AU737132B2 - Mammalian neuro-growth factor like protein - Google Patents

Description

MAMMALIAN NEURO-GROWTH FACTOR LIKE PROTEIN Background of the Invention Proliferation and differentiation of cells of multicellular organisms are controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair and regenerate damaged tissue. Examples of hormones and growth factors include the steroid hormones estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.

Hormones and growth factors influence cellular metabolism by binding to proteins.

Proteins may be integral membrane proteins that are linked to signalling pathways within the cell, such as second messenger systems. Other classes of proteins are soluble molecules, such as the transcription factors.

1 5 Summary of the Invention 9 According to a first embodiment of the invention, there is provided an isolated polynucleotide which encodes a mammalian Zneul polypeptide wherein said polynucleotide encodes a polypeptide selected from the group SEQ ID Nos: 2-3, 19 and 24 or a polypeptide which is at least 90% identical to the polypeptides of said group and which retain the activity of said polypeptides.

According to a second embodiment of the invention, there is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a chimeric polypeptide, wherein said chimeric 25 polypeptide consists essentially of a first portion and a second portion joined by a peptide bond, said first portion being comprised of a mammalian polypeptide, said polypeptide being the amino acid sequences of SEQ ID Nos: 2-3, 8, 9, 11-16, and 19-24 or a polypeptide which is at least 90% identical to said amino acid sequences and said second portion being a second polypeptide or protein; a transcription terminator.

[I:\DayLib\LIBFF]28603spec.doc:gcc la According to a third embodiment of the invention, there is provided an isolated Zneul polypeptide selected from the group of amino acid sequences consisting of SEQ ID Nos: 2, 3, 19 and 24 or a polypeptide which is at least 90% identical to said polypeptides.

According to a fourth embodiment of the invention, there is provided an antibody, antibody fragment or single-chain antibody that specifically binds to a mammalian polypeptide, said polypeptide being defined by the amino acid sequences of SEQ ID Nos: 2-3, 8, 9, 11-16, and 19-24.

According to a fifth embodiment of the invention, there is provided a method for producing an antibody which binds to a peptide or polypeptide defined by SEQ ID Nos: 2-3, 8, 9, 11-16, and 19-24 or to a peptide or polypeptide which is at least identical to said peptide or polypeptide comprising inoculating an animal with said peptide or polypeptide or with a nucleic acid which encodes said peptide or polypeptide, wherein said animal produces antibodies to said peptide or polypeptide; and isolating said antibody.

According to a sixth embodiment of the invention, there is provided an antiidiotypic antibody, anti-idiotypic antibody fragment or anti-idiotypic single-chain antibody which binds to an antibody, an antibody fragment or single-chain antibody of peptide or polypeptide defined by SEQ ID Nos: 2-3, 8, 9, 11-16 and 19-24 or to a peptide or polypeptide which is at least 90% identical to said peptide or polypeptide.

20 The present invention provides a novel neuro-growth factor like polypeptide called Zneul and related compositions and methods. Within one aspect, the present invention provides an isolated polynucleotide encoding a mammalian polypeptide termed Zneul.

The mature human Zneul polypeptide is comprised of a sequence of amino acids approximately 254 amino acids *ooo °ooo* [I:\DayLi b\LI B FF] 2 8603 spec.doc:gcc WO 98/57983 PCT/US98/12763- 2 long. Amino acid residue 20 of SEQ ID NO: 2, a threonine, is the initial amino acid of the mature polypeptide. Thus, it is believed that amino residues 1-19 comprise a signal sequence, and the mature Zneul polypeptide is represented by the amino acid sequence comprised of residues 20-254.

The mature Zneul polypeptide is further represented by SEQ ID NO: 3. Mouse Zneul is defined by SEQ ID NOs:18 and 19.

Having a signal sequence of amino acid residues 1-23, and the mature mouse Zneul is from 24-278 represented by SEQ ID NO: 24. Within an additional embodiment, the polypeptide further comprises an affinity tag. Within a further embodiment, the polynucleotide is DNA.

Within a second aspect of the invention there is provided an expression vector comprising a transcription promoter; a DNA segment encoding Zneul polypeptide, and a transcription terminator, wherein the promoter, DNA segment, and terminator are operably linked.

Within a third aspect of the invention there is provided a cultured eukaryotic cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a protein polypeptide encoded by the DNA segment.

Within a further aspect of the invention there is provided a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond. The first portion of the chimeric polypeptide consists essentially of a Zneul polypeptide as shown in SEQ ID NO: 2 allelic variants of SEQ ID NO:2; and protein polypeptides that are at least 90% identical to or The second portion of the chimeric polypeptide consists essentially of another polypeptide such as an affinity tag. Within one WO 98/57983 PCT/US98/12763 3 embodiment the affinity tag is an immunoglobulin Fc polypeptide. The invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.

Within an additional aspect of the invention there is provided an antibody that specifically binds to a Zneul polypeptide as disclosed above, and also an antiidiotypic antibody which neutralizes the antibody to a Zneul polypeptide.

In addition to the above, the present invention is also directed domains of the polypeptide including SEQ ID NOs:8, 9, 10, 11, 12, 13, 14, 15, and 16.

An additional embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a Zneul polypeptide having an amino acid sequence described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zneul polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least and more preferably at least 30 to 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention. Specific examples of said polypeptides are defined by the amino acid sequences of SEQ ID NOs:20-23. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule.

Another embodiment of the present invention relates to a method for producing an antibody which binds to a peptide or polypeptide defined by SEQ ID NOs: 2-3,8,

I

WO 98/57983 PCTIUS98/12763 4 9, 11-16, and 19-24 or to a peptide or polypeptide which is at least 90% identical to said peptide or polypeptide comprising inoculating an animal with said peptide or polypeptide or with a nucleic acid which encodes said peptide or polypeptide, wherein said animal produces antibodies to said peptide or polypeptide; and isolating said antibody.

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

DETAILED DESCRIPTION OF THE INVENTION The teachings of all of the references cited herein are incorporated in their entirety by reference.

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 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 WO 98/57983 PCT/US98/12763 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.

"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.

A "polynucleotide" is a single- or doublestranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.

The term "promoter" is used herein for its artrecognized 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 non-coding regions of genes.

A "soluble protein" is a protein polypeptide that is not bound to a cell membrane.

WO 98/57983 PCT/US98/12763 6 Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:1, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5 0 C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are those in which the salt concentration is about 0.02 M or less at pH 7 and the temperature is at least about 60 0 C. As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. Total RNA can be prepared using guanidine HC1 extraction followed by isolation by centrifugation in a CsCl gradient, Chirgwin et al., Biochemistry 18:52-94 (1979). Poly RNA is prepared from total RNA using the method of Aviv and Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412 (1972).

Complementary DNA (cDNA) is prepared from poly(A) RNA using known methods. Polynucleotides encoding Zneul polypeptides are then identified and isolated by, for example, hybridization or PCR.

The polynucleotides of the present invention can be synthesized using DNA synthesizer. Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing WO 98/57983 PCT/US98/12763 7 them. For the production of longer genes (>300 bp), however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. See Glick, Bernard R. and Jack J. Pasternak, Molecular Biotechnology, Principles Applications of Recombinant DNA,(ASM Press, Washington, D.C. 1994), Itakura, K. et al. Synthesis and use of synthetic oligonucleotides. Annu. Rev. Biochem. 53 323- 356 (1984), and Climie, S. et al. Chemical synthesis of the thymidylate synthase gene. Proc. Natl. Acad. Sci. USA 87 :633-637 (1990).

Those skilled in the art will recognize that the sequences disclosed in SEQ ID NOS:1, 2 and 3 represent a single allele of the human. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.

The present invention further provides counterpart proteins and polynucleotides from other species ("species orthologs"). Of particular interest are Zneul polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primates. Species orthologs of the human Zneul protein can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the protein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell WO 98/57983 PCT/US98/12763 8 line. A protein-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human or mouse cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the sequences disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the protein. Similar techniques can also be applied to the isolation of genomic clones. As used and claimed the language "an isolated polynucleotide which encodes a polypeptide, said polynucleotide being defined by SEQ ID NO: 2" includes all allelic variants and species orthologs of the polypeptide of SEQ ID NO:2.

The present invention also provides isolated protein polypeptides that are substantially homologous to the polypeptide of SEQ ID NO: 3 and its species orthologs.

By "isolated" is meant a protein or polypeptide 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. The term "substantially homologous" is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequence shown in SEQ ID NO:2,or its species orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:3,or its species orthologs. Percent sequence identity is determined by conventional methods. See, for example, WO 98/57983 PCT/US98/12763 9 Altschul et al., Bull. Math. Bio. 48: 603-616 (1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blossom 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as: Total number of identical matches x 100 [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences] Table 1 00 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 E -1 0 0 2 -4 2 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 o I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 Lo WO 98/57983 PCT/US98/12763 11 Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.

Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag), such as 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), 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 Pharmacia Biotech, Piscataway, NJ).

Table 2 Conservative amino acid substitutions Basic: arginine lysine histidine Acidic: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine WO 98/57983 PCT/US98/12763 12 Table 2, continued valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis, Cunningham and Wells, Science 244, 1081-1085, (1989); Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, (1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity ligand binding and signal transduction) to identify amino acid residues that are critical to the activity of the molecule. Sites of ligand-protein interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al., Science 255:306-312, (1992); Smith et al., J. Mol. Biol. 224:899-904, (1992); Wlodaver et al., FEBS Lett. 309:59-64, (1992). The identities of essential amino acids can also be inferred from analysis of homologies with related proteins.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer, Science 241:53-57, (1988) or Bowie and Sauer, Proc.

WO 98/57983 PCT/US98/12763 13 Natl. Acad. Sci. USA 86:2152-2156, (1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display, Lowman et al., Biochem. 30:10832-10837, (1991); Ladner et al., U.S.

Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204, and region-directed mutagenesis, Derbyshire et al., Gene 46:145, (1986); Ner et al., DNA 7:127, (1988) Mutagenesis methods as disclosed above can be combined with high-throughput screening methods to detect activity of cloned, mutagenized proteins in host cells.

Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays, which are described below. Mutagenized DNA molecules that encode active proteins or portions thereof ligand-binding fragments) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art can prepare a variety of polypeptides that are substantially homologous to SEQ ID NO:3 or allelic variants thereof and retain the properties of the wild-type protein. As expressed and claimed herein the language, "a polypeptide as defined by SEQ ID NO: 2" includes all allelic variants and species orthologs of the polypeptide.

WO 98/57983 PCT/US98/12763 14 The protein polypeptides of the present invention, including full-length proteins, protein fragments ligand-binding fragments), and fusion polypeptides can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred.

Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989), and Ausubel et al., ibid.

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

Another embodiment of the present invention provides for a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.

WO 98/57983 PCT/US98/12763 The epitope of the this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. A region of a protein to which an antibody can bind is defined as an "antigenic epitope". See for instance, Geysen, H.M. et al., Proc. Natl. Acad Sci. USA 81:3998-4002 (1984).

As to the selection of peptides or polypeptides bearing an antigenic epitope that contain a region of a protein molecule to which an antibody can bind), it is well known in the art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See Sutcliffe, J.G.

et al. Science 219:660-666 (1983) Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer soluble peptides, especially those containing proline residues, usually are effective.

Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention.

Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, WO 98/57983 PCT/US98/12763 16 containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that react with the protein. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents the sequence includes relatively hydrophilic residues and hydrophobic residues are preferably avoided); and sequences containing proline residues are particularly preferred. All of the polypeptides shown in the sequence listing contain antigenic epitopes to be used according to the present invention, however, specifically designed antigenic epitopes include the peptides defined by SEQ ID NOs:20-24.

Polynucleotides, generally a cDNA sequence, of the present invention encode the above-described polypeptides. A cDNA sequence which encodes a polypeptide of the present invention is comprised of a series of codons, each amino acid residue of the polypeptide being encoded by a codon and each codon being comprised of three nucleotides. The amino acid residues are encoded by their respective codons as follows.

Alanine (Ala) is encoded by GCA, GCC, GCG or

GCT;

Cysteine (Cys) is encoded by TGC or TGT; Aspartic acid (Asp) is encoded by GAC or GAT; Glutamic acid (Glu) is encoded by GAA or GAG; Phenylalanine (Phe) is encoded by TTC or TTT; Glycine (Gly) is encoded by GGA, GGC, GGG or

GGT;

Histidine (His) is encoded by CAC or CAT; Isoleucine (Ile) is encoded by ATA, ATC or ATT; Lysine (Lys) is encoded by AAA, or AAG; WO 98/57983 PCT/US98/12763 17 Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT; Methionine (Met) is encoded by ATG; Asparagine (Asn) is encoded by AAC or AAT; Proline (Pro) is encoded by CCA, CCC, CCG or

CCT;

Glutamine (Gln) is encoded by CAA or CAG; Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT; Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT; Threonine (Thr) is encoded by ACA, ACC, ACG or

ACT;

Valine (Val) is encoded by GTA, GTC, GTG or GTT; Tryptophan (Trp) is encoded by TGG; and Tyrosine (Tyr) is encoded by TAC or TAT.

It is to be recognized that according to the present invention, when a cDNA is claimed as described above, it is understood that what is claimed are both the sense strand, the anti-sense strand, and the DNA as double-stranded having both the sense and anti-sense strand annealed together by their respective hydrogen bonds. Also claimed is the messenger RNA (mRNA) which encodes the polypeptides of the present invention, and which mRNA is encoded by the above-described cDNA. A messenger RNA (mRNA) will encode a polypeptide using the same codons as those defined above, with the exception that each thymine(T) is replaced by a uracil nucleotide To direct a Zneul polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of the protein, or WO 98/57983 PCT/US98/12763 18 may be derived from another secreted protein t-PA) or synthesized de novo. The secretory signal sequence is joined to the Zneul DNA sequence in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).

Cultured mammalian cells are preferred hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, Wigler et al., Cell 14:725, (1978); Corsaro and Pearson, Somatic Cell Genetics 7:603, (1981): Graham and Van der Eb, Virology 52:456, (1973), electroporation, Neumann et al., EMBO J. 1:841- 845, (1982), DEAE-dextran mediated transfection, Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, (1987), and liposome-mediated transfection, Hawley-Nelson et al., Focus 15:73, (1993); Ciccarone et al., Focus 15:80, (1993). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No.

CRL 10314), 293, ATCC No. CRL 1573; Graham et al., J. Gen.

Virol. 36:59-72, (1977) and Chinese hamster ovary (e.g.

CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or WO 98/57983 PCT/US98/12763 19 cytomegalovirus. See, U.S. Patent No. 4,956,288.

Other suitable promoters include those from metallothionein genes Patent Nos. 4,579,821 and 4,601,978,and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin.

Selection is carried out in the presence of a neomycintype drug, such as G-418 or the like. Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.

Other drug resistance genes hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used.

Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222; Bang et al., U.S. Patent No. 4,775,624; and WIPO publication WO 94/06463. The use of Agrobacterium rhizogenes as a vector for expressing WO 98/57983 PCT/US98/12763 genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, (1987).

Fungal cells, including yeast cells, and particularly cells of the genus Saccharomyces, can also be used within the present invention, such as for producing protein fragments or polypeptide fusions. Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No.

4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient leucine). A preferred vector system for use in yeast is the POT1 vector system disclosed by Kawasaki et al. (U.S.

Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.

Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S.

Patent No. 4,615,974; and Bitter, U.S. Patent No.

4,977,092 )and alcohol dehydrogenase genes. See also U.S.

Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art.

See, for example, Gleeson et al., J. Gen. Microbiol.

132:3459-3465, (1986) and Cregg, U.S. Patent No.

4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349.

Methods for transforming Acremonium chrysogenum are WO 98/57983 PCT/US98/12763 21 disclosed by Sumino et al., U.S. Patent No. 5,162,228.

Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.

Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or cotransfected into the host cell.

Within one aspect of the present invention, a novel protein is produced by a cultured cell, and the cell is used to screen for a receptor or receptors for the protein, including the natural receptor, as well as agonists and antagonists of the natural ligand.

PROTEIN ISOLATION: Expressed recombinant polypeptides (or chimeric polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable anion exchange media include derivatized dextrans, agarose, cellulose, polyacrylamide, WO 98/57983 PCT/US98/12763 22 specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, NJ) being particularly preferred.

Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, Nhydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art.

Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, (1988).

The polypeptides of the present invention can be isolated by exploitation of their properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins. Briefly, a gel is first charged with divalent WO 98/57983 PCTIUS98/1 2763 23 metal ions to form a chelate, E. Sulkowski, Trends in Biochem. 3:1-7, (1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography, Methods in Enzymol., Vol. 182, "Guide to Protein Purification", M.

Deutscher, Acad. Press, San Diego, (1990), pp.529- 39. Alternatively, a fusion of the polypeptide of interest and an affinity tag polyhistidine, maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification.

Physical Structure of Zneul The Zneul polypeptide shown in SEQ ID NO: 2 has a signal peptide including amino acid residues 1-19. Amino acid residues 20-104 define a hydrophilic domain homologous to an HSMHC3W5A domain, SEQ ID NO: 17, (GenBank No. g1401159). Amino acid residues 105-135 define a domain homologous to an Epidermal Growth Factor (EGF) domain.

Amino acid residues 136-177 define another domain homologous to an EGF domain; and amino acid residues 178- 273 define a domain also homologous to an domain.

However, the first EGF-like domain (EGF1) of Zneul, SEQ ID NO: 9 which corresponds to amino acid residues 105 to 135 of SEQ ID NO: 2, is distinct from any other EGF domain in the prior art. The EGF1 in Zneul is about 56% similar to the HSMHC3W5A_6 domain, its closest human relative.

WO 98/57983 PCT/US98/12763 24 The second EGF-like domain (EGF2) of Zneul, SEQ ID NO: 10 which corresponds to amino acid residues 136 to 177 of SEQ ID NO: 2,is distinct from any other EGF domain in the prior art. EGF2 of Zneul is about 48% similar to PIR S31101 fibrillin, its closest human relative.

The first HSMHC3W5A-like (HSM1) domain of Zneul, SEQ ID NO: 8 which corresponds to amino acid residues 104 of SEQ ID NO: 2. SEQ ID NO: 8 is approximately 38% similar to HSMHC3W5A,its closest human relative.

The second HSMHC3W5A-like domain (HSM2) of Zneul, SEQ ID NO: 11 which corresponds to amino acid residues 178-273 of SEQ ID NO: 2, is distinct from any other polypeptide in the prior art. It is about 32% similar to HSMHC3W5A 6.

Uses The tissue specificity of Zneul expression indicates that Zneul can be used as a growth, maintenance, or differentiation factor in the spinal cord, heart, spleen, testis, thyroid and lymph nodes.

The present invention also provides reagents which will find use in diagnostic applications. For example, the Zneul gene has been mapped on chromosome 9q34.3. A Zneul nucleic acid probe could be used to check for abnormalities in chromosome 9. In a normal chromosome 9, one would predict that a Zneul nucleic acid probe would hybridize to chromosome 9. If the probe does not hybridize to chromosome 9, this would indicate an abnormality in chromosome 9.

Zneul's closest human homolog is HSMHC3W5A a gene in the HLA class III region, which is contained in a WO 98/57983 PCTIUS98/12763 cosmid which contains Notch 4. Zneul is also homologous to Notch 4 in its EGF-like domains. Zneul may be involved in EGF-receptor pathways.

Notch Structure/Function The original member of this gene family was the Drosophila gene Notch which controls cell fate decisions in the development of the peripheral nervous system.

Notch is a cell surface receptor with a single transmembrane domain. Homologues have now been found in C.

elegans (linl2 and glpl), Xenopus, mouse and human. All members of the Notch family have large numbers of EGF-like motifs (29-39 in mouse, 10-13 in C. elegans) and three or more copies of LNR (linl2/ Notch repeats) in the extracellular domain. Notch family members also contain six copies of the cdclO/SWI6 motif (also called ankyrin repeats) and a PEST protein degradation sequence in the intracellular domain. Specific EGF repeats (Drosophila repeats 11 and 12) are involved in ligand binding. LNR may be regulatory domains which bind ligand when high ligand concentrations exist and cause decreased activity of Notch. CdclO/SWI6 domains are involved in proteinprotein interactions with components of the Notchactivated signal transduction pathway.

Notch Biology Two different translocations led to formation of altered Notch genes resulting in an oncogenic state. The TAN-1 oncogene is a fusion of part of the P T cell receptor with a small region of the human Notch 1 extracellular domain and the entire intracellular domain.

TAN-1 is an activated form of Notch which causes Tlymphoblastic leukemias. The int-3 oncogene is caused byintegration of the mouse mammary tumor virus into the WO 98/57983 PCT/US98/12763 26 Notch 4 gene resulting in expression of the intact intracellular domain. Int-3 also is an activated form of Notch which leads to mammary carcinoma.

The function of Notch family members has been extensively studied in Drosophila and C. elegans. These proteins control binary decisions that depend on cell-cell interactions. Notch proteins act consistent with their proposed role as a receptor. Gain-of-function and lossof-function Notch alleles result in opposite cell fate decisions. Notch receptors and their ligands play important roles in lateral inhibition, the process whereby signaling between neighboring cells is amplified by a feedback loop between Notch and its ligand. This process results in increased receptor activity in some cells and increased ligand activity in others leading to the distinction between signaling cells and receiving cells.

It has recently been shown that the expression of an activated form of Notchl in developing T cells of the mouse leads to both an increase in CD8 lineage T cells and a decrease in CD4 lineage T cells. Expression of activated Notch permits the development of mature CD8 lineage thymocytes even in the absence of class I major histocompatability complex (MHC) proteins, ligands that are normally required for the development of these cells.

However, activated Notch is not sufficient to promote CD8 when both class I and class II MHC are absent. These results implicate Notch as a participant in the CD4 versus CD8 lineage decision. Robey, E. et al. Cell 87: 483-492 (1996).

Mutations in a gene region called CADASIL (for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) on chromosome 19 are associated with a type of stroke and dementia whose key WO 98/57983 PCT/US98/12763 27 features include recurrent subcortical ischaemic events and vascular dementia. Notch3 has been mapped to this region, and mutations in CADASIL patients indicate that Notch3 could be the defective protein in CADASIL patients, Joutel, A. et al. Nature 383:707-710 (1996) Notch Ligands There is also a conserved family of ligands for the Notch receptor family. Multiple ligands are able to activate the same receptor. For example, delta and serrate each act as ligands for Drosophila Notch. These ligands all contain EGF repeats (from 1-14), a DSL domain (delta, serrate, lag-2) and a transmembrane domain.

Therefore, receptor and ligand are homologous to one another. In addition, receptor and ligand are often coexpressed and are associated with each other in vesicles.

Zneul Structure Zneul is similar to Notch and its ligands in having two EGF repeats. However, it has a small number of EGF repeats and lacks a membrane spanning domain, linl2/Notch domains and ankyrin repeats. Based on structure/function experiments of Notch, one would predict that Zneul would antagonize Notch function. If the EGF repeats in zneul could bind receptor, it could inhibit ligand binding on neighboring cells. Furthermore, Zneul may have its own target receptor for which it would be an agonist.

Zneul Tissue Distribution/Multiple mRNA sizes Zneul is widely expressed in adult human tissues. Zneul is most highly expressed in heart, WO 98/57983 PCT/US98/12763 28 placenta, spleen, testis, thyroid, spinal cord and lymph node. Dot blots indicate that Zneul is also expressed in a variety of fetal tissues. There are at least three mRNA sizes: 1.3 kb mRNA only in brain and testis 1.7 kb only in lymph node 1.3 1.7 in multiple tissues 2.4 kb only in placenta Since the sequence of Zneul is from the 1.3 kb mRNA in brain, it is difficult to predict what types of molecules the larger transcripts encode. It is possible that larger forms could encode soluble Zneul proteins with more EGF repeats and other domains observed in Notch or Notch ligands. Alternatively, the extra sequences could encode transmembrane and intracellular domains.

Possible relationship to Notch -function It is difficult to predict whether Zneul will act as a Notch ligand or to antagonize the activity of other Notch ligands by competing for receptor binding.

Zneul may alter the binary decisions in differentiation of stem cells into specific lineages or may alter the cell fate decisions of adjacent cells.

Alternatively, Zneul may have nothing to do with Notch. Many proteins have EGF repeats. Zneul may act as a growth factor for a different class of receptor.

Other Possible Roles role in breast cancer (EGF-receptor is overexpressed in many breast cancers) WO 98/57983 PCT/US98/12763 29 role in glioblastomas, pituitary adenomas.

Mapping Data Zneul maps to human chromosome 9q34.3, in the same chromosomal band as Notchl. It is of interest that Notch4 and HSMHC3W5A are also linked at the MHC III locus, duplication of an authentic Notch receptor and a 2 EGF-repeat novel protein.

Therapeutic utility Zneul and its antagonists can be used as therapeutic reagents for the following.

1. Alzheimer's disease The Sell2 gene was identified as a suppresser of a linl2 gain-of-function mutant. Sell2 is a homolog of a positional cloned human early-onset familial Alzheimer's disease gene. Therefore, Zneul could affect a pathway affecting this disease and it is expressed in brain, albeit at lower levels than most other tissues.

2. Cancer There are a number of chromosomal rearrangements associated with breakpoints at 9q34 including Non- Hodgkin's lymphoma and acute myeloid leukemia. A probe for Zneul which does not properly hybridize to chromosome 9q34 would indicate an abnormality of chromosome 9 and would indicate a possible predilection of the individual for developing cancer.

WO 98/57983 PCT/US98/12763 Given the possible association with Notch 4, an endothelial-specific gene, Zneul could be involved in promoting or inhibiting endothelial cell tumors such as hemangiopericytomas? Another possibility is in angiogenesis since blocking a tumor's blood supply would be an effective cancer treatment.

Given the tissues where Zneul is highly expressed, the most prevalent forms of cancer would be in the testis and lymph nodes.

3. Hematopoiesis Moore et al (PNAS 94:4011-4016, 1997) implicated delta-like (a mammalian Notch ligand) in promoting both high-proliferative potential progenitors and in stem cell repopulation. Since Zneul is highly expressed in lymph node and spleen, it could either be involved in inhibiting differentiation to promote stem cell self-renewal or in determination of progenitor populations. Possible use in repopulating blood cells after chemotherapy treatment or in vitro expansion of stem cells.

4. Heart Stimulation of myofibroblast proliferation or migration in the repair process after myocardial infarction. Recently, a frizzled homolog has been implicated in this process. There is evidence for interactions between the frizzled and Notch pathways in Drosophila.

Placenta Stimulation or inhibition of various growth factor made in placenta.

WO 98/57983 PCT/US98/12763 31 6. Testis Role in fertility or contraception 7. Spinal cord Zneul may play a role in Nerve regeneration since Notch plays a role in neurogenesis in both flies and mammalian cells.

The present invention also provides reagents with significant therapeutic value. The Zneul polypeptide (naturally occurring or recombinant), fragments thereof, antibodies and anti-idiotypic antibodies thereto, along with compounds identified as having binding affinity to the Zneul polypeptide, should be useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions. For example, a disease or disorder associated with abnormal expression or abnormal signaling by a Zneul polypeptide should be a likely target for an agonist or antagonist of the Zneul polypeptide.

Antibodies to the Zneul polypeptide can be purified and then administered to a patient. These reagents can be combined for therapeutic use with additional active or inert ingredients, in pharmaceutically acceptable carriers or diluents along with physiologically innocuous stabilizers and excipients.

These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations. This invention also contemplates use of antibodies, binding fragments WO 98/57983 PCT/US98/12763 32 thereof or single-chain antibodies of the antibodies including forms which are not complement binding.

The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medications administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in vivo administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Methods for administration include oral, intravenous, peritoneal, intramuscular, or transdermal administration. Pharmaceutically acceptable carriers will include water, saline, buffers to name just a few. Dosage ranges would ordinarily be expected from l1g to 1000Lg per kilogram of body weight per day. However, the doses by be higher or lower as can be determined by a medical doctor with ordinary skill in the art. For a complete discussion of drug formulations and dosage ranges see Remington's Pharmaceutical Sciences,17 th Ed., (Mack Publishing Co., Easton, Penn., 1990), and Goodman and Gilman's: The Pharmacological Bases of Therapeutics,9 th Ed. (Pergamon Press 1996).

Nucleic Acid-based Therapeutic Treatment If a mammal has a mutated or lacks a Zneul gene, the Zneul gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a Zneul 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, WO 98/57983 PCT/US98/12763 33 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 :320-330 (1991)], an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin.

Invest., 90 :626-630 (1992), and a defective adenoassociated virus vector [Samulski et al., J. Virol., 61:3096-3101 (1987); Samulski et al. J. Virol., 63:3822- 3828 (1989)].

In another embodiment, the gene can be introduced in a retroviral vector, 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; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al.; and Blood, 82:845 (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 [Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987); see Mackey et al., Proc. Natl. Acad. Sci. USA, 85:8027-8031 (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 WO 98/57983 PCT/US98/12763 34 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, 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, Wu et al., J. Biol. Chem., 267:963-967 (1992); Wu et al., J. Biol. Chem., 263:14621- 14624 (1988)].

ANTIBODIES

ZNEU1 polypeptides can also be used to prepare antibodies that specifically bind to Zneul epitopes, peptides or polypeptides. The Zneul polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.

Suitable antigens would be the Zneul polypeptide encoded by SEQ ID NO:2 or 3 or at least a contiguous 9 amino acid fragment thereof. Antibodies generated from this immune response can be isolated and purified as described herein.

Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for WO 98/57983 PCT/US98/12763 example, Current Protocols in Immunology, Cooligan, et al.

National Institutes of Health, (John Wiley and Sons, Inc., 1995); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor, NY, 1989); and Hurrell, J. G. 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, polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a Zneul polypeptide or a fragment thereof.

The immunogenicity of a Zneul polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.

Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Zneul or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a fulllength 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.

As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab') 2 and Fab proteolytic fragments.

Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigenbinding peptides and polypeptides, are also included.

WO 98/57983 PCT/US98/12763 36 Non-human antibodies may be humanized by grafting nonhuman 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.

Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to Zneul protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zneul protein or peptide). Genes encoding polypeptides having potential Zneul 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 WO 98/57983 PCT/US98/12763- 37 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 Zneul sequences disclosed herein to identify proteins which bind to Zneul. These "binding proteins" which interact with Zneul 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 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 Zneul "antagonists" to block Zneul binding and signal transduction in vitro and in vivo. These anti-Zneul binding proteins would be useful for down regulating the effect of Zneul.

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. First, antibodies herein specifically bind if they bind to a Zneul polypeptide, peptide or epitope with a binding affinity (Ka) of 10 M or greater, preferably 10 M or greater, 8 more preferably 10 M or greater, and most preferably 9 -1 or greater. The binding affinity of an antibody M or greater. The binding affinity of an antibody WO 98/57983 PCT/US98/12763 38 can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis.

Second, antibodies are determined to specifically bind if they do not significantly cross-react with related polypeptides. Antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect Zneul 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 IL-16), Zneul polypeptides, and non-human Zneul. 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 Zneul are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to Zneul 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. (Academic Press Ltd., 1996); Benjamin et al., Ann. Rev. Immunol. 2: 67-101 (1984).

WO 98/57983 PCT/US98/12763 39 A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to Zneul proteins or peptides.

Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.) (Cold Spring Harbor Laboratory Press, 1988). Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant Zneul protein or polypeptide.

Antibodies to Zneul may be used for tagging cells that express Zneul; for isolating Zneul by affinity purification; for diagnostic assays for determining circulating levels of Zneul polypeptides; for detecting or quantitating soluble Zneul as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block Zneul in vitro and in vivo.

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 use of biotin-avidin or other complement/anti-complement pairs as intermediates. Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Moreover, antibodies to Zneul or fragments thereof may be used in vitro to detect denatured Zneul or fragments thereof in assays, for example, Western Blots or other assays known in the art.

WO 98/57983 PCT/US98/12763 An additional embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a Zneul polypeptide having an amino acid sequence described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zneul polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least and more preferably at least 30 to 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention. Examples of said polypeptides are defined by the amino acid sequences of SEQ ID NOs:20-23. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule.

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

Example 1. Cloning of Zneul Zneul was identified from expressed sequence tag (EST) SEQ ID NO: 4. The cDNA clone containing the EST was discovered in a brain cDNA library which contained the EST. The cDNA was isolated from E. coli transfected with the plasmid and then streaked out on an LB 100 jig/ml ampicillin and 100 pg/ml methicillin plate. The cDNA insert was sequenced. The insert was determined to be 1514 base pairs long with a 274 amino acid open reading frame and a putative 19 amino acid signal peptide.

WO 98/57983 PCT/US98/12763 41 Example 2 Northern Blot Analysis Human multiple tissue blots 1,2,3 (Clontech)were probed to determine the tissue distribution of Zneul. A HindIII/NotI fragment containing the entire Zneul coding region was generated from the isolated cDNA clone and used for the probe. A plasmid prep of the clone was prepared from a 5 ml LB 100 pg/ml ampicillin overnight culture at 370 using the QIAprep Spin Miniprep Kit (Qiagen). 20 pl out of 100 pl were digested with 3 pl of NEB Buffer 3, units of HindIII (Gibco BRL) and 10 units Notl (New England Biolabs) in a 30 il reaction at 370C for 2 hours.

The digest was electrophoresed on a 0.8% TBE agarose gel and the fragment was cut out. The DNA was extracted from the gel slab with a QIAquick Gel Extraction Kit (Qiagen) ng of this DNA was labeled with P 32 using the Multiprime DNA Labeling System (Amersham) and unincorporated radioactivity was removed with a NucTrap Probe Purification Column (Stratagene). Multiple tissue northerns and a human RNA master blot were prehybridized 3 hours with 10 ml ExpressHyb Solution and added to blots.

Hybridization was carried out overnight at 420C with a ml solution of probe containing a concentration of 2 x 6 /ml of probe to which 1 mg of salmon sperm DNA was added which had been boiled for 5 minutes and then iced 1 minute and added to 10 ml of ExpressHyb Solution (Clontech).

Initial wash conditions were as follows: 2X SSC, 0.05% SDS RT for 40 minutes with several changes of solution then 0.1X SSC, 0.1% SDS at 650C for 40 minutes, 1 solution change. Blots were than exposed to film a -800C. There was cross hybridization/background so blots were further washed at 720C then 650C with 0.1% X SSC, 0.1% SDS for 1 hour each.

WO 98/57983 PCT/US98/12763 42 The results showed that Zneul is widely expressed in adult tissues. Zneul is highly expressed in heart, placenta, spleen, testis, thyroid, spinal cord and lymph node. There are at least three mRNA sizes: 1.3 kb mRNA only in brain and testis; 1.4 kb only in lymph node; 1.7 kb in multiple tissues; and 2.4 kb only in placenta.

Example 3 Chromosomal Assignment and Placement of Zneul.

Zneul was mapped to chromosome 9 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/cgibin/contig/rhmapper.pl) allows mapping relative to the Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the "WICGR" radiation hybrid map) which was constructed with the GeneBridge 4 Radiation Hybrid Panel.

For the mapping of Zneul with the "GeneBridge 4 RH Panel", 20 pl reactions were set up in a PCRable 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 pl 10X KlenTaq PCRreaction buffer (CLONTECH Laboratories, Inc., Palo Alto, WO 98/57983 PCT/US98/12763 43 CA), 1.6 p1 dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA), 1 pl sense primer, SEQ ID NO: 6, 1 pl antisense primer, SEQ ID NO: 7, 2 pl "RediLoad" (Research Genetics, Inc., Huntsville, AL), 0.4 p1 50X Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and x p1 for a total volume of 20 pl. The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle minute denaturation at 95 0 C, 35 cycles of a 1 minute denaturation at 95 0 C, 1 minute annealing at 70 0 C and minute extension at 72 0 C, followed by a final 1 cycle extension of 7 minutes at 72 0 C. The reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).

The results showed that Zneul maps 529.80 cR 3000 from the top of the human chromosome 9 linkage group on the WICGR radiation hybrid map, 7.90 cR 3000 distal of framework marker D9S158. This positions Zneul in the 9q34.3 region on the integrated LDB chromosome 9 map (The Genetic Location Database, University of Southhampton, WWW server: http://cedar.genetics. soton.ac.uk/public html/) WO 98/57983 PCT/US98/12763 44 SEQUENCE LISTING GENERAL INFORMATION APPLICANT: ZymoGenetics, Inc.

1201 Eastlake Ave East Seattle

WA

USA

98102 (ii) TITLE OF THE INVENTION: MAMMALIAN NEURO-GROWTH FACTOR LIKE

PROTEIN

(iii) NUMBER OF SEQUENCES: 24 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Zymogenetics STREET: 1201 Eastlake Ave East CITY: Seattle STATE: WA COUNTRY: USA ZIP: 98102 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SOFTWARE: FastSEQ for Windows Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: FILING DATE: (viii) ATTORNEY/AGENT INFORMATION: NAME: Lunn, Paul G REGISTRATION NUMBER: 32,743 WO 98/57983 PCT/US98/12763- REFERENCE/DOCKET NUMBER: 97-28PC (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 206-442-6627 TELEFAX: 206-442-6678

TELEX:

INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1297 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 69...887 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AAGCTTGGCA CGAGGTGGCA CGAGGCCTCG CACAGGCC ATG AGG GGC TCT CAG GAG Met Arg Gly Ser Gin Glu TGCCAAGCTT GGCACGAGGC CGCCTGGAGG GTG CTG CTG ATG TGG CTT CTG GTG Val Leu Leu Met Trp Leu Leu Val 110

TTG

Leu GCA GTG GGC GGC Ala Val Gly Gly

ACA

Thr 20 GAG CAC GCC TAC CGG Glu His Ala Tyr Arg 25 CCC GGC CGT AGG GTG Pro Gly Arg Arg Val 158 TGT GCT GTC CGG GCT CAC GGG GAT Cys Ala Val Arg Ala His Gly Asp CCT GTC Pro Val 40 TCC GAG TCG TTC GTG CAG Ser Glu Ser Phe Val Gln 206 CGT GTG TAC CAG CCC Arg Val Tyr Gln Pro TTC CTC ACC ACC TGC GAC GGG CAC CGG GCC TGC Phe Leu Thr Thr Cys Asp Gly His Arg Ala Cys 55 ATC TAT AGG ACC GCC TAC CGC CGC AGC CCT GGG Ile Tyr Arg Thr Ala Tyr Arg Arg Ser Pro Gly 70 254 302 AGC ACC TAC Ser Thr Tyr CGA ACC Arg Thr WO 98/57983 PCT/US98/1 2763 CTG GCC Leu Ala CCI GCC AGG CCI CGC Pro Ala Arg Pro Arg 85 TAG GCG TGC TGC CCC GGC TGG AAG AGG Tyr Ala Cys Cys Pro Gly Trp Lys Arg 350 398 ACC Thr AGC GGG CTT CCT GGG Sen Gly Leu Pro Gly 100 GCC TGI GGA GGA GCA ATA TGC GAG Ala Gys Gly Ala Ala Ile Gys Gln 105 CCG CCA Pro Pro 110 TGC CGG AAC GGA GGG Gys Arg Asn Gly Gly 115 AGG TGT GTC CAG CCT Ser Cys Val Gln Pro 120 GGC CGC TGC CGG TGG CCI Gly Ang Cys Ang Gys Pro 125 446

GCA

Ala GGA IGG CGG Gly Irp Arg 130 GGT GAG ACT TGC CAG TCA Gly Asp Thr Gys Gln Ser 135 GAT GIG GAT Asp Val Asp GAA TGC AGI Glu Cys Sen 140 494 GCT AGG AGG GGG Ala Ang Arg Gly 145 GGC TGT CCC GAG Gly Gys Pro Gln 150 CGC TGC GTC AAC ACC GCG GGG AGI Arg Gys Val Asn Thr Ala Gly Ser 155 542 TAC TGG Tyr Trp 160 TGC CAG TGT TGG GAG Cys Gln Cys lrp Glu 165 GGG CAC AGC CTG TCT GGA GAG GGT ACA Gly His Ser Leu Sen Ala Asp Gly Thr 170 590

CTC

Leu 175 TGT GIG CCC AAG GGA GGG Cys Val Pro Lys Gly Gly 180 CCC CCC AGG Pro Pro Arg GTG GCC CCC AAC CCG ACA Val Ala Pro Asn Pro Thr 185 190 GAG AGG CTG GAG ICC AGG Gln Ang Leu Gln Sen Arg 205 638 686 GGA GIG GAG AGI GCA Gly Val Asp Sen Ala 195 ATG AAG GAA GAA GTG Met Lys Glu Glu Val 200 GTG GAC CTG CTG GAG GAG AAG Val Asp Leu Leu Glu Glu Lys 210 CTG GAG Leu Gln 215 CTG GIG CTG GGC CCA CTG CAC Leu Val Leu Ala Pro Leu His 220 GGG CTC CCG GAG CCC GGG AGG Gly Leu Pro Asp Pro Gly Ser 235 AGC CTG GCG Sen Leu Ala 225 ICG GAG GCA CTG GAG CAT Sen Gln Ala Leu Glu His 230 CTC CTG Leu Leu 240 GTG CAC TCC TIC GAG Val His Sen Phe Gln 245 GAG CTC GGG CGC AIC GAC TCC CTG AGG Gln Leu Gly Arg Ile Asp Sen Leu Sen 250 830 WO 98/57983 PCT/US98/12763 GAG CAG ATT TCC TTC CTG GAG GAG Glu Gin Ile Ser Phe Leu Glu Glu 255 260 CAG CTG GGG TCC TGC TCC TGC AAG Gin Leu Gly Ser Cys Ser Cys Lys 265 270 878 936 AAA GAC TCG TGACTGCCCA GCGCCCCAGG CTGGACTGAG CCCCTCACGC CGCCCTGCA Lys Asp Ser

GCCCCCATGC

GAAGGCCAGG

CCTGGCATGG

GCTACCCCAA

CTGCTGGAGC

GTGGGGGCTG

A

CCCTGCCCAA

CAGGGCCTTC

GATGGGCTGG

CGGCATCCCA

CTGGGACCCA

CTGCCTGACC

CATGCTGGGG

CTCCTCTTCC

GATCTTCTCT

AGGCCAGGTG

TGGCACAGGC

CCCAGCACAA

GTCCAGAAGC

TCCTCCCCTT

GTGAATCCAC

GGCCCTCAGC

CAGGCAGCCC

TAAAAATGAA

CACCTCGGGG

CCTCAGGAGG

CCCTGGCTAC

TGAGGGAAGG

GGAGGCTGGG

ACGTGAAAAA

TGACTGAGCG

CTCCCCAGAC

CCCCACCCTG

TACGAGCTCC

TGGGGCCTCA

AAAAAAAAAA

996 1056 1116 1176 1236 1296 1297 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 273 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met 1 Arg Gly Ser Gin Glu Val Leu Leu Val Gly Gly Val Arg Ala Thr His Glu His Ala Tyr Arg 25 Ser Met Trp Leu 10 Pro Gly Arg Glu Ser Phe Leu Val Leu Ala Gly Asp Pro Val 40 Cys Arg Val Ala Val Cys Ala Gin Arg Val Cys Ser Thr Tyr Gin Tyr Arg Pro Phe Leu Thr Thr Ile Tyr Arg 70 Arg Pro Arg Tyr Thr 55 Thr Asp Gly His Arg Ser Ala Tyr Arg Pro Arg 75 Gly Pro Gly Leu Ala Ser Al a Ala Cys Cys Pro Trp Lys Arg Ala Gly Leu Pro Asn Gly Gly 115 Gly 100 Ser Cys Gly Ala Ala 105 Gly Ile Cys Gin Pro Pro Cys Arg 110 Pro Ala Gly Cys Val Gin Pro 120 Arg Cys Arg Cys 125 WO 98/57983 PCT/US98/12763 48 Trp Arg Gly Asp Thr Cys Gin Ser Asp Val Asp Glu Cys Ser Ala Arg 130 135 140 Arg Gly Gly Cys Pro Gin Arg Cys Val Asn Thr Ala Gly Ser Tyr Trp 145 150 155 160 Cys Gin Cys Trp Glu Gly His Ser Leu Ser Ala Asp Gly Thr Leu Cys 165 170 175 Val Pro Lys Gly Gly Pro Pro Arg Val Ala Pro Asn Pro Thr Gly Val 180 185 190 Asp Ser Ala Met Lys Glu Glu Val Gin Arg Leu Gin Ser Arg Val Asp 195 200 205 Leu Leu Glu Glu Lys Leu Gin Leu Val Leu Ala Pro Leu His Ser Leu 210 215 220 Ala Ser Gin Ala Leu Glu His Gly Leu Pro Asp Pro Gly Ser Leu Leu 225 230 235 240 Val His Ser Phe Gin Gin Leu Gly Arg Ile Asp Ser Leu Ser Glu Gin 245 250 255 Ile Ser Phe Leu Glu Glu Gin Leu Gly Ser Cys Ser Cys Lys Lys Asp 260 265 270 Ser INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 254 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Thr Glu His Ala Tyr Arg Pro Gly Arg Arg Val Cys Ala Val Arg Ala 1 5 10 His.Gly Asp Pro Val Ser Glu Ser Phe Val Gin Arg Val Tyr Gin Pro 25 Phe Leu Thr Thr Cys Asp Gly His Arg Ala Cys Ser Thr Tyr Arg Thr 40 Ile Tyr Arg Thr Ala Tyr Arg Arg Ser Pro Gly Leu Ala Pro Ala Arg 55 Pro Arg Tyr Ala Cys Cys Pro Gly Trp Lys Arg Thr Ser Gly Leu Pro 70 75 Gly Ala Cys Gly Ala Ala Ile Cys Gin Pro Pro Cys Arg Asn Gly Gly 90 WO 98/57983 PCT/US98/12763 Ser Cys Val Gin Pro Gly Arg Cys Arg 105 Glu Cys Pro Ala Gly Asp Thr Cys 115 Cys Pro Gin Gin Ser Asp Val Asp 120 Thr Cys Ser Ala Arg 125 Trp Trp Arg Gly 110 Arg Gly Gly Cys Gin Cys Arg Cys Val 130 Trp Glu Asn 135 Ser Ala Gly Ser Gly His Ser 145 Gly Leu 150 Val Ala Asp Gly Thr 155 Thr Cys Val Pro Lys 160 Gly Pro Pro Ala Pro Asn Gly Val Asp Ser Ala 175 Met Lys Glu Glu Lys Leu 195 Ala Leu Glu Gin Arg Leu Gln 185 Pro Arg Val Asp Leu Val Leu Leu His Ser Leu 205 Leu Leu Leu Glu 190 Ala Ser Gin Val His Ser His Gly Leu Pro 215 Ile Pro Gly Ser Phe 225 Leu Gin Leu Gly Asp Ser Leu Ser 235 Lys G1n Ile Ser Phe 240 Glu Glu Gin Leu 245 Ser Cys Ser Lys Asp Ser INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 284 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GGCGGCGGCG

TGGAGGCACA

CAGTGGGCGG

ACGGGGACCC

GCGACGGGCA

CGTGCGCGCC

GGCCATGAGG

CACAGAGCAC

TGTCTCCGAG

CCGGGCCTGC

CCGGATCCGG

GGCTCTCAGG

GCCTACCGGC

TCGTTCGTGC

AGCACCTACC

CGGCCACCCA

AGGTGCTGCT

CCGGCCGTAG

AGCGTGTGTA

GAACCATCTA

GAGGAGAAGG

GATGTGGCTT

GGTGTGTGCT

CCAGCCCTTC

TAGG

CCACCCCGCC

CTGGTGTTGG

GTCCGGGCTC

CTCACCACCT

120 180 240 284 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 98/57983 PCT/US98/12763 TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID TGCGGCGGTA GGCGGTCCTA TAGATGGTTC GGTAGGTGCT INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCTGATGTGG CTTCTGGT 18 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Other (iv) ANTISENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGTAGGCGTG CTCTGTGC 18 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 708 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/57983 PCT/US98/12763 (ii) MOLECULE (xi) SEQUENCE Thr His Arg Gly Leu TYPE: protein DESCRIPTION: SEQ ID NO:8: 1 Gly Tyr Ser Leu Val Thr Ile Arg Ala 145 Arg Arg Leu Leu Glu 225 Leu Pro Tyr Leu Ala 305 Pro Ser Gly Ser Ala His Ser Thr 130 Arg Gly Ala Ala Tyr 210 Pro Ala Arg Ser Tyr 290 Leu Arg Ala Leu Glu Leu Arg Ala 115 Tyr Gly Ser Leu Cys 195 Ser Arg Ala Cys Glu 275 Ala Ala Gly Leu Tyr Arg Thr Thr 100 Arg Arg Thr Glu Ala 180 Tyr Ala Gly Leu Tyr 260 Arg Arg Gly 5 Leu Ala Ala Pro Tyr His Gly Ala His Arg 165 Ala Ser Arg Leu Ala 245 Ser Cys Gly Leu His Tyr Val Ser His 70 Arg Arg Ala Arg Arg 150 Pro Arg Cys Gly Tyr 230 Ile Ala Tyr Cys Tyr 310 Ile Ala Ala Pro 55 Glu Gly Cys Leu Gly 135 Ala Arg Gly Tyr Thr 215 Ala Leu Arg Ser Tyr 295 Thr Ser Arg Leu 40 Pro Val Leu Tyr Ala 120 Thr Leu Gly Pro Ser 200 His Leu Glu Gly Val 280 Ser Arg Ala Gly 25 Ala Arg Ala Asn Ser 105 Cys His Ala Leu Arg 185 Pro Arg Ala Cys Al a 265 Al a Ala Pro Leu Ala Arg Val Leu Pro Ala Tyr Arg Thr Tyr 170 Ala Arg Ser Cys Tyr 250 Ser Leu Arg Al a Ala Arg Gly Ala Gly 75 Arg Ser Ser Ile Tyr 155 Leu Arg Gly Glu Tyr 235 Ser Asn Gly Gly Arg 315 Thr Gly Ala Leu Leu Pro Pro Ser Leu 140 Arg Glu Gly Leu Arg 220 Ser Gly Gly Leu Cys 300 Gly Tyr Val Leu Ser Asn His Gly Glu 125 Glu Ala Ala Thr Tyr 205 Gly Gly Leu Leu Asn 285 Tyr Gly Arg Ala Ala Glu Ala Glu Leu 110 Arg Thr Arg Leu Tyr 190 Thr Leu Leu Asn Tyr 270 Pro Ser Leu Arg Cys Ile Gly Gly Glu His His Arg Ala 160 Pro Ala Pro Leu Ala 240 Arg Leu Gly Arg Ala 320 WO 98/57983 PCT/US98/12763 52 Ser Pro Thr His Arg Cys Tyr Ser Gly Leu Asn Ser Glu Arg Ala Ser 325 330 335 Pro Val Ala Leu Ala Ser Pro Gly Leu Cys Tyr Ser Ser Glu Arg Ala 340 345 350 Leu Ala Ala Arg Gly Ala Arg Gly Gly Leu Tyr Gly Leu Tyr Cys Tyr 355 360 365 Ser Pro Arg Gly Leu Asn Ala Arg Gly Cys Tyr Ser Val Ala Leu Ala 370 375 380 Ser Asn Thr His Arg Ala Leu Ala Gly Leu Tyr Ser Glu Arg Thr Tyr 385 390 395 400 Arg Thr Arg Pro Cys Tyr Ser Gly Leu Asn Cys Tyr Ser Thr Arg Pro 405 410 415 Gly Leu Gly Leu Tyr His Ile Ser Ser Glu Arg Leu Glu Ser Glu Arg 420 425 430 Ala Leu Ala Ala Ser Pro Gly Leu Tyr Thr His Arg Leu Glu Cys Tyr 435 440 445 Ser Val Ala Leu Pro Arg Leu Tyr Ser Gly Leu Tyr Gly Leu Tyr Pro 450 455 460 Arg Pro Arg Ala Arg Gly Val Ala Leu Ala Leu Ala Pro Arg Ala Ser 465 470 475 480 Asn Pro Arg Thr His Arg Gly Leu Tyr Val Ala Leu Ala Ser Pro Ser 485 490 495 Glu Arg Ala Leu Ala Met Glu Thr Leu Tyr Ser Gly Leu Gly Leu Val 500 505 510 Ala Leu Gly Leu Asn Ala Arg Gly Leu Glu Gly Leu Asn Ser Glu Arg 515 520 525 Ala Arg Gly Val Ala Leu Ala Ser Pro Leu Glu Leu Glu Gly Leu Gly 530 535 540 Leu Leu Tyr Ser Leu Glu Gly Leu Asn Leu Glu Val Ala Leu Leu Glu 545 550 555 560 Ala Leu Ala Pro Arg Leu Glu His Ile Ser Ser Glu Arg Leu Glu Ala 565 570 575 Leu Ala Ser Glu Arg Gly Leu Asn Ala Leu Ala Leu Glu Gly Leu His 580 585 590 Ile Ser Gly Leu Tyr Leu Glu Pro Arg Ala Ser Pro Pro Arg Gly Leu 595 600 605 Tyr Ser Glu Arg Leu Glu Leu Glu Val Ala Leu His Ile Ser Ser Glu 610 615 620 Arg Pro His Glu Gly Leu Asn Gly Leu Asn Leu Glu Gly Leu Tyr Ala 625 630 635 640 Arg Gly Ile Leu Glu Ala Ser Pro Ser Glu Arg Leu Glu Ser Glu Arg 645 650 655 Gly Leu Gly Leu Asn Ile Leu Glu Ser Glu Arg Pro His Glu Leu Glu 660 665 WO 98/57983 PCT/US98/12763 Gly Leu Gly Leu Gly Leu Asn Leu Glu Gly Leu Tyr 675 680 Tyr Ser Ser Glu Arg Cys Tyr Ser Leu Tyr Ser Leu 690 695 700 Pro Ser Glu Arg 705 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 31 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Ser Glu Arg Cys 685 Tyr Ser Ala Ser Ala Ile Cys Gin Pro Pro Cys Arg Asn 1 5 Gly Arg Cys Arg Cys Pro Ala Gly Trp 25 Gly Ser Cys Gly Asp Thr Val Gin Pro Cys G1n INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 42 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Asp Val Asp Glu Cys Ser Ala Arg 1 5 10 Cys Val Asn Thr Ala Gly Ser Tyr Trp Cys 25 Ser Leu Ser Ala Asp Gly Thr Leu Cys Val Gly Gly Cys Gin Cys Trp Pro Gin Arg Glu Gly His INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 256 amino acids WO 98/57983 WO 987983PCTIUS98/1 2763 TYPE: amino acid STRANDEDNESS: single

TOPOLOGY

(ii) MOLECULE (xi) SEQUENCE Pro Arg Leu Tyr Ser :linear TYPE: protein DESCRIPTION: SEQ ID NO:11: Arg His Al a Asn Al a Leu Arg Arg Ty r 145 Leu Gi y Gi u Asn Gly 225 Arg Gi y Arg Met Al a Leu Gi u Leu Gly 130 Leu Gi u Leu Al a Ile 210 Leu Cys Val1 Gi y Glu Arg Al a Gi y Gi u 115 Leu Gi u Leu Asn Ser 195 Leu Asr Ty r AlaI Leu Th r Gly Ser Leu 100 His Asn Pro Gl u Gl y 180 Pro IGlu ILeu Ser Leu Ty r Leu Leu Pro Asn Ile Al a Arg Val1 165 Leu Ser Ser Gl L LeL Gly Ala Val Tyr Glu 70 Leu Leu Ser Leu Al a 150 Al a As n Gi u Glu IGly 230 Tyr Leu Leu A~la Se r 55 81 y 81 u Gi u Ser Al a 135 Ser Leu Leu Arg Arg 215 Leu Ser Ty r Al a Leu 40 Gi y Leu Leu Val1 Gi u 120 Leu Pro His Gi u Leu 200 Pro ITyr Leu Gl y Pro 25 Al a Leu Asn 81 u Al a 105 Arg Gi u Pro Ilie Gl y 185 Gi u His Ser Ty r Leu Arg Se r Gly Ser Gi y 90 Leu Leu Gly Arg Se r 170 Leu Se r Gi u Glu Ser 250 Ty r Al a Pro Leu Gi u 75 Leu Leu 81 u L eu Gly 155 Ser Ty r Gl u Leu Arg 235 Al a Pro Ser Ser Val1 Arg Gly Glu Ala His 140 Leu Glu Al a Arg Glu 220 Cy s Ser Arg Asn 81 u Al a Al a Leu Al a Leu 125 Ile Tyr Arg Arg Gl y 205 Gi y Ty r Pro Pro Pro Arg Leu Arg Leu Leu 110 Al a Se r Ser Pro 81 y 190 Leu Leu Ser Ser Arg Arg Al a Gi y Gi y Ty r Al a Ser Gi y Glu His 175 Ile 81 y Gly Ser 81 u 255 Al a Thr Leu Leu Val1 Ser Pro Gi u Leu Arg 160 Glu Leu Leu Leu GI u 240 Arg INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 331 amino acids WO 98/57983 PCT/US98/12763 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) M (xi) S Thr His Arg 1 Gly Pro Arg Tyr Ser Ala Ser Gly Leu Leu Ser Glu Val Ala Leu Thr His Arg Ile Ser Ala 115 Arg Thr Tyr 130 Ala Arg Gly 145 Arg Gly Ser Arg Ala Leu Leu Ala Cys 195 Leu Tyr Ser 210 Glu Pro Arg 225 Leu Ala Ala Pro Arg Cys

OLECULE

EQUENCE

Gly Leu 5 Gly Leu Leu Ala Tyr Ala Arg Pro Thr Tyr Thr His 100 Arg Gly Arg Ala Thr His Glu Arg 165 Ala Ala 180 Tyr Ser Ala Arg Gly Leu Leu Ala 245 Tyr Ser 260 TYPE: protein DESCRIPTION: SEQ ID NO:12: His Tyr Val Ser His 70 Arg Arg Ala Arg Arg 150 Pro Arg Cys Gly Tyr 230 Ile Ala Ile Ala Ala Pro 55 Glu Gly Cys Leu Gly 135 Ala Arg Gly Tyr Thr 215 Ala Leu Arg Ser Arg Leu 40 Pro Val Leu Tyr Ala 120 Thr Leu Gly Pro Ser 200 His Leu Glu Gly Ala Gly 25 Ala Arg Ala Asn Ser 105 Cys His Ala Leu Arg 185 Pro Arg Ala Cys Ala 265 Leu 10 Ala Arg Val Leu Pro 90 Ala Tyr Arg Thr Tyr 170 Ala Arg Ser Cys Tyr 250 Ser Ala Thr Tyr Arg Ala Arg Arg Gly Ala Gly 75 Arg Ser Ser Ile Tyr 155 Leu Arg Gly Glu Tyr 235 Ser Asn Gly Al a Leu Leu Pro Pro Ser Leu 140 Arg Glu Gly Leu Arg 220 Ser Gly Gly Val Leu Ser Asn His Gly Glu 125 Glu Ala Ala Thr Tyr 205 Gly Gly Leu Leu Ala Ala Glu Ala Glu Leu 110 Arg Thr Arg Leu Tyr 190 Thr Leu Leu Asn Tyr 270 Leu His Arg Arg Leu Tyr Thr Tyr Gly Ala 175 Arg Arg Tyr Tyr Pro 255 Gly Cys Ile Gly Gly Glu His His Arg Ala 160 Pro Ala Pro Leu Ala 240 Arg Leu Tyr Ser Glu Arg Cys Tyr Ser Val Ala Leu Gly Leu Asn Pro Arg Gly 275 280 285 WO 98/57983 PCT/US98/12763 Leu Tyr Ala Arg Gly Cys Tyr Ser Ala 290 295 Ala Leu Ala Gly Leu Tyr Thr Arg Pro 305 310 Ser Pro Thr His Arg Cys Tyr Ser Gly 325 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 158 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear Arg Gly Cys Tyr Ser Pro Arg 300 Ala Arg Gly Gly Leu Tyr Ala 315 320 Leu Asn 330 NO:13: (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:13: Thr Glu His Ala Tyr 1 His Phe Ile Pro Gly Ser Asp Cys Trp Gly Leu Tyr Arg Ala Cys Thr Pro 130 Glu Asp Thr Arg Tyr Cys Val Cys 115 Gin Gly Pro Thr Thr Ala Gly Gin 100 Gin Arg His 5 Val Cys Ala Cys Ala Pro Ser Cys Ser Arg Ser Asp Tyr Cys 70 Ala Gly Asp Val Leu 150 Pro Glu Gly Arg 55 Pro Ile Arg Val Asn 135 Ser Gly Ser His 40 Arg Gly Cys Cys Asp 120 Thr Ala Arg Phe 25 Arg Ser Trp Gin Arg 105 Glu Al a Asp Arg 10 Val Ala Pro Lys Pro 90 Cys Cys Gly Gly Val Gin Cys Gly Arg 75 Pro Pro Ser Ser Thr 155 Cys Arg Ser Leu Thr Cys Ala Ala Tyr 140 Leu Ala Val Thr Ala Ser Arg Gly Arg 125 Trp Cys Val Tyr Tyr Pro Gly Asn Trp 110 Arg Cys Val Arg Gin Arg Ala Leu Gly Arg Gly Gin Ala Pro Thr Arg Pro Gly Gly Gly Cys INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 73 amino acids TYPE: amino acid WO 98/57983 PCT/US98/12763 57 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Ala Ile Cys Gln Pro Pro Cys Arg Asn Gly Gly Ser Cys Val Gln Pro 1 5 10 Gly Arg Cys Arg Cys Pro Ala Gly Trp Arg Gly Asp Thr Cys Gln Ser 25 Asp Val Asp Glu Cys Ser Ala Arg Arg Gly Gly Cys Pro Gin Arg Cys 40 Val Asn Thr Ala Gly Ser Tyr Trp Cys Gin Cys Trp Glu Gly His Ser 55 Leu Ser Ala Asp Gly Thr Leu Cys Val INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 169 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Ile Cys Gln Pro Pro Cys Arg Asn Gly Gly Ser Cys Val Gln Pro 1 5 10 Gly Arg Cys Arg Cys Pro Ala Gly Trp Arg Gly Asp Thr Cys Gin Ser 25 Asp Val Asp Glu Cys Ser Ala Arg Arg Gly Gly Cys Pro Gin Arg Cys 40 Val Asn Thr Ala Gly Ser Tyr Trp Cys Gln Cys Trp Glu Gly His Ser 55 Leu Ser Ala Asp Gly Thr Leu Cys Val Pro Lys Gly Gly Pro Pro Arg 70 75 Val Ala Pro Asn Pro Thr Gly Val Asp Ser Ala Met Lys Glu Glu Val 90 G1n Arg Leu Gin Ser Arg Val Asp Leu Leu Glu Glu Lys Leu Gin Leu 100 105 110 WO 98/57983 PCT/US98/12763 Val Le Leu Pr 13 Arg Il 145 Gly Se u Ala Pro Leu His Ser Leu Ala Ser Gl 115 120 o Asp Pro Gly Ser Leu Leu Val His Se 0 135 e Asp Ser Leu Ser Glu Gin Ile Ser Ph 150 15 r Cys Ser Cys Lys Lys Asp Ser 165 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 181 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear n Ala Leu Glu His Gly 125 r Phe Gin Gin Leu Gly 140 e Leu Glu Glu Gin Leu 5 160 Thr 1 His Phe Ile Pro Gly Pro Ser Leu Gly 145 Leu Cys (ii) M (xi) S Glu His Gly Asp Leu Thr Tyr Arg Arg Tyr Ala Cys Thr Gly Arg Val 115 His Ser 130 Ser Leu Ser Glu Lys Lys

OLECULE

EQUENCE

Ala Tyr 5 Pro Val Thr Cys Thr Ala Ala Cys Gly Ala Val Asp 100 Asp Leu Leu Ala Leu Val Gin Ile 165 Asp Ser 180 TYPE: protein DESCRIPTION: SEQ ID NO:16: Arg Ser Asp Tyr Cys 70 Pro Ser Leu Ser His 150 Pro Glu Gly Arg 55 Pro Lys Ala Glu Gin 135 Ser Gly Ser His 40 Arg Gly Gly Met Glu 120 Ala Phe Arg Phe 25 Arg Ser Trp Gly Lys 105 Lys Leu Gin Arg Val Ala Pro Lys Pro Glu Leu Glu Gin Val Cys Gln Arg Cys Ser Gly Leu Arg Thr 75 Pro Arg Glu Val Gln Leu His Gly 140 Leu Gly 155 Ala Val Thr Ala Ser Val G1n Val 125 Leu Arg Val Tyr Tyr Pro Gly Al a Arg 110 Leu Pro Ile Ala Pro Thr Arg Pro Asn Gin Pro Pro Ser 160 Ser Phe Leu Glu Glu Gin Leu Gly Ser Cys Ser WO 98/57983 PCT/US98/12763 59 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 293 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) (xi)

MOLECULE

SEQUENCE

TYPE: protein DESCRIPTION: SEQ ID NO:17: Met Gly Ser Arg Ala 1 Leu Glu Tyr Cys Met Cys Ile Gin Val 145 Asn Gly Ser Arg Leu 225 Leu Ser Asn Ala Trp Gin Cys Cys 130 Asp Thr Val Ala Al a 210 Glu Leu Gin Glu Gly Arg Gly Ala 115 Glu Glu Al a Asp Ser 195 Leu Gin Leu Gly Ser Arg Glu Trp 100 Lys Cys Cys Gly Gly 180 Ile Lys Trp 5 Ile Val Tyr Arg Val Lys Pro Ala Arg Ser 165 Arg Leu Gin Ala Glu 245 Glu Pro Cys Ser Ile 70 Arg Lys Cys Pro Thr 150 Phe Thr Ser Glu Gly 230 Leu Gly Ser Gin 55 Cys Arg Arg Leu Gly 135 Ser Thr Cys Val Ile 215 G1n Cys Glu Lys 40 Pro Ser Glu His Asn 120 Trp Ile Cys Met Ala 200 His Al a Thr Gly 25 Gin Val Thr Val Pro 105 Gly Gly Thr Gly Glu 185 Val Glu Gly Leu 10 Ala Thr Tyr Tyr Gin 90 Gly Gly Gly Leu Cys 170 Gly Arg Leu Ala Glu 250 Leu Lys Leu Lys Arg 75 Gin Ala Val Lys Cys 155 Pro Ser Glu Arg Trp 235 Gly Gly Val Pro Thr Thr Leu Cys His 140 Ser His Pro Ala Gly 220 Val Gly Gly Val Tyr Met His Thr Val 125 Cys His Asp Glu Glu 205 Arg Arg Phe Ser Pro Leu Tyr Ala Cys 110 Arg His His Leu Pro 190 Lys Leu Al a Ser Leu Leu Thr Arg Val Glu Pro Val Cys Val 175 Pro Asp Glu Val Phe Arg His Leu Val Cys Ala Asp Asp Phe 160 Leu Thr Glu Arg Leu 240 Pro Val Pro Pro Glu Leu Gin Pro Gln Val Ala Glu Leu Trp 255 WO 98/57983 PCT/US98/12763 Gly Arg Gly Asp Arg Ile Glu Ser Leu 260 265 Glu Glu Arg Leu Gly Ala Cys Ser Cys 275 280 Gly Val Asn His Arg 290 Ser Asp Gin Val Leu Leu Leu 270 Glu Asp Asn Ser Leu Gly Leu 285 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 1339 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 261...1094 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

GTAGGGCTCT

GTGGTGAGGG

CTGTCCCTGT

AACAGCCACC

GAAGGCTACC

GCCGGGACCT

GTCCAAGGAG

GGGAAGCCCC

ACCAGTACCC

CCACTTACAG

GGGTCTTCCC TCTCCTGGAG CTGCAGAGGC AGTCCGGGGA GACCAGGGAG GCTCTGTCCA CGGCAGCAGC AAGACGCTGG CTGTTCCACC AGGGGATGAC AAGCGGCCGG ACCACAGGCC ATG CAG ACC ATG TGG GGC TCC GGA Met Gin Thr Met Trp Gly Ser Gly

CAGAAGTTCA

TCCCCTGTCC

TGCCCACAAG

ACAAAAAGAA

GAA CTG Glu Leu 120 180 240 290 CTT GTA GCA TGG TTT CTA GTG TTG GCA GCA GAT GGT ACT ACT GAG CAT Leu Val Ala Trp Phe Leu Val Leu Ala Ala Asp Gly Thr Thr Glu His 20 GTC TAC AGA CCC AGC CGT AGA GTG TGT ACT GTG GGG ATT TCC GGA GGT Val Tyr Arg Pro Ser Arg Arg Val Cys Thr Val Gly Ile Ser Gly Gly 35 338 386 TCC ATC TCG GAG ACC TTT GTG CAG CGT GTA Ser Ile Ser Glu Thr Phe Val Gin Arg Val 50 TAC CAG CCT TAC CTC ACC Tyr Gin Pro Tyr Leu Thr 434 WO 98/57983 PCT/US98/12763 ACT TGC GAC GGA CAC AGA GCC TGC AGC ACC TAC Cys Ser Thr Tyr Thr Cys Asp Gly His Arg Ala 65

ACT

Thr GCC TAT CGC CGT AGC Ala Tyr Arg Arg Ser 80 CCT GGG GTG ACT CCC Pro Gly Val Thr Pro 85 CGA ACC ATC TAC CGG Arg Thr Ile Tyr Arg GCA AGG CCT CGC TAT Ala Arg Pro Arg Tyr CTC CCT GGG GCT TGT Leu Pro Gly Ala Cys 105 GCT TGC TGC CCT GGT Ala Cys Cys Pro Gly

GGA

Gly GCA GCA ATA Ala Ala Ile 110 TGG AAG AGG ACC AGT Trp Lys Arg Thr Ser 100 CAG CCT CCA TGT GGG Gin Pro Pro Cys Gly 115 CGC TGC CCT GTG GGA Arg Cys Pro Val Gly 130 482 530 578 626 674 AAT GGA GGG AGT TGC ATC Asn Gly Gly Ser Cys Ile 120 TGG CAG GGA GAT ACT TGC Trp Gin Gly Asp Thr Cys 135 CGC CCA GGA CAC Arg Pro Gly His 125 CAG ACA Gin Thr 140 GAT GTT GAT GAA TGC Asp Val Asp Glu Cys 145 AGT ACA GGA GAG GCC AGT TGT CCC CAG Ser Thr Gly Glu Ala Ser Cys Pro Gin 150

CGC

Arg 155 TGT GTC AAT ACT GTG GGA Cys Val Asn Thr Val Gly 160 AGT TAC TGG Ser Tyr Trp CAA AGC CCA TCT GCA Gin Ser Pro Ser Ala 175 TCC CCG GTG GCC CCA Ser Pro Val Ala Pro 190 GAG GAG GTG TAC AGG Glu Glu Val Tyr Arg 205 GAT GGG ACG CGC TGC Asp Gly Thr Arg Cys 180 AAC CCC ACA GCA GGA Asn Pro Thr Ala Gly 195 TGC CAG GGA TGG GAG GGA Cys Gin Gly Trp Glu Gly 165 170 CTG TCT AAG GAG GGG CCC Leu Ser Lys Glu Gly Pro 185 GTG GAC AGC ATG GCG AGA Val Asp Ser Met Ala Arg 200 770 818 866 914 CTG CAG GCT Leu Gin Ala 210 CGG GTT GAT GTG CTA GAA CAG AAA Arg Val Asp Val Leu Glu Gin Lys 215 CTG CAG Leu Gin 220 TTG GTG CTG GCC CCA Leu Val Leu Ala Pro 225 CTG CAC Leu His AGC CTG GCC Ser Leu Ala 230 TCT CGG TCC ACA Ser Arg Ser Thr 962 WO 98/57983 PCT/US98/12763 GAG CAT GGG CTA CAA GAT CCT GGC AGC CTG CTG GCC Glu His Gly Leu Gln Asp Pro Gly Ser Leu Leu Ala 235 240 245 CAT TCC TTC CAG His Ser Phe Gin 250 1010 CAG CTG GAC Gin Leu Asp CGA ATT GAT TCA CTG AGT GAG Arg Ile Asp Ser Leu Ser Glu 255 260 CAG GTG TCC TTC TTG GAG Gln Val Ser Phe Leu Glu 265 1058 GAA CAT CTG GGG TCC Glu His Leu Gly Ser 270 TGC TCC TGC AAA AAA GAT CTG TGATAACCTC TCACCA 1110 Cys Ser Cys Lys Asp Leu

CCCAGGCTGG

GGTGGTGCCT

GACTTGGGCA

GTAAGGTGGG

ATAGAGCAGT

ATGAGCAGAA

TGGGGAGTGG

GATTGTCTCC

CATCCCTAGA

GGCCCTGCCT

GGTCTTGTGT

ATCTTTGTCA

TCCCTTGTAG

CATTGTCCCT

GACTCTTCAG

TAATAAAGCT

CCAGAGTTCA

CTTTCTTAGG

TGGGGCTCCC

GAGACTTGA

GGCGCTGTCT

AGGTTCCTAG

TGTCTAAGTG

1170 1230 1290 1339 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 278 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Met 1 Val Arq Gin Thr Met Leu Ala Ala Val Cvs Thr Trp 5 Gly Ser Gly Glu Leu 10 Leu Val Ala Trp Phe Leu Asp Gly Thr Thr Val Gly Ile Ser Glu 25 Gly His Val Tyr Arg Gly Ser Ile Val Gin Arg Ser Asp Pro Ser Arg Glu Thr Phe Gly His Arg Val Tyr Gln Pro 55 Thr Tyr Leu Thr Thr Cys Ala Cys Ser Thr Tyr Arg Ile Tyr Arg Thr 75 Tyr Arg Arg Ser Pro Gly Val Thr Pro Ala Arg Pro Arg Gly Leu Pro Gly Ala 105 Tyr 90 Cys Ala Cys Cys Pro Gly Trp Cys Gin Lys Arg Thr Gly Ala Ala WO 98/57983 PCT/US98/12763 63 Pro Pro Cys Gly Asn Gly Gly Ser Cys Ile Arg Pro Gly His Cys Arg 115 120 125 Cys Pro Val Gly Trp Gin Gly Asp Thr Cys Gin Thr Asp Val Asp Glu 130 135 140 Cys Ser Thr Gly Glu Ala Ser Cys Pro Gin Arg Cys Val Asn Thr Val 145 150 155 160 Gly Ser Tyr Trp Cys Gin Gly Trp Glu Gly Gin Ser Pro Ser Ala Asp 165 170 175 Gly Thr Arg Cys Leu Ser Lys Glu Gly Pro Ser Pro Val Ala Pro Asn 180 185 190 Pro Thr Ala Gly Val Asp Ser Met Ala Arg Glu Glu Val Tyr Arg Leu 195 200 205 Gin Ala Arg Val Asp Val Leu Glu Gin Lys Leu Gin Leu Val Leu Ala 210 215 220 Pro Leu His Ser Leu Ala Ser Arg Ser Thr Glu His Gly Leu Gin Asp 225 230 235 240 Pro Gly Ser Leu Leu Ala His Ser Phe Gin Gin Leu Asp Arg Ile Asp 245 250 255 Ser Leu Ser Glu Gin Val Ser Phe Leu Glu Glu His Leu Gly Ser Cys 260 265 270 Ser Cys Lys Lys Asp Leu 275 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 29 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Thr Cys Asp Gly His Arg Ala Cys Ser Thr Tyr Arg Thr Ile Tyr Arg 1 5 10 Thr Ala Tyr Arg Arg Ser Pro Gly Leu Ala Pro Ala Arg INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid WO 98/57983 PCT/US98/12763 64 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Gin 1 Gin Pro Gly Arg Cys 5 Ser Asp Val Asp Arg Cys Pro Ala Glu Cys Ser Ala Trp Arg Gly Asp Thr Cys Pro Gin Arg Arg Gly Gly INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 37 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Cys Val Pro Lys Gly Gly Pro Pro Arg 1 5 Val Asp Ser Ala Met Lys Glu Glu Val Asp Leu Leu Glu Glu Val 10 Gin Ala Pro Asn Pro Thr Gly Arg Val Arg Leu Gin INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 29 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Gln Gln Leu Gly Arg Ile Asp Ser Leu Ser Glu Gin Ile 1 5 10 Glu Glu Gin Leu Gly Ser Cys Ser Cys Lys Ser Phe Leu Lys Asp Ser WO 98/57983 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 255 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear PCT/US98/12763 (ii) (xi)

MOLECULE

SEQUENCE

TYPE: protein DESCRIPTION: SEQ ID NO:24: Thr Glu His Val Tyr 1 Ser Tyr Ile Pro Gly Ser Asp Cys Trp 145 Glu Met Glu Arg Ser 225 Gly Leu Tyr Arg Ala Cys Thr Pro 130 Glu Gly Ala Gin Ser 210 Phe Gly Thr Arg Tyr Cys Ile Cys 115 Gin Gly Pro Arg Lys 195 Thr Gin Ser Thr Thr Ala Gly Arg 100 Gin Arg Gin Ser Glu 180 Leu Glu Gin 5 Ile Cys Ala Cys Ala Pro Thr Cys Ser Pro 165 Glu Gin His Leu Arg Ser Asp Tyr Cys 70 Ala Gly Asp Val Pro 150 Val Val Leu Gly Asp 230 Pro Glu Gly Arg 55 Pro Ile His Val Asn 135 Ser Ala Tyr Val Leu 215 Arg Ser Arg Thr Phe 25 His Arg 40 Arg Ser Gly Trp Cys Gln Cys Arg 105 Asp Glu 120 Thr Val Ala Asp Pro Asn Arg Leu 185 Leu Ala 200 Gln Asp Ile Asp Arg 10 Val Ala Pro Lys Pro 90 Cys Cys Gly Gly Pro 170 Gin Pro Pro Ser Val Gln Cys Gly Arg 75 Pro Pro Ser Ser Thr 155 Thr Ala Leu Gly Leu 235 Cys Arg Ser Val Thr Cys Val Thr Tyr 140 Arg Ala Arg His Ser 220 Ser Thr Val Thr Thr Ser Gly Gly Gly 125 Trp Cys Gly Val Ser 205 Leu Glu Val Tyr Tyr Pro Gly Asn Trp 110 Glu Cys Leu Val Asp 190 Leu Leu Gin Gly Gin Arg Ala Leu Gly Gin Ala Gin Ser Asp 175 Val Al a Ala Val Ile Pro Thr Arg Pro Gly Gly Ser Gly Lys 160 Ser Leu Ser His Ser 240 Phe Leu Glu Glu His Leu Gly Ser Cys Ser Cys Lys Lys Asp Leu 245 250 255

Claims (14)

1. An isolated polynucleotide which encodes a mammalian Zneul polypeptide wherein said polynucleotide encodes a polypeptide selected from the group SEQ ID Nos: 2-3, 19 and 24 or a polypeptide which is at least 90% identical to the polypeptides of said group and which retain the activity of said polypeptides.

2. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a chimeric polypeptide, wherein said chimeric polypeptide consists essentially of a first portion and a second portion joined by 0o a peptide bond, said first portion being comprised of a mammalian polypeptide, said polypeptide being the amino acid sequences of SEQ ID Nos: 2-3, 8, 9, 11-16, and 19-24 or a polypeptide which is at least 90% identical to said amino acid sequences and said second portion being a second polypeptide or protein; a transcription terminator.

3. An isolated Zneul polypeptide selected from the group of amino acid sequences consisting of SEQ ID Nos: 2, 3, 19 and 24 or a polypeptide which is at least 90% identical to said polypeptides.

4. An isolated peptide or polypeptide having at least 15 amino acid residues comprised of an epitope-bearing portion of a polypeptide of SEQ ID Nos: 2-3, 8, 9, 20 11-16, and 19-24 or is at least 90% identical to said epitope bearing portion.

An isolated peptide or polypeptide of claim 4 wherein the epitope-bearing portion is selected from the group of amino acid sequence consisting of SEQ ID Nos: 20-23 or a peptide or polypeptide which is at least 90% identical to said epitope bearing portion. 25

6. An antibody, antibody fragment or single-chain antibody that specifically binds to a mammalian polypeptide, said polypeptide being defined- by the amino acid sequences of SEQ ID Nos: 2-3, 8, 9, 11-16, and 19-24.

7. An antibody, antibody fragment or single-chain antibody of claim 6 wherein said antibody, antibody fragment or single-chain antibody is humanised.

8. A method for producing an antibody which binds to a peptide or polypeptide defined by SEQ ID Nos: 2-3, 8, 9, 11-16, and 19-24 or to a peptide or polypeptide which is at least 90% identical to said peptide or polypeptide comprising inoculating an animal with said peptide or polypeptide or with a nucleic acid which encodes said peptide or Spolypeptide, wherein said animal produces antibodies to said peptide or polypeptide; and [I:\DayLib\LIBFF]28603spec.doc:gcc 67 isolating said antibody.

9. An anti-idiotypic antibody, anti-idiotypic antibody fragment or anti-idiotypic single-chain antibody which binds to an antibody, an antibody fragment or single-chain antibody of peptide or polypeptide defined by SEQ ID Nos: 2-3, 8, 9, 11-16 and 19-24 or to a peptide or polypeptide which is at least 90% identical to said peptide or polypeptide.

The antibodies of claims 6-9 wherein said antibodies are either polyclonal or monoclonal.

11. An isolated polynucleotide which encodes a mammalian Zneul polypeptide, substantially as hereinbefore described with reference to any one of the examples.

12. An antibody, antibody fragment or single-chain antibody that specifically binds to a mammalian polypeptide encoded by the polynucleotide of claim 11.

13. A method for producing an antibody which binds to a peptide or polypeptide defined by SEQ ID Nos: 2-3, 8, 9, 11-16, and 19-24 or to a peptide or polypeptide which is at least 90% identical to said peptide or polypeptide comprising inoculating an animal with said peptide or polypeptide or with a nucleic acid which encodes said peptide or polypeptide, substantially as hereinbefore described with reference to any one of the examples.

14. An antibody produced by the method of claim 8 or 13. 20 Dated 30 May, 2001 ZymoGenetics Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON *g* [I:\DayLib\LIBFF]28603spec.doc:gcc