ZA200102310B

ZA200102310B - Secretory protein - 48.

Info

Publication number: ZA200102310B
Application number: ZA200102310A
Authority: ZA
Inventors: Si Lok; Paul O Sheppard
Original assignee: Zymogenetics Inc
Priority date: 1998-10-01
Filing date: 2001-03-20
Publication date: 2001-10-26
Also published as: JP2002525100A; WO2000018796A2; CA2344712A1; EP1117793A2; AU6506999A; WO2000018796A3; IL142193A0

Description

. )

SECRETORY PROTEIN - 48

TECHNICAL FIELD

The present invention relates generally to a new cytokine having diagnostic and therapeutic uses. In particular, the present invention relates to a novel secreted protein designated “Secretory Protein - 48' or ,

Zsig48 for short, and to nucleic acid molecules encoding

Zsig4s.

BACKGROUND OF THE INVENTION ) . 15

Proliferation, maintenance, survival and differentiation of cells of multicellular organisms are : controlled by hormones and polypeptide growth factors.

These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair and regenerate damaged tissue. Examples of hormecnes 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.

Hormones and growth factors influence cellular metabolism by binding to proteins. Proteins may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of proteins are soluble molecules, such as the transcription factors.

Of particular interest are cytokines, molecules that promote the proliferation, maintenance, survival or differentiation of cells. Examples of 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. These cytokines are useful in restoring normal blood cell levels in patients suffering from anemia or receiving chemotherapy for cancer. The demonstrated in vivo activities of these cytokines illustrates the enormous clinical potential of, and need for, other cytckines, cytokine agonists, and cytokine antagonists.

SUMMARY OF THE INVENTION .

The present invention fills this need by providing for a secretory protein, designated “Zsig48' . which can stimulate the proliferation of peripheral blood mononuclear cells, i.e., T-cells, B-cells and monocytes, (PBMNCs). The present invention also provides Zsig48 polypeptides and Zsig48 fusion proteins, as well as nucleic acid molecules encoding such polypeptides and proteins.

The human Zsig48 polypeptide with signal sequence is comprised of a sequence which is 105 amino acid residues as shown in SEQ ID NOs: 1 and 2. The signal sequence is comprised of amino acid residues 1-26 of SEQ

ID NO:2. A mature Zsig4B polypeptide thus is comprised of amino acid residues 27, a leucine, to and including amino acid residue 105, a histidine, of SEQ ID NO:2 also defined by SEQ ID NO:3. An alternative mature Zsig48 polypeptide is comprised of amino acid residues 29, a leucine, to and including amino acid residue 105 of SEQ ID NO: 2, also defined by SEQ ID NO:4. In yet a third alternative signal peptidase cleavage site, the signal sequence is comprised of amino acid residues 1-40, the mature sequence being comprised of amino acid residue 41, a proline, to and including amino acid residue 105 of SEQ ID NO:2. This mature sequence is also defined by SEQ ID NO:5. Another mature sequence of Zsig48 extends from amino acid residue 26 to and including amino acid residue 105 of SEQ ID NO:2, also represented by SEQ ID NO:16.

The present invention further provides pharmaceutical compositions that comprise such polypeptides, and a pharmaceutically acceptable carrier.

The present invention also includes variant } human Zsig48 polypeptides, wherein the variant polypeptide shares an identity with the amino acid sequence of SEQ ID

NOs:2, 3, 4, 5 or 16 selected from the group consisting of : at least 70% ldentity, at least 80% identity, at least 90% identity, at least 95% identity, or greater than 95% identity, and wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NOs:2, 3, 4 and 5 is due to one or more conservative amino acid substitutions.

An additional embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion cof a

Zsig48 polypeptide having an amino acid sequence described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zsig48 polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although epitcpe-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule. Examples of such polypeptides are those polypeptides comprised of one or more of SEQ ID NOs: 8, 9, 10, 11, or 12.

The present invention also provides for isolated polynucleotides which encode the above-described polypeptides.

The present invention also provides isolated nucleic acid molecules that encode a Zsig48 polypeptide, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, (b) a nucleic acid molecule which encodes isclated polypeptides having an amino acid sequence that is at least 70%, 80%, 90%, 95% or 99% identical to an amino acid sequence selected from the group consisting of the polypeptide defined by SEQ ID -

NOs:2, 3, 4 or 5; (c) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule consisting cf the nucleotide sequence of SEQ ID NO:1, or the complement of SEQ ID NO:1, and (d) a nucleic acid molecule that remains hybridized fellowing stringent wash conditions to a nucleic acid molecule which encodes an isolated polypeptides having an amino acid sequence that is at least 70%, 80%, 90%, 95% or 99% identical to an amino acid sequence selected from the group consisting of the polypeptide defined by SEQ ID

NOs:2, 3, 4 and 5. SEQ ID NO:14 shows a genomic sequence of Zsig4s.

The present invention also provides vectors and expression vectors comprising such nucleic acid molecules, recombinant host cells comprising such vectors and expression vectors, and recombinant viruses comprising such expression vectors. These expression vectors and recombinant host cells can be used to prepare Zsig48 polypeptides. In addition, the present invention provides pharmaceutical compositions, comprising a pharmaceutically acceptable carrier and at least one of such an expression vector or recombinant virus. Preferably, such pharmaceutical compositions comprise a human Zsig48 gene, or a variant thereof.

The present invention further contemplates antibodies and antibody fragments that specifically bind with Zsig48 polypeptides. Such antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies. Examples of antibody fragments include F(ab'),, F(ab),, Fab', Fab, Fv, scFv, and minimal recognition units. : 15

In addition, the presence of Zsig48 polypeptide . in a biological sample can be detected by methods that comprise the steps of (a) contacting the biological sample with an antibody, or an antibody fragment, that specifically binds with a polypeptide having the amino acid sequence of either SEQ ID NOs:2 3, 4 or 5, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.

The present invention also contemplates isolated nucleic acid molecules comprising a nucleotide sequence that encodes an Zsig48 secretion signal sequence and a nucleotide sequence that encodes a biologically active polypeptide, wherein the Zsig48 secretion signal sequence comprises an amino acid sequence of residues 1 to 26, 1 to 28 or 1 to 40 of SEQ ID NO:2. Illustrative biologically active polypeptides include Factor VIIa, proinsulin, insulin, follicle stimulating hormone, tissue type plasminogen activator, tumor necrosis factor, interleukin, colony stimulating factor, interferon, erythropoietin, and thrombopoietin. Moreover, the present invention provides fusion proteins comprising a Zsig48 secretion signal sequence and a polypeptide, wherein the Zsig48 secretion signal sequence comprises an amino acid sequence of residues 1 to 26, 1 to 28 or 1 to 40 of SEQ ID NO:2.

The present invention also contemplates anti- idiotype antibodies, or anti-idiotype antibody fragments, that specifically bind with an anti-Zsig48 antibody or antibody fragment, wherein the anti-idiotype antibody, or anti-idiotype antibody fragment, possesses the ability to cause proliferation of T-cells, B-cells or monocytes.

The present invention further includes methods for detecting an alteration in a chromosome containing

Zsig48. Illustrative chromosomal aberrations at the Zsig48 gene locus include aneuploidy, gene copy number changes, . insertions, deletions, restriction site changes and rearrangements. These aberrations can occur within flanking sequences, including upstream promoter and regulatory regions, and can be manifested as physical alterations within a coding sequence or changes in gene expression level. Such methods are effected by examining the Zsig48 gene and its gene products. In general, suitable assay methods include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing polymerase chain reaction techniques, ligation chain reaction, ribonuclease protection assays, use of single-nucleotide polymorphisms, protein truncation assays, and other genetic linkage techniques known in the art.

In particular, the present invention provides methods for diagnosing an alteration in the Zsig48 gene of an individual, comprising: (a) amplifying nucleic acid molecules that encode Zsig48 from RNA isolated from a

. N biological sample of the individual, and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates an alteration in the

Zsig48 gene. Similarly, methods of detecting a chromosome abnormality in a subject comprise: (a) amplifying nucleic acid molecules that encode Zsig48 from RNA isclated from a biological sample of the subject, and (b) detecting a mutation in the amplified nucleic acid molecules, wherein the presence of a mutation indicates a chromosome abnormality. In variations of these methods, the detecting step is performed by comparing the nucleotide sequence of the amplified nucleic acid molecules to the nucleotide sequence of SEQ ID NOs:1. Alternatively, the detecting step can be performed by fractionating the ) 15 amplified nucleic acid molecules and control nucleic acid molecules that encode the amino acid sequence of SEQ ID . NOs:2, and comparing the lengths of the fractionated amplified and control nucleic acid molecules. Exemplary methods for amplification include polymerase chain reaction or reverse transcriptase-polymerase chain reaction.

The present invention further provides for a method for promoting the proliferation of leukocytes comprising bringing the leukocytes into contact with

Zsig48.

These and other aspects of the invention will become evident upon reference to the following detailed description and the attached drawings. In addition, various references are identified below and are incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

The teachings of all the references cited herein are incorporated in their entirety herein by reference.

Definitions

In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the invention.

As used herein, "nucleic acid" or "nucleic acid molecule" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonuclectides, fragments generated by the polymerase chain reaction (PCR}, and fragments generated by any of ligation, scission, endonuclease action, and exonuclease . action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as

DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., a-enantiomeric forms of naturally- occurring nucleotides), or a combination of both.

Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.

Sugar mcdifications include, for example, replacement of one or more hydroxyl groups with halcgens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithicate, phosphoroselenocate, phosphorodiselencate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which comprise naturally- occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.

The term "complement of a nucleic acid molecule” refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5° CCCGTGCAT 3.

The term "contig" denotes a nucleic acid ) 15 molecule that has a contiguous stretch of identical or complementary sequence to another nucleic acid molecule. . Contiguous sequences are said to "overlap" a given stretch of a nucleic acid molecule either in their entirety or along a partial stretch cof the nucleic acid molecule.

The term “degenerate nucleotide sequence! denotes a sequence of nucleotides that includes one or more degenerate codons as compared to a reference nucleic acid 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).

The term "structural gene" refers to a nucleic acid molecule that is transcribed into messenger RNA (mRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide.

An "isolated nucleic acid molecule" is a nucleic acid molecule that is not integrated in the genomic DNA of an organism. For example, a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule. Another example of an isolated nucleic acid molecule is a chemically- synthesized nucleic acid molecule that is not integrated in the genome of an organism. A nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.

A "nucleic acid molecule construct" is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature. "Linear DNA" denotes non-circular DNA molecules having free 5' and 3' ends. Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by . enzymatic digestion or physical disruption. "Complementary DNA (cDNA)" is a single-stranded

DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription. Those skilled in the art also use the term "cDNA" to refer to a double-stranded

DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand. The term "cDNA" also refers to a clone of a cDNA molecule synthesized from an RNA template.

A "promoter" is a nucleotide sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites,

TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 {1923)), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer

Biol. 1:47 (1990)), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye et al., J. Biol. Chem. 269:25728 (1994)),

SPl, cAMP response element binding protein (CREB; Loeken,

Gene Expr. 3:253 (1993)) and octamer factors (see, in general, Watson et al., eds., Molecular Biology of the

Gene, 4th ed. (The Benjamin/Cummings Publishing Company,

Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1 ) 15 (1994)). If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing . agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Repressible promoters are also known.

A "core promoter" contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription. By this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity.

A "regulatory element" is a nucleotide sequence that modulates the activity of a core promoter. For example, a regulatory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific," "tissue-specific," or "organelle-specific" manner. For example, the Zsig48 regulatory element preferentially induces gene expression in placenta, kidney, heart or leukocytes.

An "enhancer" is a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription. "Heterologous DNA" refers to a DNA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell. DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e., endogenous DNA) so long as that host DNA is combined with non-host DNA (i.e., exogenous DNA). For example, a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a . transcription promoter is considered to be a heterologous

DNA molecule. Conversely, a heterolcgous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter. As another illustration, a DNA molecule comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell that lacks the wild-type dene.

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."

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.

A peptide or polypeptide encoded by a non-host

DNA molecule is a "heterologous" peptide or polypeptide.

An "integrated genetic element" is a segment of

DNA that has been incorporated into a chromosome of a host cell after that element is introduced into the cell through human manipulation. Within the present invention, integrated genetic elements are most commonly derived from linearized plasmids that are introduced into the cells by ) 15 electroporation or other techniques. Integrated genetic elements are passed from the original host cell to its . progeny.

A "cloning vector" is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, that has the capability of replicating autonomously in a host cell.

Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites that allow insertion of a nucleic acid molecule in a determinable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.

Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.

An "expression vector" is a nucleic acid molecule encoding a gene that is expressed in a host cell.

Typically, an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity cf the core promoter.

A "recombinant host" is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector. In the present context, an example of a recombinant host is a cell that produces

Zsig48 from an expression vector. In contrast, Zs8ig48 can be produced by a cell that is a "natural source" of 2sig48, and that lacks an expression vector. “Integrative transformants" are recombinant host cells, in which heterologous DNA has become integrated into the genomic DNA of the cells.

A "fusion protein" is a hybrid protein expressed : by a nucleic acid molecule comprising nucleotide sequences of at least two genes. For example, a fusion protein can comprise at least part of an Zsig4s polypeptide fused with a polypeptide that binds an affinity matrix. Such a fusion protein provides a means to isolate large quantities of Zsig48 using affinity chromatography.

The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand." This interaction mediates the effect of the ligand on the cell. Receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor). Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.

In general, the binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell, which in turn leads to an alteration in the metabolism of the cell. Metabolic events that are often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and ’ 15 hydrolysis of phospholipids. . The term "secretory signal sequence" denotes a

DNA sequence that encodes a peptide (a "secretory peptide”) that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

An "isolated polypeptide" is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature. Typically, a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. One way toc show that a particular protein preparation contains an isolated polypeptide is by the appearance of a single band following sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel. However, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.

The terms "amino-terminal or N-terminal” and "carboxyl-terminal or C-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.

The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.

The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene.

Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed

RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a polypeptide encoded by a splice variant of an mRNA transcribed from a gene.

As used herein, the term "immunomodulator" includes cytokines, stem cell growth factors,

lymphotoxins, co-stimulatory molecules, hematopoietic factors, and synthetic analogs of these molecules.

The term "complement/anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair.

Other exemplary complement/anti-complement pairs include rzreceptor/ligand pairs, antibody/antigen (or hapten or epitope} pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement /anti-complement pair is desirable, the complement /anti-complement pair preferably has a binding affinity of less than 10° M. - An "anti-idiotype antibody" is an antibody that binds with the variable region domain of an immunoglobulin. In the present context, an anti-idiotype antibody binds with the variable region of an anti-

Zsig48-antibody, and thus, an anti-idiotype antibody mimics an epitope of Zsig4s.

An "antibody fragment" is a portion of an antibody such as F(ab'),, F(ab),, Fab', Fab, and the like.

Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody.

For example, a Zsig48 monoclonal antibody fragment binds with an epitope of Zsig48.

The term "antibody fragment" also includes a synthetic or a genetically engineered polypeptide that binds tc a specific antigen, such as polypeptides consisting of the light chain variable region, "Fv" fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable regicn.

A "chimeric antibody" is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody. "Humanized antibodies" are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain.

As used herein, a "therapeutic agent" is a : molecule or atom which is conjugated to an antibody moiety to produce a conjugate which is useful for therapy.

Examples of therapeutic agents include drugs, toxins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes.

A "detectable label" is a molecule or atom which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.

The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.

Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al.,

Methods Enzymol. 198:3 (13%1)), glutathione S transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P, FLAG peptide (Hopp et al.,

Biotechnology 6:1204 (1888)), streptavidin binding peptide, or other antigenic epitope or binding domain.

See, in general, Ford et al., Protein Expression and

Purification 2:95 (1991). DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia

Biotech, Piscataway, NJ).

A "naked antibody" is an entire antibody, as opposed to an antibody fragment, which is not conjugated with a therapeutic agent. Naked antibodies include both - polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric and humanized antibodies.

As used herein, the term "antibody component" includes both an entire antibody and an antibody fragment.

An "immunoconjugate" is a conjugate of an antibody component with a therapeutic agent or a detectable label.

As used herein, the term "antibody fusion protein” refers to a recombinant molecule that comprises an antibody component and a therapeutic agent. Examples of therapeutic agents suitable for such fusion proteins include immunomodulators ("antibody-immunomedulator fusion protein”) and toxins ("antibody-toxin fusion protein").

A "tumor associated antigen" is a protein normally not expressed, or expressed at lower levels, by a normal counterpart cell. Examples of tumor associated

- antigens include alpha-~fetopreotein, carcinoembryonic antigen, and Her-2/neu. Many other illustrations of tumor associated antigens are known to those of skill in the art. See, for example, Urban et al., Ann. Rev. Immunol. 10:617 (1992).

As used herein, an "infectious agent" denotes both microbes and parasites. A "microbe" includes viruses, bacteria, rickettsia, mycoplasma, protozoa, fungi and like microorganisms. A "parasite" denotes infectious, generally microscopic or very small multicellular invertebrates, or ova or juvenile forms thereof, which are susceptible tc immune-mediated clearance or lytic or phagocytic destruction, such as malarial parasites, spirochetes, and the like.

An "infectious agent antigen" is an antigen - associated with an infectious agent.

A "target polypeptide" or a "target peptide" is an amino acid sequence that comprises at least one epitope, and that is expressed on a target cell, such as a tumor cell, or a cell that carries an infectious agent antigen. T cells recognize peptide epitopes presented by a major histocompatibility complex molecule to a target polypeptide or target peptide and typically lyse the target cell or recruit other immune cells to the site of the target cell, thereby killing the target cell.

An "antigenic peptide" is a peptide which will bind a major histocompatibility complex molecule to form an MHC-peptide complex which is recognized by a T cell, thereby inducing a cytotoxic lymphocyte response upon presentation to the T cell. Thus, antigenic peptides are capable of binding to an appropriate major histocompatibility complex molecule and inducing a cytotoxic T cells response, such as cell lysis or specific cytokine release against the target cell which binds or expresses the antigen. The antigenic peptide can be bound in the context of a class I or clasg II major histocompatibility complex molecule, on an antigen presenting cell or on a target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA. A nucleic acid molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a : sequence that is complementary to that of a specific mRNA.

The RNA transcript is termed an ‘anti-sense RNA' and a nucleic acid molecule that encodes the anti-sense RNA is termed an "anti-sense gene." Anti-sense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.

An "anti-sense oligonucleotide specific for

Zsig48" or an "Zsig48 anti-gense oligonucleotide! is an oligonucleotide having a sequence (a) capable of forming a stable triplex with a portion of a Zsig48 gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the Zsig48 gene.

A "ribozyme" is a nucleic acid molecule that contains a catalytic center. The term includes RNA enzymes, self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions. A nucleic acid molecule that encodes a ribozyme is termed a "ribozyme gene."

An "external guide sequence" is a nucleic acid molecule that directs the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acid molecule that encodes an external guide sequence is termed an "external guide sequence gene."

The term "variant human Zsig48 gene" refers to nucleic acid molecules that encode a polypeptide having an amino acid sequence that is a modification of SEQ ID NO:2.

Such variants include naturally-occurring polymorphisms of

Z251g48 genes, as well as synthetic genes that contain conservative amino acid substitutions of the amino acid sequence of SEQ ID NOs:2, 3, 4 or 5. Additional variant forms of Zsig48 genes are nucleic acid molecules that contain insertions or deletions of the nucleotide sequences described herein. A variant Zsig48 gene can be identified by determining whether the gene hybridizes with a nucleic acid molecule having the nucleotide sequence of

SEQ ID NO:1, or its complement, under stringent conditions.

Alternatively, variant Zsigd48genes can be ‘ identified by sequence comparison. Two amino acid sequences have "100% amino acid sequence identity" if the amino acid residues of the two amino acid sequences are the same when aligned for maximal correspondence.

Similarly, two nucleotide sequences have "100% nucleotide sequence identity" if the nucleotide residues of the two nucleotide sequences are the same when aligned for maximal correspondence. Sequence comparisons can be performed using standard software programs such as those included in the LASERGENE bicinformatics computing suite, which is produced by DNASTAR (Madison, Wisconsin). Other methods for comparing two nucleotide or amino acid sequences by determining optimal alignment are well-known to those of skill in the art (see, for example, Peruski and Peruski,

The Internet and the New Biology: Tools for Genomic and

Molecular Research (ASM Press, Inc. 1997), Wu et al. (eds.), "Information Superhighway and Computer Databases of Nucleic Acids and Proteins," in Methods in Gene

Biotechnology, pages 123-151 (CRC Press, Inc. 1997), and

Bishop (ed.), Guide to Human Genome Computing, 2nd Edition

(Academic Press, Inc. 1998)). Particular methods for determining sequence identity are described below.

Regardless of the particular method used to identify a variant Zsig48 gene or variant Zsig4s polypeptide, a variant gene or polypeptide encoded by a variant gene is functionally characterized by its ability to bind specifically to an anti-Zsig48 antibody.

The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locug. 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.

The term "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, a-globin, B- globin, and myoglobin are paralogs of each other.

Due to the imprecision of standard analytical methods, molecular weights and lengths of polymers are understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to +10%.

Claims

CLAIMS We claim:

1. A polypeptide comprised of a sequence that is at least 90% identical with an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 3, 4, 5, 15 and 16.

2. A polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9 10, 11 and 12.

3. A polynucleotide which encodes a polypeptide that ie at least 90% identical with an amino acid sequence ) selected from the group consisting of SEQ ID NOs:2, 3, 4, 5, and 16.

4. A polynucleotide which encodes a polypeptide containing an amino acid seguence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11 and 12.

5. An antibody which specifically binds to a polypeptide comprised of a sequence that is at least 90% identical with an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 3, 4, 5, 8, 9, 10, 11, 12, 15 and

16.

6. An anti-idiotypic antibody which binds to an epitope of an antibody which specifically binds to a polypeptide comprised of a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 8, 9, 10, 11, 12 and 15.

7. A method for promoting proliferation of leukocytes comprising bringing said leukocytes into contact with a polypeptide comprised of an amino acid sequence that is at least 90% identical with an amino acid sequence selected from the group consisting of 2, 3, 4, 5, 15 and 16.

8. The use of a polypeptide which is at least 90% identical to a polypeptide comprised of an amino acid sequence selected from the group consisting of 2, 3, 4, 5, 15 and 16 or a polynucleotide which encodes said polypeptide for the treatment of low leukocyte counts within an individual.

9. The use of a polypeptide which is at least 90% identical to a polypeptide comprised of an amino acid sequence selected from the group consisting of 2, 3, 4, 5, 15 and 16 or a polynucleotide which encodes said polypeptide for the manufacture of a medicament for the treatment of low leukocyte counts within an individual.