medium from cells expressing zalpha11 Ligand, were combined with 50 μl of TBS-B (20 mM Tris, 150 mM NaCl, 1 mg / ml BSA, pH 7.2) containing 400 ng / ml of Ru-zalpha11 and Bio-zalpha11 receptor, or 400 ng / ml of Ru-zalpha11 receptor and Bio-gp130 or 400 ng / ml of Ru-IL2Ry and Bio-zAlf11. After incubation for one hour at room temperature, 30 µg of 2.8 mm magnetic beads, coated with streptavidin (Dynal, Inc., Oslo, Norway) were added and the reaction was incubated an additional hour at room temperature. 200 µl ORIGEN Assay Buffer (Igen, Inc., Gaithersburg, MD) was then added and the extent of receptor association was measured using an M8 ORIGEN analyzer (Igen, Inc.).

The conditioned medium containing zalpha11 Ligand caused Bio-zalpha11 receptor heterodimerization with Ru-IL2Ry. No receptor dimerization was observed in the presence of control medium. The conditioned medium containing zalpha11 Ligand did not cause Ru-zalpha11 receptor homodimerization with Bio-zalpha11 receptor, nor Ru-zalpha11 receptor heterodimerization with Bio-gp130.

B. Assay Using Purified Human Zalpha11 Ligand

To assess the ligand specificity of zalpha11 and IL2Ry receptor heterodimerization, 50 μl of TBS-B containing 400 ng / ml of Ru-zalpha11 and Bio-zAlf11 receptor, or 400 ng / ml of Ru-IL2Ry and BiozAlf11 were combined. , with 50 μl of TBS-B containing IL-2, IL-4, IL-15 or purified human zalpha11 Ligand (commonly owned US Patent Application No. 09 / 522,217) with concentrations of 133 pg / ml to 300 ng / ml. After incubation for one hour at room temperature, 3 µg of streptavidin-coated 2.8 mm magnetic beads (Dynal, Inc.) were added and the reaction was incubated an additional hour at room temperature. 200 µl of Origlo Assay Buffer (Igen, Inc.) was then added and the extent of receptor association was measured using an M8 Origen analyzer (Igen, Inc.). Human zalpha11 Ligand caused Bio-zalpha11 receptor heterodimerization with RuIL-2Ry in a dose-dependent manner with a half-maximum concentration of 10 ng / ml. Ru-zalpha11 receptor homodimerization with Bio-zalpha11 was not observed with any concentration of zalpha11 Ligand tested. Ru-zalpha11 receptor homodimerization with Bio-zalpha11 receptor or Bio-zalpha11 receptor heterodimerization with IL-2, IL-4 or IL-15 was not observed at any of the concentrations tested. Thus, the results show that the human zalpha11 receptor specifically heterodimerizes with the IL-2-y receptor in the presence of human zalpha11 Ligand, and that the zalpha11 receptor does not homodimerize or heterodimerize in the presence of other cytokines tested.

Example 28

Construction to generate human zalpha11 receptor / IL-2Ry heterodimer

A vector was constructed that expresses a secreted human hzalpha11 / hIL2Rgamma heterodimer. In this construct, the extracellular domain of hzalfa11 was fused to the heavy chain of IgG gamma 1 (IgGy 1 (IgGy 1) (SEQ ID NO: 16), while the extracellular portion of hIL-2Ry was fused to a kappa light chain human (human κ light chain) (SeQ ID NO: 18).

A. Construction of IgG gamma 1 and human κ light chain fusion vectors

The IgGy 1 heavy chain was cloned into the mammalian expression vector Zem229R (ATCC deposit # 69447) such that any extracellular portion of a receptor having a 5 'EcoRI and 3' NheI site can be cloned to produce a N-lerimnal-IgGy 1 C-terminal extracellular domain fusion. The IgGy 1 fragment used in this construct was made using PCR to isolate the IgGy1 sequence from a Clontech hFetal Liver cDNA library as a template. A PCR reaction was performed as follows using oligos ZC11,450 (SEQ ID NO: 50) and ZC11,443 (SEQ ID NO: 51): 40 cycles of 94 ° C for 60 s, 53 ° C for 60 s, and 72 ° C for 120 s; and 72 ° C for 7 min. The PCR products were separated by agarose gel electrophoresis and purified using a QiaQuick gel extraction kit.^® (Qiagen). The isolated 990 bp DNA fragment was digested with Mlu I and EcoRI (boerhinger Mannheim), ethanol precipitated and ligated with oligos ZC11,440 (SEQ ID NO: 52) and ZC11,441 (SEQ ID NO: 53 ), comprising a MluI / EcoRI linker, in Zem229R previously digested with, and EcoRI, using standard molecular biology techniques described herein. The generic cloning vector was named Vector # 76 hIgGgamma1 w / Ch1 # 786 Zem229R (Vector # 76). The polynucleotide sequence of the extracellular domain of hzalpha11 fused to the heavy chain of IgG gamma 1 is shown in SeQ ID NO: 15 and the corresponding polypeptide sequence is shown in SEQ ID NO: 16.

The human κ light chain was cloned into the mammalian expression vector Zem228R (ATCC deposit # 69446) such that any extracellular portion of a receptor having a 5 'EcoRI site and a 3' KpnI site can be cloned to produce an N-terminal extracellular domain-C-terminal human κ light chain fusion. The human κ light chain fragment used in this construct was made using PCR to isolate the light chain sequence

ES 2 279 809 human T3 κ from the same Clontech hFetal Liver cDNA library used above. A PCR reaction was carried out under the conditions described above using oligos ZC11,501 (SEQ ID NO: 54) and ZC11,451 (SEQ ID NO: 55). The PCR products were separated by agarose gel electrophoresis and purified using a QiaQuick® gel extraction set (Qiagen). The isolated 315 bp DNA fragment was digested with Mlu I and EcoRI (boerhinger-Mannheim), ethanol precipitated and ligated with the MluI / EcoRI linker described above in Zem228R previously digested with, and EcoRI, using techniques molecular biology standards described here. This generic cloning vector was named Vector # 77 hKlight # 774 Zem228R (Vector # 77). The polynucleotide sequence of the extracellular domain of hIL-2Ry was fused to a human kappa light chain as shown in SEQ ID NO: 17 and the corresponding polypeptide sequence is shown in SEQ ID NO: 18.

B. Insertion of zalpha11 or IL-2Ry receptor extracellular domains into fusion vector constructs

Using the above construction vectors, a construct was made having human zalpha11 fused to IgGy1. This construction was made by subjecting human zalpha11 receptor PCR of a CD4 + bone marrow library (selected and made in our own facilities) with oligos ZC24,052 (SEQ ID NO: 56) and ZC24,053 (SEQ ID NO: 57), under conditions described as follows: 30 cycles of 94 ° C for 60 s, 57 ° C for 60 s and 72 ° C for 120 s; and 72 ° C for 7 min. The resulting PCR product was digested with EcoRI and NheI, gel purified as described above and ligated to Vector # 76 (above) digested with EcoRI and NheI previously and band purified. The resulting vector sequence was determined to confirm that the human zalpha11 / IgG gamma 1 (hzalpha11 / Ch1 IgG) fusion was correct. The hzalfa11 / Ch1 IgG gamma 1 vector was named Vector # 190.

A separate construct was also constructed that had IL-2Ry lightly fused κ. The IL2Ry / human κ light chain construction was performed as before by subjecting to PCR from the same CD4 + library mentioned above with oligos ZC12,834 (SEQ ID NO: 58) and ZC12,831 (SEQ ID NO: 59), digesting the resulting band with EcoRI and KpnI and then ligating this product to a Vec # 77 (above) previously digested with EcoRI and KpnI and band purified. The sequence of the resulting vector was determined to confirm that the human IL-2Ry / human κ light chain (hIL-2Ry / Kligera) fusion was correct. This vector was named hIL-2 gamma / Kligera # 1052 Zem228R Vector # 101.

D. Co-expression of human zalpha11 and human IL-2Ry receptors

Approximately 16 µg of each of Vectors # 190 and # 101 above were co-transfected into BHK570 cells (ATCC No. CRL-10314) using LipofectaminePlus® reagent (Gibco / BRL) according to the manufacturer's instructions. The transfected cells were selected for 10 days in DMEM + 5% FBS (Gibco / BRL) containing 1 juM methotrexate (MTX) (Sigma, St. Louis, MO) and 0.5 mg / ml G418 (Gibco / BRL ) for 10 days. The resulting pool of transfectants was selected again in 10 µM MTX and 0.5 mg / ml G418 for 10 days.

The resulting pool of doubly selected cells was used to generate protein. Three Factories (Nunc, Denmark) from this pool were used to generate 10 µl of serum-free conditioned medium. This conditioned medium was passed over a 1 ml protein-A column and eluted in 750 microliter fractions (10). 4 of these fractions found to have the highest concentration were pooled and dialyzed (10 kD Mw cutoff) against PBS. Finally, the dialyzed material was subjected to amino acid analysis (AAA) and found to have a concentration of 227.17 jug / ml of AAA. A total of 681.5 µg was obtained from this purification of 101. The human zalpha11 receptor / purified IL-2Ry receptor was used to assess its ability to compete with the human zalpha11 Ligand in a BaF3 proliferation assay (Example 29 below ).

Example 29

Soluble human zalpha11 receptor / human IL2 gamma receptor-Fc as antagonist of zalpha11 Ligand

BaF3 cells stably expressing the human zalpha11 receptor (Example 2) were plated at 5500 cells per well in standard 96-well tissue culture plates in base medium plus 3ng / ml human zylpha11 Ligand. The base medium is 500 ml of RPMI 1640 (JRH Biosciences), 5 ml of 10x sodium pyruvate (Gibco BRL), 5 ml of 100x L-glutamine (Gibco BRL) and 50 ml of fetal calf serum (FBS) inactivated by heat (Hyclone Laboratories). A decreasing dose of purified soluble human zalpha11 receptor-Fc homodimer (Example 6C) or purified soluble human zalpha11 receptor / human IL2 gamma receptor heterodimer (Example 27) was added to the cells. An Alamar Blue proliferation assay was performed and fluorimetry was done as in Example 2B.

The zalpha11 receptor / IL2 gamma-Fc receptor heterodimer inhibited the activity of human zalpha11 Ligand in a dose-dependent manner, with 0.312 jug / ml being able to completely inhibit the activity of 3 ng / ml human zalpha11 Ligand. The soluble zalpha11-Fc receptor homodimer was also capable of inhibiting zalpha11 Ligand activity in a dose-dependent manner, although it required approximately 10 jug / ml of soluble homodimer to fully inhibit the activity of 3 ng / ml of zalpha11 Ligand. . These data suggested that the soluble zalpha11 receptor / IL2 gamma-Fc receptor heterodimer is approximately 30 to 100 times more potent than the homodimer soluble zalpha11 receptor in inhibiting human zalpha11 Ligand.

ES 2 279 809 T3

Example 30

Zalfa11 receptor layout

To assess the distribution of the zalpha11 receptor in various cell types, rabbit polyclonal and mouse monoclonal antibodies (mAbs) directed against the human receptor were generated (Example 24 and Example 10) and these antibodies were conjugated to biotin for use in cytometry. flow. Polyclonal antibodies, which were of relatively low affinity, were initially used to stain a panel of cell lines: IL-3 dependent mouse pre-B cell line wild-type BaF3 cells (Palacios and Steinmetz, ibid .; Mathey- Prevot et al., Ibid.); BaF3 cells transfected with human zalpha11 (Example 2); Human Burkitt's lymphoma cell lines Raji (ATCC No. CCL-86), Ramos (ATCC No. CRL-1596), RPMI 8226 (ATCC No. CCL-155) and Daudi (ATCC No. CCL-213); Jurkat human T-cell leukemia cell line (ATCC No. TIB-152); human myelomonocytic leukemia cell lines Thp-1 (ATCC No. TIB-202) and U937 (ATCC No. CRL-1593.2); human pro-myelomonocytic cells HL-60 (ATCC No. CCL-240); Mouse B-cell lymphoma cell line A20 (ATCC No. TIB-208); and mouse thymoma cell line EL4 (ATCC No. TIB-39).

Cells were harvested and washed once with serum FACS wash buffer (WBS). The WBS consisted of Hank's Balanced Salt Solution (Gibco / BRL) + 10 mM HEPES (Gibco / BRL) + 1% BSA (Sigma) + 10% normal goat serum (Gemini Bioproducts, Woodland, CA) + 10% from normal rabbit serum (Sigma); the wash buffer (WB) was identical to WBS except that it is serum free. After washing, cells were resuspended in 100 µl of WB containing 10 µg / ml of rabbit anti-zalpha11 polyclonal antibodies (Example 10). Cells were kept on ice with Ab for 20 min, then washed with WB and resuspended in WB containing rabbit, goat anti-FITC (BioSource, International), incubated a further 20 min on ice, and then washed and resuspended in 400 µl WB for analysis on a FACSCalibur flow cytometer (Becton Dickinson). Control samples were stained with secondary rabbit, goat, anti-FITC Ab only. Although the polyclonal antibodies were of low affinity, it was reasonably expected that zalpha11 expression would be detected in the BaF3 / zalpha11 transfectant, in the four human Burkitt lymphomas (Raji, Ramos, Daudi, and RPMI 8226), and in T cells from Jurkat. The data with the monocytic cell lines were more ambiguous. HL-60 cells at rest (undifferentiated) did not bind to anti-zalpha11 antibodies, but a positive signal was detected in HL-60 cells activated for 24 hours with PMA (Calbiochem, La Jolla, CA) that induces differentiation of HL-60 cells into a monocyte-like cell. A positive signal was also seen in U937 and Thp-1 cells, although this signal may have been due to non-specific binding. Polyclonal antibodies were weakly cross-reactive in the mouse B cell line A20, but EL4 mouse thymoma staining was not seen.

The four anti-zalpha11 monoclonal antibodies (Example 24) were conjugated to biotin, and the zalpha11 receptor expression of a subset of the cells described above (BaF3, BaF3 / zalpha11, Raji, Jurkat and resting HL-60) was examined. Cells were harvested, washed, and then resuspended in 100 µl WB containing 15 µg / ml of one of each of the 4 biotinated mAbs. Cells were incubated with mAb for 20 min on ice, then washed with 1.5 ml of WB, and globulized in a centrifuge. The supernatant was removed by aspiration and the beads were resuspended in 100 µl of CyChrome-conjugated streptavidin (CyC-SA; PharMingen), then incubated on ice for another 20 min and washed and globulized as before. Control tubes contained cells stained only with CyC-SA. The beads were resuspended in 400 µl of WB and flow cytometry was performed as before. Positive staining was defined as a signal that exceeds the background level of staining with CyC-SA alone. Using the BaF3 / zalpha11 transfectant as a control, the 4 mAbs could be classified in terms of their respective mean fluorescence intensities (MFI), which may reflect the affinity of the antibody and / or the extent of biotination of the mAbs. The mAbs were ranked as follows, from highest to lowest MFIs: 249.28.2.1.2.2, 247.10.2.15.4.6, 249.19.2.2.3.5, and 249.15.2.4.2.7. This pattern was essentially the same in both Raji and Jurkat cells, indicating that zalpha11 is expressed in these B and T cell lines. The staining patterns in non-activated HL60 cells were identical for all mAbs, and the signal was very weak. It is speculated that this does not reflect the actual expression of zalpha11 by this cell lineage, but rather is a function of non-specific binding of mouse mAbs to human cells, probably via Fc receptors.

Example 31

Reconstitution of human zalpha11 receptor in vitro

To identify components involved in the zalpha11 signaling complex, receptor reconstitution studies were performed as follows. BHK 570 cells (ATCC No. CRL-10314) transfected, using methods described herein, with the luciferase reporter plasmid KZ134 (Example 19) served as a bioassay cell line to measure the signal transduction response of a transfected zalpha11 receptor complex. to the luciferase reporter in the presence of zalpha11 ligand. BHK cells do not endogenously express the zalpha11 receptor. The bioassay cell line was transfected with the zalpha11 receptor alone, or co-transfected with the zalpha11 receptor along with one of a variety of other known receptor subunits. Each receptor subunit was cloned using PCR followed by ligation into appropriate expression vectors; the correct sequence of each construct was confirmed prior to transfection. Cell lineage receptor expression was assayed by RT / PCR prior to assays. The receptor complexes tested included: zalpha11 sol receptor: zalpha11 receptor with IL-2Ry; zalpha11 receptor with IL-2Ry and IL-2Re; zalpha11 receptor with IL-2Ry and IL-13Ra; zalpha11 receptor with IL-2Ry and IL-2Ra; and zalpha11 receptor with IL2Ry and IL-4Ra. Each independent receptor complex cell line was assayed in the presence of zalpha11 Ligand

ES 2 279 809 T3 human and luciferase activity was measured as described in Example 19. The untransfected bioassay cell line served as a control for background luciferase activity, and was used as a baseline to compare the signaling by the various receptor complex combinations. In each cell line containing zalpha11 receptor and IL-2Ry, the maximum luciferase activity was approximately two-fold over background in the presence of zalpha11 Ligand. No signal enhancement was observed in the presence of any other receptor subunit tested (IL-2Re, IL-2Ra, IL-4Ra, or IL-13Ra).

Other zalpha11 receptor complexes that can be assessed by this method include combinations of the zalpha11 receptor with one or more of the receptor components of the IL-4 / IL-13 receptor family (IL-13Ra '), as well as other interleukin receptors. (eg IL-15Ra, IL-7Ra, IL-9Ra).

Example 32

Binding study of human zalphaIÍ Ligand labeled with ¹²⁵1 in cell lines

25 micrograms of purified human zalpha11 Ligand (commonly owned US Patent Application No. 09 / 522,217) were labeled with 2 mCi of ¹²⁵I using iodine beads (Pierce, Rockford, Illinois) according to the manufacturer's instructions. This labeled protein was used to assess the binding of human zalpha11 Ligand to human Raji cells (ATCC No. CCL-86), using binding to wild-type mouse BaF3 cells, and zalpha11 receptor transfected BaF3 cells (BaF3 / hzalpha11 cells ) as witnesses. Binding of zalpha11 Ligand to BaF3 / hzalpha11 cells (positive control) was expected, while binding to wild-type BaF3 cells (negative control) was not expected, based on proliferation assay results (Example 2). Each was placed in 96-well plates approximately 5 x 10⁵ Raji cells / well, 1 x 10⁶ BaF3 / hzalpha11 cells / well and 1 x 10⁶ BaF3 cells / well. Ten ng / ml of labeled human zalpha11 Ligand was added in duplicate to the wells, with a series of competitor dilutions of unlabeled human zalpha11 Ligand added from 250-fold molar excess in 1: 4 dilutions to 0.061 molar excess times. Each point was made in duplicate. After adding the labeled human zalpha11 Ligand to the wells, it was allowed to incubate at 4 ° C for 2 h to allow binding of Ligand to cells. Cells were then washed 3X in binding buffer (RPMI-1710 (JRH Biosciences) with 1% BSA (Sigma)) and counted in the COBRA II AUTO-GAMMA gamma counter (Packard Instrument Company, Meriden, CT).

Cell-labeled zalpha11 Ligand binding was evident in Raji and BaF3 / hzalpha11 cells. In addition, for Raji cells, a 250-fold mean molar excess of unlabeled zalpha11 Ligand decreased binding 3-fold in the presence of a non-specific non-labeled competitor (Interferon gamma from R&amp;D Systems, Minneapolis, MN) and 3.7-fold with relationship to non-competitor. Competition was observed in a dose-dependent manner for the specific unlabeled competitor, human zalpha11 Ligand. Thus, the binding of zalpha11 Ligand to Raji cells was specific. Similarly, for positive control BaF3 / zalpha11 cells, the 250-fold molar excess of unlabeled zalpha11 Ligand decreased binding 2-fold relative to non-specific competitor and 3.06-fold relative to non-competitor. Thus, the binding of zalpha11 Ligand to BaF3 / zalpha11 cells was also specific. No competitive binding was observed with wild-type BaF3 cells. Thus, the zalpha11 Ligand was shown to bind specifically to Raji cells and BaF3 / hzalpha11 cells, but not to negative control BaF3 cells.

The bound radiolabeled zalpha11 Ligand is then crosslinked with the molecule it binds to on the cell surface of Raji cells using standard crosslinking methods, to identify the receptor complex to which it binds on these cells. In addition, anti-zalpha11 receptor antibodies (Example 24 and Example 10) and other anti-cytokine receptor subunit antibodies are used to assess which components of the subunit comprise a functional hzalpha11 receptor complex, for example, in Raji cells and other lineages. cells to which zalpha11 Ligand binds. Such antibodies can be used to compete for zalpha11 Ligand in a binding assay as described above, and thereby show which receptor subunits are present on the surface of Raji cells, and in other cell lines to which Ligand of zalfa11. In addition, such antibodies can be used to immunoprecipitate radiolabeled zalpha11 Ligand cross-linked material using methods known in the art and described herein. In addition, anti-zalpha11 Ligand antibodies (commonly owned US Patent Application No. 09 / 522,217) can be used to immunoprecipitate radiolabeled zalpha11 Ligand cross-linked material.

Example 33

Expression of the zalphaII receptor in human blood cells

A. Preparation and culture of human peripheral blood cells

Freshly drawn human blood was diluted 1: 1 with PBS (GIBCO BRL) and layered on Ficoll / Hypaque Plus (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) and centrifuged for 30 minutes at 1800 rpm and allowed to stand still. The interface layer was separated and transferred to a new 50 ml Falcon tube (Falcon, VWR; Seattle, WA), brought to a final volume of 40 ml with PBS and centrifuged for 10 minutes at 1200 rpm with the brake. Market Stall. Isolated cells were assayed for viability using Trypan Blue (GIBCO BRL) and cells were resuspended with a final concentration of 1 x 10⁶ cells / ml cell medium (RPMI 1640 medium, 10% heat inactivated fetal calf serum, 5% L-glutamine, 5% penicillin / streptomycin) (GIBCO BRL).

ES 2 279 809 T3

Cells were grown in 6-well plates (Falcon, VWR) for 0, 4, or 24 hours with a variety of different stimuli described below. Anti-IgM, anti-CD40 and anti-CD3 stimulation was done as in Example 26. Phorbol myristate acetate (PMA) and ionomycin (Sigma, St. Louis, MO) were added to appropriate wells at 10 ng / ml and 0.5 mg / ml respectively. Cells were incubated at 37 ° C in a humidified incubator for various periods of time.

B. Antibody staining and testing

Cells were collected from the plates, washed, and resuspended in ice-cold staining medium (HBSS, 1% fetal calf serum, 0.1% sodium azide) with a concentration of approximately ten million cells per milliliter. . Blockade of the Fc receptor and non-specific binding of antibodies to cells were achieved by adding 10% normal goat serum (Gemini Bioproducts, Woodland, CA) and 10% normal human serum (Ultraserum, Gemini) to the cell suspension. . Aliquots of the cell suspensions were mixed with a FITC-labeled monoclonal antibody against one of the lineage markers CD3, CD19, or CD14 (PharMingen, La Jolla, CA) and a biotinylated monoclonal antibody against the human zalpha11 receptor (hu-zalpha11 ) (Example 24). After incubation on ice for 60 minutes, cells were washed twice with ice cold staining medium and resuspended in 50 ml of staining medium containing streptavidin-PE (Caltag, Burlingame, CA). After a 30 minute incubation on ice, cells were washed twice with ice cold wash buffer (PBS, 1% fetal calf serum, 0.1% sodium azide) and resuspended in wash buffer which contained 1 mg / ml 7-AAD (Molecular Probes, Eugene, OR) as a viability marker. Live cell flow data was obtained using a FACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose, CA). Collection and analysis were performed using CellQuest software (BD Immunocytometry Systems).

The results showed that the human zalpha11 receptor is expressed in human peripheral blood cells that express CD3, CD19 or CD14. Activation of T cells with anti-CD3 or B cells with anti-CD40 produced an increased level of cell surface zalpha11 at 24 hours. No increase in zalpha11 expression level was seen at 4 hours with any stimulus in any population of cells. Treatment of cells with zalpha11 Ligand produced a decrease in zalpha11 staining in CD3-positive and CD19-positive cells but not in CD14-positive cells at 4 and 24 hours.

Example 34

Human zalpha11 Ligand activity is blocked with anti-IL-2Ry antibodies in a BaF3 / zalpha11 proliferation assay

The role of the IL-2y receptor was investigated using anti-IL-2y receptor monoclonal antibodies to assess whether they would block zalpha11 Ligand activity in a BaF3 / zalpha11 proliferation assay (Example 2). Conditioned medium from BHK570 cells transfected with human zalpha11 Ligand in concentrations of 5%, 2.5%, 1.25% and 0.625%, with or without IL-2 receptor antibodies, was added to the assay.

The following anti-mouse IL-2 receptor monoclonal antibodies from PharMingen International, San Diego, California were added at 50 µg / ml each: (a) 4G3 + TUGm2 or (b) TM-61. 4G3 and TUGm2 are anti-chain antibodies and_c TM- / H is a purified mouse anti-CD122 antibody (IL-2 receptor chain). The test results demonstrated almost complete inhibition of the zalpha11 Ligand response with the 4G3 + TUGm2 antibody combination compared to the control without antibody. The TM- / H antibody had no effect. These results suggest a role for the IL-2 γ receptor in the zalpha11 Ligand proliferative response, and further support that IL-2Ry heterodimerizes with the zalpha11 receptor to elicit that response. Example 35

Post-translational mannosylation of zalpha11 receptor polypeptide at a highly conserved Trp residue

Mannosylation of the human zalpha11 receptor was assessed using the method for C-2 mannosylation of tryptophan as described in Hofsteenge, J. et al., Biochemistry 33: 13524-13530, 1994, and Loeffler, A. et al., Biochemistry 35 : 12005-14, 1996. Furthermore, these investigators showed that in an amino acid motif WXXW (SEQ ID NO: 67), Trp can be mannosylated.

A soluble zalpha11 receptor bearing a C-terminal Glu-Glu (CEE) (SEQ ID NO: 14) or FLAG (SEQ ID NO: 23) tag was expressed in BHK cells and purified by anti-Flag or anti-affinity chromatography. -EE (Example 4A). A soluble zalpha11 receptor C-terminally tagged with an Fc4 tag (SEQ ID NO: 25) and expressed in CHO cells was affinity purified by anti-Fc4 affinity chromatography (Example 4B). These polypeptides were enzymatically cleaved to generate peptide fragments for study.

All enzyme digestions were carried out overnight with a protein concentration of 1.0 mg / ml. Digestion with PNGaseF (Oxford GlycoSciences, Abingdon, Oxford, UK) was performed by diluting each soluble zalpha11 receptor polypeptide in a 50 mM EDTA buffer, 20 mM sodium phosphate, pH 7.5, and incubating with 0.4 U of enzyme per μg of protein. Digestion with Glu-C (Roche Molecular Biochemicals, Indianapolis, Indiana) was performed with a 1:50 ratio of enzyme to protein by exchanging buffer the sample in NH₄HCO3 25 mM pH 7.8

ES 2 279 809 T3 and incubating it at 25 ° C, except for the Fc4-tagged material, which was digested in 50 mM sodium phosphate pH 7.8 + 5% acetonitrile (EM Science, Darmstadt, Germany) at 37 ° C. Glu-C digestion generated a peptide containing zalpha11 WSXWS as shown from amino acid 178 (Leu) to amino acid 199 (Ser) of SeQ ID NO: 6 (197 (Leu) to amino acid 218 (Ser) of SEQ ID NO : 2). Digestion with Asp-N (Roche Molecular Biochemicals, Indianapolis, Indiana) was performed by buffering the protein in 50 mM sodium phosphate pH 7.7 and incubating it at 37 ° C with enzyme in a 1:50 ratio to zalpha11 receptor polypeptide. Digestion with Asp-N generated a peptide containing WSXWS from zalpha11 as shown from amino acid 179 (Glu) to amino acid 210 (Ser) of SEQ ID NO: 6 (198 (Leu) to amino acid 229 (Glu) of SEQ ID NO : 2).

LCMS and LCMS-MS analyzes were performed on a Magic HPLC (Michrom Bioresources, Auburn, CA) connected online to a Finnigan LCQ mass spectrometer (Finnigan MAT, San Jose, CA). LC separation was done on a Vydac C4 5 μ 300 A column (Michrom Bioresources) with a gradient elution of 20% -80% solvent B over 80 minutes in which solvent A was 2% acetonitrile + 0.1 % TFA and solvent B was 90% acetonitrile + 0.095% TFA (EM Science; Sigma, St. Louis, MO). The LCQ mass spectrometer was set to collect MS spectra for the duration of the test. LCMS-MS analysis of polypeptide digests was performed on the same instrument system using a Vydac C18 5 μ 300 A column (Michrom Bioresources) with an elution gradient of 5-65% solvent B for 80 minutes with the same system. of solvent described above for LCMS analysis. The LCQ mass spectromer was set up to collect MS, zoom scan, and MS-MS spectra for each ion for a minimum threshold.

The extent of tryptophan mannosylation was estimated by comparing ionic intensities for the 2+ and 3+ ions of the peptides containing the WSXWS motif (SEQ ID NO: 13) from the Glu-C and Asp-N digestion described above. Peak composition was first determined using MS data and a peptide map was generated. Next, a mean spectrum was created starting about 1 minute before the early-eluting WSXWS (SEQ ID NO: 13) containing peptide and ending about 1 minute after its late-eluting non-mannosylated companion peptide. The normalized intensities of the ions corresponding to mannosylated and nonmannosylated peptide were compared and used to generate a percentage occupancy number. The values ​​generated for both loading states 2+ and 3+ were averaged to generate a percentage occupancy value for each digest. This value was then averaged with the value of the companion digest for each batch of protein to generate a final value.

Table 7 summarizes the data that was calculated for each peptide-tag and host cell used for soluble zalpha11 receptor expression.

TABLE 7

<td>Label C-terminal</td><td>Host of expression</td><td>% of WSXWS mannosylated</td>

<td>Glu Glu</td><td>BHK</td><td> -46%</td>

<td>FLAG</td><td>BHK</td><td> -35%</td>

<td>Fc4</td><td>CHO</td><td> -11%</td>

One skilled in the art would appreciate that mannosylation or non-mannosylation of the WSXWS motif (SEQ ID NO: 13) of the zalpha11 receptor may affect the ability of the zalpha11 receptor or soluble zalpha11 receptor to homodimerize, heterodimerize and / or its ability to bind to the Ligand. by zalfa11. As zalpha11 receptor mannosylation appears to differ depending on the type of cell in which the receptor is expressed, optimization of the expression and production of zalpha11 receptor and soluble receptor polypeptides can take into consideration whether the cell-produced zalpha11 receptor is mannosylated. or not handsylated. As such, one skilled in the art would appreciate that the polypeptides of the present invention may be mannosylated or nonmannosylated.

As the mannosylation event is within the WSXWS (SEQ ID NO: 13) motif of the class I cytokine receptor zalpha11, Trp mannosylation or lack thereof can functionally affect the polypeptide. For example, insertions or deletions in the WSXWS (SEQ ID NO: 13) motif of EPOR can abrogate cell surface expression, destroy or reduce proliferative response, decrease receptor internalization, and affect EPO binding (Yoshimura, A. et al. ., J. Biol. Chem. 267: 11619-11625, 1992; Quelle, DE et al., Mol. Cell. Biol. 12: 4553-4561, 1992; Hilton, DJ et al., Proc. Natl. Acad. Sci. USA 92: 190-194, 1995). However, mutation in the WSXWS motif (SEQ ID NO: 13) can also result in more efficient ER export and increased cell surface receptor expression (Hilton, DJ et al., Supra). Effects on cell surface expression, ligand binding, and stimulatory response have also been seen with studies on the WSXWS motif (SEQ ID NO: 13) and related motifs in mutational analysis on IL-2R /, GM-CSFR, and GHR. (Miyazaki et al., EMBO J. 10: 3191-3197, 1991; Ronco, LV et al., J. Biol. Chem. 269: 277-283, 1994; Baumgartner, JW et al., J. Biol. Chem 269: 29094-29101, 1994).

Similarly, mannosylation of the Trp residue in the WSXWS motif (SEQ ID NO: 13) of zalpha11 receptor polypeptides, including full-length and soluble receptors described herein, may have implications64

ES 2 279 809 Important structural and functional T3s such as having effects on overall receptor stability, rate of proteolysis, intracellular processing, antigenicity, cell surface expression, dimerization or multimerization, co-receptor binding, signaling or internalization , effects on zalpha11 ligand binding and receptor-ligand interaction stability. A comparison of mannosylated and nonmannosylated zalpha11 receptors can be made using X-ray or NMR crystallography on purified zalpha11 polypeptides (eg, soluble receptors), or functional studies comparing zalpha11 expressed in cell lines that are mannosylated (eg, BHK or other cell lineage) or are defective or reduced in mannosylation (eg, CHO or other cell lineage) and comparing the receptors in the various assays described herein.

Example 36

BHK transfectant binding studies

He was iodized with ¹²⁵I (Amersham) using iodine-beads (Pierce) purified human zalpha11 Ligand protein (25 μβ) (commonly owned US Patent Application No. 09 / 522,217) and purified on a Sephadex G25 PD column -10 (Pharmacia). BHK transfectants (Example 31) expressing human zalpha11 alone or human zalpha11 + human IL-2Ry receptor were plated at 30K / well in a 24-well plate 24 hours prior to binding study. BHK transfectants were incubated for 2 hours at 4 ° C with 2.5 ng (0.147 pmol) of ¹²⁵I zalpha11 ligand (specific activity 6.4 x 10⁷ cpm / ug) in the presence of various concentrations of cold zalpha11 Ligand (in a range of approximately 10,884-fold excess to non-competition in 15 4-fold dilutions). Cells were washed three times with binding buffer before lysis in 0.8 M NaOH, followed by gamma emission counting. Analysis of these data produced an affinity of approximately 1 nM for the zalpha11 transfectants and approximately 0.1 nM for the human zalpha11 receptor + human IL-2Ry transfectants. This result suggested that the zalpha11 Ligand has high affinity for the homodimer human zalpha11 receptor or human zalpha11 + heterodimer human IL2Ry, and that the affinity is higher for the heterodimer.

Example 37

Mouse homodimer zalpha11-mG2a receptor fusion protein

The expression vector pEZE2 was used to express the mouse zalpha11 receptor-mouse IgGamma2A Fc fusion protein (zalpha11m-mG2a). The DNA sequence of the mouse zalpha11 extracellular domain fusion protein mouse immunoglobulin gamma 2a heavy chain Fc region (zalpha11m-mG2a) is shown in SEQ ID NO: 72, and the corresponding polypeptide sequence is shown in SEQ ID NO: 73.

Vector pEZE2 is a plasmid derived from pDC312 (Immunex Corp., Seattle, WA), and contains an EASE segment as described in WIPO Publication WO 97/25420. The presence of the EASE segment in an expression vector can enhance the expression of recombinant proteins approximately two to eight-fold in stable cell pools. Plasmid pEZE2 is a dicistronic expression vector that can be used to express two different proteins in mammalian cells, such as Chinese Hamster Ovary (CHO) cells. The pEZE2 expression unit contains a CMV enhancer / promoter; an adenovirus tripartite leader sequence; a multiple cloning site (MCS) for insertion of the coding region for the recombinant protein of interest; an internal ribosome entry site of the encephalomyocarditis virus; a coding segment for mouse dihydrofolate reductase; and the SV40 transcription terminator. In addition, pEZE2 contains an origin of replication of E. coli and the bacterial beta lactamase gene.

The zalpha11m-mG2a fusion protein is a disulfide-linked homodimer consisting of two mouse zalpha11 extracellular domain chains fused to a wild-type mouse immunoglobulin gamma 2a Fc region. Mouse Fc gamma 2a immunoglobulin confers effector functions, FcyRI binding, and C1q complement fixation. The mouse zalpha11 extracellular domain fusion construct of mouse immunoglobulin gamma 2a Fc constant region was generated by overlap PCR of three separate DNA fragments, each generated by separate PCR amplification reactions. The first fragment contained an optimized tPA (tissue plasminogen activator) signal sequence (SEQ ID NO: 80). The optimized tPA signal sequence (otPA) was multiplied using oligonucleotide primers ZC26,644 (SEQ ID NO: 74) and ZC26,641 (SEQ ID NO: 75) using as template an expression vector previously generated in-house. The PCR reaction mix contained 20 pmol of each primer, 10 ng template cDNA, each 20 µM dNTP, 1X Taq buffer (Life Technologies, Gaithersburg, MD), 0.5 µl Taq polymerase in a 100 µΕ reaction PCR conditions: 1 cycle, 94 ° C, 2 minutes, 25 cycles, 94 ° C, 30 seconds, 60 ° C, 30 seconds, 72 ° C, 30 seconds, 1 cycle, 72 ° C, 5 minutes. The second fragment contained the mouse zalpha11 extracellular domain coding region from amino acids 20 to 257 of SEQ ID NO: 12. The oligonucleotide primers ZC26,642 (SEQ ID NO: 76) and ZC26,662 (SEQ ID NO: 77) were used to multiply this segment of mouse zalpha11 using as a template a previously generated clone of mouse zalpha11 (SEQ ID NO : eleven). This PCR fragment was made using the same PCR reaction mix specified above. The PCR reaction conditions were as follows: 1 cycle, 94 ° C, 2 minutes, 25 cycles, 94 ° C, 30 seconds, 50 ° C, 30 seconds, 72 ° C, 45 seconds, 1 cycle, 72 ° C, 5 minutes.

The mouse gamma 2a heavy chain Fc region was generated from a previously generated clone of mouse Ig gamma 2a heavy chain cDNA. The segment containing the hinge domains, C_H2 and C_H3 of the mouse immunoglobulin gamma 2a heavy chain constant region was generated by PCR amplification

ES 2 279 809 T3 using oligonucleotide primers ZC26,643 (SEQ ID NO: 78) and ZC26,645 (SEQ ID NO: 79). This PCR fragment was made using the same reaction mix specified above. The PCR conditions were as follows: 1 cycle, 94 ° C, 2 minutes, 25 cycles, 94 ° C, 30 seconds, 60 ° C, 30 seconds, 72 ° C, 30 seconds, 1 cycle, 72 ° C, 5 minutes.

To prepare the fusion protein coding segment, three protein-encoding domains were linked by overlap PCR using oligonucleotides ZC26,644 (SEQ ID NO: 74) and ZC26,662 (SEQ ID NO: 77) to link the first two PCR products and ZC26,644 (SEQ ID NO: 74) and ZC26,645 (SEQ ID NO: 79) to bind in the Fc region. Two reactions were established: The first ran 25 cycles of 94 ° C for 2 min, 55 ° C for 30 seconds, and 72 ° C for 1 min and 30 s. The other reaction ran 25 cycles of 94 ° C for 2 min, 60 ° C for 30 seconds, and 72 ° C for 1 min and 30 seconds. The PCR products from the two reactions were pooled and purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's method. The product was eluted in 60 µl of buffer. 30 µl of this eluate was digested with restriction enzymes Fse1 and Asc1 in diluted 10X NEB buffer # 4 (New England Biolabs, Beverly, MA) according to the manufacturer's instructions. The material was then run on a 1% TAE agarose gel and the approximately 1,500 bp band was cut and the DNA was purified using a Qiagen agarose gel extraction set (Qiagen) according to the manufacturer's instructions. The fragment was eluted in 30 µl of H₂OR.

To prepare the receptor vector for the insert, approximately 3 pg of pEZE2 vector was digested with Asc1 and Fse1 in the same manner as above, with the exception of 1 pl of calf intestinal phosphatase (CIP) (New England Biolabs) added after Restriction enzyme digestion (reaction was allowed to run for an additional 2 hours). The vector was then run on an agarose gel and purified as before. The material eluted in 30 μl of H₂OR.

The coding segment of the fusion protein was cloned into the MCS of pEZE2 from the FseI site to the AscI site on the polylinker, and ligated at 20 µl using standard molecular biology reagents and procedures. The ligation reaction was incubated overnight at 16 ° C. Approximately 4 µl of this ligation mixture was electroporated into 50 µl of electrocompetent E. coli DH12s (Life Technologies, Rockville, MD) and cells were rescued in 1 ml of LB medium and allowed to shake / incubate for 1 h and spread 100 µl on Amp 100 agar plates. Plates were allowed to incubate overnight at 37 ° C. The sequence of a single colony was analyzed. A mutation that would produce a change from Glu to Lys was found at position 25 of SEQ ID NO: 73. This amino acid substitution is within the otPA leader, and may have produced improper signal peptide treatment, because the N-terminus showed that the leader sequence was incompletely cleaved and began at a pyroglutamine residue upstream of the predicted start. However, this homodimeric construct was still active in inhibiting the zalpha11 Ligand (Example 40).

A large prep was created using the Qiagen Maxi prep set (Qiagen) according to the manufacturer's instructions. The plasmid was used to transfect CHO cells. Cells were selected in medium without hypoxanthine or thymidine and the transgene was multiplied using methotrexate (Example 38). The presence of protein was assayed by Western blotting using an anti human gamma 1 heavy chain constant region and anti human kappa light chain antibodies (Rockland Immunochemicals, Gilbertsville, PA).

Example 38

Production of zalpha11m-mG2A in CHO DG-44 cells

20 pg of a zalpha11m-mG2A / pEZE2 construct was digested with 40 units of Pvu I at 37 ° C for three hours and then precipitated with isopropanol and glubulized in a 1.5 ml microcentrifuge tube. The supernatant was aspirated off the bead and the bead was resuspended in 100 µl of water. Approximately 200 pg (20 µl) of sheared salmon sperm DNA was added to the digested zalpha11m-mG2A / pEZE2 construct. The DNA mixture was co-precipitated using 0.1 volumes of sodium acetate (pH 5.2) and 2.2 volumes of ethanol. The tube was placed on dry ice for 15 minutes and then centrifuged in a microfuge at 14,000 rpm for 15 minutes, forming a DNA bead. The supernatant was aspirated off the bead, and the bead was washed with 1 ml of 70% ethanol and allowed to incubate for 5 minutes at room temperature. The tube was centrifuged in a microcentrifuge for 10 minutes at 14,000 rpm and the supernatant was aspirated off the bead. The bead was allowed to air dry for 30 minutes. The bead was then resuspended in 100 µl of water and allowed to incubate at room temperature for 10 minutes. 500 μl containing approximately 5 x 10 was added to the DNA in the microcentrifuge tube.⁶ CHO DG-44 cells, and then the DNA / cell mixture was placed in a 0.4 cm opening cuvette and electroporated using the following parameters: 1070 pF, high capacity and 376 v. The contents of the cuvette were then removed and diluted to 25 ml with PF-CHO EX-CELL protein-free medium.^® 325 (JRH Biosciences, Lenexa, KS) with 3 mM L-glutamine and put into a 125 ml shake flask. The flask was placed in an incubator on a shaker at 37 ° C and 6% CO2 and was shaken at 120 rpm.

The CHO DG-44 zalpha11m-mG2A culture was multiplied with methotrexate (MTX) using standard methods to a final MTX level of MTX 50 nm. The culture was cloned with dilution and examined using a series of Western blots. A final clone was chosen and further selected on MTX to a level of 200 nM MTX and then extrapolated for production. The production of each batch of the clone was achieved by seeding 8X 4-L rotary flasks with 2 L of culture at a rate of approximately 5 x 10⁵ cells / ml. The cultures were centrifuged at 70 rpm, kept at 37 ° C and 6% CO2, and allowed to incubate for 72 or 96 hours. Cells were precipitated by centrifugation and supernatants filtered through 0.2 pm. Sufficient number of cells were recovered to

ES 2 279 809 T3 seed the next series of flasks. Four total batches were produced in this way for protein purification Example 39).

Example 39

Purification of the homodimeric zalpha11m-mG2A soluble receptor protein

All procedures were carried out at 4 ° C, unless otherwise indicated. Conditioned medium (Example 38) was directly captured on an appropriately sized POROS 50 A column (coupled protein A; PerSeptive BioSystems, Framingham, MA) with an optimal capture rate. The column was washed with 20 column volumes (CV) of loading buffer, and then rapidly eluted with 3 CV of 0.1M glycine pH 2.5. The collected fractions had a predetermined volume of 2M Tris pH 8.0 added prior to elution to neutralize the pH to about 7.2.

NuPAGE gels stained with Brilliant Blue (Sigma) were run to analyze elution. The fractions of interest were pooled and concentrated against a 30 kD MWCO centrifugal concentrator to nominal volume. The concentrated Protein A pool was injected onto an appropriately sized Phamicia Sephacryl 200 column (Pharmacia) to separate aggregates and to exchange the protein buffer to PBS pH 7.2.

NuPAGE gels (NOVEX) stained with Brilliant Blue (Sigma) were used again to analyze the elution. Fractions were pooled and concentrated as before to ~ 1-2 mg / ml. NuPAGE gels (NOVEX) stained with Western and Brilliant Blue (Sigma) were run to confirm purity and content. In addition, the protein was subjected to amino acid analysis (AAA) and N-terminal sequence determination for further analysis. Example 40

Soluble homodimeric zalpha11m-mg2A fusion protein as antagonist of zalpha11 Ligand

BaF3 cells stably expressing the mouse zalpha11 receptor (constructed according to Example 2 using primers for SEQ ID NO: 11) were plated at 5,500 cells per well in standard 96-well tissue culture plates in base medium. plus 3 ng / ml human zalpha11 Ligand. The base medium is 500 ml of RPMI 1640 (JRH Biosciences), 5 ml of 100X sodium pyruvate (Gibco BRL), 5 ml of 100X L-glutamine (Gibco BRL) and 50 ml of fetal calf serum (FBS) inactivated by heat (Hyclone Laboratories). A decreasing dose of purified homodimeric zalpha11m-mg2A (Example 39) was added to the cells. A proliferation assay was performed with Alamar Blue and fluorimetry was performed as in Example 2B.

The homodimer zalpha11m-mg2A fusion protein inhibited the activity of human zalpha11 Ligand in a dose-dependent manner, with 1-5 jug / ml being capable of inhibiting the activity of 1.25 ng / ml of human zalpha11 Ligand.