WO1999045114A2 - Human semaphorin zsmf-7 - Google Patents

Abstract

Semaphorin polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods are disclosed. The polypeptides are expressed in neuronal and lymphatic tissues. The polypeptides may be used within methods for detecting receptors that mediate neurite outgrowth, modulate cellular proliferation and/or differentiation, and immune response.

Description

DESCRIPTION HUMAN SEMAPHORIN ZSMF-7

BACKGROUND OF THE INVENTION Neuronal cell outgrowths, known as processes, grow away from the cell body to form synaptic connections. Long, thin processes which carry information away from the cell body are called axons, and short, thicker processes which carry information to and from the cell body are called dendrites. Axons and dendrites are collectively referred to as neurites. Neurites are extended by means of growth cones, the growing tip of the neurite, which is highly motile and is ultimately responsible for increasing and extending the neuronal network in the body. The growth cones are able to navigate their way to their targets using environmental cues or signals, which encourage or discourage the growth cone from extending the neurite in a particular direction. Such cues and signals include older neurons and orienting glial fibers, chemicals such as nerve growth factor released by astrocytes and other attracting or repelling substances released by target cells. The membrane of the growth cone bears molecules such as N-CAM (nerve cell adhesion molecule) which are attracted or repelled by environmental cues and thus influence the direction and degree of neurite growth. The growth cone also engulfs molecules from the environment which are transported to the cell body and influence growth. A number of proteins from vertebrates and invertebrates have been identified as influencing the guidance of neurite growth, either through repulsion or chemoattraction. Among those molecules are netrins, EPH-related receptor tyrosine kinases and their ligands, vitronectin, thrombospondin, human neuronal attachment factor-1 (NAF-1) , connectin, adhesion molecules such as CAM (cell adhesion molecule) and the semaphorins/collapsins (Neugebauer et al . , Neuron 6_.:345-58, 1991; 0 ' Shea et al . , Neuron 7:231-7, 1991; Osterhout et al . , Devel. Biol . 150:256-65, 1992; Goodman, Cell 78:353-6, 1993; DeFreitas et al . , Neuron 15:333-43 , 1995; Dodd and Schuchardy Cell 81:471-4, 1995; Keynes and Cook, Cell 83_.: 161-9, 1995; Mύller et al . , Cur. Opin. Genet, and Devel . 6:469-74, 1996, Goodman, Annu. Rev. Neurosci. 19:341-77, 1996; WIPO Patent Application No: 97/29189 and Goodman et al . , US Patent No. 5,639,856). Semaphorins/collapsins are a family of related transmembrane and secreted molecules. Invertebrate, vertebrate and viral semaphorins are known (Kolodkin et al., Cell 75:1389-99, 1993; Luo et al . , Cell 75:217-27, 1993; Ensser and Fleckenstein, J. Gen. Virol. 76 : 1063-7, 1995; Luo et al , Neuron 14:1131-40, 1995; Adams et al . , Mech. Devel. 57:33-45, 1996; Hall et al . , Proc . Natl. Acad. Sci . USA 93:11780-8, 1996; Roche et al . , Oncoqene 12:1289- 97, 1996; Skeido et al . , Proc. Natl. Acad. Sci. USA 93:4120-5, 1996; Xiang et al . , Genomics 32:39-48, 1996; Eckhardt et al . , Mol . Cell Neurosci. 9_.:409-19, 1997 and Zhou et al., Mol . Cell . Neurosci . 9:26-41, 1997).

The semaphorins generally comprise an N-terminal variable region of 30-60 amino acids that includes a secretory signal sequence, followed by a conserved region of about 500 amino acid residues called the semaphorin or sema domain. The extracellular semaphorin domain contains between 13-16 conserved cysteine residues, an N-linked glycosylation site and numerous blocks of amino acid residues which are conserved though-out the family. Classification into five subgroups within the semaphorin family has made based on the sequence of the region C- terminal to the semaphorin domain. Both soluble (lacking a transmembrane domain) and membrane-bound (having a transmembrane domain and localized to a membrane) semaphorins have been described. See, for example, Kolodkin et al . , ibid. ; Adams et al . , ibid, and Goodman et al., US Patent No : 5, 639, 856. Group I semaphorins include semaphorins having a transmembrane domain followed by a cytoplasmic domain. Most insect semaphorins are membrane bound proteins and belong to Group I. G-Sema I, T-Sema I and D-Sema I have a region of 80 amino acid residues following the semaphorin domain, which is followed by a transmembrane domain and an 80-110 amino acid cytoplasmic domain. Murine Sema IVa has a transmembrane domain followed by a 216 amino acid cytoplasmic domain. Groups II and III have no transmembrane domain or membrane association, but have a region with Ig homology. Group II secreted proteins, such as D-sema II, have a region of less than 20 amino acids between the semaphorin domain and an Ig-like domain followed by a short region of amino acid residues. Also included is alcelaphine herpesvirus type 1 semaphorin-like gene (avh-sema, Ensser and Fleckenstein, J. Gen. Virol. 76:1063-7, 1995) which ends with an Ig-like domain. Group III proteins, such as H-Sema III, are similar to Group II with the exception that the C-terminal amino acid region following the Ig-like domain is longer.

Group IV has a region of Ig homology C-terminal of the semaphorin domain followed by a transmembrane and cytoplasmic domain and includes semaphorins such as Sem B. Group V has a series of thrombospondin repeats C- terminal of the semaphorin domain followed by a transmembrane and cytoplasmic domain and include murine sema F and G.

Other viral semaphorins such as vaccinia virus sema IV and variola virus sema IV, have a truncated, 441 amino acid residue, semaphorin domain and no Ig region. See Kolodkin et al . , ibid. ; Adams et al . ibid, and Zhou et al . ibid.

Overall semaphorins share the greatest degree of homology within the semaphorin domain, between, 25-93%, and a greater degree of divergence in all other regions and domains, suggesting distinct roles for various sub-groups within the semaphorin family. The viral semaphorins are the most diverse, sharing only 25% identity with vertebrate semaphorins . Between vertebrate and invertebrate semaphorins, the percent identity varies between 30-40%. Neurite growth cues are of great therapeutic value. Isolating and characterizing novel semaphorins would be of value for example, in modulating neurite growth and development; treatment of peripheral neuropathies; for use as therapeutics for the regeneration of neurons following strokes, brain damage caused by head injuries and paralysis caused by spinal injuries; diagnosing neurological diseases and in treating neurodegenerative diseases such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. In addition, semaphorins are also being found in non-neuronal tissues and their usefulness for modulating cellular proliferation and differentiation as well as mediating immunological responses is now being reported. The present invention addresses these needs and others by providing novel semaphorins and related compositions and methods.

SUMMARY OF THE INVENTION

The present invention provides a novel semaphorin polypeptide and related compositions and methods.

Within one aspect is provided an isolated semaphorin polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 69 through 541 of SEQ ID NO : 2 , said polypeptide comprising cysteine residues at positions corresponding to residues 126, 143, 152, 266, 291, 335, 366, 493, 500, 503, 511, 518 and 541 of SEQ ID NO : 2. Within one embodiment the sequence of amino acid residues is at least 90% identical. Within another embodiment the polypeptide further comprises an Ig-like domain. Within a related embodiment the Ig-like domain comprises a sequence of amino acids from residue 561-620 of SEQ ID NO: 2. Within another embodiment the polypeptide comprises residues 45- 666 of SEQ ID NO : 2. Within yet another embodiment the sequence of amino acid residues comprises residues 1-666 of SEQ ID NO: 2. Within another embodiment the sequence of amino acid residues is from 473-624 amino acid residues. The invention further provides an isolated semaphorin polypeptide selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues from amino acid residue 45 to residue 666 of SEQ ID NO : 2 ; b) a polypeptide comprising a sequence of amino acid residues from amino acid residue 69 to residue 666 of SEQ ID NO: 2; c) a polypeptide comprising a sequence of amino acid residues from amino acid residue 69 to residue 541 of SEQ ID NO: 2 and d) a polypeptide comprising a sequence of amino acid residues from amino acid residue 1 to residue 666 of SEQ ID NO : 2. Within yet another embodiment any difference between said amino acid sequence of said isolated polypeptide and said corresponding amino acid sequence of SEQ ID NO: 2 is due to a conservative amino acid substitution. Within another embodiment the polypeptide is covalently linked to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores . Within a related embodiment the moiety is an affinity tag selected from the group consisting of polyhistidine, FLAG, Glu-Glu, glutathione S transferase and an immunoglobulin heavy chain constant region. Within a further related embodiment the polypeptide further comprises a proteolytic cleavage site between said sequence of amino acid residues and said affinity tag.

Within another aspect the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a semaphorin polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 69 through 541 of SEQ ID NO: 2, said polypeptide comprising cysteine residues at positions corresponding to residues 126, 143, 152, 266, 291, 335, 366, 493, 500, 503, 511, 518 and 541 of SEQ ID NO : 2 ; and a transcriptional terminator. Within one embodiment the expression vector further comprises a secretory signal sequence operably linked to said DNA segment. Within a related embodiment the secretory signal sequence encodes residues 1-44 of SEQ ID NO: 2. Within another embodiment the sequence of amino acid residues is at least 90% identical. Within another embodiment the DNA segment encodes a semaphorin polypeptide comprising an Ig-like domain. Within a related embodiment the Ig-like domain comprises a sequence of amino acids from residue 561-620 of SEQ ID NO: 2. Within another embodiment the sequence of amino acid residues comprises residues 45-666 of SEQ ID NO: 2. Within yet another embodiment the DNA segment encodes a semaphorin polypeptide covalently linked to an affinity tag selected from the group consisting of polyhistidine, FLAG, Glu-Glu, glutathione S transferase and an immunoglobulin heavy chain constant region. The invention further provides a cultured cell into which has been introduced an expression vector as described above, wherein said cell expresses the polypeptide encoded by the DNA segment . The invention also provides a method of producing a semaphorin protein comprising: culturing a cell into which has been introduced an expression vector as described above, whereby said cell expresses said semaphorin protein encoded by said DNA segment; and recovering said expressed semaphorin protein.

Within another aspect the invention provides a pharmaceutical composition comprising a polypeptide as described above, in combination with a pharmaceutically acceptable vehicle.

Within another aspect the invention provides an antibody or antibody fragment that specifically binds to an epitope of a semaphorin polypeptide as described above. Within one embodiment the antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b) ; and d) human monoclonal antibody. Within a related embodiment the antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit. Within a related embodiment is provided an anti-idiotype antibody that specifically binds to the antibody described above.

Within another aspect the invention provides a binding protein that specifically binds to an epitope of a semaphorin polypeptide as described above.

Within yet another aspect the invention provides an isolated polynucleotide encoding a semaphorin polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 69 through 541 of SEQ ID NO : 2 , said polypeptide comprising cysteine residues at positions corresponding to residues 126, 143, 152, 266, 291, 335, 366, 493, 500, 503, 511, 518 and 541 of SEQ ID NO : 2. Within one embodiment the sequence of amino acid residues is at least 90% identical. Within another embodiment the semaphorin polypeptide comprises an Ig-like domain. Within a related embodiment the Ig-like domain comprises a sequence of amino acids from residue 561-620 of SEQ ID NO : 2. Within another embodiment the sequence of amino acid residues comprises residues 45- 666 of SEQ ID NO : 2. Within yet another embodiment the sequence of amino acid residues comprises residues 1-666 of SEQ ID NO: 2. Within another embodiment the polynucleotide comprises nucleotide 1 to nucleotide 1998 of SEQ ID NO: 5. Also provided by the invention is an isolated polynucleotide selected from the group consisting of: a) a polynucleotide sequence consisting of the polynucleotide sequence from nucleotide 152 to nucleotide 2017 of SEQ ID NO : 1 ; b) a polynucleotide sequence consisting of the polynucleotide sequence from nucleotide 244 to nucleotide 2017 of SEQ ID NO:l; c) a polynucleotide sequence consisting of the polynucleotide sequence from nucleotide 244 to nucleotide 1640 of SEQ ID NO : 1 ; d) a polynucleotide sequence consisting of the polynucleotide sequence from nucleotide 20 to nucleotide 2017 of SEQ ID NO:l; and e) a complementary polynucleotide sequence of a, b, c or d. Within another aspect the invention provides a method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID N0:1 or the complement of SEQ ID NO:l, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; comparing said first reaction product to a control reaction product, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.

These and other aspects of the invention will become evident upon reference to the following detailed description and the attached drawing.

BRIEF DESCRIPTION OF THE DRAWING The figure shows an alignment of ZSMF-7 (SEQ ID N0:2), alcelaphine herpesvirus type 1 semaphorin-like gene (AHU18243) (SEQ ID N0:31), mouse semaA (SEQ ID N0:33), mouse semaB (SEQ ID NO: 3), mouse semaC (SEQ ID NO: 30), mouse semaD (SEQ ID NO: 32), mouse semaE (SEQ ID NO: 29) and mouse semaF (SEQ ID NO: 23) is shown in the Figure. There are clusters of conserved or highly homologous amino acids throughout the semaphorin domains of these semaphorin proteins . Conserved amino acid residues are indicated by "*" and residues with a high degree of homology are indicated by " : " and " . " .

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter:

The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al . , EMBO J . 4:1075, 1985; Nilsson et al . , Methods Enzymol . 198 : 3 , 1991), glutathione S transferase

(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al . , Proc. Natl. Acad. Sci. USA 82:7952-4, 1995), substance P, Flag™ peptide (Hopp et al . , Biotechnology 6_:1204-10, 1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al . , Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ) .

The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic 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 allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.

The terms "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.

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

The term "contig" denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5 ' -ATGGCTTAGCTT-3 ' are 5 ' -TAGCTTgagtct-3 ' and 3 ' -gtcgacTACCGA-5 ' .

The term "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) .

The term "expression vector" is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

The term "isolated", when applied to a polynucleotide, 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 . Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and 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. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.

The term "operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.

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.

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 vi tro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double- stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded 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. Such unpaired ends will in general not exceed 20 nt in length.

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" as used herein 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. For gross analysis of genes, 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, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art. Examples of ZSMF-7 probes and primers include, but are not limited to, the sequences disclosed herein as SEQ ID NOs: 4, 6, 7, 9-21, 24, 25, 26 and 28.

The term "promoter" is used herein for its art- recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA 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. The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell. Membrane-bound receptors are characterized by a multi- domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids . In general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor) .

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

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 PAGES 15-50

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directing and defining the growth of developing tissue, in particular, defining the margins of a particular organ or tissue. ZSMF-7 polypeptides would be useful in the defining and directing development of various tissues and organs including those associated with muscle, fibroblasts, reproductive, endocrine and lymphatic.

Semaphorins have also been associated with non- neuronal functions. Viral semaphorins have been speculated to act as modulators of the immune system, as natural immunosuppressants reducing the immune response by mimicking the function of a particular subfamily of semaphorins that can modulate immune functions (Kolodkin et al . , ibid. , and Ensser and Fleckenstein, ibid. ) . Other non- viral semaphorins are also associated with the immune system. Human semaphorin E, which is homologous to viral cytokine inhibiting proteins, contains conserved regions of amino acid residues that have been found in the viral semaphorins . Semaphorin E was found to be upregulated in rheumatoid synovial fibroblastoid cells which suggests that it may have a role as a regulator of inflammatory processes and an involvement in the development of rheumatoid arthritis (Mangasser-Stephan et al . , Biochem. Biophys . Res. Comm. 234 : 153-6, 1997). Semaphorin CD100 has been reported to promote B-cell growth and aggregation and may be involved in lymphocyte activation (Hall et al . , Proc. Natl. Acad. Sci. USA 93: 11780-5, 1996) and its mouse homologue, mSema G, is expressed on lymphocytes and is suggested to play a role in the immune system as well (Furuyama et al . , J. Biol. Chem. 271:33376-81, 1996) . ZSMF-7 shares the greatest homology with a viral semaphorin, alcelaphine herpesvirus type 1 semaphorin-like gene (ahv-sema) and coupled with the strong mRNA expression in activated T lymphocytes suggests that ZSMF-7 plays a role as a mediator of immunosuppression, in particular the activation and regulation of T lymphocytes. ZSMF-7 polypeptides would be useful additions to therapies for treating immunodeficiencies. ZSMF-7 was expressed in 52

activated lymphocytes (MRL cells) and not in resting lymphocyte cells (CD4⁺ and CD8⁺) suggesting that it would be useful tool for diagnosis and treatment of conditions where selective elimination of inappropriately activated T cells would be benificial, such as in autoimmune diseases, in particular insulin dependent diabetes mellitus, rheumatoid arthritis and multiple sclerosis. Such polypeptides could be used to screen serum samples from patients suffering from such conditions. Inappropriately activated T cells would include those specific for self-peptide/self-major histocompatibility complexes and those specific for non- self antigens from transplanted tissues. Use could also be made of these polypeptides in blood screening for removal of inappropriately activated T cells before returning the blood to the donor. Those skilled in the art will recognize that conditions related to ZSMF-7 underexpression or overexpression may be amenable to treatment by therapeutic manipulation of ZSMF-7 protein levels.

ZSMF-7 polypeptides can be used in vivo as an anti-inflammatory, for inhibition of antigen in humoral and cellular immunity and for immunosuppression in graft and organ transplants.

ZSMF-7 polynucleotides and/or polypeptides can be used for regulating the proliferation and stimulation of a wide variety of cells, such as T cells, B cells, lymphocytes, peripheral blood mononuclear cells, fibroblasts and hematopoietic cells. ZSMF-7 polypeptides will also find use in mediating metabolic or physiological processes in vivo . Proliferation and differentiation can be measured in vi tro using cultured cells. Suitable cell lines are available commercially from such sources as the American Type Culture Collection (Rockville, MD) . Bioassays and ELISAs are available to measure cellular response to ZSMF-7, in particular are those which measure changes in cytokine production as a measure of cellular response (see for example, Current Protocols in Immunology ed. John Coligan et al . , NIH, 1996) . Also of interest are 53

apoptosis assays, such as the DNA fragmentation assay described by Wiley et al . (Immunity, 2:673-82, 1995, and the cell death assay described by Pan et al . , Science, 276_.: 111-13, 1997). Assays to measure other cellular responses, including antibody isotype, monocyte activation, NK cell formation and antigen presenting cell function are also known. The ZSMF-7 polypeptides may also be used to stimulate lymphocyte development, such as during bone marrow transplantation and as therapy for some cancers. In vivo response to ZSMF-7 polypeptides can also be measured by administering polypeptides of the claimed invention to the appropriate animal model. Well established animal models are available to test in vivo efficacy of ZSMF-7 polypeptides for certain disease states. In particular, ZSMF-7 polypeptides can be tested in vivo in a number of animal models of autoimmune disease, such as the NOD mice, a spontaneous model system for insulin- dependent diabetes mellitus (IDDM) , to study induction of non-responsiveness in the animal model. Administration of ZSMF-7 polypeptides prior to or after onset of disease can be monitored by assay of urine glucose levels in the NOD mouse. Alternatively, induced models of autoimmune disease, such as experimental allergic encephalitis (EAE) , can be administered ZSMF-7 polypeptides. Administration in a preventive or intervention mode can be followed by monitoring the clinical symptoms of EAE. In addition, ZSMF-7 polypeptides can be tested in vivo in animal models for cancer, where suppression or apoptosis of introduced tumor cells can be monitored following administration of ZSMF-7.

The present invention also provides reagents for use in diagnostic applications. For example, the ZSMF-7 gene, a probe comprising ZSMF-7 DNA or RNA, or a subsequence thereof can be used to determine if the ZSMF-7 gene is present on chromosome 15 or if a mutation has occurred. Detectable chromosomal aberrations at the ZSMF-7 gene locus include, but are not limited to, aneuploidy, 54

gene copy number changes, insertions, deletions, restriction site changes and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level. Deletion of the region 3p21, associated with human semaphorin III/F (also known as human semaphorin IV) , is correlated with small cell lung cancer (Roche et al . , Oncocrene 12:1289-97, 1996 and Xiang et al . , Genomics 32:39-48, 1996).

In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product . A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ ID NO:l, or an RNA equivalent thereof. Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction

(Barany, PCR Methods and Applications 1:5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ; Ausubel et . al . , ibid. ; Marian, Chest 108_.: 255-65, 1995). Ribonuclease protection assays (see, e.g., Ausubel et al . , ibid., ch. 4) comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction 55

product (RNA-RNA hybrid) is exposed to RNase . Hybridized regions of the RNA are protected from digestion. Within

PCR assays, a patient's genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient. Another PCR-based technique that can be employed is single strand conformational polymorphism

(SSCP) analysis (Hayashi, PCR Methods and Applications 1:34-8, 1991) .

As a ligand, the activity of ZSMF-7 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 Cytosensor™ Microphysiometer (Molecular Devices, Sunnyvale, CA) . A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell et al., Science ^57:1906-12, 1992; Pitchford et al . , Meth. Enzymol. 228:84-108, 1997; Arimilli et al . , J. Immunol. Meth. 212:49-59, 1998; Van Liefde et al . , Eur . J. Pharmacol . 346 : 87-95, 1998. The microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including ZSMF-7 polypeptide, its agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a ZSMF-7-responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to ZSMF-7 polypeptide. ZSMF-7-responsive eukaryotic cells comprise cells into which a receptor for 56

ZSMF-7 has been transfected creating a cell that is responsive to ZSMF-7; or cells naturally responsive to ZSMF-7 such as cells derived from neurological, endrocrinological or tumor tissue. Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to ZSMF-7 polypeptide, relative to a control not exposed to ZSMF-7, are a direct measurement of ZSMF-7- modulated cellular responses. Moreover, such ZSMF-7- modulated responses can be assayed under a variety of stimuli. Using the microphysiometer, there is provided a method of identifying agonists of ZSMF-7 polypeptide, comprising providing cells responsive to a ZSMF-7 polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change extracellular acidification rate. Moreover, culturing a third portion of the cells in the presence of ZSMF-7 polypeptide and the absence of a test compound can be used as a positive control for the ZSMF-7-responsive cells, and as a control to compare the agonist activity of a test compound with that of the ZSMF-7 polypeptide. Moreover, using the microphysiometer, there is provided a method of identifying antagonists of ZSMF-7 polypeptide, comprising providing cells responsive to a ZSMF-7 polypeptide, culturing a first portion of the cells in the presence of ZSMF-7 and the absence of a test compound, culturing a second portion of the cells in the presence of ZSMF-7 and the presence of a test compound, and detecting a change, for example, an increase or a diminution in a cellular 57

response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change extracellular acidification rate. Antagonists and agonists, for ZSMF-7 polypeptide, can be rapidly identified using this method.

Moreover, ZSMF-7 can be used to identify cells, tissues, or cell lines which respond to a ZSMF-7-stimulated pathway. The microphysiometer, described above, can be used to rapidly identify ligand-responsive cells, such as cells responsive to ZSMF-7 of the present invention. Cells can be cultured in the presence or absence of ZSMF-7 polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of ZSMF-7 are responsive to ZSMF-7. Such cell lines, can be used to identify antagonists and agonists of ZSMF-7 polypeptide as described above.

ZSMF-7 polypeptides can also be used to identify inhibitors (antagonists) of its activity. ZSMF-7 antagonists include anti-ZSMF-7 antibodies and soluble ZSMF-7 receptors, as well as other peptidic and non- peptidic agents (including ribozymes) . Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of ZSMF-7. In addition to those assays disclosed herein, samples can be tested for inhibition of ZSMF-7 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of ZSMF-7-dependent cellular responses. For example, ZSMF-7-responsive cell lines can be transfected with a reporter gene construct that is responsive to a ZDMF- 7-stimulated cellular pathway.

Reporter gene constructs of this type are known in the art, and will generally comprise a ZSMF-7-DNA response element operably linked to a gene encoding an assayable protein, such as luciferase. DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE) , hormone response elements (HRE) insulin response element (IRE) (Nasrin et al . , Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al . Cell 56: 563-72, 1989). Cyclic AMP response elements are reviewed in Roestler et al . , J. Biol . Chem. 263 (19):9063- 6; 1988 and Habener, Molec . Endocrinol . 4 (8) : 1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 56_.: 335-44; 1989. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of ZSMF-7 on the target cells as evidenced by a decrease in ZSMF-7 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block ZSMF-7 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of ZSMF-7 binding to receptor using ZSMF-7 tagged with a detectable label (e.g., ¹²⁵I, biotin, horseradish peroxidase, FITC, and the like) . Within assays of this type, the ability of a test sample to inhibit the binding of labeled ZSMF-7 to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays . Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors. ZSMF-7 antagonists would find use to modulate or down regulate one or more detrimental biological processes in cells, tissues and/or biological fluids, such as over- responsiveness, unregulated or inappropriate growth, and inflammation or allergic reaction. ZSMF-7 antagonists would have beneficial therapeutic effect in diseases where the inhibition of activation of certain B lymphocytes and/or T cells would be effective. In particular, such diseases would include autoimmune diseases, such as multiple sclerosis, insulin-dependent diabetes and systemic lupus erythematosus . Also, benefit would be derived from using ZSMF-7 antagonists for chronic inflammatory and 59

infective diseases. Antagonists could be used to dampen or inactivate ZSMF-7 during activated immune response.

The activity of semaphorin polypeptides, agonists, antagonists and antibodies of the present invention can be measured, and compounds screened to identify agonists and antagonists, using a variety of assays, such as assays that measure axon guidance and growth. Of particular interest are assays that indicate changes in neuron growth patterns, see for example, Hastings, WIPO Patent Application No: 97/29189 and Walter et al . , Development 101 : 685-96 , 1987. Assays to measure the effects of semaphorins on neuron growth are well known in the art. For example, the C assay (see for example, Raper and Kapfhammer, Neuron 4:21-9, 1990 and Luo et al . , Cell 75:217-27, 1993), can be used to determine collapsing activity semaphorins on growing neurons. Other methods which assess semaphorin induced inhibition of neurite extension or divert such extension are also known, see Goodman, Annu . Rev. Neurosci. 19:341-77, 1996. Conditioned media from cells expressing a semaphorin, semaphorin agonist or semaphorin antagonist, or aggregates of such cells, can by placed in a gel matrix near suitable neural cells, such as dorsal root ganglia (DRG) or sympathetic ganglia explants, which have been cocultured with nerve growth factor. Compared to control cells, semaphorin- induced changes in neuron growth can be measured (see for example, Messersmith et al . , Neuron 14:949-59, 1995; Puschel et al . , Neuron 14_.: 941-8, 1995) . Likewise neurite outgrowth can be measured using neuronal cell suspensions grown in the presence of molecules of the present invention see for example, O'Shea et al . , Neuron 7:231-7, 1991 and DeFreitas et al . , Neuron 15 : 333-43 , 1995.

Also available are assay systems that use 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 (BIAcore™, Pharmacia Biosensor, Piscataway, NJ) . As used 60

herein, "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 receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <10⁹ M^_1. Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol . 234 : 554-63 , 1993. A receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding. 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: 660-72, 1949) and calorimetric assays

(Cunningham et al . , Science 253 :545-8, 1991; Cunningham et al., Science 245:821-5, 1991).

Proteins of the present invention may also be assayed using viral delivery systems. Exemplary viruses 61

for this purpose include adenovirus, herpesvirus, 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 a review, see T.C. Becker et al . , Meth. Cell Biol . 41:161-89, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44-53, 1997) . The adenovirus system offers several advantages: adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary) . When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.

The adenovirus system can also be used for protein production in vi tro . By culturing adenovirus- infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the 62

adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al., Cytotechnol . 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293S cell production protocol, non- secreted proteins may also be effectively obtained.

ZSMF-7 polypeptides can also be used to prepare antibodies that specifically bind to ZSMF-7 polypeptides. As used herein, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like, including genetically engineered antibodies . For particular uses, it may be desirable to prepare fragments of anti-ZSMF-7 antibodies. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. As an illustration, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')₂. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. patent No. 4,331,647, Nisonoff et al . , Arch Biochem. Biophys. 89:230, 1960, Porter, Biochem. J. 73:119, 1959, 63

Edelman et al . , in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan, ibid.

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

For example, Fv fragments comprise an association of V_H and V_L chains. This association can be noncovalent, as described by Inbar et al . , Proc. Natl. Acad. Sci. USA _69:2659, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as gluteraldehyde (see, for example, Sandhu, Crit. Rev. Biotech. 12:437, 1992).

The Fv fragments may comprise V_H and V_L chains which are connected by a peptide linker. These single- chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V_H and V_L domains which are connected by an oligonucleotide . The structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E . coli . The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described, for example, by Whitlow et al . , Methods : A Companion to Methods in Enzymology 2_:97, 1991, also see, Bird et al . , Science 242:423, 1988, Ladner et al., U.S. Patent No. 4,946,778, Pack et al . , Bio/Technology 11:1271, 1993, and Sandhu, supra .

As an illustration, a scFV can be obtained by exposing lymphocytes to ZSMF-7 polypeptide in vi tro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ZSMF-7 protein or peptide) . Genes encoding polypeptides having potential ZSMF-7 polypeptide binding domains can be obtained by screening random peptide 64

libraries displayed on phage (phage display) or on bacteria, such as E . coli . Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al . , U.S. Patent No. 5,223,409, Ladner et al . , U.S. Patent No. 4,946,778, Ladner et al . , U.S. Patent No. 5,403,484, Ladner et al., U.S. Patent No. 5,571,698, and Kay et al . , Phage Display of Peptides and Proteins (Academic Press, Inc. 1996) ) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA) , Invitrogen Inc. (San Diego, CA) , New England Biolabs, Inc. (Beverly, MA), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ) . Random peptide display libraries can be screened using the ZSMF-7 sequences disclosed herein to identify proteins which bind to ZSMF-7.

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR) . CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody- producing cells (see, for example, Larrick et al., Methods : A Companion to Methods in Enzymology 2_:106, 1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al . (eds.), page 166 (Cambridge University Press 1995) , and Ward et al . , "Genetic Manipulation and Expression of Antibodies," 65

in Monoclonal Antibodies: Principles and Applications, Birch et al . , (eds.), page 137 (Wiley-Liss, Inc. 1995)).

Non-human antibodies can 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). In some instances, 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. One skilled in the art can generate humanized antibodies with specific and different constant domains (i.e., different Ig subclasses) to facilitate or inhibit various immune functions associated with particular antibody constant domains. 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 . Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to ZSMF-7 polypeptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ZSMF-7 polypeptide) .

Antibodies are determined to be specifically binding if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with related polypeptide molecules. Antibodies herein specifically bind if they bind to a human ZSMF-7 polypeptide, peptide or epitope with a binding affinity

(K_a) of 10⁶ mol^"1 or greater, preferably 10⁷ mol^"1 or 66

greater, more preferably 10 mol^" or greater, and most preferably 10 mol^" 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, ibid. ) . Antibodies of the current invention do not significantly cross-react with related polypeptide molecules, for example, if they detect ZSMF-7 but not known related polypeptides using a standard Western blot analysis (Ausubel et al . , ibid.). Examples of known related polypeptides are orthologs; proteins from the same species that are members of a protein family such as other known semaphorins (Sema A-Sema G, Sema IV and CD 100) ; mutant semaphorin polypeptides; and non-human semaphorins (G Sema I, D Sema I and II and T Sema I) . Moreover, antibodies may be "screened against" known related polypeptides to isolate a population that specifically binds to the inventive polypeptides. For example, antibodies raised to ZSMF-7 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to ZSMF-7 will flow through the matrix under the proper buffer conditions. Such screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to closely related polypeptides

(Antibodies: A Laboratory Manual , Harlow and Lane (eds.),

Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al . (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art (see, Fundamental Immunology. Paul (eds.), Raven Press, 1993; Getzoff et al . , Adv . in Immunol . 43_.: 1- 98, 1988; Monoclonal Antibodies: Principles and Practice , Goding, J.W. (eds.), Academic Press Ltd., 1996; Benjamin et al., Ann . Rev . Immunol . 2:67-101, 1984).

Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Hurrell, Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982) . As would be evident to one of ordinary skill in the art, 67

polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity of a ZSMF-7 polypeptide can 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 a ZSMF-7 polypeptide 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.

Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to ZSMF-7 polypeptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ZSMF-7 polypeptide) .

Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-ZSMF-7 antibodies or antibody fragments, using standard techniques. See, for example, Green et al . , "Production of Polyclonal Antisera," in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan, ibid. at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can be prepared using anti-ZSMF-7 antibodies or antibody fragments as immunogens with the techniques, described above. As another alternative, humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques. Methods for producing anti- idiotype antibodies are described, for example, by Irie, U.S. Patent No. 5,208,146, Greene, et. al . , U.S. Patent No. 68

5,637,677, and Varthakavi and Minocha, J. Gen. Virol. 77:1875, 1996.

A variety of assays known to those skilled in the art can be utilized to detect antibodies that specifically bind to ZSMF-7 polypeptides. 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, radio- immunoassays, radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA) , dot blot assays, Western blot assays, inhibition or competition assays, and sandwich assays. In addition, antibodies can be screened for binding to wild-type versus mutant ZSMF-7 protein or peptides.

Antibodies to ZSMF-7 can be used for affinity purification of ZSMF-7 polypeptides; within diagnostic assays for determining circulating levels of ZSMF-7 polypeptides; for detecting or quantitating soluble ZSMF-7 polypeptide as a marker of underlying pathology or disease; for immunolocalization within whole animals or tissue sections, including immunodiagnostic applications; for immunohistochemistry; and as antagonists to block protein activity in vi tro and in vivo . Antibodies to ZSMF-7 can also be used for tagging cells that express ZSMF-7; for affinity purification of ZSMF-7 polypeptides; in analytical methods employing FACS; for screening expression libraries; and for generating anti-idiotypic antibodies. Antibodies can be linked to other compounds, including therapeutic and diagnostic agents, using known methods to provide for targeting of those compounds to cells expressing receptors for ZSMF-7. For certain applications, including in vi tro and in vivo diagnostic uses, it is advantageous to employ labeled antibodies. Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors , inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature 69

use of biotin-avidin or other complement/anti-complement pairs as intermediates. Antibodies of the present invention 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 .

Genes encoding polypeptides having potential

ZSMF-7 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli .

Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al . , US Patent NO. 5,223,409; Ladner et al . , US Patent NO. 4,946,778; Ladner et al . , US Patent NO. 5,403,484 and Ladner et al . , US Patent NO. 5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA) , Invitrogen Inc. (San Diego, CA) , New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ) . Random peptide display libraries can be screened using the ZSMF-7 sequences disclosed herein to identify proteins which bind to ZSMF-7. These "binding proteins" which interact with ZSMF-7 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays 70

for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. These binding proteins can also act as ZSMF-7 "antagonists" to block ZSMF-7 binding and signal transduction in vi tro and in vivo . These anti-ZSMF-7 binding proteins would be useful for inhibiting ZSMF-7 binding.

ZSMF-7 polypeptides and polynucleotides may be used within diagnostic systems. Antibodies or other agents that specifically bind to ZSMF-7 may be used to detect the presence of circulating ligand or receptor polypeptides.

Such detection methods are well known in the art and include, for example, enzyme-linked immunosorbent assay

(ELISA) and radioimmunoassay . Immunohistochemically labeled ZSMF-7 antibodies can be used to detect ZSMF-7 receptor and/or ligands in tissue samples and identify ZSMF-7 receptors. ZSMF-7 levels can also be monitored by such methods as RT-PCR, where ZSMF-7 mRNA can be detected and quantified. The information derived from such detection methods would provide insight into the significance of

ZSMF-7 polypeptides in various diseases and biological processes, and as a would serve as diagnostic tools for diseases for which altered levels of ZSMF-7 are significant . Nucleic acid molecules disclosed herein can be used to detect the expression of a ZSMF-7 gene in a biological sample. Such probe molecules include double- stranded nucleic acid molecules comprising the nucleotide sequences of SEQ ID NOs : 1 or 5, or fragments thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequences of SEQ ID NOs : 1 or 5, or a fragment thereof. Probe molecules may be DNA, RNA, oligonucleotides, and the like.

As an illustration, suitable probes include nucleic acid molecules that bind with a portion of a ZSMF-7 domain or motif, such as the ZSMF-7 semaphorin domain 71

(nucleotides 69-541 of SEQ ID NO : 1 or nucleotides 205-1623 of SEQ ID NO: 5). Other probes include those to the Ig-like domain .

In a basic assay, a single-stranded probe molecule is incubated with RNA, isolated from a biological sample, under conditions of temperature and ionic strength that promote base pairing between the probe and target ZSMF-7 RNA species. After separating unbound probe from hybridized molecules, the amount of hybrids is detected. Well-established hybridization methods of RNA detection include northern analysis and dot/slot blot hybridization, see, for example, Ausubel ibid, and Wu et al . (eds.), "Analysis of Gene Expression at the RNA Level," in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc. 1997), and methods described herein. Nucleic acid probes can be detectably labeled with radioisotopes such as

³²P or ³⁵S. Alternatively, ZSMF-7 RNA can be detected with a nonradioactive hybridization method (see, for example, Isaac

(ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Humana Press, Inc., 1993). Typically, nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates. Illustrative non-radioactive moieties include biotin, fluorescein, and digoxigenin. ZSMF-7 oligonucleotide probes are also useful for in vivo diagnosis. As an illustration, ¹⁸F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al., Nature Medicine 4:467, 1998). Numerous diagnostic procedures take advantage of the polymerase chain reaction (PCR) to increase sensitivity of detection methods. Standard techniques for performing PCR are well-known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols; Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), 72

Hanausek and Walaszek (eds.), Tumor Marker Protocols

(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in

Bioanalysis (Humana Press, Inc. 1998)). PCR primers can be designed to amplify a sequence encoding a particular

ZSMF-7 domain or motif, such as the ZSMF-7 semaphorin domain (nucleotides 69-541 of SEQ ID N0:1 or nucleotides

205-1623 of SEQ ID NO:5).

One variation of PCR for diagnostic assays is reverse transcriptase-PCR (RT-PCR) . In the RT-PCR technique, RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with ZSMF-7 primers (see, for example, Wu et al . (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR," in Methods in Gene Biotechnology, CRC Press, Inc., pages 15-28, 1997). PCR is then performed and the products are analyzed using standard techniques.

As an illustration, RNA is isolated from biological sample using, for example, the guanidinium- thiocyanate cell lysis procedure described herein. Alternatively, a solid-phase technique can be used to isolate mRNA from a cell lysate. A reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or ZSMF-7 anti- sense oligomers . Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences. ZSMF-7 sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically at least 5 bases in length.

PCR amplification products can be detected using a variety of approaches. For example, PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining. Alternatively, fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled ZSMF-7 probe, and examined by 73

autoradiography . Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay. Another approach is real time quantitative PCR

(Perkin-Elmer Cetus, Norwalk, Ct . ) . A fluorogenic probe, consisting of an oligonucleotide with both a reporter and a quencher dye attached, anneals specifically between the forward and reverse primers. Using the 5' endonuclease activity of Taq DNA polymerase, the reporter dye is separated from the quencher dye and a sequence-specific signal is generated and increases as amplification increases. The fluorescence intensity can be continuously monitored and quantified during the PCR reaction. Another approach for detection of ZSMF-7 expression is cycling probe technology (CPT) , in which a single-stranded DNA target binds with an excess of DNA-RNA- DNA chimeric probe to form a complex, the RNA portion is cleaved with RNase H, and the presence of cleaved chimeric probe is detected (see, for example, Beggs et al . , J. Clin. Microbiol . 34:2985, 1996 and Bekkaoui et al . , Biotechniques 20:240, 1996) . Alternative methods for detection of ZSMF-7 sequences can utilize approaches such as nucleic acid sequence-based amplification (NASBA) , cooperative amplification of templates by cross-hybridization (CATCH) , and the ligase chain reaction (LCR) (see, for example, Marshall et al . , U.S. Patent No. 5,686,272 (1997), Dyer et al., J. Virol. Methods 60:161, 1996; Ehricht et al . , Eur . J. Biochem. 243:358, 1997 and Chadwick et al . , J. Virol. Methods 7_0:59, 1998). Other standard methods are known to those of skill in the art.

ZSMF-7 probes and primers can also be used to detect and to localize ZSMF-7 gene expression in tissue samples. Methods for such in situ hybridization are well- known to those of skill in the art (see, for example, Choo

(ed.), In Situ Hybridization Protocols, Humana Press, Inc.,

1994; Wu et al . (eds.), "Analysis of Cellular DNA or 74

Abundance of mRNA by Radioactive In Si tu Hybridization (RISH)," in Methods in Gene Biotechnology, CRC Press, Inc., pages 259-278, 1997 and Wu et al . (eds.), "Localization of DNA or Abundance of mRNA by Fluorescence In Si tu Hybridization (RISH)," in Methods in Gene Biotechnology, CRC Press, Inc., pages 279-289, 1997).

Various additional diagnostic approaches are well-known to those of skill in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics Humana Press, Inc., 1991; Coleman and Tsongalis, Molecular Diagnostics, Humana Press, Inc., 1996 and Elles, Molecular Diagnosis of Genetic Diseases, Humana Press, Inc., 1996) .

The ZSMF-7 polynucleotides and/or polypeptides disclosed herein can be useful as therapeutics, wherein ZSMF-7 agonists and antagonists could modulate one or more biological processes in cells, tissues and/or biological fluids. ZSMF-7 antagonists provided by the invention, bind to ZSMF-7 polypeptides or, alternatively, to a receptor to which ZSMF-7 polypeptides bind, thereby inhibiting or eliminating the function of ZSMF-7. Such ZSMF-7 antagonists would include antibodies; oligonucleotides which bind either to the ZSMF-7 polypeptide or to its ligand; natural or synthetic analogs of ZSMF-7 ligands which retain the ability to bind the receptor but do not result in either ligand or receptor signaling. Such analogs could be peptides or peptide-like compounds. Natural or synthetic small molecules which bind to ZSMF-7 polypeptides and prevent signaling are also contemplated as antagonists. As such, ZSMF-7 antagonists would be useful as therapeutics for treating certain disorders where blocking signal from either a ZSMF-7 receptor or ligand would be beneficial.

The invention also provides nucleic acid-based therapeutic treatment. If a mammal lacks or has a mutated ZSMF-7 gene, the ZSMF-7 gene can be introduced into the cells of the mammal. Using such methods, cells altered to 75

express the nerve growth factor neurotrophin-3 (NT-3) were grafted to a rat model for spinal injury and stimulated axon regrowth at the lesion site and the rats thus treated recovered some ability to walk (Grill et al . , J__^ Neuroscience 17: 5560-72, 1997) . In one embodiment, a gene encoding a ZSMF-7 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV) , papillomavirus, Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV) , and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell . Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al . , Molec . Cell. Neurosci. 2_.:320-30, 1991), an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al . (J. Clin. Invest. _.90:626-30, 1992), and a defective adeno- associated virus vector (Samulski et al . , J. Virol. 61:3096-101, 1987; Samulski et al . , J. Virol. 63.-3822-8,

1989) . In another embodiment, the gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al . , Cell 33 :153,

1983; Temin et al . , U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al . , J. Virol. 62:1120, 1988; Temin et al . , U.S. Patent No. 5,124,263; Dougherty et al . , WIPO Publication WO 95/07358; and Kuo et al., Blood 82:845-52, 1993.

Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al . , Proc. Natl. Acad. Sci. USA .84:7413-7, 1987; and Mackey et 76

al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically. It is possible to remove the cells from the body and introduce the vector as a naked DNA plasmid and then re-implant the transformed cells into the body. Naked DNA vector for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter (see, for example, Wu et al . , J. Biol . Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263.-14621-4, 1988). Another aspect of the present invention involves antisense polynucleotide compositions that are complementary to a segment of the polynucleotide set forth in SEQ ID N0:1. Such synthetic antisense oligonucleotides are designed to bind to mRNA encoding ZSMF-7 polypeptides and to inhibit translation of such mRNA. Such antisense oligonucleotides are used to inhibit expression of ZSMF-7 polypeptide-encoding genes in cell culture or in a subject.

Transgenic mice, engineered to express the ZSMF-7 gene, and mice that exhibit a complete absence of ZSMF-7 gene function, referred to as "knockout mice" (Snouwaert et al . , Science 257:1083, 1992), may also be generated (Lowell et al . , Nature 366.: 740-2, 1993). These mice may be 77

employed to study the ZSMF-7 gene and the protein encoded thereby in an in vivo system.

For pharmaceutical use, the proteins 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. In general, pharmaceutical formulations will include a ZSMF-7 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. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed. , 1995. Determination of dose is within the level of ordinary skill in the art.

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

78

EXAMPLES

Example 1 Identification of ZSMF-7

Novel ZSMF-7 encoding polynucleotides and polypeptides of the present invention were initially identified by querying an EST database for sequences homologous to conserved motifs within the semaphorin family. Expressed sequence tags (ESTs) from human retina, human placenta and human fibroblasts cDNA libraries that corresponded the 5' end of the gene were identified.

To obtain the complete cDNA sequence of ZSMF-7, a human testis library was screened. The construction of the cDNA libraries is known in the art and such libraries may be purchased from commercial suppliers such as Clontech Laboratories, Inc. (Palo Alto, CA) . The library was plated in pools of 5000 colonies/pool. Plasmid DNA was prepared from the plated bacteria using a Qiagen^* plasmid purification column (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's instructions. DNA from these pools were combined into larger pools. Oligonucleotides ZC16,189 (SEQ ID NO: 24) and ZC16188 (SEQ ID NO: 25) were designed from an incomplete clone obtained from a human placenta library for use as PCR primers. Using the pooled human testis library DNA as a template, amplification was carried out as follows: 35 cycles of 94°C for 30 seconds, 58°C for 30 seconds and 72°C for 30 seconds. Positive clones were identified by the presence of a 583 bp PCR fragment (SEQ ID NO: 26) . Two pools of 5000 colonies were found to contain this fragment. These pools were used to transform E. coli which were plated to agar. The colonies were transferred to nylon membrane and probed with the 583 bp PCR fragment (SEQ ID NO: 26) . The fragment was gel purified using a Qiaquick kit (Qiagen, Inc., Chatsworth, CA) and radioactively labeled using the random priming MULTIPRIME DNA labeling system (Amersham, Arlington 79

Heights, IL) , according to the manufacturer's specifications. The probe was purified using a NUCTRAP push column (Stratagene, La Jolla, CA) . ExpressHyb (Clontech) solution was used for prehybridization and as a hybridizing solution for the colony lifts. The filters were hybridized with the labeled probe at 65°C, overnight, and then washed with an SSC/SDS buffer under appropriately stringent conditions and positive colonies detected upon exposure to film. Plasmid DNA from colonies producing signal was then isolated and submitted for sequence analysis. The plasmid DNA from a positive colony was used as template and oligos ZC694 (SEQ ID NO: 8) and ZC2681 (SEQ ID NO: 22) to the vector were used as sequencing primers. Oligonucleotides ZC16820 (SEQ ID NO:9), ZC16087 (SEQ ID NO:10), ZC16818 (SEQ ID NO: 11), ZC15394 (SEQ ID NO:12),

ZC16819 (SEQ ID NO:13), ZC16460 (SEQ ID NO:14), ZC16548

(SEQ ID NO:15), ZC16807 (SEQ ID NO:16), ZC16806 (SEQ ID

NO:17), ZC16667 (SEQ ID NO:18), ZC16729 (SEQ ID NO:19),

ZC16728 (SEQ ID NO: 20) and ZC16666 (SEQ ID NO: 21) were used to complete the sequence. Sequencing reactions were carried out in a Hybaid OmniGene Temperature Cycling System

(National Labnet Co., Woodbridge, NY) . Sequencher™ 3.0 sequence analysis software (Gene Codes Corporation, Ann

Arbor, MI) was used for data analysis. The resulting 3,377 bp sequence is disclosed in SEQ ID NO:l.

Example 2 Tissue Distribution

Human Multiple Tissue Northern Blots (MTN I, MTN II, and MTN III; Clontech) were probed to determine the tissue distribution of human ZSMF-7 expression. An approximately 234 bp probe (SEQ ID NO: 4) was amplified from a human retina derived Marathon™-ready cDNA library. Oligonucleotide primers ZC14298 (SEQ ID NO: 27) and ZC14299 (SEQ ID NO: 28) were designed based on an EST sequence. The Marathon™-ready cDNA library was prepared according to 80

manufacturer's instructions (Marathon™ cDNA Amplification Kit; Clontech) using human retina poly A+ RNA (Clontech) . The probe was amplified in a polymerase chain reaction as follows: 1 cycle at 94°C for 1 minute,- 35 cycles of 94°C for 30 seconds and 68°C for 1 minute 30 seconds, followed by 1 cycle at 72°C for 10 minutes. The resulting DNA fragment was electrophoresed on a 2% low melt agarose gel (SEA PLAQUE GTG low melt agarose, FMC Corp., Rockland, ME), the fragment was purified using the QIAquick™ method (Qiagen, Chatsworth, CA) , and the sequence was confirmed by sequence analysis. The probe was radioactively labeled and purified as described herein. ExpressHyb™ (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 65°C using 1.0 x 10⁶ cpm/ml of labeled probe. The blots were then washed 4 times at room temperature in 2X SSC, 0.05% SDS followed by 2 washes at 50°C in 0. IX SSC, 0.01% SDS for 20 minutes each. A transcript of approximately 4.0 kb was seen in testis, spleen, spinal cord and placenta, a weak signal was detected in brain, thymus, ovary, lymph node and bone marrow.

Additional analysis was carried out on Northern blots made with poly (A) RNA from the human vascular cell lines HUVEC (human umbilical vein endothelial cells; Cascade Biologies, Inc., Portland, OR), HPAEC (human pulmonary artery endothelial cells; Cascade Biologies, Inc.), HAEC (human aortic endothelial cells; Cascade Biologies, Inc.), AoSMC (aortic smooth muscle cells; Clonetics, San Diego, CA) , UASMC (umbilical artery smooth muscle cells; Clonetics) , HISM (human intestinal smooth muscle cells; ATCC CRL 7130), SK-5 (human dermal fibroblast cells; obtained from Dr. Russell Ross, University of Washington, Seattle, WA) , NHLF (normal human lung fibroblast cells; Clonetics) , and NHDF-NEO (normal human dermal fibroblast-neonatal cells; Clonetics) . The probe was prepared and labeled and prehybridization and hybridization were carried out essentially as disclosed above. The blots 81

were then washed at 50°C in 0. IX SSC, 0.05% SDS. A transcript of approximately 4.0 kb was seen in was seen in VASMC, AoSMC, SK-5, NHLF and NHDF-Neo cells. Signal intensity was highest in NHLF cells. Additional analysis was carried out on Northern blots made with poly (A) RNA from K-562 cells (erythroid, ATCC CCL 243), HUT78 cells (T cell, ATCC TIB-161) , Jurkat cells (T cell), DAUDI (Burkitt ' s human lymphoma, Clontech, Palo Alto, CA) , RAJI (Burkitt 's human lymphoma, Clontech) and HL60 (Monocyte) . The probe preparation and hybridization were carried out as above. Two transcripts, approximately, ~4.5 and 4.0, were seen in DAUDI, RAJI, JRUKAT, HUT78 and HL60 cells. Signal intensity was highest in RAJI and JURKAT. Additional analysis was carried out on Northern blots made with poly (A) RNA from CD4⁺, CD8⁺, CD19⁺ and mixed lymphocyte reaction cells (CellPro, Bothell, WA) using probes and hybridization conditions described above. A transcript of approximately 4.0 kb was seen in the mixed lymphocytes and CD19+ cells. Signal intensity was highest in the mixed lymphocyte cells.

Additional analysis was carried out on Human Brain Multiple Tissue Northern Blots II and III (Clontech) using the probe and hybridization conditions described above. A transcript of 4.0 kb was seen in all tissue tested.

Example 3 Chromosomal Assignment and Placement of ZSMF-7

ZSMF-7 was mapped to chromosome 15 using the commercially available GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL) . The GeneBridge 4 Radiation Hybrid Panel contains PCRable DNAs 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.pl) allows mapping relative to the Whitehead 82

Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the "WICGR" radiation hybrid map) which was constructed with the GeneBridge 4 Radiation

Hybrid Panel . For the mapping of ZSMF-7 with the GeneBridge 4

RH Panel, 20 μl reactions were set up in a 96-well microtiter plate (Stratagene, La Jolla, CA) and used in a

RoboCycler Gradient 96 thermal cycler (Stratagene) . Each of the 95 PCR reactions consisted of 2 μl 10X KlenTaq PCR reaction buffer (Clontech), 1.6 μl dNTPs mix (2.5 mM each,

PERKIN-ELMER, Foster City, CA) , 1 μl sense primer, ZC 16086

(SEQ ID NO:6), 1 μl antisense primer, ZC 16,085 (SEQ ID

NO-.7), 2 μl . e iLoad (Research Genetics, Inc.), 0.4 μl 50X

Advantage KlenTaq Polymerase Mix (Clontech) , 25 ng of DNA from an individual hybrid clone or control and ddH₂0 for a total volume of 20 μl . The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 95°C, 35 cycles of a 1 minute denaturation at 95°C, 1 minute annealing at 66°C and 1.5 minute 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 (Life Technologies, Gaithersburg, MD) .

The results showed that ZSMF-7 maps 3.98 cR_3000 from the framework marker CHLC.GATA85D02 on the WICGR radiation hybrid map. Proximal and distal framework markers were CHLC.GATA85D02 and CHLC.GCT7C09, respectively. The use of surrounding markers positions ZSMF-7 in the 15q24.3 region on the integrated LDB chromosome 15 map (The Genetic Location Database, University of Southhampton, WWW server: http: //cedar . genetics.soton.ac.uk/public_html/) .

Example 4 ZSMF-7 Anti-peptide Antibodies

Polyclonal anti-peptide antibodies were prepared by immunizing two female New Zealand white rabbits and 5 83

mice with the peptide, huzsmf7-2 NIGSTKGSCLDKRDC ENYITLLERRSEGLLACGTNA (SEQ ID NO: 35) from the N-terminal region of the semaphorin domain or huzsmf7-3 SINPAEPHKECPNPKPDKC (SEQ ID NO: 36) from the C-terminal portion of the semaphorin domain. The peptides were synthesized using an Applied Biosystems Model 431A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA) according to manufacturer's instructions. The peptides were then conjugated to the carrier protein maleimide- activated keyhole limpet hemocyanin (KLH) . The rabbits were each given an initial intraperitoneal (ip) injection of 200 μg of peptide in Complete Freund's Adjuvant followed by booster ip injections of 100 μg peptide in Incomplete Freund's Adjuvant every three weeks. Seven to ten days after the administration of the second booster injection, the animals were bled and the serum was collected. The animals were then boosted and bled every three weeks.

The mice were each given an initial ip injection of 20 μg of peptide in Complete Freund's Adjuvant followed by booster ip injections of 10 μg peptide in Incomplete Freund's Adjuvant every two weeks. Seven to ten days after the administration of the second booster injection, the animals were bled and the serum was collected. Than animals were then boosted and bled every three weeks. The ZSMF-7 peptide-specific seras were characterized by an ELISA titer check using 1 μg/ml of the peptide used to make the antibody (SEQ ID NOs: 35 and 36) as an antibody target. All 5 mouse seras to huzsmf7-2 and huzsmf7-3 have titer to their specific peptides at a dilution of 1 x 10⁵. A single rabbit sera to huzsmf7-2 had titer to its specific peptide at a dilution of 1 x 10^s and to recombinant full-length protein at a dilution of 1 x 10^s.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit 84

and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

85CLAIMS We claim:

1. An isolated semaphorin polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 69 through 541 of SEQ ID NO : 2 , said polypeptide comprising cysteine residues at positions corresponding to residues 126, 143, 152, 266, 291, 335, 366, 493, 500, 503, 511, 518 and 541 of SEQ ID NO : 2.

2. An isolated semaphorin polypeptide according to claim 1, wherein said sequence of amino acid residues is at least 90% identical.

3. An isolated semaphorin polypeptide according to claim 1, further comprising an Ig-like domain.

4. An isolated semaphorin polypeptide according to claim 3, wherein said Ig-like domain comprises a sequence of amino acids from residue 561-620 of SEQ ID NO : 2.

5. An isolated semaphorin polypeptide according to claim 1, wherein said polypeptide comprises residues 45-666 of SEQ ID NO: 2.

6. An isolated semaphorin polypeptide according to claim 1, wherein said sequence of amino acid residues comprises residues 1-666 of SEQ ID NO: 2.

7. An isolated semaphorin polypeptide according to claim 1, wherein said sequence of amino acid residues is from 473-624 amino acid residues.

8. An isolated semaphorin polypeptide selected from the group consisting of: 86

a) a polypeptide comprising a sequence of amino acid residues from amino acid residue 45 to residue 666 of SEQ ID NO: 2; b) a polypeptide comprising a sequence of amino acid residues from amino acid residue 69 to residue 666 of SEQ ID NO: 2; c) a polypeptide comprising a sequence of amino acid residues from amino acid residue 69 to residue 541 of SEQ ID NO: 2 and d) a polypeptide comprising a sequence of amino acid residues from amino acid residue 1 to residue 666 of SEQ ID NO : 2.

9. An isolated semaphorin polypeptide according to claim 1, wherein any difference between said amino acid sequence of said isolated polypeptide and said corresponding amino acid sequence of SEQ ID NO: 2 is due to a conservative amino acid substitution.

10. An isolated semaphorin polypeptide according to claim 1, covalently linked to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores .

11. An isolated semaphorin polypeptide according to claim 10, wherein said moiety is an affinity tag selected from the group consisting of polyhistidine, FLAG, Glu-Glu, glutathione S transferase and an immunoglobulin heavy chain constant region.

12. An isolated semaphorin polypeptide according to claim 11 further comprising a proteolytic cleavage site between said sequence of amino acid residues and said affinity tag. 87

13. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a semaphorin polypeptide according to claim 1; and a transσriptional terminator.

14. An expression vector according to claim 13 further comprising a secretory signal sequence operably linked to said DNA segment .

15. An expression vector according the claim 14, wherein said secretory signal sequence encodes residues 1-44 of SEQ ID N0:2.

16. An expression vector according to claim 13, wherein said sequence of amino acid residues is at least 90% identical .

17. An expression vector according to claim 13, wherein said DNA segment encodes a semaphorin polypeptide comprising an Ig-like domain.

18. An expression vector according to claim 17, wherein said Ig-like domain comprises a sequence of amino acids from residue 561-620 of SEQ ID N0:2.

19. An expression vector according to claim 13, wherein said sequence of amino acid residues comprises residues 45-666 of SEQ ID N0:2.

20. An expression vector according to claim 13, wherein said DNA segment encodes a semaphorin polypeptide covalently linked to an affinity tag selected from the group consisting of polyhistidine, FLAG, Glu-Glu, glutathione S transferase and an immunoglobulin heavy chain constant region. 88

21. A cultured cell into which has been introduced an expression vector according to claim 13, wherein said cell expresses the polypeptide encoded by the DNA segment .

22. A method of producing a semaphorin protein comprising: culturing a cell into which has been introduced an expression vector according to claim 13, whereby said cell expresses said semaphorin protein encoded by said DNA segment ,- and recovering said expressed semaphorin protein.

23. A pharmaceutical composition comprising a polypeptide according to claim 1, in combination with a pharmaceutically acceptable vehicle.

24. An antibody or antibody fragment that specifically binds to an epitope of a semaphorin polypeptide according to claim 1.

25. An antibody according to claim 24, wherein said antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b) ; and d) human monoclonal antibody.

26. An antibody fragment according to claim 25, wherein said antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit.

27. A binding protein that specifically binds to an epitope of a semaphorin polypeptide according to claim 1. 89

28. An anti-idiotype antibody that specifically binds to said antibody of claim 24.

29. An isolated polynucleotide encoding a semaphorin polypeptide according to claim 1.

30. An isolated polynucleotide according to claim 29, wherein said sequence of amino acid residues is at least 90% identical.

31. An isolated polynucleotide according to claim 29, wherein said semaphorin polypeptide comprises an Ig-like domain .

32. An isolated polynucleotide according to claim 31, wherein said Ig-like domain comprises a sequence of amino acids from residue 561-620 of SEQ ID NO: 2.

33. An isolated polynucleotide according to claim 29, wherein said sequence of amino acid residues comprises residues 45-666 of SEQ ID N0:2.

34. An isolated polynucleotide according to claim 29, wherein said sequence of amino acid residues comprises residues 1-666 of SEQ ID NO: 2.

35. An isolated polynucleotide according to claim 29 comprising nucleotide 1 to nucleotide 1998 of SEQ ID NO: 5.

36. An isolated polynucleotide selected from the group consisting of : a) a polynucleotide sequence consisting of the polynucleotide sequence from nucleotide 152 to nucleotide 2017 of SEQ ID NO:l; 90

b) a polynucleotide sequence consisting of the polynucleotide sequence from nucleotide 244 to nucleotide 2017 of SEQ ID N0:1; c) a polynucleotide sequence consisting of the polynucleotide sequence from nucleotide 244 to nucleotide 1640 of SEQ ID N0:1; d) a polynucleotide sequence consisting of the polynucleotide sequence from nucleotide 20 to nucleotide 2017 of SEQ ID NO:l; and e) a complementary polynucleotide sequence of a, b, c or d.

37. A method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:l or the complement of SEQ ID NO:l, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; comparing said first reaction product to a control reaction product, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.