Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]

Definitions

• Hormones and polypeptide growth factors control proliferation and differentiation of cells of multicellular organisms. These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair damaged tissue.
• hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.
• cytokines molecules that promote the proliferation and/or differentiation of cells.
• cytokines include erythropoietin (EPO), which stimulates the development of red blood cells; thrombopoietin (TPO), which stimulates development of cells of the megakaryocyte lineage; and granulocyte-colony stimulating factor (G-CSF), which stimulates development of neutrophils.
• EPO erythropoietin
• TPO thrombopoietin
• G-CSF granulocyte-colony stimulating factor
• the demonstrated in vivo activities of these cytokines illustrate the enormous clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists.
• the present invention addresses these needs by providing new a hematopoietic cytokine receptor, as well as related compositions and methods.
• affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
• affinity tag 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.
• allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
• allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
• amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
• complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
• biotin and avidin are prototypical members of a complement/anti-complement pair.
• Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
• the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 9 M ⁇ 1 .
• polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence.
• sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.
• degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide).
• Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
• expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
• 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.
• isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
• isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
• Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5′ and 3′ untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
• an “isolated” polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
• the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
• the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers, multimers, or alternatively glycosylated or derivatized forms.
• operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
• ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
• “Paralogs” are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ -globin, ⁇ -globin, and myoglobin are paralogs 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 may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases (“kb”). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded.
• double-stranded molecules When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term “base pairs”. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired.
• a “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides”.
• Probes and/or primers can be RNA or DNA.
• DNA can be either cDNA or genomic DNA.
• Polynucleotide probes and primers are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences or its complements.
• Analytical probes will generally 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 a small region of the gene is targeted for analysis.
• a polynucleotide probe may comprise an entire exon or more. Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, Oreg., and Amersham Corp., Arlington Heights, Ill., using techniques that are well known in the art.
• promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5′ non-coding regions of genes.
• a “protein” is a macromolecule comprising one or more polypeptide chains.
• a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
• receptor is used herein to denote a cell-associated protein, or a 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 ligand to receptor results in a conformational change in the receptor (and, in some cases, receptor multimerization, i.e., association of identical or different receptor subunits) that causes interactions between the effector domain(s) and other molecule(s) in the cell. These interactions in turn lead to alterations in the metabolism of the cell.
• Cytokine receptor subunits are characterized by a multi-domain structure comprising an extracellular domain, a transmembrane domain that anchors the polypeptide in the cell membrane, and an intracellular domain.
• the extracellular domain is typically a ligand-binding domain
• the intracellular domain is typically an effector domain involved in signal transduction, although ligand-binding and effector functions may reside on separate subunits of a multimeric receptor.
• the ligand-binding domain may itself be a multi-domain structure.
• Multimeric receptors include homodimers (e.g., PDGF receptor ⁇ and ⁇ isoforms, erythropoietin receptor, MPL, and G-CSF receptor), heterodimers whose subunits each have ligand-binding and effector domains (e.g., PDGF receptor ⁇ isoform), and multimers having component subunits with disparate functions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSF receptors). Some receptor subunits are common to a plurality of receptors.
• the AIC2B subunit which cannot bind ligand on its own but includes an intracellular signal transduction domain, is a component of IL-3 and GM-CSF receptors.
• Many cytokine receptors can be placed into one of four related families on the basis of the 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:5). Cytokine receptor structure has been reviewed by Urdal, Ann. Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine 5:95-106, 1993.
• the cytokine receptor superfamily is subdivided into several families, for example, the immunoglobulin family (including CSF-1, MGF, IL-1, and PDGF receptors); the hematopoietin family (including IL-2 receptor ⁇ -subunit, GM-CSF receptor ⁇ -subunit, GM-CSF receptor ⁇ -subunit; and G-CSF, EPO, IL-3, IL-4, IL-5, IL-6, IL-7, and IL-9 receptors); TNF receptor family (including TNF (p80) TNF (p60) receptors, CD27, CD30, CD40, Fas, and NGF receptor).
• the immunoglobulin family including CSF-1, MGF, IL-1, and PDGF receptors
• the hematopoietin family including IL-2 receptor ⁇ -subunit, GM-CSF receptor ⁇ -subunit, GM-CSF receptor ⁇ -subunit; and G-CSF, EPO, IL-3,
• receptor polypeptide is used to denote complete receptor polypeptide chains and portions thereof, including isolated functional domains (e.g., ligand-binding domains).
• ligand-binding domain(s) and “cytokine-binding domain(s)” can be used interchangeably.
• 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 larger peptide is commonly cleaved to remove 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 that lack transmembrane and cytoplasmic domains. Soluble receptors can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to 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.
• splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
• Cytokine receptor subunits are characterized by a multi-domain structure comprising a ligand-binding domain and an effector domain that is typically involved in signal transduction.
• Multimeric cytokine receptors include homodimers (e.g., PDGF receptor ⁇ and ⁇ isoforms, erythropoietin receptor, MPL (thrombopoietin receptor), and G-CSF receptor); heterodimers whose subunits each have ligand-binding and effector domains (e.g., PDGF receptor ⁇ isoform); and multimers having component subunits with disparate functions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSF receptors).
• Some receptor subunits are common to a plurality of receptors.
• the AIC2B subunit which cannot bind ligand on its own but includes an intracellular signal transduction domain, is a component of IL-3 and GM-CSF receptors.
• Many cytokine receptors can be placed into one of four related families on the basis of their structures and functions.
• Class I hematopoietic receptors for example, are characterized by the presence of a domain containing conserved cysteine residues and the WSXWS motif (SEQ ID NO:5).
• Additional domains including protein kinase domains; fibronectin type III domains; and immunoglobulin domains, which are characterized by disulfide-bonded loops, are present in certain hematopoietic receptors.
• Cytokine receptor structure has been reviewed by Urdal, Ann. Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine 5:95-106, 1993. It is generally believed that under selective pressure for organisms to acquire new biological functions, new receptor family members arose from duplication of existing receptor genes leading to the existence of multi-gene families. Family members thus contain vestiges of the ancestral gene, and these characteristic features can be exploited in the isolation and identification of additional family members.
• Cell-surface cytokine receptors are further characterized by the presence of additional domains. These receptors are anchored in the cell membrane by a transmembrane domain characterized by a sequence of hydrophobic amino acid residues (typically about 21-25 residues), which is commonly flanked by positively charged residues (Lys or Arg). On the opposite end of the protein from the extracellular domain and separated from it by the transmembrane domain is an intracellular domain.
• the Zcytor19 receptor of the present invention is a class II cytokine receptor. These receptors usually bind to four-helix-bundle cytokines. Interleukin-10 and the interferons have receptors in this class (e.g., interferon-gamma, alpha and beta chains and the interferon-alpha/beta receptor alpha and beta chains). Class II cytokine receptors are characterized by the presence of one or more cytokine receptor modules (CRM) in their extracellular domains. Other class II cytokine receptors include zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4 (Genbank Accession No.
• IL-10R Genbank Accession No.s U00672 and NM — 001558
• DIRS1 zcytor7
• zcytor16 tissue factor, and the like.
• the CRMs of class II cytokine receptors are somewhat different than the better-known CRMs of class I cytokine receptors. While the class II CRMs contain two type-III fibronectin-like domains, they differ in organization.
• Zcytor19 like all known class II receptors except interferon-alpha/beta receptor alpha chain, has only a single class II CRM in its extracellular domain.
• Zcytor19 is a receptor for a helical cytokine of the interferon/IL-10 class. As was stated above, Zcytor19 is similar to other Class II cytokine receptors such as zcytor11 and zcytor16. Due to its ability to bind IL28A, IL28B, and IL29 ligands, the Zcyto19 receptor (ZcytoR19) has been designated IL29RA.
• the extracellular ligand-binding domain there are two fibronectin type III domains and a linker region.
• the first fibronectin type III domain comprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:19
• the linker comprises residues 120 (Leu) to 124 (Glu) of SEQ ID NO:19
• the second fibronectin type III domain comprises residues 125 (Pro) to 223 (Pro) of SEQ ID NO:19.
• a polypeptide comprising amino acids 21 (Arg) to 223 (Pro) of SEQ ID NO:19 (SEQ ID NO:4) is considered a ligand binding fragment.
• Trp Tryptophan residues comprising residues 43 (Trp) and 68 (Trp) as shown in SEQ ID NO:19, and conserved Cysteine residues at positions 74, 82, 195, 217 of SEQ ID NO:19.
• the extracellular ligand-binding domain there are two fibronectin type III domains and a linker region.
• the first fibronectin type III domain comprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:2
• the linker comprises residues 120 (Leu) to 124 (Glu) of SEQ ID NO:2
• the second fibronectin type III domain is short, and comprises residues 125 (Pro) to 223 (Pro) of SEQ ID NO:2.
• a polypeptide comprising amino acids 21 (Arg) to 223 (Pro) of SEQ ID NO:2 (SEQ ID NO:4) is considered a ligand binding fragment.
• Trp Tryptophan residues comprising residues 43 (Trp) and 68 (Trp) as shown in SEQ ID NO:2, and conserved Cysteine residues at positions 74, 82, 195, 217 of SEQ ID NO:2.
• a truncated soluble form of the zyctor19 receptor mRNA appears to be naturally expressed.
• Analysis of a human cDNA clone encoding the truncated soluble Zyctor19 (SEQ ID NO:20) revealed an open reading frame encoding 211 amino acids (SEQ ID NO:21) comprising a secretory signal sequence (residues 1 (Met) to 20 (Gly) of SEQ ID NO:21) and a mature truncated soluble zyctor19 receptor polyptide (residues 21 (Arg) to 211 (Ser) of SEQ ID NO:21) a truncated extracellular ligand-binding domain of approximately 143 amino acid residues (residues 21 (Arg) to 163 (Trp) of SEQ ID NO:21), no transmembrane domain, but an additional domain of approximately 48 amino acid residues (residues 164 (Lys) to 211 (Ser)
• the first fibronectin type III domain comprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:21
• the linker comprises residues 120 (Leu) to 124 (Glu) of SEQ ID NO:21
• the second fibronectin type III domain comprises residues 125 (Pro) to 163 (Trp) of SEQ ID NO:21.
• a polypeptide comprising amino acids 21 (Arg) to 163 (Trp) of SEQ ID NO:21 is considered a ligand binding fragment.
• Trp Tryptophan residues comprising residues 43 (Trp) and 68 (Trp) as shown in SEQ ID NO:21
• conserved Cysteine residues in this truncated soluble form of the zyctor19 receptor are at positions 74, and 82 of SEQ ID NO:21.
• the zyctor19 polypeptide of the present invention can be naturally expressed wherein the extracellular ligand binding domain comprises an additional 5-15 amino acid residues at the N-terminus of the mature polypeptide, or extracellular cytokine binding domain or cytokine binding fragment, as described above.
• domain boundaries are approximate and are based on alignments with known proteins and predictions of protein folding. Deletion of residues from the ends of the domains is possible.
• regions, domains and motifs described above in reference to SEQ ID NO:2 are also as shown in SEQ ID NO:1; domains and motifs described above in reference to SEQ ID NO:19 are also as shown in SEQ ID NO:18; and domains and motifs described above in reference to SEQ ID NO:21 are also as shown in SEQ ID NO:20.
• transmembrane regions and conserved and low variance motifs generally correlates with or defines important structural regions in proteins.
• Regions of low variance e.g., hydrophobic clusters
• regions of structural importance Sheppard, P. et al., Gene 150:163-167, 1994.
• Such regions of low variance often contain rare or infrequent amino acids, such as Tryptophan.
• the regions flanking and between such conserved and low variance motifs may be more variable, but are often functionally significant because they may relate to or define important structures and activities such as binding domains, biological and enzymatic activity, signal transduction, cell-cell interaction, tissue localization domains and the like.
• zyctor19 sequence has revealed that it is a member of the same receptor subfamily as the class II cytokine receptors, for example, interferon-gamma, alpha and beta chains and the interferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4 (Genbank Accession No. Z17227), DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511) receptors.
• ⁇ -subunit a second subunit to bind ligand and transduce a signal.
• ⁇ -subunits associate with a plurality of specific cytokine receptor subunits.
• Zyctor19 has been shown to form a heterodimer with CRF2-4.
• CRF2-4 has also been shown to be a binding partner with zcytor11 (IL-22R) to bind the IL-10, and the binding partner for zcytor11 to bind cytokine IL-TIF (See, WIPO publication WO 00/24758; Dumontier et al., J. Immunol. 164:1814-1819, 2000; Spencer, S D et al., J. Exp. Med. 187:571-578, 1998; Gibbs, V C and Pennica Gene 186:97-101, 1997 (CRF2-4 cDNA); Xie, M H et al., J. Biol. Chem.
• Receptors in this subfamily may also associate to form heterodimers that transduce a signal.
• class II receptor complexes can be heterodimeric, or multimeric.
• monomeric, homodimeric, heterodimeric and multimeric receptors comprising a zyctor19 subunit are encompassed by the present invention.
• a zyctor19 “soluble receptor” by preparing a variety of polypeptides that are substantially homologous to the extracellular cytokine-binding domain (residues 21 (Arg) to 226 (Asn) of SEQ ID NO:2 or SEQ ID NO:19), a cytokine-binding fragment (e.g., residues 21 (Arg) to 223 (Pro) of SEQ ID NO:2 or SEQ ID NO:19; SEQ ID NO:4) or allelic variants or species orthologs thereof) and retain ligand-binding activity of the wild-type zyctor19 protein.
• variant zyctor19 soluble receptors can be isolated.
• Such polypeptides may include additional amino acids from, for example, part or all of the transmembrane and intracellular domains.
• Such polypeptides may also include additional polypeptide segments as generally disclosed herein such as labels, affinity tags, and the like.
• the receptors of the present invention have been shown to form complexes with a genus of polynucleotide and polypeptide molecules that have functional and structural similarity to the interferons.
• this new family which includes molecules designated zcyto20 (SEQ ID NOS: 51 and 52), zcyto21 (SEQ ID NOS: 54 and 55), zcyto22 (SEQ ID NOS: 56 and 57), zcyto24 (SEQ ID NOS: 59 and 60), zcyto25 (SEQ ID NOS: 61 and 62), zcyto20, 21, and 22 are human sequences and zcyto24 and 25 are mouse sequences.
• certain biological activities have been shown to be exhibited by each molecule in the family. These activities include, for example, antiviral activities and increasing circulating myeloid cell levels. While not wanting to be bound by theory, these molecules appear to all signal through zyctor19 receptor via the same pathway.
• Table 2 is an illustration of the sequence identity between zcyto20, zcyto21, zcyto22, IFN ⁇ , IFN ⁇ , IFN ⁇ , and IL10 at the amino acid level.
• Table 2 amino acid sequence identity
• Zcyto20 gene encodes a polypeptide of 205 amino acids, as shown in SEQ ID NO:52.
• the signal sequence for Zcyto20 can be predicted as comprising amino acid residue 1 (Met) through amino acid residue 21 (Ala) of SEQ ID NO: 52.
• the mature peptide for Zcyto20 begins at amino acid residue 22 (Val).
• Zcyto21 gene encodes a polypeptide of 200 amino acids, as shown in SEQ ID NO:55.
• the signal sequence for Zcyto21 can be predicted as comprising amino acid residue 1 (Met) through amino acid residue 19 (Ala) of SEQ ID NO: 55.
• the mature peptide for Zcyto21 begins at amino acid residue 20 (Gly).
• Zcyto21 has been described in PCT application WO 02/02627.
• Zcyto22 gene encodes a polypeptide of 205 amino acids, as shown in SEQ ID NO:57.
• the signal sequence for Zcyto22 can be predicted as comprising amino acid residue 1 (Met) through amino acid residue 21 (Ala) of SEQ ID NO: 57.
• the mature peptide for Zcyto22 begins at amino acid residue 22 (Val).
• Zcyto24 gene encodes a polypeptide of 202 amino acids, as shown in SEQ ID NO:60.
• Zcyto24 secretory signal sequence comprises amino acid residue 1 (Met) through amino acid residue 28 (Ala) of SEQ ID NO:60.
• An alternative site for cleavage of the secretory signal sequence can be found at amino acid residue 24 (Thr).
• the mature polypeptide comprises amino acid residue 29 (Asp) to amino acid residue 202 (Val).
• Zcyto25 gene encodes a polypeptide of 202 amino acids, as shown in SEQ ID NO:62.
• Zcyto25 secretory signal sequence comprises amino acid residue 1 (Met) through amino acid residue 28 (Ala) of SEQ ID NO:62.
• An alternative site for cleavage of the secretory signal sequence can be found at amino acid residue 24 (Thr).
• the mature polypeptide comprises amino acid residue 29 (Asp) to amino acid residue 202 (Val).
• CRF2-4 SEQ ID NOS: 63 and 64
• zyctor19 provides further support that the receptor plays an important role in the immunomodulatory system, affecting physiologies such as the innate immune system and the inflammatory response system.
• Localizing the expression of a receptor for a ligand/receptor pair may have significance for identifying the target cell or tissue at which the ligand acts. This is particularly useful when the receptor/ligand complex involves a heterodimeric receptor in which one of the subunits is expressed widely and another of the subunits is expressed in a limited manner, either spatially or temporally restricted.
• zyctor19 Using in situ hybridization expression of zyctor19 has been identified in a skin carcinoma sample, where the cancerous granular epithelium was strongly positive, while no positive signal is observed in normal skin.
• zyctor19 Other tissues identified as expressing zyctor19 included fetal liver, where signal was observed in a mixed population of mononuclear cells in sinusoid spaces; in lung expression was observed in type II alveolar epithelium; and in macrophage-like mononuclear cells in the interstitial tissue.
• RAJI Burkitt's lymphoma
• zyctor19 regions of conserved amino acid residues in zyctor19, described above, can be used as tools to identify new family members. For instance, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding the conserved regions from RNA obtained from a variety of tissue sources or cell lines.
• RT-PCR reverse transcription-polymerase chain reaction
• highly degenerate primers designed from the zyctor19 sequences are useful for this purpose. Designing and using such degenerate primers may be readily performed by one of skill in the art.
• the present invention provides polynucleotide molecules, including DNA and RNA molecules that encode the zyctor19 polypeptides disclosed herein.
• SEQ ID NO:3 is a degenerate DNA sequence that encompass all DNAs that encode the zyctor19 polypeptide of SEQ ID NO:2
• SEQ ID NO:28 is a degenerate DNA sequence that encompass all DNAs that encode the zyctor19 polypeptide of SEQ ID NO:19
• SEQ ID NO:29 is a degenerate DNA sequence that encompass all DNAs that encode the zyctor19 polypeptide of SEQ ID NO:21.
• zyctor19 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 1473 of SEQ ID NO:3, 1 to nucleotide 1560 of SEQ ID NO:28, 1 to nucleotide 633 of SEQ ID NO:29, and their RNA equivalents are contemplated by the present invention.
• subfragments of these degenerate sequences such as the mature forms of the polypeptides, extracellular, cytokine binding domains, intracellular domains, and the like, as described herein are included in the present invention.
• SEQ ID NO:2 SEQ ID NO:19 and SEQ ID NO:21 and the subfragments thereof described herein could readily determine the respective nucleotides in SEQ ID NO:3, SEQ ID NO:28 or SEQ ID NO:29, that encode those subfragments.
• Table 3 sets forth the one-letter codes used within SEQ ID NO:3, to denote degenerate nucleotide positions. “Resolutions” are the nucleotides denoted by a code letter.
• “Complement” indicates the code for the complementary nucleotide(s).
• the code Y denotes either C or T
• its complement R denotes A or G, A being complementary to T, and G being complementary to C.
• TABLE 3 Nucleotide Resolution Complement Resolution A A T T C C G G G G C C T T A A R A
• degenerate codon representative of all possible codons encoding each amino acid.
• WSN can, in some circumstances, encode arginine
• MGN can, in some circumstances, encode serine
• 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 sequence of SEQ ID NO:2, SEQ ID NO:19, and/or SEQ ID NO:21. Variant sequences can be readily tested for functionality as described herein.
• preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2).
• the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
• Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
• preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO:3 serves as templates for optimizing expression of zyctor19 polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
• the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20, or a sequence complementary thereto, under stringent conditions.
• stringent conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
• T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
• Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, Calif.), as well as sites on the Internet, are available tools for analyzing a given sequence and calculating T m based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences (e.g., >50 base pairs) is performed at temperatures of about 20-25° C. below the calculated T m . For smaller probes (e.g., ⁇ 50 base pairs) hybridization is typically carried out at the T m or 5-10° C. below.
• Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42° C. in a solution comprising: about 40-50% formamide, up to about 6 ⁇ SSC, about 5 ⁇ Denhardt's solution, zero up to about 10% dextran sulfate, and about 10-20 ⁇ g/ml denatured commercially-available carrier DNA.
• such stringent conditions include temperatures of 20-70° C.
• hybridization is then followed by washing filters in up to about 2 ⁇ SSC.
• a suitable wash stringency is equivalent to 0.1 ⁇ SSC to 2 ⁇ SSC, 0.1% SDS, at 55° C. to 65° C. Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence.
• the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes.
• Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art.
• the isolated polynucleotides of the present invention include DNA and RNA.
• Methods for preparing DNA and RNA are well known in the art.
• RNA is isolated from a tissue or cell that produces large amounts of zyctor19 RNA.
• tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include PBLs, spleen, thymus, bone marrow, and lymph tissues, human erythroleukemia cell lines, acute monocytic leukemia cell lines, B-cell and T-cell leukemia tissue or cell lines, other lymphoid and hematopoietic cell lines, and the like.
• Total RNA can be 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. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zyctor19 polypeptides are then identified and isolated by, for example, hybridization or polymerase chain reaction (PCR) (Mullis, U.S. Pat. No. 4,683,202).
• a full-length clone encoding zyctor19 can be obtained by conventional cloning procedures.
• Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron.
• Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library.
• Expression libraries can be probed with antibodies to zcytor19, receptor fragments, or other specific binding partners.
• the polynucleotides of the present invention can also be synthesized using DNA synthesis machines.
• the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
• An alternative way to prepare a full-length gene is to synthesize a specified set of overlapping oligonucleotides (40 to 100 nucleotides). See Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990.
• other sequences are generally added that contain signals for proper initiation and termination of transcription and translation.
• the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are zyctor19 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zyctor19 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
• SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20 represents one allele of human zyctor19 and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1, SEQ ID NO:18 or SEQ ID NO:20 including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21.
• cDNAs generated from alternatively spliced mRNAs, which retain the properties of the zyctor19 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
• Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
• the present invention also provides isolated zyctor19 polypeptides that are substantially similar to the polypeptides of SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 and their orthologs.
• the term “substantially similar” is used herein to denote polypeptides having at least 70%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 or their orthologs.
• Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 or its orthologs.) Percent sequence identity is determined by conventional methods.
• the percent identity is then calculated as: Total ⁇ ⁇ number ⁇ ⁇ of ⁇ ⁇ identical ⁇ ⁇ matches [ length ⁇ ⁇ of ⁇ ⁇ the ⁇ ⁇ longer ⁇ ⁇ sequence ⁇ ⁇ plus ⁇ ⁇ the number ⁇ ⁇ of ⁇ ⁇ gaps ⁇ ⁇ introduced ⁇ ⁇ into ⁇ ⁇ the ⁇ ⁇ longer sequence ⁇ ⁇ in ⁇ ⁇ order ⁇ ⁇ to ⁇ ⁇ align ⁇ ⁇ the ⁇ ⁇ two ⁇ ⁇ sequences ] ⁇ 100 TABLE 5 A R N D C Q E G H I L K M F P S T W Y V A 4 R ⁇ 1 5 N ⁇ 2 0 6 D ⁇ 2 ⁇ 2 1 6 C 0 ⁇ 3 ⁇ 3 ⁇ 3 9 Q ⁇ 1 1 0 0 ⁇ 3 5 E ⁇ 1 0 0 2 ⁇ 4 2 5 G 0 ⁇ 2 0 ⁇ 1 ⁇ 3 ⁇ 2 ⁇ 2 6 H ⁇ 2 0 1 ⁇ 1 ⁇
• Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.
• the “FASTA” similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zyctor19.
• the FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
• the ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are “trimmed” to include only those residues that contribute to the highest score.
• the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
• 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 for amino acid insertions and deletions.
• FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
• the ktup value can range between one to six, preferably from three to six, most preferably three, with other FASTA program parameters set as default.
• the BLOSUM62 table (Table 3) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (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 may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed below), the language “conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than ⁇ 1.
• an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
• preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
• Variant zyctor19 polypeptides or substantially homologous zyctor19 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 6) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of 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 polypeptides that comprise 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:2, SEQ ID NO:19 or SEQ ID NO:21, excluding the tags, extension, linker sequences and the like.
• Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zyctor19 polypeptide and the affinity tag. Suitable sites include thrombin cleavage sites and factor Xa cleavage sites.
• the present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions.
• a zyctor19 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos. 5,155,027 and 5,567,584.
• Preferred dimerizing proteins in this regard include immunoglobulin constant region domains.
• Immunoglobulin-zyctor19 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric zyctor19 analogs.
• Auxiliary domains can be fused to zyctor19 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen).
• a zyctor19 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting 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 can 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.
• coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the 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 the protein in place of its natural counterpart. 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 modification. 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).
• a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zyctor19 amino acid residues.
• 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).
• 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.
• single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g. ligand binding and signal transduction) as disclosed 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.
• Sites of ligand-receptor, protein-protein or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. 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 analysis of homologies with related receptors.
• Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule.
• Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e.g., the Insight II® viewer and homology modeling tools; MSI, San Diego, Calif.), secondary structure propensities, binary patterns, complementary packing and buried 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 to molecules or identifying specific fragments determination of structure will be accompanied by evaluating activity of modified molecules.
• Amino acid sequence changes are made in zyctor19 polypeptides so as to minimize disruption of higher order structure essential to biological activity.
• the zyctor19 polypeptide comprises one or more structural domains, such as Fibronectin Type III domains
• changes in amino acid residues will be made so as not to disrupt the domain structure and geometry and other components of the molecule where changes in conformation ablate some critical function, for example, binding of the molecule to its binding partners.
• the effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995).
• CD circular dichroism
• a Hopp/Woods hydrophilicity profile of the zyctor19 protein sequence as shown in SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 can be generated (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. Buried G, S, and T residues and exposed H, Y, and W residues were ignored.
• hydrophilic regions include amino acid residues 295 through 300 of SEQ ID NO:2; 451 through 456 of SEQ ID NO:2; 301 through 306 of SEQ ID NO:2; 244 through 299 of SEQ ID NO:2; and 65 through 70 of SEQ ID NO:2.
• zyctor19 hydrophilic regions including antigenic epitope-bearing polypeptides can be predicted by a Jameson-Wolf plot, e.g., using DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.).
• hydrophobic residues selected from the group consisting of Val, Leu and Ile or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp.
• residues tolerant of substitution could include such residues as shown in SEQ ID NO:2.
• Cysteine residues at positions 74, 82, 195, and 217 of SEQ ID NO:2 or SEQ ID NO:19, and corresponding Cys residues in SEQ ID NO:4 are relatively intolerant of substitution.
• Cysteine residues at positions 74, 82, of SEQ ID NO:21 are relatively intolerant of substitution.
• the identities of essential amino acids can also be inferred from analysis of sequence similarity between class II cytokine receptor family members with zyctor19. Using methods such as “FASTA” analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions.
• An alternative approach to identifying a variant zyctor19 polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zyctor19 polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20 as discussed above.
• the present invention also includes functional fragments of zyctor19 polypeptides and nucleic acid molecules encoding such functional fragments.
• a “functional” zyctor19 or fragment thereof defined herein is characterized by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti-zyctor19 antibody or zyctor19 ligand (either soluble or immobilized).
• functional fragments also include the signal peptide, intracellular signaling domain, and the like.
• zyctor19 is characterized by a class II cytokine receptor structure.
• the present invention further provides fusion proteins encompassing: (a) polypeptide molecules comprising an extracellular domain, cytokine-binding domain, or intracellular domain described herein; and (b) functional fragments comprising one or more of these domains.
• the other polypeptide portion of the fusion protein may be contributed by another class II cytokine receptor, for example, interferon-gamma, alpha and beta chains and the interferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511), and the like, or by a non-native and/or an unrelated secretory signal peptide that facilitates secretion of the fusion protein.
• Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a zyctor19 polypeptide.
• DNA molecules having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20 or fragments thereof can be digested with Bal31 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for zyctor19 activity, or for the ability to bind anti-zyctor19 antibodies or zyctor19 ligand.
• exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired zyctor19 fragment.
• particular fragments of a zyctor19 polynucleotide can be synthesized using the polymerase chain reaction.
• Variants of the disclosed zyctor19 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994 and WIPO Publication WO 97/20078.
• Mutagenesis methods as disclosed herein can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized zyctor19 receptor polypeptides in host cells.
• Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays, which are described below.
• Mutagenized DNA molecules that encode active receptors or portions thereof e.g., ligand-binding fragments, signaling domains, and the like
• These methods allow the routine and rapid determination of the importance of individual amino acid residues in a polypeptide of interest.
• proteins of the present invention can be joined to other bioactive molecules, particularly cytokine receptors, to provide multi-functional molecules.
• cytokine receptors particularly cytokine receptors
• one or more domains from zyctor19 soluble receptor can be joined to other cytokine soluble receptors to enhance their biological properties or efficiency of production.
• the present invention thus provides a series of novel, hybrid molecules in which a segment comprising one or more of the domains of zyctor19 is fused to another polypeptide. Fusion is preferably done by splicing at the DNA level to allow expression of chimeric molecules in recombinant production systems. The resultant molecules are then assayed for such properties as improved solubility, improved stability, prolonged clearance half-life, improved expression and secretion levels, and pharmacodynamics.
• Such hybrid molecules may further comprise additional amino acid residues (e.g. a polypeptide linker) between the component proteins or polypeptides.
• any zyctor19 polypeptide including variants, soluble receptors, and fusion polypeptides or proteins
• one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.
• the zyctor19 polypeptides of the present invention 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 bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred.
• a DNA sequence encoding a zyctor19 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription 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 may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. 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 such elements are described in the literature and are available through commercial suppliers.
• a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
• the secretory signal sequence may be that of zyctor19, or may be derived from another secreted protein (e.g., t-PA) or synthesized de-novo.
• the secretory signal sequence is operably linked to the zyctor19 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
• Secretory signal sequences are commonly positioned 5′ to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
• the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
• the present invention provides for such fusion polypeptides.
• a signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid 1 (Met) to amino acid 20 (Gly) of SEQ ID NO:2 or SEQ ID NO:19 is operably linked to another polypeptide using methods known in the art and disclosed herein.
• the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
• 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.
• Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (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 (e.g. CHO-K1; ATCC No. CCL 61) cell lines.
• COS-1 ATCC No. CRL 1650
• COS-7 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
• Chinese hamster ovary e.g. CHO-K1; ATCC No. CCL 61
• Suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection (ATCC), Rockville, Md.
• ATCC American Type Culture Collection
• strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288.
• Other suitable promoters include those from metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
• Drug selection is generally used to select for 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 able to pass 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 carried out in the presence of a neomycin-type drug, such as G-418 or the like.
• Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as “amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
• a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
• Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
• hygromycin resistance e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
• Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
• Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
• Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. ( Bangalore ) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Pat. 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, L. A.
• This system which utilizes transfer vectors, is sold in the Bac-to-BacTM kit (Life Technologies, Rockville, Md.).
• This system utilizes a transfer vector, pFastBac1TM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zyctor19 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a “bacmid.” See, Hill-Perkins, M. S. and Possee, R. D., J Gen Virol 71:971-6, 1990; Bonning, B. C. et al., J Gen Virol 75:1551-6, 1994; and, Chazenbalk, G.
• transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zyctor19 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985).
• the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994.
• Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).
• Commercially available serum-free media are used to grow and maintain the cells.
• Suitable media are Sf900 IITM (Life Technologies) or ESF 92TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, Kans.) or Express FiveOTM (Life Technologies) for the T. ni cells. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification of the zyctor19 polypeptide from the supernatant can be achieved 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 disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat.
• Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
• a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
• Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No.
• Prokaryotic host cells including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.).
• Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
• suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
• the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
• P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25° C.
• Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
• a preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% BactoTM Peptone (Difco Laboratories, Detroit, Mich.), 1% BactoTM yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
• a zyctor19 cytokine receptor (including transmembrane and intracellular domains) is produced by a cultured cell, and the cell is used to screen for ligands for the receptor, including the natural ligand, as well as agonists and antagonists of the natural ligand.
• a cDNA or gene encoding the receptor is combined with other genetic elements required for its expression (e.g., a transcription promoter), and the resulting expression vector is inserted into a host cell.
• Cells that express the DNA and produce functional receptor are selected and used within a variety of screening systems.
• Mammalian cells suitable for use in expressing the novel receptors of the present invention and transducing a receptor-mediated signal include cells that express a ⁇ -subunit, such as a class II cytokine receptor subunit, for example, interferon-gamma, alpha and beta chains and the interferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511) receptors.
• a ⁇ -subunit such as a class II cytokine receptor subunit, for example, interferon-gamma, alpha and beta chains and the interferon-alpha/beta receptor alpha and beta chains
• zcytor11 commonly owned U.S. Pat. No. 5,965,704
• CRF2-4 CRF2-4
• DIRS1 zcytor7
• Such subunits can either naturally be
• An exemplary cell system for class I cytokine receptors is to use cells that express gp130, and cells that co-express gp130 and LIF receptor (Gearing et al., EMBO J. 10:2839-2848, 1991; Gearing et al., U.S. Pat. No. 5,284,755).
• a cell that is responsive to other cytokines that bind to receptors in the same subfamily, such as IL-6 or LIF because such cells will contain the requisite signal transduction pathway(s).
• Preferred cells of this type include BaF3 cells (Palacios and Steinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell.
• suitable host cells can be engineered to produce a ⁇ -subunit or other cellular component needed for the desired cellular response.
• the murine cell line BaF3 (Palacios and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol.
• a baby hamster kidney (BHK) cell line or the CTLL-2 cell line (ATCC TIB-214) can be transfected to express individual class II subunits such as, interferon-gamma, alpha and beta chains and the interferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511) receptors in addition to zyctor19.
• individual class II subunits such as, interferon-gamma, alpha and beta chains and the interferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511) receptors in addition to zyctor19.
• a host cell and receptor(s) from the same species it is generally preferred to use a host cell and receptor(s) from the same species, however this approach allows cell lines to be engineered to express multiple receptor subunits from any species, thereby overcoming potential limitations arising from species specificity.
• species homologs of the human receptor cDNA can be cloned and used within cell lines from the same species, such as a mouse cDNA, in the BaF3 cell line.
• Cell lines that are dependent upon one hematopoietic growth factor, such as IL-3 can thus be engineered to become dependent upon a zyctor19 ligand or anti-zyctor19 antibody.
• Cells expressing functional zyctor19 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.
• One such assay is a cell proliferation assay. Cells are cultured in the presence or absence of a test compound, and cell proliferation is detected by, for example, measuring incorporation of tritiated thymidine or by colorimetric assay based on the reduction or metabolic breakdown of Alymar BlueTM (AccuMed, Chicago, Ill.) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Mosman, J. Immunol. Meth.
• 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 is responsive to the receptor-linked pathway, e.g, JAK/STAT pathway, and the assay detects activation of transcription of the reporter gene.
• a preferred promoter element in this regard is a serum response element, SRE (see, for example, Shaw et al., Cell 56:563-572, 1989).
• a preferred such reporter gene is a luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). Expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner et al., J.
• Luciferase assay kits are commercially available from, for example, Promega Corp., Madison, Wis.
• Target cell lines of this type can be used to screen libraries of chemicals, cell-conditioned culture media, fungal broths, soil samples, water samples, and the like.
• a secretion trap method employing zyctor19 soluble receptor polypeptide was used to isolate a zyctor19 ligand (Aldrich, et al, Cell 87: 1161-1169, 1996), as explained in the Examples.
• Other methods for identifying natural ligand for zyctor19 include mutagenizing a cytokine-dependent cell line expressing zyctor19 and culturing it under conditions that select for autocrine growth. See WIPO publication WO 95/21930.
• the activity of zyctor19 polypeptide can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
• An exemplary device is the CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, Calif. Additional assays provided by the present invention include the use of hybrid receptor polypeptides. These hybrid polypeptides fall into two general classes.
• the intracellular domain of zyctor19 comprising approximately residues 250 (Lys) to 491 (Arg) of SEQ ID NO:2 or residues 250 (Lys) to 520 (Arg) of SEQ ID NO:19), is joined to the ligand-binding domain of a second receptor.
• the second receptor be a hematopoietic cytokine receptor, such as mpl receptor (Souyri et al., Cell 63:1137-1147, 1990).
• the hybrid receptor will further comprise a transmembrane domain, which may be derived from either receptor. A DNA construct encoding the hybrid receptor is then inserted into a host cell.
• Cells expressing the hybrid receptor are cultured in the presence of a ligand for the binding domain and assayed for a response.
• This system provides a means for analyzing signal transduction mediated by zyctor19 while using readily available ligands. This system can also be used to determine if particular cell lines are capable of responding to signals transduced by zyctor19.
• a second class of hybrid receptor polypeptides comprise the extracellular (ligand-binding) cytokine-binding domain (residues 21 (Arg) to 226 (Asn) of SEQ ID NO:2 or SEQ ID NO:19), or cytokine-binding fragment (e.g., residues 21 (Arg) to 223 (Pro) of SEQ ID NO:2 or SEQ ID NO:19; SEQ ID NO:4) with a cytoplasmic domain of a second receptor, preferably a cytokine receptor, and a transmembrane domain.
• the transmembrane domain may be derived from either receptor.
• Hybrid receptors of this second class are expressed in cells known to be capable of responding to signals transduced by the second receptor. Together, these two classes of hybrid receptors enable the use of a broad spectrum of cell types within receptor-based assay systems.
• Cells found to express a ligand for zyctor19 are then used to prepare a cDNA library from which the ligand-encoding cDNA may be isolated as disclosed above.
• the present invention thus provides, in addition to novel receptor polypeptides, methods for cloning polypeptide ligands for the receptors.
• Agonist ligands for zyctor19, or anti-zyctor19 antibodies may be useful in stimulating cell-mediated immunity and for stimulating lymphocyte proliferation, such as in the treatment of infections involving immunosuppression, including certain viral infections. Additional uses include tumor suppression, where malignant transformation results in tumor cells that are antigenic. Agonist ligands or anti-zyctor19 antibodies could be used to induce cytotoxicity, which may be mediated through activation of effector cells such as T-cells, NK (natural killer) cells, or LAK (lymphoid activated killer) cells, or induced directly through apoptotic pathways. For example, zyctor19 antibodies could be used for stimulating cytotoxicity or ADCC on zyctor19-bearing cancer cells. Agonist ligands may also be useful in treating leukopenias by increasing the levels of the affected cell type, and for enhancing the regeneration of the T-cell repertoire after bone marrow transplantation.
• Antagonist ligands, compounds, soluble zyctor19 receptors, or anti-zyctor19 antibodies may find utility in the suppression of the immune system, such as in the treatment of autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, diabetes mellitis, inflammatory bowel disease, Crohn's disease, etc. Immune suppression can also be used to reduce rejection of tissue or organ transplants and grafts and to treat T-cell specific leukemias or lymphomas by inhibiting proliferation of the affected cell type.
• the present invention contemplates the use of naked anti-zyctor19 antibodies (or naked antibody fragments thereof), as well as the use of immunoconjugates to effect treatment of various disorders, including B-cell malignancies and other cancers described herein wherein zyctor19 is expressed.
• immunoconjugates as well as anti-zyctor19 antibodies can be used for stimulating cytotoxicity or ADCC on zyctor19-bearing cancer cells.
• Immunoconjugates can be prepared using standard techniques. For example, immunoconjugates can be produced by indirectly conjugating a therapeutic agent to an antibody component (see, for example, Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J.
• one standard approach involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of drug, toxin, chelator, boron addends, or other therapeutic agent. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
• the carrier polymer can be an aminodextran or polypeptide of at least 50 amino acid residues, although other substantially equivalent polymer carriers can also be used.
• the final immunoconjugate is soluble in an aqueous solution, such as mammalian serum, for ease of administration and effective targeting for use in therapy.
• solubilizing functions on the carrier polymer will enhance the serum solubility of the final immunoconjugate.
• the therapeutic agent is coupled to aminodextran by glutaraldehyde condensation or by reaction of activated carboxyl groups on the polypeptide with amines on the aminodextran.
• Chelators can be attached to an antibody component to prepare immunoconjugates comprising radiometals or magnetic resonance enhancers.
• Illustrative chelators include derivatives of ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid. Boron addends, such as carboranes, can be attached to antibody components by conventional methods.
• Immunoconjugates can also be prepared by directly conjugating an antibody component with a therapeutic agent.
• the general procedure is analogous to the indirect method of conjugation except that a therapeutic agent is directly attached to an oxidized antibody component.
• a therapeutic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation.
• the tetanus toxoid peptides can be constructed with a single cysteine residue that is used to attach the peptide to an antibody component.
• such peptides can be attached to the antibody component using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)proprionate. Yu et al., Int. J. Cancer 56:244 (1994). General techniques for such conjugation are well-known in the art.
• carbohydrate moieties in the Fc region of an antibody can be used to conjugate a therapeutic agent.
• the Fc region is absent if an antibody fragment is used as the antibody component of the immunoconjugate.
• the engineered carbohydrate moiety is then used to attach a therapeutic agent.
• the carbohydrate moiety can be used to attach polyethyleneglycol in order to extend the half-life of an intact antibody, or antigen-binding fragment thereof, in blood, lymph, or other extracellular fluids.
• One type of immunoconjugate comprises an antibody component and a polypeptide cytotoxin.
• An example of a suitable polypeptide cytotoxin is a ribosome-inactivating protein.
• Type I ribosome-inactivating proteins are single-chain proteins, while type II ribosome-inactivating proteins consist of two nonidentical subunits (A and B chains) joined by a disulfide bond (for a review, see Soria et al., Targeted Diagn. Ther. 7:193 (1992)).
• Useful type I ribosome-inactivating proteins include polypeptides from Saponaria officinalis (e.g., saporin-1, saporin-2, saporin-3, saporin-6), Momordica charantia (e.g, momordin), Byronia dioica (e.g., bryodin, bryodin-2), Trichosanthes kirilowii (e.g., trichosanthin, trichokirin), Gelonium multiflorum (e.g., gelonin), Phytolacca americana (e.g., pokeweed antiviral protein, pokeweed antiviral protein-II, pokeweed antiviral protein-S), Phytolacca dodecandra (e.g., dodecandrin, Mirabilis antiviral protein), and the like. Ribosome-inactivating proteins are described, for example, by Walsh et al., U.S. Pat. No. 5,635,384.
• Suitable type II ribosome-inactivating proteins include polypeptides from Ricinus communis (e.g., ricin), Abrus precatorius (e.g., abrin), Adenia digitata (e.g., modeccin), and the like. Since type II ribosome-inactiving proteins include a B chain that binds galactosides and a toxic A chain that depurinates adensoine, type II ribosome-inactivating protein conjugates should include the A chain.
• Additional useful ribosome-inactivating proteins include bouganin, clavin, maize ribosome-inactivating proteins, Vaccaria pyramidata ribosome-inactivating proteins, nigrine b, basic nigrine 1, ebuline, racemosine b, luffin-a, luffin-b, luffin-S, and other ribosome-inactivating proteins known to those of skill in the art. See, for example, B perfumesi and Stirpe, international publication No. WO98/55623, Colnaghi et al., international publication No. WO97/49726, Hey et al., U.S. Pat. No. 5,635,384, B perfumesi and Stirpe, international publication No.
• Ribosome-inactivating proteins can be produced using publicly available amino acid and nucleotide sequences. As an illustration, a nucleotide sequence encoding saporin-6 is disclosed by Lorenzetti et al., U.S. Pat. No. 5,529,932, while Walsh et al., U.S. Pat. No. 5,635,384, describe maize and barley ribosome-inactivating protein nucleotide and amino acid sequences. Moreover, ribosome-inactivating proteins are also commercially available.
• Additional polypeptide cytotoxins include ribonuclease, DNase I, Staphylococcal enterotoxin-A, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641 (1986), and Goldenberg, CA—Cancer Journal for Clinicians 44:43 (1994).
• tyrosine kinase inhibitor Another general type of useful cytotoxin is a tyrosine kinase inhibitor. Since the activation of proliferation by tyrosine kinases has been suggested to play a role in the development and progression of tumors, this activation can be inhibited by anti-zyctor19 antibody components that deliver tyrosine kinase inhibitors.
• Suitable tyrosine kinase inhibitors include isoflavones, such as genistein (5,7,4′-trihydroxyisoflavone), daidzein (7,4′-dihydroxyisoflavone), and biochanin A (4-methoxygenistein), and the like. Methods of conjugating tyrosine inhibitors to a growth factor are described, for example, by Uckun, U.S. Pat. No. 5,911,995.
• immunomodulator includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, and the like, as well as synthetic analogs of these molecules.
• immunomodulators include tumor necrosis factor, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-28A, IL-28B, and IL-29), colony stimulating factors (e.g., granulocyte-colony stimulating factor and granulocyte macrophage-colony stimulating factor), interferons (e.g., interferons- ⁇ , - ⁇ , - ⁇ , - ⁇ , and - ⁇ ), the stem cell growth factor designated “S1 factor,” erythropoietin, and thrombopoietin.
• Illustrative immunomodulator moieties include IL-2, IL-1,
• Immunoconjugates that include an immunomodulator provide a means to deliver an immunomodulator to a target cell, and are particularly useful against tumor cells.
• the cytotoxic effects of immunomodulators are well known to those of skill in the art. See, for example, Klegerman et al., “Lymphokines and Monokines,” in Biotechnology And Pharmacy, Pessuto et al. (eds.), pages 53-70 (Chapman & Hall 1993).
• interferons can inhibit cell proliferation by inducing increased expression of class I histocompatibility antigens on the surface of various cells and thus, enhance the rate of destruction of cells by cytotoxic T lymphocytes.
• tumor necrosis factors such as tumor necrosis factor- ⁇ , are believed to produce cytotoxic effects by inducing DNA fragmentation.
• the present invention also includes immunocongugates that comprise a nucleic acid molecule encoding a cytotoxin.
• immunocongugates that comprise a nucleic acid molecule encoding a cytotoxin.
• Hoganson et al., Human Gene Ther. 9:2565 (1998) describe FGF-2 mediated delivery of a saporin gene by producing an FGF-2-polylysine conjugate which was condensed with an expression vector comprising a saporin gene.
• Other suitable toxins are known to those of skill in the art.
• Conjugates of cytotoxic polypeptides and antibody components can be prepared using standard techniques for conjugating polypeptides.
• Lam and Kelleher U.S. Pat. No. 5,055,291
• Lam and Kelleher U.S. Pat. No. 5,055,291
• the general approach is also illustrated by methods of conjugating fibroblast growth factor with saporin, as described by Lappi et al., Biochem. Biophys. Res. Commun. 160:917 (1989), Soria et al., Targeted Diagn. Ther. 7:193 (1992), Buechler et al., Eur. J. Biochem.
• fusion proteins comprising an antibody component and a cytotoxic polypeptide can be produced using standard methods. Methods of preparing fusion proteins comprising a cytotoxic polypeptide moiety are well-known in the art of antibody-toxin fusion protein production. For example, antibody fusion proteins comprising an interleukin-2 moiety are described by Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et al., Cancer Gene Ther. 2:161 (1995), Becker et al., Proc. Nat'l Acad. Sci. USA 93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and Hu et al., Cancer Res.
• immunoconjugates can comprise a radioisotope as the cytotoxic moiety.
• an immunoconjugate can comprise an anti-zyctor19 antibody component and an ⁇ -emitting radioisotope, a ⁇ -emitting radioisotope, a ⁇ -emitting radioisotope, an Auger electron emitter, a neutron capturing agent that emits ⁇ -particles or a radioisotope that decays by electron capture.
• Suitable radioisotopes include 198 Au, 199 Au, 32 P, 33 P, 125 I, 131 I, 123 I, 90 Y, 186 Re, 188 Re, 67 Cu, 211 At, 47 Sc, 103 Pb, 109 Pd, 212 Pb, 71 Ge, 77 As, 105 Rh, 113 Ag, 119 Sb, 121 Sn, 131 Cs, 143 Pr, 161 Tb, 177 Lu, 191 Os, 193M Pt, 197 Hg, and the like.
• a radioisotope can be attached to an antibody component directly or indirectly, via a chelating agent.
• a chelating agent for example, 67 Cu, which provides ⁇ -particles and ⁇ -rays, can be conjugated to an antibody component using the chelating agent, p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid.
• chelating agent p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid.
• 90 Y which emits an energetic ⁇ -particle, can be coupled to an antibody component using diethylenetriaminepentaacetic acid.
• boron addends such as carboranes can be attached to antibody components, using standard techniques.
• chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, antibiotics, epipodophyllotoxins, platinum coordination complexes, and the like.
• chemotherapeutic drugs include methotrexate, doxorubicin, daunorubicin, cytosinarabinoside, cis-platin, vindesine, mitomycin, bleomycin, melphalan, chlorambucil, maytansinoids, calicheamicin, taxol, and the like.
• Suitable chemotherapeutic agents are described in Remington: The Science and Practice of Pharmacy, 19th Edition (Mack Publishing Co. 1995), and in Goodman And Gilman's The Pharmacological Basis Of Therapeutics, 9th Ed. (MacMillan Publishing Co. 1995).
• Other suitable chemotherapeutic agents are known to those of skill in the art.
• immunoconjugates are prepared by conjugating photoactive agents or dyes to an antibody component.
• Fluorescent and other chromogens, or dyes, such as porphyrins sensitive to visible light have been used to detect and to treat lesions by directing the suitable light to the lesion.
• This type of “photoradiation,” “phototherapy,” or “photodynamic” therapy is described, for example, by Mew et al., J. Immunol. 130:1473 (1983), Jori et al. (eds.), Photodynamic Therapy Of Tumors And Other Diseases (Libreria Progetto 1985), Oseroff et al., Proc. Natl. Acad. Sci.
• Antibodies disclosed herein include antibodies that bind the zyctor19/CRF2-4 heterodimeric complex, including the heterodimeric soluble receptor.
• Anti-zyctor19 antibodies, and multispecific antibody compositions can be used to modulate the immune system by preventing the binding of zyctor19 ligands (for example, zcyto20, zcyto21, zcyto22, zcyto24,a nd zcyto25) with endogenous zyctor19 receptors.
• zyctor19 ligands for example, zcyto20, zcyto21, zcyto22, zcyto24,a nd zcyto25
• Such antibodies can be administered to any subject in need of treatment, and the present invention contemplates both veterinary and human therapeutic uses.
• Illustrative subjects include mammalian subjects, such as farm animals, domestic animals, and human patients.
• Multispecific antibody compositions and dual reactive antibodies that bind zyctor19 can be used for the treatment of autoimmune diseases, B cell cancers, immuomodulation, and other pathologies (e.g., ITCP, T cell-mediated diseases, cattleman's disease, autoimmune disease, myelodysplastic syndrome, and the like), renal diseases, graft rejection, and graft versus host disease.
• the antibodies of the present invention can be targeted to specifically regulate B cell responses during the immune response. Additionally, the antibodies of the present invention can be used to modulate B cell development, antigen presentation by B cells, antibody production, and cytokine production.
• Antagonistic anti-zyctor19 antibodies can be useful to neutralize the effects of zyctor19 ligands for treating B cell lymphomas and leukemias, chronic or acute lymphocytic leukemia, myelomas such as multiple myeloma, plasma cytomas, and lymphomas such as non-Hodgkins lymphoma, for which an increase in zyctor19 ligand polypeptides is associated, or where zyctor19 ligand is a survival factor or growth factor.
• Anti-zyctor19 antibodies can also be used to treat Epstein Barr virus-associated lymphomas arising in immunocompromised patients (e.g., AIDS or organ transplant).
• Anti-zyctor19 antibodies that induce a signal by binding with zyctor19 may inhibit the growth of lymphoma and leukemia cells directly via induction of signals that lead to growth inhibition, cell cycle arrest, apoptosis, or tumor cell death.
• Zyctor19 antibodies that initiate a signal are preferred antibodies to directly inhibit or kill cancer cells.
• agonistic anti-zyctor19 monoclonal antibodies may activate normal B cells and promote an anticancer immune response.
• Anti-zyctor19 antibodies may directly inhibit the growth of leukemias, lymphomas, and multiple myelomas, and the antibodies may engage immune effector functions.
• Anti-zyctor19 monoclonal antibodies may enable antibody-dependent cellular cytotoxicity, complement dependent cytotoxicity, and phagocytosis.
• zyctor19 ligand may be expressed in neutrophils, monocytes, dendritic cells, and activated monocytes.
• B cells might exacerbate autoimmunity after activation by zyctor19 ligand.
• Immunosuppressant proteins that selectively block the action of B-lymphocytes would be of use in treating disease.
• Autoantibody production is common to several autoimmune diseases and contributes to tissue destruction and exacerbation of disease. Autoantibodies can also lead to the occurrence of immune complex deposition complications and lead to many symptoms of systemic lupus erythematosus, including kidney failure, neuralgic symptoms and death.
• Modulating antibody production independent of cellular response would also be beneficial in many disease states.
• B cells have also been shown to play a role in the secretion of arthritogenic immunoglobulins in rheumatoid arthritis.
• inhibition of zyctor19 ligand antibody production would be beneficial in treatment of autoimmune diseases such as myasthenia gravis and rheumatoid arthritis.
• Immunosuppressant therapeutics such as anti-zyctor19 antibodies that selectively block or neutralize the action of B-lymphocytes would be useful for such purposes.
• the invention provides methods employing anti-zyctor19 antibodies, or multispecific antibody compositions, for selectively blocking or neutralizing the actions of B-cells in association with end stage renal diseases, which may or may not be associated with autoimmune diseases. Such methods would also be useful for treating immunologic renal diseases. Such methods would be would be useful for treating glomerulonephritis associated with diseases such as membranous nephropathy, IgA nephropathy or Berger's Disease, IgM nephropathy, Goodpasture's Disease, post-infectious glomerulonephritis, mesangioproliferative disease, chronic lymphocytic leukemia, minimal-change nephrotic syndrome.
• diseases such as membranous nephropathy, IgA nephropathy or Berger's Disease, IgM nephropathy, Goodpasture's Disease, post-infectious glomerulonephritis, mesangioproliferative disease, chronic lymphocytic leukemia,
• Such methods would also serve as therapeutic applications for treating secondary glomerulonephritis or vasculitis associated with such diseases as lupus, polyarteritis, Henoch-Schonlein, Scleroderma, HIV-related diseases, amyloidosis or hemolytic uremic syndrome.
• the methods of the present invention would also be useful as part of a therapeutic application for treating interstitial nephritis or pyelonephritis associated with chronic pyelonephritis, analgesic abuse, nephrocalcinosis, nephropathy caused by other agents, nephrolithiasis, or chronic or acute interstitial nephritis.
• the invention provides methods employing anti-zyctor19 antibodies, or multispecific antibody compositions, for selectively blocking or neutralizing the viral infection associated with the liver.
• anti-zyctor19 antibodies or multispecific antibody compositions, for selectively blocking or neutralizing the viral infection associated with the liver.
• Example 24 while normal and diseased liver specimens show expression of zcytoR19 mRNA, there is specific expression of the receptor in liver specimens that are positive for Hepatitis Virus C, and Hepatitis B.
• liver disease When liver disease is inflammatory and continuing for at least six months, it is generally considered chronic hepatitis.
• Hepatitis C virus (HCV) patients actively infected will be positive for HCV-RNA in their blood, which is detectable by reverse transcritptase/polymerase chain reaction (RT-PCR) assays.
• RT-PCR reverse transcritptase/polymerase chain reaction
• the methods of the present invention will slow the progression of the liver disease, and can be measured, for example, as improved serum alanine transaminase (ALT) levels, improved levels of aspartate aminotrasnferase (AST), decreased portal inflammation as determined by biopsy, or decrease in hepatocytic necrosis. Histological improvement can be measured using the Histological Activity Index (Davis et al., New Eng.
• the present invention also provides methods for treatment of renal or urological neoplasms, multiple myelomas, lymphomas, leukemias, light chain neuropathy, or amyloidosis.
• the invention also provides methods for blocking or inhibiting activated B cells using anti-zyctor19 antibodies, or multispecific antibody compositions, for the treatment of asthma and other chronic airway diseases such as bronchitis and emphysema.
• anti-zyctor19 antibodies, or multispecific, antibody compositions for immunosuppression, in particular for such therapeutic use as for graft-versus-host disease and graft rejection.
• anti-zyctor19 antibodies, or multispecific antibody compositions would be useful in therapeutic protocols for treatment of such autoimmune diseases as insulin dependent diabetes mellitus (IDDM), multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (IBD), and Crohn's Disease.
• IDDM insulin dependent diabetes mellitus
• IBD inflammatory bowel disease
• Methods of the present invention would have additional therapeutic value for treating chronic inflammatory diseases, in particular to lessen joint pain, swelling, anemia and other associated symptoms as well as treating septic shock.
• B cell responses are important in fighting infectious diseases including bacterial, viral, protozoan and parasitic infections.
• Antibodies against infectious microorganisms can immobilize the pathogen by binding to antigen followed by complement mediated lysis or cell mediated attack.
• Agonistic, or signaling, anti-zyctor19 antibodies may serve to boost the humoral response and would be a useful therapeutic for individuals at risk for an infectious disease or as a supplement to vaccination.
• anti-zyctor19 antibodies can be tested in vivo in a number of animal models of autoimmune disease, such as MRL-Lpr/Lpr or NZB ⁇ NZW F1 congenic mouse strains which serve as a model of systemic lupus erythematosus.
• animal models are known in the art.
• anti-zyctor19 antibodies, or multispecific antibody compositions will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition and previous medical history.
• anti-zyctor19 antibodies, or multispecific antibody compositions can be administered at low protein doses, such as 20 to 100 milligrams protein per dose, given once, or repeatedly.
• anti-zyctor19 antibodies, or multispecific antibody compositions can be administered in doses of 30 to 90 milligrams protein per dose, or 40 to 80 milligrams protein per dose, or 50 to 70 milligrams protein per dose, although a lower or higher dosage also may be administered as circumstances dictate.
• Administration of antibody components to a subject can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection.
• the administration may be by continuous infusion or by single or multiple boluses. Additional routes of administration include oral, mucosal-membrane, pulmonary, and transcutaneous.
• a pharmaceutical composition comprising an anti-zyctor19 antibody, or bispecific antibody components, can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier.
• a composition is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient.
• Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
• Other suitable carriers are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
• anti-zyctor19 antibodies, or bispecific antibody components, and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount.
• a combination of anti-zyctor19 antibodies, or bispecific antibody components, and a pharmaceutically acceptable carrier is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant.
• An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
• an agent used to treat inflammation is physiologically significant if its presence alleviates the inflammatory response.
• an agent used to inhibit the growth of tumor cells is physiologically significant if the administration of the agent results in a decrease in the number of tumor cells, decreased metastasis, a decrease in the size of a solid tumor, or increased necrosis of a tumor.
• a pharmaceutical composition comprising anti-zyctor19 antibodies, or bispecific antibody components, can be furnished in liquid form, in an aerosol, or in solid form.
• Liquid forms are illustrated by injectable solutions and oral suspensions.
• Exemplary solid forms include capsules, tablets, and controlled-release forms. The latter form is illustrated by miniosmotic pumps and implants (Bremer et al., Pharm. Biotechnol.
• liposomes provide a means to deliver anti-zyctor19 antibodies, or bispecific antibody components, to a subject intravenously, intraperitoneally, intrathecally, intramuscularly, subcutaneously, or via oral administration, inhalation, or intranasal administration.
• Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments (see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 ( Suppl.
• Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable. Depending on the method of preparation, liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 ⁇ m to greater than 10 ⁇ m.
• a variety of agents can be encapsulated in liposomes: hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s) (see, for example, Machy et al., Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover, it is possible to control the therapeutic availability of the encapsulated agent by varying liposome size, the number of bilayers, lipid composition, as well as the charge and surface characteristics of the liposomes.
• target cells can be prelabeled with biotinylated anti-zyctor19 antibodies. After plasma elimination of free antibody, streptavidin-conjugated liposomes are administered.
• This general approach is described, for example, by Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998). Such an approach can also be used to prepare multispecific antibody compositions.
• the present invention also contemplates chemically modified antibody components, in which an antibody component is linked with a polymer.
• the polymer is water soluble so that an antibody component does not precipitate in an aqueous environment, such as a physiological environment.
• a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation. In this way, the degree of polymerization can be controlled.
• An example of a reactive aldehyde is polyethylene glycol propionaldehyde, or mono-(C 1 -C 10 ) alkoxy, or aryloxy derivatives thereof (see, for example, Harris, et al., U.S. Pat. No. 5,252,714).
• the polymer may be branched or unbranched.
• a mixture of polymers can be used to produce conjugates with antibody components.
• Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono-(C 1 -C 10 )alkoxy-PEG, aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonate PEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers.
• Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000.
• a conjugate can also comprise a mixture of such water-soluble polymers.
• Polypeptide cytotoxins can also be conjugated with a soluble polymer using the above methods either before or after conjugation to an antibody component.
• Soluble polymers can also be conjugated with antibody fusion proteins.
• naked anti-zyctor19 antibodies, or antibody fragments can be supplemented with immunoconjugate or antibody fusion protein administration.
• naked anti-zyctor19 antibodies or naked antibody fragments
• naked anti-zyctor19 antibodies are administered with low-dose radiolabeled anti-zyctor19 antibodies or antibody fragments.
• naked anti-zyctor19 antibodies or antibody fragments
• naked anti-zyctor19 antibodies are administered with low-dose radiolabeled anti-zyctor19 antibodies-cytokine immunoconjugates.
• naked anti-zyctor19 antibodies (or antibody fragments) are administered with anti-zyctor19-cytokine immunoconjugates that are not radiolabeled.
• a preferable dosage is in the range of 15 to 40 mCi, while the most preferable range is 20 to 30 mCi.
• a preferred dosage of 90 Y-labeled immunoconjugates is in the range from 10 to 30 mCi, while the most preferable range is 10 to 20 mCi.
• bispecific antibody components can be supplemented with immunoconjugate or antibody fusion protein administration.
• Immunoconjugates having a boron addend-loaded carrier for thermal neutron activation therapy will normally be effected in similar ways. However, it will be advantageous to wait until non-targeted immunoconjugate clears before neutron irradiation is performed. Clearance can be accelerated using an antibody that binds to the immunoconjugate. See U.S. Pat. No. 4,624,846 for a description of this general principle.
• the present invention also contemplates a method of treatment in which immunomodulators are administered to prevent, mitigate or reverse radiation-induced or drug-induced toxicity of normal cells, and especially hematopoietic cells.
• Adjunct immunomodulator therapy allows the administration of higher doses of cytotoxic agents due to increased tolerance of the recipient mammal.
• adjunct immunomodulator therapy can prevent, palliate, or reverse dose-limiting marrow toxicity.
• suitable immunomodulators for adjunct therapy include granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, thrombopoietin, IL-1, IL-3, IL-12, and the like.
• the method of adjunct immunomodulator therapy is disclosed by Goldenberg, U.S. Pat. No. 5,120,525.
• the efficacy of anti-zyctor19 antibody therapy can be enhanced by supplementing naked antibody components with immunoconjugates and other forms of supplemental therapy described herein.
• the supplemental therapeutic compositions can be administered before, concurrently or after administration of naked anti-zyctor19 antibodies.
• Multimodal therapies of the present invention further include immunotherapy with naked anti-zyctor19 antibody components supplemented with administration of anti-zcytor19 immunoconjugates.
• subjects receive naked anti-zyctor19 antibodies and standard cancer chemotherapy.
• the antibodies and antibody fragments of the present invention can be used as vaccines to treat the various disorders and diseases described above.
• an antibody component of a dual reactive zyctor19 receptor monoclonal antibody can provide a suitable basis for a vaccine.
• Cysteine-rich regions of zyctor19 receptors can also provide useful components for a vaccine.
• a vaccine can comprise at least one of the following polypeptides: a polypeptide comprising amino acid residues 8 to 41 of SEQ ID NO:2, a polypeptide comprising amino acid residues 34 to 66 of SEQ ID NO:4, and a polypeptide comprising amino acid residues 71 to 104 of SEQ ID NO:4.
• compositions may be supplied as a kit comprising a container that comprises anti-zyctor19 antibody components, or bispecific antibody components.
• Therapeutic molecules can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection.
• a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of an anti-zyctor19 antibody component.
• Such a kit may further comprise written information on indications and usage of the pharmaceutical composition. Moreover, such information may include a statement that the composition is contraindicated in patients with known hypersensitivity to exogenous antibodies.
• Zyctor19 polypeptides such as soluble zyctor19 receptors, may also be used within diagnostic systems for the detection of circulating levels of ligand.
• antibodies or other agents that specifically bind to zyctor19 receptor polypeptides can be used to detect circulating receptor polypeptides. Elevated or depressed levels of ligand or receptor polypeptides may be indicative of pathological conditions, including cancer. Soluble receptor polypeptides may contribute to pathologic processes and can be an indirect marker of an underlying disease.
• 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, B-cell lymphomas, chronic lymphocytic leukemia, colon cancer, breast cancer, and ovarian cancer (Heaney et al., Blood 87:847-857, 1996).
• zyctor19 is expressed in B-cell leukemia cells, an increase of zyctor19 expression can even serve as a marker of an underlying disease, such as leukemia.
• a ligand-binding polypeptide of a zyctor19 receptor can be prepared by expressing a truncated DNA encoding the zyctor19 extracellular cytokine-binding domain (residues 21 (Arg) to 226 (Asn) of SEQ ID NO:2 or SEQ ID NO:19), cytokine-binding fragment (e.g., residues 21 (Arg) to 223 (Pro) of SEQ ID NO:2 or SEQ ID NO:19; SEQ ID NO:4), the soluble version of zyctor19 variant, or the corresponding region of a non-human receptor.
• the extracellular domain be prepared in a form substantially free of transmembrane and intracellular polypeptide segments.
• ligand-binding polypeptide fragments within the zyctor19 cytokine-binding domain, described above, can also serve as zyctor19 soluble receptors for uses described herein.
• the receptor DNA is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide or a zyctor19 secretory peptide.
• a C-terminal extension such as a poly-histidine tag, Glu-Glu tag peptide, substance P, FlagTM peptide (Hopp et al., Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., New Haven, Conn.) or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the receptor polypeptide.
• a receptor extracellular domain can be expressed as a fusion with immunoglobulin heavy chain constant regions, typically an F c fragment, which contains two constant region domains and lacks the variable region.
• immunoglobulin heavy chain constant regions typically an F c fragment
• Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two receptor polypeptides are arrayed in close proximity to each other. Fusions of this type can be used to affinity purify the cognate ligand from solution, as an in vitro assay tool, to block signals in vitro by specifically titrating out ligand, and as antagonists in vivo by administering them parenterally to bind circulating ligand and clear it from the circulation.
• a zyctor19-Ig chimera is added to a sample containing the ligand (e.g., cell-conditioned culture media or tissue extracts) under conditions that facilitate receptor-ligand binding (typically near-physiological temperature, pH, and ionic strength).
• the chimera-ligand complex is then separated by the mixture using protein A, which is immobilized on a solid support (e.g., insoluble resin beads).
• the ligand is then eluted using conventional chemical techniques, such as with a salt or pH gradient.
• the chimera itself can be bound to a solid support, with binding and elution carried out as above. Collected fractions can be re-fractionated until the desired level of purity is reached.
• zyctor19 soluble receptors can be used as a “ligand sink,” i.e., antagonist, to bind ligand in vivo or in vitro in therapeutic or other applications where the presence of the ligand is not desired.
• zyctor19 soluble receptors can be used as a direct antagonist of the ligand in vivo, and may aid in reducing progression and symptoms associated with the disease.
• zyctor19 soluble receptor can be used to slow the progression of cancers that over-express zyctor19 receptors, by binding ligand in vivo that would otherwise enhance proliferation of those cancers. Similar in vitro applications for a zyctor19 soluble receptor can be used, for instance, as a negative selection to select cell lines that grow in the absence of zyctor19 ligand.
• zyctor19 soluble receptor can be used in vivo or in diagnostic applications to detect zyctor19 ligand-expressing cancers in vivo or in tissue samples.
• the zyctor19 soluble receptor can be conjugated to a radio-label or fluorescent label as described herein, and used to detect the presence of the ligand in a tissue sample using an in vitro ligand-receptor type binding assay, or fluorescent imaging assay.
• a radiolabeled zyctor19 soluble receptor could be administered in vivo to detect ligand-expressing solid tumors through a radio-imaging method known in the art.
• zcytor19 polynucleotides, polypeptides, anti-zyctor19 andibodies, or peptide binding fragments can be used to detect zyctor19 receptor expressing cancers.
• zyctor19 polynucleotides, polypeptides, anti-zyctor19 andibodies, or peptide binding fragments can be used to detect leukemias, more preferably B-cell leukemias, and most preferably pre-B-cell acute lymphoblastic leukemia.
• polypeptides of the present invention it is preferred to purify the polypeptides of the present invention to ⁇ 80% purity, more preferably to ⁇ 90% purity, even more preferably ⁇ 95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
• a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
• Expressed recombinant zyctor19 polypeptides can be purified using fractionation and/or conventional purification methods and media.
• Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
• Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
• Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
• Exemplary chromatographic media include those media derivatized 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 that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. 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.
• the polypeptides of the present invention can be isolated by exploitation of their biochemical, structural, and biological properties.
• immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
• IMAC immobilized metal ion adsorption
• a fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification.
• an affinity tag e.g., maltose-binding protein, an immunoglobulin domain
• polypeptide fusions, or hybrid zyctor19 proteins are constructed using regions or domains of the inventive zyctor19 in combination with those of other human cytokine receptor family proteins, or heterologous proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest.
• Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.
• Fusion polypeptides or proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them.
• a polynucleotide encoding one or more components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain(s) conferring a biological function may be swapped between zyctor19 of the present invention with the functionally equivalent domain(s) from another cytokine family member.
• Such domains include, but are not limited to, the secretory signal sequence, extracellular cytokine binding domain, cytokine binding fragment, fibronectin type III domains, transmembrane domain, and intracellular signaling domain, as disclosed 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. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
• Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the zyctor19 polypeptide and those polypeptides to which they are fused.
• a DNA segment that encodes a domain of interest e.g., a zyctor19 domain described herein, is operably linked in frame to at least one other DNA segment encoding an additional polypeptide (for instance a domain or region from another cytokine receptor, such as, interferon-gamma, alpha and beta chains and the interferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No.
• DNA constructs are made such that the several DNA segments that encode the corresponding regions of a polypeptide are operably linked in frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof.
• a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a cytokine binding domain, followed by a transmembrane domain, followed by an intracellular signaling domain.
• Such fusion proteins can be expressed, isolated, and assayed for activity as described herein. Moreover, such fusion proteins can be used to express and secrete fragments of the zyctor19 polypeptide, to be used, for example to inoculate an animal to generate anti-zyctor19 antibodies as described herein.
• a secretory signal sequence can be operably linked to extracellular cytokine binding domain, cytokine binding fragment, individual fibronectin type III domains, transmembrane domain, and intracellular signaling domain, as disclosed herein, or a combination thereof (e.g., operably linked polypeptides comprising a fibronectin III domain attached to a linker, or zyctor19 polypeptide fragments described herein), to secrete a fragment of zyctor19 polypeptide that can be purified as described herein and serve as an antigen to be inoculated into an animal to produce anti-zyctor19 antibodies, as described herein.
• viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
• Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for review, see T. C. Becker et al., Meth. Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science & Medicine 4:44-53, 1997).
• adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with a large number of different promoters including ubiquitous, tissue specific, and regulatable promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
• zyctor19 In view of the tissue distribution observed for zyctor19, agonists (including the natural ligand/substrate/cofactor/etc.) and antagonists have enormous potential in both in vitro and in vivo applications.
• Compounds identified as zyctor19 agonists are useful for stimulating growth of immune and hematopoietic cells in vitro and in vivo.
• zyctor19 soluble receptors, and agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture.
• Agonists are thus useful in specifically promoting the growth and/or development of T-cells, B-cells, and other cells of the lymphoid and myeloid lineages in culture.
• zyctor19 soluble receptor, agonist, or antagonist may be used in vitro in an assay to measure stimulation of colony formation from isolated primary bone marrow cultures. Such assays are well known in the art.
• Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction.
• Inhibitors of zyctor19 activity include anti-zyctor19 antibodies and soluble zyctor19 receptors, as well as other peptidic and non-peptidic agents (including ribozymes).
• Zyctor19 can also be used to identify modulators (e.g, antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of zyctor19. In addition to those assays disclosed herein, samples can be tested for inhibition of zyctor19 activity within a variety of assays designed to measure zyctor19 binding, oligomerization, or the stimulation/inhibition of zyctor19-dependent cellular responses.
• modulators e.g, antagonists
• a zyctor19 ligand-binding polypeptide such as the extracellular domain or cytokine binding domain disclosed herein, can also be used for purification of ligand.
• the polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like 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 fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide.
• the ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl), or pH to disrupt ligand-receptor binding.
• An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument may be advantageously employed (e.g., BIAcoreTM, Pharmacia Biosensor, Piscataway, N.J.; or SELDITM technology, Ciphergen, Inc., Palo Alto, Calif.).
• a ligand-binding receptor or an antibody, one member of a complement/anti-complement pair
• a commercially available biosensor instrument may be advantageously employed (e.g., BIAcoreTM, Pharmacia Biosensor, Piscataway, N.J.; or SELDITM technology, Ciphergen, Inc., Palo Alto, Calif.).
• BIAcoreTM Pharmacia Biosensor, Piscataway, N.J.
• SELDITM technology Ciphergen, Inc., Palo Alto, Calif.
• Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of 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).
• Zyctor19 polypeptides can also be used to prepare antibodies that bind to zyctor19 epitopes, peptides or polypeptides.
• the zyctor19 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
• antigenic, epitope-bearing 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 zyctor19 polypeptide (e.g., SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21).
• Polypeptides comprising a larger portion of a zyctor19 polypeptide, i.e., from 30 to 100 residues up to the entire length of the amino acid sequence are included.
• Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein. Suitable antigens include the zyctor19 polypeptide encoded by SEQ ID NO:2 from amino acid number 21 (Arg) to amino acid number 491 (Arg), or a contiguous 9 to 471 amino acid fragment thereof.
• Suitable antigens also include the zyctor19 polypeptide encoded by SEQ ID NO:19 from amino acid number 21 (Arg) to amino acid number 520 (Arg), or a contiguous 9 to 500 amino acid fragment thereof; and the truncated soluble zyctor19 polypeptide encoded by SEQ ID NO:21 from amino acid number 21 (Arg) to amino acid number 211 (Ser), or a contiguous 9 to 191 amino acid fragment thereof.
• Preferred peptides to use as antigens are the extracellular cytokine binding domain, cytokine binding fragment, fibronectin type III domains, intracellular signaling domain, or other domains and motifs disclosed herein, or a combination thereof; and zyctor19 hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot, determined for example, from a Hopp/Woods hydrophilicity profile.
• Zyctor19 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: (1) residues 295 through 300 of SEQ ID NO:2; (2) residues 451 through 456 of SEQ ID NO:2; (3) residues 301 through 306 of SEQ ID NO:2; (4) residues 294 through 299 of SEQ ID NO:2; and (5) residues 65 through 70 of SEQ ID NO:2.
• zyctor19 antigenic epitopes as predicted by a Jameson-Wolf plot, e.g., using DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.) are suitable antigens.
• 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. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, Fla., 1982.
• polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zyctor19 polypeptide or a fragment thereof.
• the immunogenicity of a zyctor19 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
• Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zyctor19 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
• the polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is “hapten-like”, such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
• a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
• antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab′) 2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.
• Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally “cloaking” them with a human-like surface by replacement of exposed residues, wherein the result is a “veneered” antibody).
• humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Moreover, human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
• Antibodies in the present invention include, but are not limited to, antibodies that bind the zyctor19/CRF2-4 heterodimer, as well as the heterodimeric soluble receptor complex.
• Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zyctor19 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zyctor19 protein or peptide).
• Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No.
• Random peptide display libraries can be screened using the zyctor19 sequences disclosed herein to identify proteins which bind to zyctor19.
• binding peptides which interact with zyctor19 polypeptides can be used for tagging cells, e.g., such as those in which zyctor19 is specifically expressed; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding peptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding peptides can also be used for diagnostic assays for determining circulating levels of zyctor19 polypeptides; for detecting or quantitating soluble zyctor19 polypeptides as marker of underlying pathology or disease.
• binding peptides can also act as zyctor19 “antagonists” to block zyctor19 binding and signal transduction in vitro and in vivo. These anti-zyctor19 binding peptides would be useful for inhibiting the action of a ligand that binds with zyctor19.
• Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with related polypeptide molecules.
• a threshold level of binding is determined if anti-zyctor19 antibodies herein bind to a zyctor19 polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-zyctor19) polypeptide. It is preferred that the antibodies exhibit a binding affinity (K a ) of 10 6 M ⁇ 1 or greater, preferably 10 7 M ⁇ 1 or greater, more preferably 10 8 M ⁇ 1 or greater, and most preferably 10 9 M ⁇ 1 or greater.
• 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).
• anti-zyctor19 antibodies do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody detecting zyctor19 polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.).
• known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family (e.g., class II cytokine receptors, for example, interferon-gamma, alpha and beta chains and the interferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No.
• a variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to zyctor19 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zyctor19 protein or polypeptide.
• Antibodies to zyctor19 may be used for tagging cells that express zyctor19; for isolating zyctor19 by affinity purification; for diagnostic assays for determining circulating levels of zyctor19 polypeptides; for detecting or quantitating soluble zyctor19 as marker of underlying pathology or disease; for detecting or quantitating in a histologic biopsy, or tissue sample zyctor19 receptor as marker of underlying pathology or disease; for stimulating cytotoxicity or ADCC on zyctor19-bearing cancer cells; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zyctor19 activity in vitro and in vivo.
• Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Moreover, antibodies to zyctor19 or fragments thereof may be used in vitro to detect denatured zyctor19 or fragments thereof in assays, for example, Western Blots or other assays known in the art.
• Antibodies herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
• Suitable detectable molecules may be directly or indirectly attached to polypeptides that bind zyctor19 (“binding polypeptides,” including binding peptides disclosed above), antibodies, or bioactive fragments or portions thereof.
• Suitable detectable molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
• Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance). Binding polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin.
• the detectable or cytotoxic molecule can be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the binding polypeptide or antibody portion.
• biotin/streptavidin is an exemplary complementary/anticomplementary pair.
• binding polypeptide-toxin fusion proteins or antibody-toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues, e.g., such as those specific tissues and tumors wherein zyctor19 is expressed).
• a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest.
• the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule.
• domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti-complementary-detectable/cytotoxic molecule conjugates.
• zyctor19 binding polypeptide-cytokine or antibody-cytokine fusion proteins can be used for enhancing in vivo killing of target tissues, if the binding polypeptide-cytokine or anti-zyctor19 antibody targets the hyperproliferative cell (See, generally, Hornick et al., Blood 89:4437-47, 1997).
• fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine.
• Suitable anti-zyctor19 antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediates improved target cell lysis by effector cells.
• Suitable cytokines for this purpose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), for instance.
• zyctor19 binding polypeptide or antibody fusion proteins described herein can be used for enhancing in vivo killing of target tissues by directly stimulating a zyctor19-modulated apoptotic pathway, resulting in cell death of hyperproliferative cells expressing zyctor19.
• bioactive binding polypeptide or antibody conjugates described herein can be delivered orally, intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action.
• anti-zyctor19 antibodies and binding fragments can be used for tagging and sorting cells that specifically-express Zyctor19, such as bone marrow and thyroid cells, and other cells, described herein.
• Such methods of cell tagging and sorting are well known in the art (see, e.g., “Molecular Biology of the Cell”, 3 rd Ed., Albert, B. et al. (Garland Publishing, London & New York, 1994).
• One of skill in the art would recognize the importance of separating cell tissue types to study cells, and the use of antibodies to separate specific cell tissue types.
• Antisense methodology can be used to inhibit zyctor19 gene transcription, such as to inhibit cell proliferation in vivo.
• Polynucleotides that are complementary to a segment of a zyctor19-encoding polynucleotide e.g., a polynucleotide as set forth in SEQ ID NO:1 SEQ ID NO:18, or SEQ ID NO:20
• a segment of a zyctor19-encoding polynucleotide e.g., a polynucleotide as set forth in SEQ ID NO:1 SEQ ID NO:18, or SEQ ID NO:20
• Such antisense polynucleotides are used to inhibit expression of zyctor19 polypeptide-encoding genes in cell culture or in a subject.
• zyctor19 polypeptides can be used as a target to introduce gene therapy into a cell.
• This application would be particularly appropriate for introducing therapeutic genes into cells in which zyctor19 is normally expressed, such as lymphoid tissue, bone marrow, prostate, thyroid, and PBLs, or cancer cells which express zyctor19 polypeptide.
• viral gene therapy such as described above, can be targeted to specific cell types in which express a cellular receptor, such as zyctor19 polypeptide, rather than the viral receptor.
• Antibodies, or other molecules that recognize zyctor19 molecules on the target cell's surface can be used to direct the virus to infect and administer gene therapeutic material to that target cell. See, Woo, S. L.
• a bispecific antibody containing a virus-neutralizing Fab fragment coupled to a zyctor19-specific antibody can be used to direct the virus to cells expressing the zyctor19 receptor and allow efficient entry of the virus containing a genetic element into the cells. See, for example, Wickham, T. J., et al., J. Virol. 71:7663-7669, 1997; and Wickham, T. J., et al., J. Virol. 70:6831-6838, 1996.
• the present invention also provides reagents which will find use in diagnostic applications.
• the zyctor19 gene a probe comprising zyctor19 DNA or RNA or a subsequence thereof can be used to determine if the zyctor19 gene is present on chromosome 1 or if a mutation has occurred.
• Zyctor19 is located at the 1p36.11 region of chromosome 1. Detectable chromosomal aberrations at the zyctor19 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
• Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, fluorescence in situ hybridization methods, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
• molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, fluorescence in situ hybridization methods, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
• the precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.
• the zyctor19 gene is located at the 1p36.11 region of chromosome 1.
• chromosomal aberrations in and around the 1p36 region are involved in several cancers including neuroblastoma, melanoma, breast, colon, prostate and other cancers.
• Such aberrations include gross chromosomal abnormalities such as translocations, loss of heterogeneity (LOH) and the like in and around 1p36.
• LHO loss of heterogeneity
• a marker in the 1p36.11 locus such as provided by the polynucleotides of the present invention, would be useful in detecting translocations, aneuploidy, rearrangements, LOH other chromosomal abnormalities involving this chromosomal region that are present in cancers.
• zyctor19 polynucleotide probes can be used to detect abnormalities or genotypes associated with neuroblastoma, wherein LOH between 1p36.1 and 1p36.3 is prevalent, and a breakpoint at 1p36.1 is evident.
• At least 70% of neuroblastomas have cytogenetically visible chromosomal aberrations in 1p, including translocation and deletion, and that the abnormality is most likely due to complex translocation and deletion mechanisms. See, for example Ritke, M K et al., Cytogenet. Cell Genet. 50:84-90, 1989; and Weith, A et al., Genes Chromosomes Cancer 1:159-166, 1989).
• zyctor19 is localized to 1p36.11, and falls directly within the region wherin aberrations are prevalent in neuroblastoma, one of skill in the art would apprecitate that the polynucleotides of the present invention could serve as a diagnostic for neuroblastoma, as well as aid in the elucidation of translocation and deletion mechanisms that give rise to neuroblastoma.
• LOH at 1p36 is evident in melanoma (Dracopoli, N C et al, Am. J. Hum. Genet. 45 ( suppl. ):A19, 1989; Dracopoli, N C et al, Proc. Nat. Acad. Sci.
• zyctor19 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 1p36.11 deletions and translocations associated with human diseases, and prefereably cancers, as described above.
• those for Clq complement components C1QA, B, and G) (1p36.3-p34.1); dyslexia (1p36-p34); lymphoid activation antigen CD30 (1p36); sodium channel non-voltage-gated type 1 (1p36.3-p36.2); tumor necrosis factor receptors (TNFRSF1b and TNFRS12) (1p36.3-p36.2) which like zyctor19 are cytokine receptors; phospholipase A2 (PLA2) (1p35); rigid spine muscular dystrophy (1p36-p35) all manifest themselves in human disease states as well as map to this region of the human genome.
• zyctor19 polynucleotide probes can be used to detect abnormalities or genotypes associated with these defects.
• defects in the zyctor19 gene itself may result in a heritable human disease state.
• the zyctor19 gene (1p36.11) is located near another class II receptor, the zcytor11 cytokine receptor gene (1p35.1) (commonly owned U.S. Pat. No. 5,965,704), as well as TNF receptors (1p36.3-p36.2), suggesting that this chromosomal region is commonly regulated, and/or important for immune function.
• defects in cytokine receptors are known to cause disease states in humans. For example, growth hormone receptor mutation results in dwarfism (Amselem, S et al., New Eng. J. Med.
• IL-2 receptor gamma mutation results in severe combined immunodeficiency (SCID) (Noguchi, M et al., Cell 73: 147-157, 1993), c-Mpl mutation results in thrombocytopenia (Ihara, K et al., Proc. Nat. Acad. Sci. 96: 3132-3136, 1999), and severe mycobacterial and Salmonella infections result in interleukin-12 receptor-deficient patients (de Jong, R et al., Science 280: 1435-1438, 1998), amongst others. Thus, similarly, defects in zyctor19 can cause a disease state or susceptibility to disease or infection.
• SCID severe combined immunodeficiency
• c-Mpl mutation results in thrombocytopenia (Ihara, K et al., Proc. Nat. Acad. Sci. 96: 3132-3136, 1999)
• severe mycobacterial and Salmonella infections result in interleukin-12 receptor-deficient patients (de Jong,
• zyctor19 is a cytokine receptor in a chromosomal hot spot for aberrations involved in numerous cancers and is shown to be expressed in pre-B-cell acute leukemia cells, and other cancers described herein, the molecules of the present invention could also be directly involved in cancer formation or metastasis.
• polynucleotide probes can be used to detect chromosome 1p36.11 loss, trisomy, duplication or translocation associated with human diseases, such as immune cell cancers, neuroblastoma, bone marrow cancers, thyroid, parathyroid, prostate, melanoma, or other cancers, or immune diseases.
• molecules of the present invention such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a zcytor19 genetic defect.
• Mutations associated with the zyctor19 locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
• standard methods for direct mutation analysis such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted
• mice engineered to express the zyctor19 gene referred to as “transgenic mice,” and mice that exhibit a complete absence of zyctor19 gene function, referred to as “knockout mice,” may also be generated (Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature 366:740-42, 1993; Capecchi, M. R., Science 244: 1288-1292, 1989; Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499, 1986).
• transgenic mice that over-express zyctor19, either ubiquitously or under a tissue-specific or tissue-restricted promoter can be used to ask whether over-expression causes a phenotype.
• over-expression of a wild-type zcytor19 polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which zyctor19 expression is functionally relevant and may indicate a therapeutic target for the zyctor19, its agonists or antagonists.
• a preferred transgenic mouse to engineer is one that expresses a “dominant-negative” phenotype, such as one that over-expresses the zyctor19 polypeptide comprising an extracellular cytokine binding domain with the transmembrane domain attached (approximately amino acids 21 (Arg) to 249 (Trp) of SEQ ID NO:2 or SEQ ID NO:19; or SEQ ID NO:4 attached in frame to a transmembrane domain).
• Another preferred transgenic mouse is one that over-expresses zyctor19 soluble receptors, such as those disclosed herein. Moreover, such over-expression may result in a phenotype that shows similarity with human diseases.
• knockout zyctor19 mice can be used to determine where zyctor19 is absolutely required in vivo.
• the phenotype of knockout mice is predictive of the in vivo effects of a zyctor19 antagonist, such as those described herein, may have.
• the mouse or the human zyctor19 cDNA can be used to isolate murine zyctor19 mRNA, cDNA and genomic DNA, which are subsequently used to generate knockout mice.
• These transgenic and knockout mice may be employed to study the zyctor19 gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human or animal diseases (such as those in commercially viable animal populations).
• the mouse models of the present invention are particularly relevant as tumor models for the study of cancer biology and progression. Such models are useful in the development and efficacy of therapeutic molecules used in human cancers. Because increases in zyctor19 expression, as well as decreases in zyctor19 expression are associated with specific human cancers, both transgenic mice and knockout mice would serve as useful animal models for cancer. Moreover, in a preferred embodiment, zyctor19 transgenic mouse can serve as an animal model for specific tumors, particularly esophagus, liver, ovary, rectum, stomach, and uterus tumors, and melanoma, B-cell leukemia and other lymphoid cancers. Moreover, transgenic mice expression of zyctor19 antisense polynucleotides or ribozymes directed against zyctor19, described herein, can be used analogously to transgenic mice described above.
• the soluble receptor polypeptides of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours.
• pharmaceutical formulations will include a zyctor19 soluble receptor polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like.
• Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
• Therapeutic doses will generally be in the range of 0.1 to 100 ⁇ g/kg of patient weight per day, preferably 0.5-20 mg/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art.
• the proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years.
• a therapeutically effective amount of zyctor19 soluble receptor polypeptide is an amount sufficient to produce a clinically significant effect.
• Polynucleotides and polypeptides of the present invention will additionally find use as educational tools as a laboratory practicum kits for courses related to genetics and molecular biology, protein chemistry and antibody production and analysis. Due to its unique polynucleotide and polypeptide sequence molecules of zyctor19 can be used as standards or as “unknowns” for testing purposes.
• zyctor19 polynucleotides can be used as an aid, such as, for example, to teach a student how to prepare expression constructs for bacterial, viral, and/or mammalian expression, including fusion constructs, wherein zyctor19 is the gene to be expressed; for determining the restriction endonuclease cleavage sites of the polynucleotides; determining mRNA and DNA localization of zyctor19 polynucleotides in tissues (i.e., by Northern and Southern blotting as well as polymerase chain reaction); and for identifying related polynucleotides and polypeptides by nucleic acid hybridization.
• Zyctor19 polypeptides can be used educationally as an aid to teach preparation of antibodies; identifying proteins by Western blotting; protein purification; determining the weight of expressed zyctor19 polypeptides as a ratio to total protein expressed; identifying peptide cleavage sites; coupling amino and carboxyl terminal tags; amino acid sequence analysis, as well as, but not limited to monitoring biological activities of both the native and tagged protein (i.e., receptor binding, signal transduction, proliferation, and differentiation) in vitro and in vivo.
• native and tagged protein i.e., receptor binding, signal transduction, proliferation, and differentiation
• Zyctor19 polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroism to determine conformation, especially of the four alpha helices, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance spectroscopy to reveal the structure of proteins in solution.
• a kit containing the zyctor19 can be given to the student to analyze. Since the amino acid sequence would be known by the professor, the specific protein can be given to the student as a test to determine the skills or develop the skills of the student, the teacher would then know whether or not the student has correctly analyzed the polypeptide. Since every polypeptide is unique, the educational utility of zyctor19 would be unique unto itself.
• Zyctor19 was identified as a predicted full-length cDNA from human genomic DNA AL358412 (Genbank). The sequence of the predicted full length zyctor19 polynucleotide is shown in SEQ ID NO:1 and the corresponding polypeptide is shown in SEQ ID NO:2. A variant full-length zyctor19 cDNA sequence was identified and is shown in SEQ ID NO:18 and the corresponding polynucleotides shown in SEQ ID NO:19. Moreover, a gagnated soluble form of zyctor19 cDNA sequence was identified and is shown in SEQ ID NO:20 and the corresponding polynucleotides shown in SEQ ID NO:21.
• Human Multiple Tissue Northern Blots (Human 12-lane MTN Blot I and II, and Human Immune System MTN Blot II) (Clontech) are probed to determine the tissue distribution of human zyctor19 expression.
• a PCR derived probe that hybridizes to SEQ ID NO:1 or SEQ ID NO:18 is amplified using standard PCR amplification methods.
• An exemplary PCR reaction is carried out as follows using primers designed to hybridize to SEQ ID NO:1, SEQ ID NO:18 or its complement: 30 cycles of 94° C. for 1 minute, 65° C. for 1 minute, and 72° C. for 1 minute; followed by 1 cycle at 72° C. for 7 minutes.
• the PCR product is visualized by agarose gel electrophoresis and the PCR product is gel purified as described herein.
• the probe is radioactively labeled using, e.g., the PRIME IT IITM Random Primer Labeling Kit (Stratagene) according to the manufacturer's instructions.
• the probe is purified using, e.g., a NUCTRAPTM push column (Stratagene).
• EXPRESSHYBTM (Clontech) solution is used for the prehybridization and as a hybridizing solution for the Northern blots. Prehybridization is carried out, for example, at 68° C. for 2 hours. Hybridization takes place overnight at about 68° C.
• a transcript corresponding to the length of SEQ ID NO:1 SEQ ID NO:18, or SEQ ID NO:20 or of an mRNA encoding SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 is expected to be seen in tissues that specifically express zyctor19, but not other tissues.
• Northern analysis is also performed using Human Cancer Cell Line MTNTM (Clontech). PCR and probing conditions are as described above. A strong signal in a cancer line suggests that zyctor19 expression may be expressed in activated cells and/or may indicate a cancerous disease state. Moreover, using methods known in the art, Northern blots or PCR analysis of activated lymphocyte cells can also show whether zyctor19 is expressed in activated immune cells. Based on electronic Northern information zyctor19 was shown to be expressed specifically in pre-B cell acute lymphoblastic leukemia cells.
• a panel of cDNAs from human tissues was screened for zyctor19 expression using PCR.
• the panel was made in-house and contained 94 marathon cDNA and cDNA samples from various normal and cancerous human tissues and cell lines are shown in Table 5, below.
• the cDNAs came from in-house libraries or marathon cDNAs from in-house RNA preps, Clontech RNA, or Invitrogen RNA.
• the marathon cDNAs were made using the marathon-ReadyTM kit (Clontech, Palo Alto, Calif.) and QC tested with clathrin primers ZC21195 (SEQ ID NO:6) and ZC21196 (SEQ ID NO:7) and then diluted based on the intensity of the clathrin band.
• the panel was set up in a 96-well format that included a human genomic DNA (Clontech, Palo Alto, Calif.) positive control sample. Each well contained approximately 0.2-100 pg/ ⁇ l of cDNA.
• the PCR was set up using oligos ZC37685 (SEQ ID NO:26) and ZC37681 (SEQ ID NO:27), TaKaRa Ex TaqTM (TAKARA Shuzo Co LTD, Biomedicals Group, Japan), and Rediload dye (Research Genetics, Inc., Huntsville, Ala.). The amplification was carried out as follows: 1 cycle at 94° C. for 2 minutes, 5 cycles of 94° C. for 30 seconds, 70° C.
• the correct predicted DNA fragment size was observed in adrenal gland, bladder, cervix, colon, fetal heart, fetal skin, liver, lung, melanoma, ovary, salivary gland, small intestine, stomach, brain, fetal liver, kidney, prostate, spinal cord, thyroid, placenta, testis, tumor esophagus, tumor liver, tumor ovary, tumor rectum, tumor stomach, tumor uterus, bone marrow, CD3+ library, HaCAT library, HPV library and HPVS library.
• this primer pair does not span an intron, there may be risk that some tissues that are contaminated with genomic DNA or unprocessed mRNA messages would create a false positive in this assay.
• Mouse tissue panels were also examined using another set of primer pairs: (1) ZC38706 (SEQ ID NO:49) and ZC38711 (SEQ ID NO:50) (800 bp product) using the methods described above. This panel showed a limited tissue distribution for mouse zyctor19: mouse prostate cell lines, salivary gland library, and skin.
• Zyctor19 is mapped to chromosome 1 using the commercially available “GeneBridge 4 Radiation Hybrid (RH) Mapping Panel”(Research Genetics, Inc., Huntsville, Ala.).
• the GeneBridge 4 RH panel contains DNA from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient).
• a publicly available WWW server http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.p1 allows mapping relative to the Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the “WICGR” radiation hybrid map) which is constructed with the GeneBridge 4 RH panel.
• Each of the 95 PCR reactions consisted of 2 ⁇ l 10 ⁇ KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.), 1.6 ⁇ l dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 ⁇ l sense primer, ZC27,895 (SEQ ID NO:14), 1 ⁇ l antisense primer, ZC27,899 (SEQ ID NO:24), 2 ⁇ l “RediLoad” (Research Genetics, Inc., Huntsville, Ala.), 0.4 ⁇ l 50 ⁇ Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and distilled water for a total volume of 20 ⁇ l.
• the reactions are overlaid with an equal amount of mineral oil and sealed.
• the PCR cycler conditions are as follows: an initial 1 cycle 5 minute denaturation at 94° C., 35 cycles of a 45 seconds denaturation at 94° C., 45 seconds annealing at 54° C. and 1 minute AND 15 seconds extension at 72° C., followed by a final 1 cycle extension of 7 minutes at 72° C.
• the reactions are separated by electrophoresis on a 2% agarose gel (EM Science, Gibbstown, N.J.) and visualized by staining with ethidium bromide.
• the results show that Zyctor19 maps on the chromosome 1 WICGR radiation hybrid map in the 1p36.11 chromosomal region.
• An expression vector is prepared for the expression of the soluble, extracellular domain of the zyctor19 polypeptide, pC4zyctor19CEE, wherein the construct is designed to express a zyctor19 polypeptide comprised of the predicted initiating methionine and truncated adjacent to the predicted transmembrane domain, and with a C-terminal Glu-Glu tag (SEQ ID NO:11).
• a zyctor19 DNA fragment comprising a zyctor19 extracellular or cytokine binding domain of zyctor19 described herein, is created using PCR, and purified using standard methods.
• the excised DNA is subcloned into a plasmid expression vector that has a signal peptide, e.g., the native zyctor19 signal peptide, and attaches a Glu-Glu tag (SEQ ID NO:11) to the C-terminus of the zyctor19 polypeptide-encoding polynucleotide sequence.
• a mammalian expression vector contains an expression cassette having a mammalian promoter, multiple restriction sites for insertion of coding sequences, a stop codon and a mammalian terminator.
• the plasmid can also have 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.
• Restriction digested zyctor19 insert and previously digested vector are ligated using standard molecular biological techniques, and electroporated into competent cells such as DH10B competent cells (GIBCO BRL, Gaithersburg, Md.) according to manufacturer's direction and plated onto LB plates containing 50 mg/ml ampicillin, and incubated overnight. Colonies are screened by restriction analysis of DNA prepared from individual colonies. The insert sequence of positive clones is verified by sequence analysis. A large scale plasmid preparation is done using a QIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.
• the same process is used to prepare the zyctor19 soluble receptors with a C-terminal his tag, composed of 6 His residues in a row; and a C-terminal FLAG® tag (SEQ ID NO:12), zyctor19CFLAG.
• the aforementioned vector has either the CHIS or the FLAG® tag in place of the glu-glu tag (SEQ ID NO:11).
• An expression vector, zyctor19/Fc4/pzmp20 was prepared to express a C-terminally Fc4 tagged soluble version of zyctor19 (human zyctor19-Fc4) in BHK cells.
• a fragment of zyctor19 cDNA that includes the polynucleotide sequence from extracellular domain of the zyctor19 receptor was fused in frame to the Fc4 polynucleotide sequence (SEQ ID NO:13) to generate a zyctor19-Fc4 fusion (SEQ ID NO:22 and SEQ ID NO:23).
• the pzmp20 vector is a mammalian expression vector that contains the Fc4 polynucleotide sequence and a cloning site that allows rapid construction of C-terminal Fc4 fusions using standard molecular biology techniques.
• a 630 base pair fragment was generated by PCR, containing the extracellular domain of human zyctor19 with BamHI and Bg12 sites coded on the 5′ and 3′ ends, respectively.
• This PCR fragment was generated using primers ZC37967 (SEQ ID NO:24) and ZC37972 (SEQ ID NO:25) by amplification from human brain cDNA library.
• the PCR reaction conditions were as follows: 30 cycles of 94° C. for 20 seconds, and 68° C. for 2 minutes; 1 cycle at 68° C. for 4 minutes; followed by a 10° C. soak.
• the fragment was digested with BamHI and Bg12 restriction endonucleases and subsequently purified by 1% gel electrophoresis and band purification using QiaQuick gel extraction kit (Qiagen).
• QiaQuick gel extraction kit Qiagen
• the resulting purified DNA was ligated for 5 hours at room temperature into a pzmp20 vector previously digested with Bg12 containing Fc4 3′ of the Bg12 sites.
• BHK 570 cells (ATCC NO: CRL-10314) were plated in T-75 tissue culture flasks and allowed to grow to approximately 50 to 70% confluence at 37° C., 5% CO 2 , in DMEM/FBS media (DMEM, Gibco/BRL High Glucose, (Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum, 1 mM L-glutamine (JRH Biosciences, Lenea, Kans.), 1 mM sodium pyruvate (Gibco BRL)).
• DMEM Gibco/BRL High Glucose, Gibco BRL, Gaithersburg, Md.
• fetal bovine serum 1 mM L-glutamine
• JRH Biosciences Lenea, Kans.
• Na pyruvate Gibco BRL
• the cells were then transfected with the plasmid zyctor19/Fc4/pzmp20 (Example 4B) using LipofectamineTM (Gibco BRL), in serum free (SF) media formulation (DMEM, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).
• DMEM serum free
• Ten ⁇ g of the plasmid DNA zyctor19/Fc4/pzmp20 (Example 4B) was diluted into a 15 ml tube to a total final volume of 500 ⁇ l with SF media. 50 ⁇ l of Lipofectamine was mixed with 450 ⁇ l of SF medium.
• the Lipofectamine mix was added to the DNA mix and allowed to incubate approximately 30 minutes at room temperature.
• Four ml of SF media was added to the DNA:Lipofectamine mixture.
• the cells were rinsed once with 5 ml of SF media, aspirated, and the DNA:Lipofectamine mixture was added.
• the cells were incubated at 37° C. for five hours, and then 5 ml of DMEM/10% FBS media was added.
• the flask was incubated at 37° C. overnight after which time the cells were split into the selection media (DMEM/FBS media from above with the addition of 1 ⁇ M methotrexate (Sigma Chemical Co., St. Louis, Mo.) in 150 mm plates at 1:2, 1:10, and 1:50.
• DMEM/FBS media from above with the addition of 1 ⁇ M methotrexate (Sigma Chemical Co., St. Louis, Mo.) in 150 mm plates at 1:2, 1:10, and 1:50.
• Single clones expressing the soluble receptors can also isolated, screened and grown up in cell culture media, and purified using standard techniques. Moreover, CHO cells are also suitable cells for such purposes.
• Soluble zyctor19 receptor zyctor19CFLAG (Example 4 and Example 5), or gp130 (Hibi, M. et al., Cell 63:1149-1157, 1990) are biotinylated by reaction with a five-fold molar excess of sulfo-NHS-LC-Biotin (Pierce, Inc., Rockford, Ill.) according to the manufacturer's protocol.
• Soluble zyctor19 receptor and another soluble receptor subunit for example, soluble class II cytokine receptors, for example, interferon-gamma, alpha and beta chains and the interferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonly owned U.S.
• biotinylated and Ru-BPY-NHS-labeled forms of the soluble zyctor19 receptor can be respectively designated Bio-zyctor19 receptor and Ru-zyctor19; the biotinylated and Ru-BPY-NHS-labeled forms of the other soluble receptor subunit can be similarly designated.
• Assays can be carried out using conditioned media from cells expressing a ligand that binds zyctor19 heterodimeric receptors, or using purified ligands.
• Preferred ligands are zcyto20 (SEQ ID NO:52) , zcyto21 (SEQ ID NO:55), zcyto22 (SEQ ID NO:57), zcyto24 (SEQ ID NO:60), zcyto25 (SEQ ID NO:62), and ligands that can bind class II heterodimeric cytokine receptors such as, IL-10, IL-9, IL-TIF, interferons, TSLP (Levine, S D et al., ibid.; Isaksen, D E et al., ibid.; Ray, R J et al., ibid.; Friend, S L et al., ibid.), and the like.
• class II heterodimeric cytokine receptors such as, IL-10, IL-9, IL-TIF, interferons, TSLP (Levine, S D et al., ibid.; Isaksen, D E et al
• the cytokines mentioned above, or conditioned medium are tested to determine whether they can mediate homodimerization of zyctor19 receptor and if they can mediate the heterodimerization of zyctor19 receptor with the soluble receptor subunits described above.
• TBS-B conditioned media or TBS-B containing purified cytokine
• TBS-B 20 mM Tris, 150 mM NaCl, 1 mg/ml BSA, pH 7.2
• e.g., 400 ng/ml of Ru-zyctor19 receptor and Bio-zyctor19 e.g., 400 ng/ml of Ru-zyctor19 receptor and e.g., Bio-CRF2-4, or 400 ng/ml of e.g., Ru-CRF2-4 and Bio-zyctor19.
• a vector expressing a secreted human zyctor19 heterodimer is constructed.
• the extracellular cytokine-binding domain of zyctor19 is fused to the heavy chain of IgG gamma 1 (IgG ⁇ 1) (SEQ ID NO:14 and SEQ ID NO:15), while the extracellular portion of the heteromeric cytokine receptor subunit (E.g., class II cytokine receptors, for example, CRF2-4) is fused to a human kappa light chain (human ⁇ light chain) (SEQ ID NO:16 and SEQ ID NO:17).
• the heavy chain of IgG ⁇ 1 (SEQ ID NO:14) is cloned into the Zem229R mammalian expression vector (ATCC deposit No. 69447) such that any desired cytokine receptor extracellular domain having a 5′ EcoRI and 3′ NheI site can be cloned in resulting in an N-terminal extracellular domain-C-terminal IgG ⁇ 1 fusion.
• the IgG ⁇ 1 fragment used in this construct is made by using PCR to isolate the IgG ⁇ 1 sequence from a Clontech hFetal Liver cDNA library as a template.
• PCR products are purified using methods described herein and digested with MluI and EcoRI (Boerhinger-Mannheim), ethanol precipitated and ligated with oligos that comprise an MluI/EcoRI linker, into Zem229R previously digested with and EcoRI using standard molecular biology techniques disclosed herein.
• the human ⁇ light chain (SEQ ID NO:16) is cloned in the Zem228R mammalian expression vector (ATCC deposit No. 69446) such that any desired cytokine receptor extracellular domain having a 5′ EcoRI site and a 3′ KpnI site can be cloned in resulting in a N-terminal cytokine extracellular domain-C-terminal human ⁇ light chain fusion.
• a special primer is designed to clone the 3′ end of the desired extracellular domain of a cytokine receptor into this KpnI site:
• the primer is designed so that the resulting PCR product contains the desired cytokine receptor extracellular domain with a segment of the human ⁇ light chain up to the KpnI site (SEQ ID NO:16).
• This primer preferably comprises a portion of at least 10 nucleotides of the 3′ end of the desired cytokine receptor extracellular domain fused in frame 5′ to SEQ ID NO:16.
• the human ⁇ light chain fragment used in this construct is made by using PCR to isolate the human ⁇ light chain sequence from the same Clontech human Fetal Liver cDNA library used above. PCR products are purified using methods described herein and digested with MluI and EcoRI (Boerhinger-Mannheim), ethanol precipitated and ligated with the MluI/EcoRI linker described above, into Zem228R previously digested with and EcoRI using standard molecular biology techniques disclosed herein.
• a construct having zyctor19 fused to IgG ⁇ 1 is made. This construction is done by PCRing the extracellular domain or cytokine-binding domain of zyctor19 receptor described herein from a prostate cDNA library (Clontech) or activated lymphocyte cDNA library using standard methods, and oligos that provide EcoRI and NheI restriction sites. The resulting PCR product is digested with EcoRI and NheI, gel purified, as described herein, and ligated into a previously EcoRI and NheI digested and band-purified Zem229R/IgG ⁇ 1 described above. The resulting vector is sequenced to confirm that the zyctor19/IgG gamma 1 fusion (zyctor19/Ch1 IgG) is correct.
• a separate construct having a heterodimeric cytokine receptor subunit extracellular domain, i.e., CRF2-4 (SEQ ID NO: 64) fused to ⁇ light is also constructed as above.
• the cytokine receptor/human ⁇ light chain construction is performed as above by PCRing from, e.g., a lymphocyte cDNA library (Clontech) using standard methods, and oligos that provide EcoRI and KpnI restriction sites.
• the resulting PCR product is digested with EcoRI and KpnI and then ligating this product into a previously EcoRI and KpnI digested and band-purified Zem228R/human ⁇ light chain vector described above.
• the resulting vector is sequenced to confirm that the cytokine receptor subunit/human ⁇ light chain fusion is correct.
• the transfected cells are selected for 10 days in DMEM+5% FBS (Gibco/BRL) containing 1 ⁇ M of methotrexate (MTX) (Sigma, St. Louis, Mo.) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days.
• the resulting pool of transfectants is selected again in 10 ⁇ m of MTX and 0.5 mg/ml G418 for 10 days.
• the resulting pool of doubly selected cells is used to generate protein.
• Three Factories (Nunc, Denmark) of this pool are used to generate 10 L of serum free conditioned medium. This conditioned media is passed over a 1 ml protein-A column and eluted in about 10, 750 microliter fractions. The fractions having the highest protein concentration are pooled and dialyzed (10 kD MW cutoff) against PBS. Finally the dialyzed material is submitted for amino acid analysis (AAA) using routine methods.
• AAA amino acid analysis
• BHK 570 cells (ATCC No. CRL-10314) transfected, using standard methods described herein, with a luciferase reporter mammalian expression vector plasmid serve as a bioassay cell line to measure signal transduction response from a transfected zyctor19 receptor complex to the luciferase reporter in the presence of zyctor19 Ligand.
• BHK cells would be used in the event that BHK cells do not endogenously express the zyctor19 receptor. Other cell lines can be used.
• An exemplary luciferase reporter mammalian expression vector is the KZ134 plasmid which is constructed with complementary oligonucleotides that contain STAT transcription factor binding elements from 4 genes.
• a modified c-fos Sis inducible element m67SIE, or hSIE
• m67SIE or hSIE
• hSIE c-fos Sis inducible element
• the p21 SIE1 from the p21 WAF1 gene Choin, Y. et al., Science 272:719-722, 1996)
• the mammary gland response element of the ⁇ -casein gene Schomitt-Ney, M. et al., Mol. Cell. Biol.
• oligonucleotides contain Asp718-XhoI compatible ends and are ligated, using standard methods, into a recipient firefly luciferase reporter vector with a c-Fos promoter (Poulsen, L. K. et al., J. Biol. Chem. 273:6229-6232, 1998) digested with the same enzymes and containing a neomycin selectable marker.
• the KZ134 plasmid is used to stably transfect BHK, or BaF3 cells, using standard transfection and selection methods, to make a BHK/KZ134 or BaF3/KZ134 cell line respectively.
• the bioassay cell line is transfected with zyctor19 receptor alone, or co-transfected with zyctor19 receptor along with one of a variety of other known receptor subunits.
• Receptor complexes include but are not limited to zyctor19 receptor only, various combinations of zyctor19 receptor with class II cytokine receptors, for example, interferon-gamma, alpha and beta chains and the interferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511) receptors.
• Each independent receptor complex cell line is then assayed in the presence of cytokine-conditioned media or purified cytokines and luciferase activity measured using routine methods.
• the untransfected bioassay cell line serves as a control for the background luciferase activity, and is thus used as a baseline to compare signaling by the various receptor complex combinations.
• the conditioned medium or cytokine that binds the zyctor19 receptor in the presence of the correct receptor complex is expected to give a luciferase readout of approximately 5 fold over background or greater.
• a similar assay can be performed wherein the a Baf3/zyctor19 cell line is co-transfected as described herein and proliferation is measured, using a known assay such as a standard Alamar Blue proliferation assay.
• Biotinylated zcyto21 (SEQ ID NO:55) was tested for binding to known or orphan cytokine receptors.
• the pZP7 expression vectors containing cDNAs of cytokine receptors (including human IFN ⁇ R1, IFN ⁇ R1, IFN ⁇ R2, IFN ⁇ R2, IL-10R, CRF2-4, ZcytoR7, DIRS1, Zyctor19, and Tissue Factor) were transfected into COS cells, and the binding of biotinylated zcyto20 to transfected COS cells was carried out using the secretion trap assay described below. Positive binding in this assay showed receptor-ligand pairs.
• COS cell transfections were performed as follows: COS cells were plated (1 ⁇ 10 5 cells/well) on fibronectin coated, 12-well, tissue culture plates (Becton Dickinson, Bedford, Mass.) and incubated at 37° C. overnight.
• Cytokine receptor DNA (0.75 ⁇ g) was mixed with 50 ⁇ l serum free DMEM media (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5 mg insulin, 1 ⁇ g selenium and 5 mg fetuin in 500 ml DMEM), then mixed with 5 ⁇ l LipofectamineTM (Invitrogen, Carlsbad, Calif.) in 45 ⁇ l serum free DMEM media, and incubated at room temperature for 30 minutes. An additional 400 ⁇ l serum free DMEM media was added. The cells were rinsed with serum free DMEM, and 500 ⁇ l of the DNA mixture was added.
• serum free DMEM media 55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5 mg insulin, 1 ⁇ g selenium and 5 mg fetuin in 500 ml DMEM
• LipofectamineTM Invitrogen, Carlsbad, Calif.
• the cells were incubated for 5 hours at 37° C., at which time an additional 500 ⁇ l 20% FBS DMEM media (100 ml FBS, 55 mg sodium pyruvate and 146 mg L-glutamine in 500 ml DMEM) was added and the cells were incubated overnight.
• FBS DMEM media 100 ml FBS, 55 mg sodium pyruvate and 146 mg L-glutamine in 500 ml DMEM
• the secretion trap was performed as follows: Media was aspirated and cells were rinsed twice with 1% BSA in PBS. Cells were blocked for 1 hour with TNB (0.1M Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NEN Renaissance TSA-Direct Kit, NEN Life Science Products, Boston, Mass.) in H 2 O. The cells were incubated for 1 hour with 3 ⁇ g/ml biotinylated zcyto21 protein (Example 27) in TNB. Cells were then washed 3 times with 1% BSA in PBS and were incubated for another hour with 1:300 diluted Streptavidin-HRP (NEN kit) in TNB.
• TNB 0.1M Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NEN Renaissance TSA-Direct Kit, NEN Life Science Products, Boston, Mass.) in H 2 O. The cells were incubated for 1 hour with 3 ⁇ g/ml biotinylated zcyto21
• An expression plasmid containing a polynucleotide encoding part of the human zyctor19 fused N-terminally to maltose binding protein (MBP) was constructed via homologous recombination.
• a fragment of human zyctor19 cDNA was isolated using PCR.
• Two primers were used in the production of the human zyctor19 fragment in a PCR reaction: (1) Primer ZC39204 (SEQ ID NO:30), containing 40 bp of the vector flanking sequence and 24 bp corresponding to the amino terminus of the human zyctor19, and (2) primer ZC39205 (SEQ ID NO:31), containing 40 bp of the 3′ end corresponding to the flanking vector sequence and 24 bp corresponding to the carboxyl terminus of the human zyctor19.
• the PCR reaction conditions were as follows: 1 cycle of 94 C. for 1 minute. Then 20 cycles of 94° C. for 30 seconds, 60° C. for 30 seconds, and 68° C. for 1.5 minutes; followed by 4° C. soak, run in duplicate.
• Plasmid pTAP170 was derived from the plasmids pRS316 and pMAL-c2.
• the 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 driving MalE (gene encoding MBP) followed by a His tag, a thrombin cleavage site, a cloning site, and the rrnB terminator.
• the vector pTAP170 was constructed using yeast homologous recombination.
• yeast/DNA mixture was electropulsed at 0.75 kV (5 kV/cm), infinite ohms, 25 ⁇ F.
• To each cuvette was added 600 ⁇ l of 1.2 M sorbitol. The yeast was then plated in two 300 ⁇ l aliquots onto two-URA D plates and incubated at 30° C.
• the Ura+yeast transformants from a single plate were resuspended in 1 ml H 2 O and spun briefly to pellet the yeast cells.
• the cell pellet 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).
• 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 ⁇ l acid washed glass beads and 500 ⁇ l phenol-chloroform, vortexed for 1 minute intervals two or three times, followed by a 5 minute spin in a Eppendorf centrifuge at maximum speed.
• Transformation of electrocompetent E. coli cells was done with 1 ⁇ l yeast DNA prep and 40 ⁇ l of MC1061 cells.
• the cells were electropulsed at 2.0 kV, 25 ⁇ F and 400 ohms.
• 0.6 ml SOC 2% BactoÎ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose
• LB Kan plates LB broth (Lennox), 1.8% BactoTM Agar (Difco), 30 mg/L kanamycin).
• a positive clone was used to inoculate an overnight starter culture of Superbroth II (Becton Dickinson) with 30 ⁇ g/ml of kanamycin.
• the starter culture was used to inoculate 4 2 L-baffled flasks each filled with 500 ml of Superbroth II+Kan. Cultures shook at 37° C. at 250 rpm until the OD 600 reached 4.1. At this point, the cultures were induced with 1 mMIPTG. Cultures grew for two more hours at 37° C., 250 rpm at which point 2 ml was saved for analysis and the rest was harvested via centrifugation. Pellet was saved at ⁇ 80° C. until transferred to protein purification.
• the conditioned media was concentrated first 20 times by using an Amicon/Millipore Spiral cartridge, 10 kD MWCO. (at ambient temperature) The concentrated media was then applied to an appropriately sized POROS 50 A (coupled protein A) column at an optimal capture flow rate. The column was washed with 10 column volumes (CV) of equilibration buffer, then rapidly eluted with 3 CV of 0.1 M Glycine pH 3. The collected fractions had a predetermined volume of 2M TRIS pH 8.0 added prior to the elution to neutralize the pH to about 7.2.
• POROS 50 A coupled protein A
• Gene expression of zyctor19 was examined using commercially available normalized multiple tissue first-strand cDNA panels (OriGene Technologies, Inc. Rockville, Md.; BD Biosciences Clontech, Palo Alto, Calif.). These included OriGene's Human Tissue Rapid-ScanTM Panel (containing 24 different tissues) and the following BD Biosciences Clontech Multiple Tissue cDNA (MTCTM) Panels: Human MTC Panel I (containing 8 different adult tissues), Human MTC Panel II (containing 8 different adult tissues), Human Fetal MTC Panel (containing 8 different fetal tissues), Human Tumor MTC Panel (containing carcinomas from 7 different organs), Human Blood Fractions MTC Panel (containing 9 different blood fractions), and Human Immune System MTC Panel (containing 6 different organs and peripheral blood leukocyte).
• OriGene Technologies, Inc. Rockville, Md.; BD Biosciences Clontech, Palo Alto, Calif. included OriGene's Human Tissue Rapid-ScanTM Panel (containing 24 different tissues) and the
• PCR reactions were set up using zyctor19 specific oligo primers ZC40285 (SEQ ID NO:35) and ZC40286 (SEQ ID NO:36) which yield a 426 bp product, Qiagen HotStarTaq DNA Polymerase (Qiagen, Inc., Valencia, Calif.) and RediLoadTM dye (Research Genetics, Inc., Huntville, Ala.).
• the PCR cycler conditions were as follows: an initial 1 cycle 15 minute denaturation at 95° C., 35 cycles of a 45 second denaturation at 95° C., 1 minute annealing at 63° C.
• a DNA fragment of the correct size was observed in the following human adult tissues: adrenal gland, bone marrow, colon, heart, liver, lung, lymph node, muscle, ovary, pancreas, placenta, prostate, salivary gland, small intestine, spleen, stomach, testis, thyroid, and tonsil.
• a DNA fragment of the correct size was observed in the following human fetal tissues: heart, liver, lung, kidney, skeletal muscle, spleen, and thymus.
• a DNA fragment of the correct size was observed in the following human blood fractions: peripheral blood leukocyte, mononuclear cells (B-cells, T-cells, and monocytes), resting CD8+ cells (T-suppressor/cytotoxic), resting CD19+ cells (B-cells), activated CD19+ cells, activated mononuclear cells, and activated CD4+ cells.
• a DNA fragment of the correct size was observed in the following tumor tissues: breast carcinoma, colon adenocarcinoma, lung carcinoma, ovarian carcinoma, pancreatic adenocarcinoma, and prostatic adenocarcinoma.
• zyctor19 is expressed in these specific tumor tissues, zyctor19 polynucleotides, polypeptides and antibodies can be used as a tumor marker as disclosed herein. Moreove, an antibody to zyctor19 could have anti-tumor activity, as well as toxin-conjugates, cytokine conjugates or other conjugates of an antibody, or the zyctor19 receptor ligand itself.
• the antagonist of zyctor19 ligand, such as anti-zyctor19 antibodies or soluble receptors can also act as anti-tumor reagents.
• BAC clone positive for mouse zyctor19 gene was identified using Incyte Genomic's (St. Louis, Miss.) Easy-to-Screen DNA Pools, BAC Mouse ES (Release I) following Manufacturer's instructions. Oligonucleotides were designed to generate a PCR fragment containing partial exon 6, complete intron 6 and partial exon 7 sequences.
• PCR reactions were carried out in 25 ⁇ l using 1.75 units of Advantage 2 polymerase (Clontech). Either 2 ⁇ l or 10 ⁇ l of BAC library DNA was used as template in buffer containing 67 mM Tris pH 8.8, 16.6 mM (NH 4 ) 2 SO 4 , 6.7 mM MgCl 2
• PCR conditions were as follows 95° C. for 1 min,; 30 cycles of 95° C. for 15 seconds, 55° C. for 30 seconds, and 68 ° C. for 30 seconds; and 68° C. for 2 minutes; followed by a 4° C. hold. PCR products were analyzed by agarose gel electrophoresis. Positive PCR products were found to be 1,149 bp.
• BAC clones positive for mouse zyctor19 gene were identified using Incyte's BAC Mouse Filter Set (Release II) following Manufacturer's instructions. Oligonucleotides were designed to generate a PCR fragment containing partial exon 6, and partial exon 7 sequences from mouse cDNA template.
• PCR reactions were carried out in 25 ⁇ l using 1.75 units of Advantage 2 polymerase (Clontech). 2 ⁇ l of Neonatal Mouse skin CDNA library (JAK 062700B) was used as template in buffer containing 67 mM Tris pH 8.8, 16.6 mM (NH 4 ) 2 SO 4 , 6.7 mM MgCl 2
• the labeled probe was used to screen Incyte's 7 filter BAC library set. Hybridizations were carried out at 55° C. overnight using ExpressHyb (Clontech). Filters were then washed 3 times for 30 minutes at 50° C. with 0.1 ⁇ SSC, 0.1%SDS, autoradiographed overnight and compared to manufacturer's grid patterns to identify positive clones.
• BAC clones Five zyctor19 mouse positive BAC clones from 129/SvJ Embryonic Stem Cell libraries (Release I and II) were obtained from Incyte Genomics. BAC clones were grown within Escherichia coli host strain DH10B in liquid media and extracted using BAC large plasmid purification kit MKB-500 (Incyte Genomics) according to manufacturer's instructions. 4 of 5 BACs were found to contain at least 2,000 bp of 5′ untranslated region, exon1, and exon 5 as determined by PCR.
• PCR reactions were carried out in 25 ⁇ l using 1.75 units of Advantage 2 polymerase (Clontech) in buffer containing 67 mM Tris pH 8.8, 16.6 mM (NH 4 ) 2 SO 4 , 6.7 mM MgCl 2 , 5 mM 2-Mercaptoethanol, 100 ⁇ g/ml gelatin, 10% Dimethyl Sulfoxide, 1 mM deoxynucleotides, 140 nM forward and 140 nM reverse primer.
• PCR conditions were as follows 95° C. for 1 min,; 30 cycles of 95° C. for 15 seconds, 55° C. for 30 seconds, and 68° C. for 30 seconds; and 68° C.
• Oligonucleotides were designed to generate a PCR fragment containing partial exon 6, complete intron 6 and partial exon 7 sequences.
• PCR reactions were carried out in 25 ⁇ l using 1.75 units of Advantage 2 polymerase (Clontech). 100 ng of 129/Sv mouse genomic DNA was used as template in buffer containing 67 mM Tris pH 8.8, 16.6 mM (NH 4 ) 2 SO 4 , 6.7 mM MgCl 2 , 5 mM 2-Mercaptoethanol, 100 ⁇ g/ml gelatin, 10% Dimethyl Sulfoxide, 1 mM deoxynucleotides, 140 nM forward primer ZC39128 (SEQ ID NO:37) and 140 nM reverse primer ZC39129 (SEQ ID NO:38). PCR conditions were as described above.
• PCR products were analyzed by agarose gel electrophoresis and found to be 1,149 bp. PCR products were then purified using Qiaquick (Qiagen) PCR purification kit. Determination of intron 6 sequence was made by sequence analysis using oligos ZC39128 (SEQ ID NO:37) and ZC 39129 (SEQ ID NO:38).
• Oligonucleotides were designed to generate a PCR fragment containing partial exon5, complete intron5 and partial exon6. PCR reactions were carried out in 25 ⁇ l using 1.75 units of Advantage 2 polymerase (Clontech). 100 ng of 129/Sv mouse genomic DNA was used as template in buffer containing 67 mM Tris pH 8.8, 16.6 mM (NH 4 ) 2 SO 4 , 6.7 mM MgCl 2 , 5 mM 2-Mercaptoethanol, 100 ⁇ g/ml gelatin, 10% Dimethyl Sulfoxide, 1 mM deoxynucleotides, 140 nM forward primer ZC39408 (SEQ ID NO:43) and 140 nM reverse primer ZC39409 (SEQ ID NO:44).
• PCR conditions were as follows 95° C. for 1 min,; 30 cycles of 95° C. for 15 seconds, 55° C. for 30 seconds, and 68° C. for 30 seconds; and 68° C. for 2 minutes; followed by a 4° C. hold.
• PCR products were analyzed by agarose gel electrophoresis and found to be 356 bp. PCR products were then purified using Qiaquick (Qiagen) PCR purification kit. Determination of intron 6 sequence was made by sequence analysis using oligos ZC39408 (SEQ ID NO:43) and ZC 39409 (SEQ ID NO:44).
• zytor19 gene To investigate biological function of zytor19 gene, a knockout mouse model is being generated by homologous recombination technology in embryonic stem (ES) cells.
• ES embryonic stem
• the coding exon 1, 2 and 3 are deleted to create a null mutation of the zyctor19 gene. This deletion removes the translation initiation codon, the signal domain and part of the extracellular domain of the zyctor19 protein, thus inactivating the zyctor19 gene.
• Kanomycin resistance cassette is used to replace introns1, 2 and 3 of zyctor19 mouse gene.
• a forward knockout oligonucleotide (SEQ ID NO:45) was designed to be 121 nucleotides in length, having 52 bp of homology to the 5′UTR of zyctor19m a 42 bp linker having SfiI, FseI, BamHI and HindIII restriction sites and 27 bp of homology to the 5′ end of the Kanomycin resistance cassette.
• a reverse knockout oligonucleotide (SEQ ID NO:46) was designed to be 125 nucleotides in length, having 50 bp of homology to intron 3 of zyctor19 mouse, a 48 bp linker having SfiI, AscI, BamHII and HindIII restriction sites and 27 bp of homology to the 3′ end of the Kanomycin resistance cassette.
• the above oligonucleotides can be used to synthesize a PCR fragment 1073 bp in length containing the entire Kanomycin resistance cassette with the first 52 bp having homology to the 5′ UTR of zyctor19 mouse and the last 50 bp having homology to intron 3.
• the fragment will then be used to construct a Knockout vector through ET Cloning, in which cytor19 mouse positive BAC cell hosts are made competent through treatment with glycerol then transfected with the plasmid pBADalpha/beta/gamma(Amp). Resistance to chloramphenical and ampicillin selects for transformed cell. Cells are then re-transformed with the Kanomycin PCR fragment containing homology arms. The Beta and gamma recombination proteins of pBADalpha/beta/gamma(Amp) are induced by the addition of arabinose to the growth media through the activation of the Red alpha gene.
• Recombinant BACs are selected for by resistance to kanomycin and ampicillin then screened by PCR. Once a recombinant BAC is identified a fragment is subcloned containing at least 1,800 bp of sequence upstream of kanomycin resistance cassette insertion and at least 6,000 bp of sequence downstream into a pGEM7 derived vector. The Kanomycin resistance cassette is then replaced by standard ligation cloning with a IRES/LacZ/Neo-MC1 cassette.
• the IRES is an internal ribosome entry sequence derived from encephalomyocarditis virus. It is fused in-frame to the reporter lacZ gene, linked to a polyA signal.
• MC1 promoter Downstream of the IRES/LacZ reporter gene, MC1 promoter drives the expression of a G418 resistance selectable marker Neo gene.
• the selectable maker cassette contains termination codons in all three reading frames.
• the drug resistance gene Neo is used for selection of homologous recombination events in embryonic stem (ES) cells.
• IRES/LacZ reporter gene will be used to monitor the expression of the replaced gene after homologous recombination Homologous recombination of the knockout vector and the target locus in ES cells leads to the replacement of a total 17,980 bp, including complete exons 1, 2 and 3, of the wild type locus with the IRES/LacZ/Neo-MC1 cassette, which is about 5,200 bp in length.
• the KO vector described above, is linearized by PmeI digestion, and electroporated into ES cells. Homologous recombination events are identified by PCR screening strategy, and confirmed by Southern Blot Analysis, using a standard KO protocol. See, A. L. Joyner, Gene Targeting. A Practical Approach. IRL Press 1993.
• ES cells will be expanded, and injected into blastocysts to generate chimeras. Chimeric males will be used to breed to C57black females to achieve germ line transmission of the null mutation, according to standard procedures. See Hogan, B. et al., Manipulating the Mouse Embryo. A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1994.
• Heterozygous KO animals will be bred to test biological functions of the zyctor19 gene. Of offspring produced, 1 ⁇ 4 should be wild type, 1 ⁇ 2 should be heterozygous, and 1 ⁇ 4 should be homozygous. Homozygous will be analyzed in details as described below.
• zyctor19 is expressed in following tissues, we will examine these tissues carefully: colon, ovary placenta, pituitary, lymph node, small intestine, salivary gland, rectum, prostate, testis, brain, lung, kidney, thyroid, spinal cord, bone marrow, and cervix.
• Spleen, thymus, and mesenteric lymph nodes are collected and prepared for histologic examination from transgenic animals expressing zyctor19.
• Other tissues which are routinely harvested included the following: Liver, heart, lung, spleen, thymus, mesenteric lymph nodes, kidney, skin, mammary gland, pancreas, stomach, small and large intestine, brain, salivary gland, trachea, esophagus, adrenal, pituitary, reproductive tract, accessory male sex glands, skeletal muscle including peripheral nerve, and femur with bone marrow.
• the tissues are harvested from homozygous animals as well as wild type controls.
• Samples are fixed in 10% buffered formalin, routinely processed, embedded in paraffin, sectioned at 5 microns, and stained with hematoxylin and eosin. The slides are examined for histological, and pathological changes, such as inflammatory reactions, and hypo-proliferation of certain cell types.
• Homozygous animals missing zyctor19 gene are to be sacrificed for flow cytometric analysis of peripheral blood, thymus, lymph node, bone marrow, and spleen.
• Peripheral blood (200 ml) is collected in heparinized tubes and diluted to 10 mls with HBSS containing 10 U Heparin/ml. Erythrocytes are removed from spleen and peripheral blood preparations by hypotonic lysis. Bone marrow cell suspensions are made by flushing marrow from femurs with ice-cold culture media. Cells are counted and tested for viability using Trypan Blue (GIBCO BRL, Gaithersburg, Md.). Cells are resuspended in ice cold staining media (HBSS, 1% fetal bovine serum, 0.1% sodium azide) at a concentration of ten million per milliliter. Blocking of Fc receptor and non-specific binding of antibodies to the cells was achieved by adding 10% normal goat sera and Fc Block (PharMingen, La Jolla, Calif.) to the cell suspension.
• HBSS 1% fetal bovine serum, 0.1% sodium azide
• the cell populations in all lymphoid organs will be analyzed to detect abnormalities in specific lineages of T cell, B cell, or other lymphocytes, and cellularity in these organs.
• One in situ probe was designed against the human zyctor19 (variant ⁇ 1) sequence (INC7128744, as shown in SEQ ID NO: 25), containing the 3′UTR of zyctor19 using standard methods. T7 RNA polymerase was used to generate an antisense probe. The probe was labeled using an In Vitro transcription System (Riboprobe® in vitro Transcription System, Promega, Madison, Wis.) as per manufacturer's instruction, except that the probes digoxigenin was used instead of radiolabeled rCTP and that the water was adjusted to accomodate the reduced volume of the rNTP's. In situ hybridization was performed with a digoxigenin-labeled zyctor19 probe (above).
• the probe was added to the slides at a concentration of 1 to 5 pmol/ml for 12 to 16 hours at 60° C. Slides were subsequently washed in 2 ⁇ SSC and 0.1 ⁇ SSC at 55° C. The signals were amplified using TSATM (Tyramide Signal Amplification; PerkinElmer Life Sciences Inc., Boston, Mass.) and visualized with VECTOR Red substrate kit (Vector Laboratories, Burlingame, Calif.) as per manufacturer's instructions. The slides were then counter-stained with hematoxylin.
• the cancerous granular epithelium is strongly positive, while no positive signal is observed in the normal skin.
• signal is observed in a mixed population of mononuclear cells in sinusoid spaces.
• zyctor19 appears to be positive in type II alveolar epithelium. Occasionally bronchial epithelium may also be weakly positive. Macrophage-like mononuclear cells in the interstitial tissue are also positive.
• myocytes are negative while some circulating mononuclear cells are positive for zyctor19.
• endothelium of the vessels may be weakly positive.
• Other tissues tested including a MFH (muscle sarcoma) sample and a Kaposi's sarcoma skin sample. There is no conclusive positive signal in these tissues.
• Plasmid DNA 100933 was digested with restriction enzyme HindIII, which covers 0.7 kb from the end of 3′UTR.
• the T-7 RNA polymerase was used to generate an antisense probe.
• the probe was labeled with digoxigenin (Boehringer) using an In Vitro transcription System (Promega, Madison, Wis.) as per manufacturer's instruction.
• In situ hybridization was performed with a digoxigenin- or biotin-labeled zyctor19 probe (above).
• the probe was added to the slides at a concentration of 1 to 5 pmol/ml for 12 to 16 hours at 60° C.
• Slides were subsequently washed in 2 ⁇ SSC and 0.1 ⁇ SSC at 55° C.
• the signals were amplified using tyramide signal amplification (TSA) (TSA, in situ indirect kit; NEN) and visualized with Vector Red substrate kit (Vector Lab) as per manufacturer's instructions.
• TSA tyramide signal amplification
• Vector Lab Vector Red substrate kit
• carcinoma epithelial cells were positive. There were also some signals in a subset of lymphocytes in the lymphoid follicles. Similarly, both carcinoma and some immune cells were positive in the colon carcinoma samples, while normal colon samples were negative. Weak staining was also in the endometrial carcinoma and ovarian carcinoma, while normal ovary and uterus were negative. There was weak staining in the cancer area of the muscle sarcoma sample. Keratinocytes were positive in the skin carcinoma and Kaposi's sarcoma samples, while no staining was observed in the normal skin. In heart and liver, a subset of cells possibly circulating WBC, were positive for zyctor19.
• zyctor19 appears to be up-regulated in carcinoma cells. There is low level of zyctor19 mRNA in a subset of lymphocytes and endothelial cells.
• zyctor19 is expressed in these specific tumor tissues, zyctor19 polynucleotides, polypeptides and antibodies can be used as a tumor marker as disclosed herein. Moreove, an antibody to zyctor19 could have anti-tumor activity, as well as toxin-conjugates, cytokine conjugates or other conjugates of an antibody, or the zyctor19 receptor ligand itself.
• the antagonist of zyctor19 ligand, such as anti-zyctor19 antibodies or soluble receptors can also act as anti-tumor reagents.
• BaF3 Cells Expressing the zyctor19 Receptor (BaF3 Zcytor19 Cells) with Puromycin Resistant and Zeomycin Resistant Vectors.
• BaF3 cells expressing the full-length zyctor19 receptor were constructed using 30 ⁇ g of zyctor19 expression vectors, one resistant to puromycin, one resistant to zeomycin described below.
• the BaF3 cells expressing the zyctor19 receptor mRNA with puromycin resistance were designated as BaF3/zyctor19-p.
• the BaF3 cells expressing the zyctor19 receptor mRNA with zeomycin resistance were designated as BaF3/zyctor19-z
• IL-3 interleukin-3
• pZP-5N/CRF2-4 was prepared and purified using a Qiagen Maxi Prep kit (Qiagen) as per manufacturer's instructions.
• BaF3 cells for electroporation were washed twice in PBS (Gibco BRL) and then resuspended in RPMI media at a cell density of 10 7 cells/ml.
• PBS Gibco BRL
• resuspended BaF3 cells was mixed with 30 ⁇ g of the pZP-7p/zyctor19 plasmid DNA, or 30 ⁇ g of the pZP-7z/zyctor19 plasmid DNA, and transferred to separate disposable electroporation chambers (GIBCO BRL).
• the cells were given two serial shocks (800 lFad/300 V.; 1180 lFad/300 V.) delivered by an electroporation apparatus (CELL-PORATORTM; GIBCO BRL), with a 1 minute rest between the shocks. After a 5 minute recovery time, the electroporated cells were transferred to 50 ml of complete media and placed in an incubator for 15-24 hours (37° C., 5% CO 2 ).
• the cells were then spun down and resuspended in 50 ml of complete media containing Puromycin (Clonetech) selection (2 ⁇ g/ml) for the cells transfected with pZP-7p/zyctor19, or Zeocin selection (1:150-1:333) for the cells transfected with pZP-7z/zyctor19, and placed in a T-162 flask to isolate the antibiotic-resistant pools. Pools of the transfected BaF3 cells, hereinafter called BaF3/zyctor19-puro and BaF3/zyctor19-zeo cells, were assayed for expression of zyctor19 by RT-PCR.
• the BaF3/zyctor19-puro and BaF3/zyctor19-zeo cells were harvested for RNA, which was then put into a reverse transcriptase reaction, and subsequently tested by PCR for the presence of zyctor19.
• PCR was then done by mixing 0.2 pmol each of primers ZC40279 and ZC37863, 0.2 mM of dNTP mix (Roche) containing equal amounts of each nucleotide, 5 ⁇ 1 of 10 ⁇ cDNA PCR Reaction Buffer (Clonetech), 3 ⁇ 1 DNA from the RT reaction, 0.5 ⁇ 1 Advantage2 Polymerase (Clonetech), made to a final volume of 50 ⁇ 1 with water.
• the reaction ran for 95° C., 5 min, then 30 cycles of 95° C. 30 sec, 60° C. 30 sec, 72° C. 1 min, then 72° C. 7 min and a 4° C., on a Perkin Elmer GeneAmp PCR System 2400.
• the samples were mixed with 3 ml loading dye, and 25 ml was run on a 1% OmniPur Agarose (Merck) gel. Zyctor19 bands were detected on the gel for both BaF3/zyctor19-puro and BaF3/zyctor19-zeo, indicating that those cells are expressing the gene.
• Polyclonal antibodies are prepared by immunizing 2 female New Zealand white rabbits with the purified recombinant protein huzyctor19/MBP-6H.
• the rabbits are each given an initial intraperitoneal (ip) injection of 200 ⁇ g of purified protein in Complete Freund's Adjuvant followed by booster ip injections of 100 ⁇ g peptide in Incomplete Freund's Adjuvant every three weeks.
• ip intraperitoneal
• the huzcyotr19/MBP-6H specific rabbit serum is pre-adsorbed of anti-MBP antibodies using a CNBr-SEPHAROSE 4B protein column (Pharmacia LKB, Peapack, N.J.) that is prepared using 10 mg of purified recombinant MBP per gram of CNBr-SEPHAROSE.
• the huzyctor19-specific polyclonal antibodies are affinity purified from the rabbit serum using a CNBr-SEPHAROSE 4B protein column that is prepared using 10 mg of the specific antigen purified recombinant protein huzyctor19/MBP-6H followed by 20 ⁇ dialysis in PBS overnight.
• Huzyctor19-specific antibodies are characterized by ELISA using 500 ng/ml of the purified recombinant proteins huzyctor19/MBP-6H or huzyctor19-Fc4 as antibody targets.
• the lower limit of detection (LLD) of the rabbit anti-huzyctor19/MBP-6H affinity purified antibody on its specific purified recombinant antigen huzyctor19/MBP-6H and on purified recombinant huzyctor19-Fc4 is determined.
• a signal transduction reporter assay can be used to determine the functional interaction of zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25 with zyctor19.
• Human embryonal kidney (HEK) cells are transfected with a reporter plasmid containing an interferon-stimulated response element (ISRE) driving transcription of a luciferase reporter gene in the presence or absence of pZP7 expression vectors containing cDNAs for class II cytokine receptors (including human DIRS1, IFNaR1, IFNaR2 and Zyctor19 (SEQ ID NO:23)).
• ISRE interferon-stimulated response element
• Luciferase activity following stimulation of transfected cells with class II ligands (including zcyto20 (SEQ ID NO:52), zcyto21 (SEQ ID NO:55), zcyto22 (SEQ ID NO:57), zcyto10, huIL10 and huIFNa-2a) reflects the interaction of the ligand with transfected and native cytokine receptors on the cell surface. The results and methods are described below.
• 293 HEK cells were transfected as follows: 700,000 293 cells/well (6 well plates) were plated approximately 18 h prior to transfection in 2 milliliters DMEM+10% fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA (Stratagene), 1 microgram cytokine receptor DNA and 1 microgram pIRES2-EGFP DNA (Clontech,) were added to 9 microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. Two micrograms pIRES2-EGFP DNA was used when cytokine receptor DNA was not included. This transfection mix was added 30 minutes later to the pre-plated 293 cells.
• transfected cells Twenty-four hours later the transfected cells were removed from the plate using trypsin-EDTA and replated at approximately 25,000 cells/well in 96 well microtiter plates. Approximately 18 h prior to ligand stimulation, media was changed to DMEM+0.5% FBS.
• the signal transduction reporter assays were done as follows: Following an 18 h incubation at 37° C. in DMEM+0.5%FBS, transfected cells were stimulated with dilutions (in DMEM+0.5% FBS) of the following class II ligands; zcyto20, zcyto21, zcyto22, zcyto10, huIL10 and huIFNa-2a. Following a 4-hour incubation at 37° C., the cells were lysed, and the relative light units (RLU) were measured on a luminometer after addition of a luciferase substrate.
• RLU relative light units
• Table 14 shows that zcyto20, zcyto21 and zcyto22 induce ISRE signaling in 293 cells transfected with ISRE-luciferase giving a 15 to 17-fold induction in luciferase activity over medium alone.
• the addition of zyctor19 DNA to the transfection mix results in a 6 to 8-fold further induction in ISRE signaling by zcyto20, zcyto21 and zcyto22 giving a 104 to 125-fold total induction. None of the other transfected class II cytokine receptor DNAs resulted in increased ISRE signaling.
• ISRE Interferon Stimulated Response Element
• a signal transduction reporter assay was used to determine the functional interaction of zcyto20, zcyto21, and zcyto22 with zyctor19 and IL10Rb (CRF2-4).
• Human embryonal kidney (HEK) cells or human embryonal kidney (HEK) cells stably overexpressing human zcytoR19 were transfected with a reporter plasmid containing an interferon-stimulated response element (ISRE) driving transcription of a luciferase reporter.
• ISRE interferon-stimulated response element
• Luciferase activity following stimulation of transfected cells with class II ligands (including zcyto20, zcyto21, zcyto22 and huIFNa-2a) in the presence or absence of a neutralizing antibody to IL10Rb (CRF2-4) reflects the interaction of the ligand with cytokine receptors on the cell surface.
• class II ligands including zcyto20, zcyto21, zcyto22 and huIFNa-2a
• CRF2-4 neutralizing antibody to IL10Rb
• 293 HEK cells stably overexpressing human zcytoR19 293 cells were transfected as follows: 300,000 293 cells/well (6 well plates) were plated approximately 6 h prior to transfection in 2 milliliters DMEM+10% fetal bovine serum. Per well, 2 micrograms of a pZP7 expression vector containing the cDNA of human zcytoR19 (SEQ ID NO:23) was added to 6 microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. This transfection mix was added 30 minutes later to the pre-plated 293 cells. Forty-eight hours later the transfected cells were placed under 2 microgram/milliliter puromicin selection. Puromicin resistant cells were carried as a population of cells.
• the 293 HEK cells (wild type or overexpressing human zcytoR19) were transfected as follows: 700,000 293 cells/well (6 well plates) were plated approximately 18 h prior to transfection in 2 milliliters DMEM+10% fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA (Stratagene) and 1 microgram pIRES2-EGFP DNA (Clontech) were added to 6 microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. This transfection mix was added 30 minutes later to the pre-plated 293 cells.
• transfected cells Twenty-four hours later the transfected cells were removed from the plate using trypsin-EDTA and replated at approximately 25,000 cells/well in 96 well microtiter plates. Approximately 18 h prior to ligand stimulation, media was changed to DMEM+0.5% FBS.
• the signal transduction reporter assays were done as follows: Following an 18 h incubation at 37 degrees in DMEM+0.5% FBS, transfected cells were pretreated with a neutralizing polyclonal goat antibody to IL10Rb (2.5 micrograms/ml for zcyto21; 8 micrograms/ml for zcyto20 and zcyto22, R&D Systems) or PBS for 1 hour at 37 C.
• Human embryonal kidney (HEK) cells stably overexpressing human zcytoR19 were also pretreated with a non-neutralizing polyclonal goat antibody to IFNAR1 (8 micrograms/ml, R&D Systems) as an antibody control for experiments involving zcyto20 and zcyto22.
• Pretreated cells were stimulated with dilutions (in DMEM+0.5% FBS) of the following class II ligands; zcyto20, zcyto21, or zcyto22.
• Tables 9 and 10 show that induction of ISRE signaling by zcyto20 is inhibited by pretreatment of wild type 293 cells or 293 cells overexpressing human zytoR19 with a neutralizing antibody to IL10Rb. No or little inhibition is seen of huIFNa-2a induction of ISRE signaling. These results indicate that zcyto20 requires interaction with IL10Rb (CRF2-4) for maximal induction of ISRE signaling and that the receptor for zcyto20 is the heterdimeric combination of zcytoR19 and IL10Rb CRF2-4).
• Tables 11 and 12 show that ISRE signaling by zcyto21 is inhibited by pretreatment of wild type 293 cells or 293 cells overexpressing human zcytoR19 with a neutralizing antibody to IL10Rb. No inhibition is seen of huIFNa-2a induction of ISRE signaling. These results indicate that zcyto21 requires interaction with IL10Rb (CRF2-4) for maximal induction of ISRE signaling and that the receptor for zcyto21 is the heterdimeric combination of zcytoR19 and IL10Rb (CRF2-4).
• Tables 13 and 14 show that induction of ISRE signaling by zcyto22 is inhibited by pretreatment of wild type 293 cells or 293 cells overexpressing human zcytoR19 with a neutralizing antibody to IL10Rb. No or little inhibition is seen of huIFNa-2a induction of ISRE signaling. These results indicate that zcyto22 requires interaction with IL10Rb (CRF2-4) for maximal induction of ISRE signaling and that the receptor for zcyto22 is the heterdimeric combination of zcytoR19 and IL10Rb (CRF2-4).
• B A: Anti-IL10Rb Antibody Blocks Antiviral Activity
• An antiviral assay was performed to determine the ability of anti-IL10Rb antibody to block the antiviral activity of zcyto20.
• the assay was carried out using 293 HEK cells (wild type or overexpressing human zcytoR19).
• antibodies anti-human IL10R beta, anti-human Leptin receptor, R&D Systems
• 50,000 cells per well into a 96-well plate were diluted into cell media at 5 micrograms/ml and then plated with 50,000 cells per well into a 96-well plate.
• zcyto20-CEE from example 3 (200 ng/ml for wild-type 293 cells, 0.5 ng/ml for 293 cells overexpressing human zcytoR19) or human interferon-a-2a (1 ng/ml for wild-type 293 cells, 100 ng/ml for 293 cells overexpressing human zcytoR19) were added to the wells and incubated overnight at 37° C. The next day, the medium was removed and replaced with medium containing encephalomyocarditis virus (EMCV) at a multiplicity of infection of 0.1. The cells were then incubated at 37° C. overnight.
• EMCV encephalomyocarditis virus
• C zcyto20, zcyto21, and zcyto22 Signaling is Enhanced by Coexpression of zcytoR19 and IL10Rb:
• a signal transduction reporter assay was used to determine the functional interaction of zcyto20, zcyto21 and zcyto22 with zyctor19 and IL10Rb (CRF2-4).
• Hamster kidney (BHK) cells were transfected with a reporter plasmid containing an interferon-stimulated response element (ISRE) driving transcription of a luciferase reporter gene in the presence or absence of pZP7 expression vectors containing cDNAs for class II cytokine receptors Zyctor19 and IL10Rb (CRF2-4).
• ISRE interferon-stimulated response element
• Luciferase activity following stimulation of transfected cells with class II ligands (including zcyto20, zcyto21 and zcyto22) reflects the interaction of the ligand with transfected and native cytokine receptors on the cell surface. The results and methods are described below.
• BHK-570 cells were transfected as follows: 200,000 BHK cells/well (6 well plates) were plated approximately 5 h prior to transfection in 2 milliliters DMEM+5% fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA (Stratagene), 1 microgram cytokine receptor DNA and 1 microgram pIRES2-EGFP DNA (Clontech) were added to 9 microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. Two micrograms pIRES2-EGFP DNA was used when cytokine receptor DNA was not included. This transfection mix was added 30 minutes later to the pre-plated BHK cells.
• transfected cells Twenty-four hours later the transfected cells were removed from the plate using trypsin-EDTA and replated at approximately 25,000 cells/well in 96 well microtiter plates. Approximately 18 h prior to ligand stimulation, media was changed to DMEM+0.5% FBS.
• Table 16 shows that zcyto20, zcyto21 and zcyto22 induce ISRE signaling in BHK cells transfected with ISRE-luciferase and zcytoR19 in a dose-dependent manner.
• the addition of IL10Rb (CRF2-4) DNA to the transfection mix results in a half-maximal induction of signaling at a 10-100 fold lower cytokine dose. No response was seen with ISRE transfection alone.
• ISRE Interferon Stimulated Response Element
• the binding of the ligands (zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25) to soluble receptors can be assayed using an iodo-bead labeling method.
• a iodo-bead labeling method For example, 125 I, labeled zcyto21-CEE is labeled (1.2 ⁇ 10 7 CPM/ml; 1.5 ng/ul; and 8.6 ⁇ 10 6 CPM/ug).
• Peripheral blood leukocytes were isolated by Ficoll Hypaque (Amersham, Sweden) separation from heparinized human blood.
• the PBLs were cultured at 37° C. in standard media at a density of 1 ⁇ 10 e 6 cells per milliliter in 6-well tissue culture plates. Following overnight incubation, the PBLs were harvested and stained with biotinylated zcyto20-cee and zcyto21-cee (See Example 18) at a concentration of 10 ug/ml. Staining was detected with Phycoerythrin-labeled streptavidin (Pharmingen, Calif., USA) that was prepared at a dilution of 1:1000.
• Northern blots were probed to determine the tissue distribution of zyctor19.
• a human zyctor19 cDNA fragment was obtained using PCR with gene specific primers, 5′ ZC40285 as shown in SEQ ID NO: 21; and 3′ ZC 40286, as shown in SEQ ID NO: 22.
• the template was cloned human zyctor19 cDNA. (SEQ ID NO: 23)
• the PCR fragment was gel purified, and ⁇ 25 ng was labeled with P 32 ⁇ -dCTP using the Prime-It® RmT random prime labeling kit (Stratagene, LaJolla, Calif.).
• a human cancer cell line blot C which contains RNA samples from each of the following cancer cell lines: promyelocytic leukemia HL-60, HELA S3, chronic myelogenous leukemia k-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma RAJI, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G-361;
• a human MTN H blot which contains mRNA from the following tissues: heart, whole brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas
• a human MTN H3 which contains mRNA from the following tissues: stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland, and bone marrow; and (4) a human MTN H4, which contains
• Hybridizations were all performed in ULTRAhybTM Ultrasensitive Hybridization Buffer (Ambion, Austin, Tex.) according the manufacturer's recommendations, which the exception that an additional 0.2 mg/ml salmon sperm DNA was added to the hybridization and prehybridization buffers to lower non-specific hybridization. Following hybridization, non-specific radioactive signal was removed by treating the blots with 0.1 ⁇ SSC/0.5% SDS at 50° C. The blots were exposed using BioMax MR Film and intensifying screens (Eastman Kodak, Rochester, N.Y.), per the manufacturer's recommendations for 3 days.
• ⁇ 4.5 kb transcript was in greatest in heart, skeletal muscle, pancreas and prostate tissue, in addition to in the Burkitt's lymphoma (RAJI) cell line. Lower levels were seen in multiple other tissues. In addition, there was an ⁇ 2 kb transcript which was generally less abundant than the larger transcript, but also present in many of the tissues and cell lines. Testis tissue, in addition to having the 2 and 4.5 kb transcripts, may also have ⁇ 4 kb and 1.4 kb transcripts. Adrenal gland demonstrated equal levels of expression of the 4.5 kb and 2 kb transcripts.
• First-strand cDNA synthesis from total RNA was carried out using a commercially available first-strand synthesis system for RT-PCR (Invitrogen life technologies, Carlsbad, Calif.). The subsequent PCR reactions were set up using zyctor19 ⁇ 1 (SEQ ID NO:1) and zyctor19 ⁇ 2 (SEQ ID NO:18) specific oligo primers ZC40288 (SEQ ID NO:65) and ZC40291 (SEQ ID NO:66) which yield a 806 bp and 892 bp product, respectively, Qiagen HotStarTaq DNA Polymerase and Buffer, (Qiagen, Inc., Valencia, Calif.), GeneAmp dNTPs (Applied Biosystems, Foster City, Calif.), RediLoadTM dye (Research Genetics, Inc., Huntville, Ala.) and 2 ⁇ l first-strand cDNA (10% of the first-strand reaction) from the respective cell types.
• zyctor19 ⁇ 1 SEQ ID NO:1
• the PCR cycler conditions were as follows: an initial 1 cycle 15 minute denaturation at 95° C., 35 cycles of a 45 second denaturation at 94° C., 1 minute annealing at 63° C. and 1 minute and 15 second extension at 72° C., followed by a final 1 cycle extension of 7 minutes at 72° C.
• the reactions were separated by electrophoresis on a 2% agarose gel (EM Science, Gibbstown, N.J.) and visualized by staining with ethidium bromide.
• a cell line expressing a secreted hzyctor19/hCRF2-4 heterodimer was constructed.
• the extracellular domain of hzyctor19 was fused to the heavy chain of IgG gamma1 (Fc4) (SEQ ID NO:14 and SEQ ID NO:15) with a Glu-Glu tag (SEQ ID NO:11) at the c-terminus, while the extracellular domain of CRF2-4 (SEQ ID NO:64) was fused to Fc4 with a His tag at the C-terminus.
• a Gly-Ser spacer of 12 amino acids was engineered between the extracellular portion of the receptor and the n-terminus of Fc4.
• a thrombin cleavage site was engineered between the Fc4 domain and the c-terminal tag to enable possible proteolytic removal of the tag.
• the extracellular portion of hzyctor19 was amplified by PCR from a brain cDNA library with oligos ZC37967 (SEQ ID NO:24) and ZC37972 (SEQ ID NO:25) with BamHI and Bgl2 restriction sites engineered at the 5′ and 3′ ends, respectively, under conditions as follows: 25 cycles of 94° C. for 60 sec., 57° C. for 60 sec., and 72° C. for 120 sec.; and 72° C. for 7 min.
• PCR products were purified using QIAquick PCR Purification Kit (Qiagen), digested with BamHI and Bgl2 (Boerhinger-Mannheim), separated by gel electrophoresis and purified using a QIAquick gel extraction kit (Qiagen).
• Qiagen QIAquick PCR Purification Kit
• the hzyctor19 BamHI/Bgl2 fragment was ligated into Fc4/pzmp20 vector that had been digested with Bgl2.
• the zyctor19 fragment is cloned between a tPA leader peptide and human Fc4 fragment.
• the DNA fragement of zyctor19 with tPA leader peptide was cut out by EcoRI and Bgl2 diestion, and then cloned into pzp9/zcytor7/Fc4-CEE vector.
• This vector has the extracellular portion of hzcytor7 fused to Fc4 with a CEE tag, and digesting with EcoRI and BamHI removes the extracellular portion of hzcytor7 and allows substitution of hzyctor19.
• Minipreps of the resulting ligation were screened for an EcoRI/BamHI insert of the correct size and positive minipreps were sequenced to confirm accuracy of the PCR reaction.
• the extracellular portion of hCRF2-4 was amplified by PCR from pZP-9 CRF with oligos ZC39319 (SEQ ID NO:68) and ZC39325 (SEQ ID NO:70) under conditions as follows: 30 cycles of 94° C. for 60 sec., 57° C. for 60 sec., and 72° C. for 120 sec; and 72° C. for 7 min. PCR product were purified as described above and then digested with EcoRI and BamHI.
• PCR product had an internal EcoRI site two bands were obtained upon digestion: a 0.101 kB EcoRI/EcoRI fragment and a 0.574 kB EcoRI/BamHI fragment.
• the 0.574 EcoRI/BamHI fragment was ligated into vector#249 pHZ-1 DR1/Fc4-TCS-cHIS that had been digested with EcoRI and BamHI.
• This vector has the extracellular portion of hDR-1 fused to Fc4 with a C-HIS tag (SEQ ID NO:[#]), and digesting with EcoRI and BamHI removes the extracellular portion of hDR-1 and allows substitution of hCRF2-4.
• Minipreps of the resulting ligation were screened for an EcoRI/BamHI insert of the correct size, and positive minipreps, were EcoRI digested and band purified for further construction.
• the 0.101 kB EcoRI/EcoRI fragment was ligated into the EcoRI digested minipreps and clones were screened for proper orientation of insertion by KpnI/NdeI restriction digestion. Clones with the correct size insertion were submitted for DNA sequencing to confirm the accuracy of the PCR reaction.
• each of the hzyctor19/Fc4-cEE and hCRF2-4/Fc-4-cHIS were co-transfected into BHK-570 (ATCC No. CRL-10314) cells using Lipofectamine (Gibco/BRL), as per manufacturer's instructions.
• the transfected cells were selected for 10 days in DMEM+5% FBS (Gibco/BRL) containing 1 ⁇ M 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 ⁇ M MTX and 0.5 mg./ml G418 for 10 days.
• Conditioned culture media zyctor19/CRF2-4 heterodimer was filtered through 0.2 ⁇ m filter and 0.02% (w/v) Sodium Azide was added. The conditioned media was directly loaded a Poros Protein A 50 Column at 10-20 ml/min. Following load the column was washed with PBS and the bound protein eluted with 0.1M Glycine pH 3.0. The eluted fractions containing protein were adjusted to pH 7.2 and Concentrated to ⁇ 80 ml using YM30 Stirred Cell Membrane (Millipore).
• the 80 ml eluate from the Protein A column was loaded onto a 318 ml Superdex 200 HiLoad 26/60 Column (Pharmacia). The column was eluted with PBS pH 7.2 at 3 ml/min. Protein containing fractions were pooled to eliminate aggregates. The Superdex 200 pool was adjusted to 0.5M NaCl, 10 mM Imidazole using solid NaCl and Imidazole and the pH was adjusted to 7.5 with NaOH. The adjusted protein solution was loaded onto a 200 ml NiNTA column (Qiagen) at 2 CV/hr.
• the bound protein was eluted, following PBS wash of the column, with five concentration steps of Imidazole : 40 mM, 100 mM, 150 mM, 250 mM, 500 mM.
• the fractions eluted at each step of imidizole were pooled and analyzed by N-terminal sequencing. Pools containing heterodimer, determined by sequencing were pooled and concentrated to 50 ml using a YM30 Stirred Cell Membrane (Millipore).
• the 50 ml eluate from the NiNTA column was loaded onto a 318 ml Superdex 200 HiLoad 26/60 Column (Pharmacia).
• the column was eluted with PBS pH 7.2 at 3 ml/min. Protein containing fractions were pooled to eliminate aggregates, as determined by SEC MALS analysis.
• Table 17 B and T cells express significant levels of IL28RA mRNA. Low levels are seen in dendritic cells and most monocytes. TABLE 17 Cell/Tissue II28RA IFNAR2 CRF2-4 Dendritic Cells unstim .04 5.9 9.8 Dendritic Cells + IFNg .07 3.6 4.3 Dendritic Cells .16 7.85 3.9 CD14+ stim'd with LPS/IFNg .13 12 27 CD14+ monocytes resting .12 11 15.4 Hu CD14+ Unact. 4.2 TBD TBD Hu CD14+ 1 ug/ml LPS act. 2.3 TBD TBD H. Inflamed tonsil 3 12.4 9.5 H.
• SMVC hep. Vein 0.00 6.46 1.45 Hep SMCA hep. Artery 0.00 7.55 2.10 Hep. Fibroblast 0.00 6.20 2.94 HuH7 hepatoma 4.20 3.05 7.24 HepG2 Hepatocellular carcinoma 3.40 5.98 2.11 SK-Hep-1 adenocar.
• ZcytoR19 is detectable in normal B cells, B lymphoma cell lines, T cells, T lymphoma cell lines (Jurkat), normal and transformed lymphocytes (B cells and T cells) and normal human monocytes.
• a signal transduction reporter assay can be used to show the inhibitor properties of zyctor19-Fc4 homodimeric and zyctor19-Fc/CRF2-4-Fc heterodimeric soluble receptors on zcyto20, zcyto21 and zcyto24 signaling.
• Human embryonal kidney (HEK) cells overexpressing the zyctor19 receptor are transfected with a reporter plasmid containing an interferon-stimulated response element (ISRE) driving transcription of a luciferase reporter gene.
• ISRE interferon-stimulated response element
• Luciferase activity following stimulation of transfected cells with ligands reflects the interaction of the ligand with soluble receptor.
• 293 HEK cells overexpressing zyctor19 were transfected as follows: 700,000 293 cells/well (6 well plates) were plated approximately 18 h prior to transfection in 2 milliliters DMEM+10% fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA (Stratagene) and 1 microgram pIRES2-EGFP DNA (Clontech,) were added to 6 microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. This transfection mix was added 30 minutes later to the pre-plated 293 cells.
• transfected cells Twenty-four hours later the transfected cells were removed from the plate using trypsin-EDTA and replated at approximately 25,000 cells/well in 96 well microtiter plates. Approximately 18 h prior to ligand stimulation, media was changed to DMEM+0.5% FBS.
• the signal transduction reporter assays were done as follows: Following an 18 h incubation at 37° C. in DMEM+0.5%FBS, transfected cells were stimulated with 10 ng/ml zcyto20, zcyto21 or zcyto24 and 10 micrograms/ml of the following soluble receptors; human zyctor19-Fc homodimer, human zyctor19-Fc/human CRF2-4-Fc heterodimer, human CRF2-4-Fc homodimer, murine zyctor19-Ig homodimer.
• the cells were lysed, and the relative light units (RLU) were measured on a luminometer after addition of a luciferase substrate. The results obtained are shown as the percent inhibition of ligand-induced signaling in the presence of soluble receptor relative to the signaling in the presence of PBS alone.
• Table 26 shows that the human zyctor19-Fc/human CRF2-4 heterodimeric soluble receptor is able to inhibit zcyto20, zcyto21 and zcyto24-induced signaling between 16 and 45% of control.
• the human zyctor19-Fc homodimeric soluble receptor is also able to inhibit zcyto21-induced signaling by 45%.

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• 2006
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