WO2000045843A1 - Novel sialoadhesins - Google Patents

Abstract

The invention is directed to purified and isolated novel siglec polypeptides, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and therapeutic and other methods employing such polypeptides, nucleic acids and antibodies.

Description

NOVEL SIALOADHESINS

BACKGROUND OF THE INVENTION

Field of the Invention

The invention is directed to purified and isolated novel polypeptides of the siglec family and fragments thereof, nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides or fragments thereof, and assays and methods employing these polypeptides, antibodies and nucleic acids.

Description of Related Art Of recent interest is a group of related polypeptides known as the sialic acid- binding immunoglobulin-like lectins, or "siglecs." The siglecs are Type I membrane proteins that are classified by sequence homology as constituting a distinct group within the immunoglobulin superfamily (Ig superfamily; for review, see Williams et al., Cold Spring Harbor Symposia on Quantitative Biology 54 (part 2):637-647, 1989). Since the identification of the macrophage-specific sialoadhesin protein (Crocker et al., EMBO J. 13: 4490-503, 1994), many other proteins have been classified into this group (for reviews, see Crocker et al., Glycoconj. J. 14:601-609, 1997; Matthews et al., Leukemia 12 (suppl. 1):S33-S36, 1998). The siglecs also are referred to as the sialoadhesin family.

Several siglec family members have been reported, including CD22 (discussed in Umansky et al., Immunology 87:303-309, 1996a and Umansky et al., J. Mol. Med. 74:353-363, 1996b); CD33 (Freeman et al., Blood 85:2005-2012, 1995); the CD33-like proteins, CD33-L1 and CD33-L2 (Takei et al., Cytogenet Cell Genet 78:295-300, 1997); OBBP1 and OBBP2 (Patel et al., J Biol Chem 274:22729-38, 1999); SAF2 (EP 924 297); SAF3 (EP 869 178; p75/AIRMl (Falco et al. Exp Med 190:793-801, 1999; Vitale et al, Proc Natl Acad Sci USA 96:15091-15096, 1999); SAF4 (WO 98/53840); the murine myelin-associated proteins, or "MAGs" (Fujita et al., Biochim. Biophys. Res. Com. 165:1162, 1989); avian Schwann cell myelin protein (SMP) (Dulac et al., Neuron 8:323, 1992); siglec-5 (Cornish et al., Blood 92:2123-32, 1998); siglec 7 (Nicoll et al., J Biol Chem 274:34089-34095, 1999); and siglec 8 (Floyd et al., J. Biol. Chem. 275:861-866, 2000). The CD33-L1 and CD33-L2 translation products appear to be transmembrane and secreted forms, respectively, of the same protein. SAF3, p75/AIRMl and siglec 7 appear to encode essentially the same protein, and humacr70 (WO 9831799) appears to be an alternate form of this same protein. OBBP1 is essentially the same as CD33-L1, and OBBP2 appears to be the same as siglec 5, except for a few amino acid differences.

The siglecs are characterized by sequence similarities and by their ability to bind to sialic acid moieties on glycoproteins and glycolipids. Generally, the extracellular portions of the siglecs are more similar, i.e., more highly conserved, than their cytoplasmic regions. The extracellular regions contain at least one V-set Ig-like domain located near the amino terminus, followed by varying numbers of C2-set Ig-like domains. For example, CD33 has two Ig-like domains in its extracellular region, while sialoadhesin has 17. The siglecs contain an unusual arrangement of conserved cysteine residues at their amino termini, resulting in a predicted intra-β-sheet disulfide bridge in the first domain, and an interdomain disulfide bond between the first and eighth domains (see Crocker et al., 1997). Numerous siglec genes have been mapped to the same region of human chromosome 19.

The structural interactions between sialoadhesin and carbohydrates have been analyzed in detail (for example, see Collins et al., J Biol Chem 272: 16889-95, 1997; see also May et al., Mol. Cell 1:719-28, 1998). Siglecs exhibit functional protein- carbohydrate recognition through specific siaylated glycoconjugates on their cognate molecules, and some of them may bind preferably with glycans that terminate in oc-2,3 linked sialic acids (Kelm et al., Curr. Biol. 4:965-72, 1994). The sialic acid-binding activity usually resides on the N-terminal V-set Ig-like domain, and may also involve the penultimate Ig-like domain. Some members of this group are reported to exhibit distinct specificities for both the type of sialic acid and its linkage to subterminal sugars.

Many proteins have been reported to contain a cytoplasmic inhibitory signaling motif that is associated with the transduction of inhibitory effector functions, i.e., the "immunoreceptor tyrosine-based inhibition motif," or "ITIM" (Renard et al., Immun Rev 155:205-221, 1997). ITIMs have the consensus sequence I/VxYxxL/V, and are found in the cytoplasmic portions of diverse signal transduction proteins of the immune system, many of which, like the siglecs, belong to the Ig superfamily or to the family of type II dimeric C-lectins (see Renard et al., 1997). Proteins that contain ITIMs include the "killer cell Ig-like receptors," or "KIRs," and some members of the leukocyte Ig-like receptor or "LIR" family of proteins (Renard et al., 1997; Cosman et al., Immunity 7:273-82, 1997; Borges et al., J Immunol 159:5192-96, 1997). The KIRs and LIRs, like the siglecs, are expressed on hematopoietic cells and map to chromosome 19. Signal transduction by an ITIM is believed to downregulate targeted cellular activities, such as expression of cell surface proteins. Renard et al. propose that the regulation of complex cellular functions is fine-tuned by the interplay of ITIM-mediated inhibitory signal transduction and activation of the same functions by a 16-18 amino acid activitory motif, or "IT AM" sequence that is present in other proteins. Some of the siglecs have been reported to contain one or more ITIMs in their cytoplasmic regions.. This includes CD22, which has been characterized as a negative regulator of B cell activation. CD33 and siglec 8 also are reported to contain ITIM motifs in their cytoplasmic domains (Ulyanova et al., Eur J Immunol 29:3440-49, 1999; Floyd et al., 2000). An ITIM also is present in the cytoplasmic tail of p75/AIRMl/siglec 7, a protein expressed at significant levels on a subset of CD8⁺ natural killer (NK) cells (Nicoll et al., 1999). Falco et al. (1999) has reported that downregulation of spontaneous NK-mediated cytotoxicity could be brought about either by cross-linking p75/AIRMl, or by triggering the NK cells via any of several activating receptors.

Siglec expression is restricted largely to myeloid cells of the immune system, and is believed to be involved in control of myeloid interactions, such as adhesions between antigen presenting cells (APCs), i.e., macrophages or dendritic cells, and other cells involved in cell-mediated immunity, such as T cells or natural killer cells. These polypeptides may function in antigen capture and uptake when expressed on APCs, and thus may provide targets for enhancing cell-based tumor vaccines. Many siglecs are observed to be expressed primarily on subsets of specific types of hematopoietic cells. CD33 expression, for example, is largely restricted to the myelomonocytic lineage, and is present on mature monocytes and tissue macrophages (Freeman et al., 1995). Other examples include CD22, which is expressed primarily on B-cells, while siglec-8 is expressed specifically on eosinophilic granulocytes (Floyd et al., J. Biol. Chem. 275:861- 866, 2000). Sialoadhesin is expressed at high levels on macrophages in chronic inflammatory conditions and in tumors, suggesting a role in host defense, and can mediate specific cell-substrate and cell-cell interactions in vitro (Crocker et al., 1994; Crocker et al., 1997). Umansky et al. have reported that sialoadhesin-positive macrophages contribute to host resistance against metastasis of tumors, that these macrophages can function as antigen-presenting cells, and also that sialoadhesion expression is responsive to corticosteroids, lymphokines and cytokines (Umansky et al., 1996a and 1996b). However, siglec expression is not entirely confined to hametopoietic cells. At least two siglecs are expressed in neuronal cells, including the avian SMP protein, which was first isolated from glial cells (Dulac et al.), and the MAGs that were isolated from a rat brain cDNA library (Fujita et al., 1989).

CD33 maps to a region of chromosome 19 that was associated with an interstitial deletion (del(9)(ql2-q22)) in several patients with acute myeloblastic leukemia (AML) or T-cell acute lymphoblastic leukemia (T-ALL) (Ferrara et al., Leukemia 10:1990-92, 1996). Two of these AML patients had exhibited a myelodysplastic syndrome prior to the onset of AML. Antibodies against CD33 are used in the diagnostic differentiation of myeloid leukemic cells from the more commonly occuring CD33-negative leukemias(e.g., see Freeman et al., 1995), and such antibodies also have been used with some success in the treatment of AML (Maloney et al., Curr. Opin. Hematol. 5: 237, 1998).

The elucidation of additional members of the siglec family can provide proteins useful for regulating the immune system and for controlling disorders associated with cells that express siglecs.

SUMMARY OF THE INVENTION

Provided are novel siglec polynucleotide and polypeptide sequences that were identified by the isolation of cDNA clones from dendritic cell or T-cell cDNA libraries. Particular embodiments of the invention are directed to isolated ss5846 and ss4823 nucleic acid molecules comprising the nucleotide sequences that are shown in SEQ ID NOS:l, 3, 5, 7, 9 and 11, and allelic variants and mutants thereof, including variants that differ only with respect to codon usage. Isolated ss5846 sequences provided herein include ss5846-p, ss5846-tl, ss5846-t2, ss5846-t3 and ss5846-c. The term "ss5846" without any suffix is used herein when referring collectively to all five ss5846 sequences. The invention encompasses both single-stranded and double-stranded RNA and DNA molecules, and oligonucleotides based on the disclosed nucleotide sequences. The oligonucleotides provide hybridization probes, polymerase chain reaction primers and antisense reagents for blocking siglec expression. The invention also encompasses recombinant vectors that contain and express the disclosed novel nucleic acid molecules as well as host cells stably or transiently transformed or transfected with these vectors. The subject recombinant proteins are recovered using standard purification procedures from extracts of the transfected cells or from the culture medium if secreted. Expression vectors may be used in which the cloned DNA is operably linked to regulatory elements capable of directing expression in a variety of cell types, including bacterial, yeast, mammalian and insect cells.

Provided also are novel siglec polypeptides as exemplified by the proteins encoded by the ss5846-p, ss5846-tl, ss5846-t2, ss5846-t3, ss5846-c and ss4623 nucleotide sequences, and whose amino acids are shown in SEQ ID NOS:2, 6, 8, 10, 12 and 4, respectively. The invention also provides fragments of the proteins shown in SEQ ID NOS:2, 6, 8, 10, 12 and 4, including soluble forms and fusion proteins. The invention further encompasses methods for the production of these polypeptides and fragments by recombinant expression of nucleic acids expressing these proteins, including expression of the disclosed nucleic acid molecules.

The invention furthermore provides therapeutic methods for using the disclosed polypeptides to treat patients who require an enhancement or an inhibition of ss4623 of ss5846 activity. These siglecs thus are useful for treating a variety of conditions, particularly those affecting the hematopoietic or nervous systems. Such conditions include: rheumatologic diseases (e.g., rheumatoid arthritis, psoriatic arthritis, seronegative spondyloarthropathies); inflammatory conditions; bone marrow or solid organ transplantation, graft-versus-host disease, autoimmune disorders (e.g., systemic lupus erythematosus, Hashimoto's thyroiditis, Sjogren's syndrome); allergies (e.g., asthma, allergic rhinitis); neurologic disorders (e.g., Alzheimer's, Parkinson's, dementia, brain cancer, Bell's palsy, post-herpetic neuralgia); hematopoietic cancers (e.g., lymphoma, B-cell, T-cell and myeloid cell leukemias), adenoma; skin cancer; trauma (e.g., head injury, spinal cord injury); infections (e.g., bacterial, parasitic, protozoal and viral infections, including AIDS and tuberculosis); chemotherapy or radiation-induced toxicity; cachexia; cardiovascular disorders (e.g., congestive heart failure, myocardial infarction, ischemia reperfusion injury, arteritis, stroke); gastrointestinal disorders (e.g., inflammatory bowel disease, Crohn's disease, celiac disease); diabetes mellitus; skin diseases (e.g., psoriasis, scleroderma, dermatomyositis); hematologic disorders (e.g., myelodysplastic syndromes, acquired or Fanconi's aplastic anemia); septic shock; liver diseases (e.g., viral hepatitis or alcohol-associated); kidney disease; and bone disorders (e.g., osteoporosis, osteopetrosis).

Provided also diagnostic assays employing the disclosed siglecs, as well as assays for identifying agonists and antagonists, cell separation methods, chromosome mapping methods, and methods in which the polypeptides are used to modify cellular processes such as inflammatory responses, immune responses against tumor cells, antigen presentation, cell-cell adhesions, macrophage and dendritic cell migration and cell death. In other methods provided herein, the subject polypeptides are used as myeloid-specific cell surface markers, more specifically as markers for macrophages, dendritic cells, neuroglial cells or T-cells.

In other methods of the invention, soluble forms of the subject siglec polypeptides are used therapeutically to block the interaction between native siglecs and their ligands. Such therapies may employ antibodies directed against the subject proteins or other molecules that antagonize siglec-ligand interactions. The subject polypeptides may be used also to modulate cell-mediated immunity by inhibiting or enhancing adhesion between antigen presenting cells (APCs) such as macrophages or dendritic cells, and other cells involved in cell-mediated immunity, such as T cells or natural killer cells. When expressed on APCs, siglecs may function in antigen capture and uptake, thus are useful in methods for enhancing cell-based tumor vaccines. The invention also provides methods for modulating the inhibitory functions associated with the biological activity of native ss4623 or ss5846. In addition, the siglec polypeptides are used in methods for identifying proteins that bind to or otherwise interact specifically with the polypeptides encoded by ss5846 or ss4623 nucleic acids.

Furthermore, the invention provides assays utilizing the disclosed polypeptides to screen for molecules that inhibit the activity associated with polypeptide ligands or binding partners of the ss4623 or ss5846 polypeptides, and methods of using these molecules as therapeutic agents for the treatment of diseases mediated by ss5846 or ss4623 polypeptide counter-structure molecules. A further aspect of the invention includes methods of using these molecules in the design of inhibitors of such binding partners.

The invention also encompasses methods for using the disclosed polypeptides as molecular weight markers that allow the estimation of the molecular weight or isoelectric points of another protein or fragments thereof, as well as methods for establishing the extent of fragmentation of another protein.

Further encompassed by this invention is the use of the ss5846 and ss4623 nucleic acid sequences, predicted amino acid sequences of the polypeptide or fragments thereof, or a combination of the predicted amino acid sequences of the polypeptide and fragments thereof for use in searching an electronic database or to aid in the identification of sample nucleic acids and/or proteins.

Isolated polyclonal or monoclonal antibodies that bind to the subject polypeptides are also encompassed, as well as antibody-based methods for purifying the disclosed novel siglec polypeptides. Encompassed also are antibody -based methods for separating from mixtures of cell types those cells expressing the subject polypeptides, e.g., dendritic cells or T cells. This is accomplished, for example, by using fluoresence-activated cell sorting (FACS), panning or other methods that involve antibody binding to cells that are expressing a transmembrane form of one of the subject polypeptides.

DETAILED DESCRIPTION OF THE INVENTION

Two cDNA clones, ss5846-p and ss4623, encoding members of the siglec family were identified initially from a human dendritic cell cDNA library by high-throughput sequencing of individual clones. The nucleotide sequence of ss5846-p (SEQ ID NO:l) includes a termination codon, thus this cDNA insert contains an intact coding region at its carboxy terminus. Further screening of a T-cell cDNA library yielded several additional clones encoding three distinct variants of the protein encoded by ss5846-p. These isolates are classified as members of the siglec family based on analysis of their predicted amino acid sequences. Both the ss5846 and ss4623 genes map to chromosome 19. More specifically, ss5846 maps to a position 276.25 cR from the top of chromosome 19. The isolated ss4623 cDNA (SEQ ID NO:3) encodes a transmembrane protein, but the ss5846 gene appears to encode both a soluble protein as well as transmembrane variants, most likely due to alternate splicing. The three T-cell derived variants of the ss5846 polypeptide (ss5846-tl, -t2, and -t3, corresponding, respectively, to SEQ ID NOS:5, 7 and 9) all encode proteins containing a transmembrane region while the protein encoded by ss5846-p clearly lacks such a region. Moreover, the 3' ends of these two types of cDNA diverge in the vicinity of the transmembrane domain coding region that is present in the variants, suggesting that this region of ss5846-p employs an alternate coding exon than the corresponding region in the T-cell derived ss5846 cDNAs. Though a complete cDNA clone corresponding to ss5846-p was not obtained, a composite sequence shown in SEQ ID NO: 11 is believed to represent the complete coding region of a naturally occurring transcript that encodes a soluble ss5846 protein. The amino acid sequence of the composite ss5846 soluble protein is shown in SEQ ID NO: 12. Other siglecs have been documented to exhibit alternate splicing, including the murine MAG proteins, the CD33-like proteins and CD33, the latter two of which are among those known siglecs most closely related to ss5846.

Several distinct regions can be discerned within the ss5846-tl, ss5846-t2 and ss5846-t3 polypeptides. A leader sequence, also called a signal peptide, is present in these polypeptides, although a few amino acids are missing at the NH terminus of the leader in ss5846-tl and -t2 translation products. However, the entire signal peptide coding region appears to be present in the ss5846-t3 clone, as the 5' nucleotide sequences in this clone include a methionine codon. A predicted cleavage site for this signal peptide is located after amino acid 17, or alternatively, after amino acid 13, of SEQ ID NO: 10. The extracellular regions of the three variants, inclusive of the leader sequence, are located at amino acids 1-256, 1-253, and 1-241, respectively, of SEQ ID NOS:6, 8 and 10. These extracellular regions contain two Ig-like domains, which for ss5846-tl are located at amino acids 20-138 and 140-233 of SEQ ID NO:6; for ss5846-t2, at amino acids 17-135 and 137-230 of SEQ ID NO:8; and for ss5846-t3, at 21-139 and 141-234 of SEQ ID NO: 10. The transmembrane regions for these polypeptides are located at amino acids 257-279, 254-276, and 242-264, respectively, of SEQ ID NOS:6, 8 and 10. The intracellular regions are located at amino acids 280-295, 277-298, and 265-286, respectively, of SEQ ID NOS:6, 8 and 10. Two of the ss5846 variants also contain a modified ITIM motif, as discussed below.

The ss5846-tl, -t2 and -t3 proteins have identical amino acid sequences throughout most of their lengths. Using SEQ ID NO: 10 as a point of reference, all three polypeptides have identical have identical amino acid sequences (i.e., after cleavage of their signal peptides) up to the amino acid corresponding to amino acid 233 of SEQ ID NO: 10. Downstream from this position, i.e., towards the carboxy terminus of the proteins, SEQ ID NO: 10 diverges from the other two by having a substitution at amino acid 234, followed by an in-frame deletion of 16 amino acids relative to SEQ ID NOS:6 and 8. Following these 16 amino acids, the three protein sequences coincide for the next 30 amino acids, after which SEQ ID NO:6 diverges from the other two by having a frameshift insertion of 26 nucleotides (nucleotides 842-867 of SEQ ID NO:5).

Interestingly, the cytoplasmic portion of the ss4623 protein contains an ITIM motif, as well as a second sequence that is a modified ITIM motif. The first of these has the sequence IQYAPL (SEQ ID NO: 13), and corresponds to amino acids 435-440 of SEQ ID NO:4. The second sequence is NEYSEI (SEQ ID NO: 14), corresponding to amino acids 458-463 of SEQ ID NO:4, and may represent a functional variant of the ITIM motif.

Two of the transmembrane variants of ss5846, ss5846-t2 and ss5846-t3, also appear to contain a modified ITIM motif that may be functional. This motif has the amino acid sequence NVYAVM (SEQ ID NO: 15), and is located at amino acid residues 292-297 and 280-285, respectively, of SEQ ID NOS: 8 and 10. In some embodiments of the invention, the inhibitory activity of ss5846-t2 or -t3 is triggered by cross-linking of cell surface proteins, stimulation by a cytokine, binding to an agonistic antibody, or binding to a ligand counterstructure.

Nucleic acid molecules of the invention include the nucleotide sequences of SEQ ID NOS:l and 3, which were determined by sequencing the inserts present in cDNA clones ss5846-p and ss4623, respectively. Clones of plasmids containing these inserts have been deposited with the American Type Culture Collection (ATCC), located at 10801 University Boulevard., Manassas, VA 10110-2209; these deposits were made in accord with the provisions of the Budapest Treaty. The clone containing the ss5846-p insert has been assigned the ATCC accession number ATCC 203634, while the clone containing the ss4623 insert has been assigned the accession number ATCC 203633. Provided also are ss5846 and ss4623 nucleic acid molecules having the nucleotide sequences shown in SEQ ID NOS:5, 7, 9 and 11.

The invention also provides nucleic acid molecules containing sequences that are highly unique to ss5846, including nucleotides 50-94 and 395-520 of SEQ ID NO:l, and nucleotides 142-167, 737-764 and 775-1392 of SEQ ID NO:9, as well as nucleic acids whose sequences are highly unique to ss4623, such as nucleotides 76-138, 181-270 and 766-825 of SEQ ID NO:3. These highly sequences have low sequence identity with other siglec family members, thus are ideal for designing ss4623-specific or ss5646-specific oligonucleotides to be used as hybridization probes or PCR primers, or for directing the synthesis of polypeptide fragments that are unique to the disclosed siglecs. Such oligonucleotides are useful, inter alia, for probing genomic DNA or cDNA under highly stringent conditions to identify naturally occurring mutants and allelic variants of ss4623 or ss5846, as well as cDNA variants that result from alternate splicing.

As used herein, "mutant" means a form of the gene or protein that cannot function normally, such as an altered form that is associated with a human disease, whereas an allelic variant may differ from the disclosed sequences, yet retain normal ss5846 or ss4623 function. The invention also encompasses man-made variants (i.e., using recombinant DNA techniques) that may or may not retain normal function. The non- highly unique sequences of ss4623 and ss5846 are useful as hybridization probes for detecting siglec DNAs other than ss4623 and ss5846, i.e., as research tools for discovering unknown members of the siglec family.

Hybridization probes generally are between 12 and 100 nucleotides in length (oligonucleotides), though longer probes also are useful for hybridization. When used as probes, DNAs longer than about 100-500 nucleotides if desired may be sheared into smaller fragments before being hybridized. In some embodiments, probes are at least about 17 contiguous nucleotides in length, and in other embodiments are at least 30 or at least 60 contiguous nucleotides in length. PCR primers are about 10-50 nucleotides long, or more preferably, 20-30 nucleotides long.

Highly stringent hybridization conditions involve a combination of buffer and incubation temperature that supports the formation of specific, i.e., well-matched, duplexes while still allowing the formation of stable duplexes at an acceptable rate. Such conditions can be optimized in accord with well-known principles by applying formulae that take into account the length and base composition of the DNA (e.g., see sections 11.45-11.47 of Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^nd ed., CSH Laboratories Press (1989), which is hereby incorporated by reference). Highly stringent hybridization conditions for oligonucleotides can be achieved, for example, by hybridizing oligonucleotides with filter-bound target nucleic acid overnight at 50-55° C in aqueous buffer containing 5 x SSC or 6 x SSC (1 x SSC=0.15 M NaCl, 0.015 M sodium citrate), followed by washes in 6 x SSC at 50-55° C. For hybridizing probes longer than about 100 nucleotides with filter-bound target

DNA or RNA, one way of achieving highly stringent conditions is to use a prewashing solution containing 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6 x SSC, and a hybridization temperature of about 42^"C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42'C ), and washing conditions employing 68^"C, 0.2 x SSC, 0.1% SDS. For moderately stringent conditions for nucleic acids longer than about 100 nucleotides, the same procedure may be used except that the washes are done at about 60 ^»C, in 0.5 x SSC, 0.1% SDS. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art (see, e.g., Sambrook et al., 1989). It should be further understood that hybridization conditions for oligonucleotide probes of defined length and sequence can be designed by applying formulae known in the art (e.g., see Sambrook et al., 1989, at 11.45-11.47). Various conditions of high or moderate stringency can be readily determined by the skilled artisan based on, for example, the length and base composition of the DNA. Equivalent degrees of stringency may be maintained under a variety of different hybridization conditions, for example by adjusting the salt concentration and temperature of the hybridization or wash buffers, or by using a different salt in the hybridization buffer, and/or or by adding formamide to the hybridization buffer and thus reducing the hybridization temperature by 1.5 C° for every 1 % formamide added.

Highly unique PCR primers based on the aforementioned highly unique sequences can be used in amplification reactions containing first strand cDNA templates from cellular sources likely to be expressing ss5846 or ss4623 mRNAs, thus providing a means for quantifying the expression of these two genes.

Also provided herein are ss4623 and ss5846 nucleic acid molecules capable of hybridizing under moderately stringent conditions to a DNA probe that is complementary to all or to a portion of a nucleotide sequence as shown in SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 11. Provided as well are nucleic acid molecules capable of hybridizing to these same DNA probes under highly stringent conditions. In one preferred embodiment, the DNA probes are derived from the aforedescribed highly unique nucleotide sequences present in ss4623 and ss5846. Other embodiments of the invention include nucleotide sequences encoding discrete domains of the polypeptides encoded by ss5846 or ss4623. Computer analysis predicts that the signal peptide for the ss4623 protein is most likely to be cleaved after residue 15 of SEQ ID NO:4, though other possible cleavage sites are located after amino acids 16 and 19. These cleavage sites predict a mature ss4623 protein comprising amino acids 16-467, 17-467 or 20-467 of SEQ ID NO:4. The two Ig-like groups located at amino acids 23-126 and 161-224 are likely to be involved in sialic acid recognition. A transmembrane region is found at amino acids 356-376, and a cytoplasmic domain at amino acids 377-467. Thus, the invention provides nucleic acid molecules encoding these discrete protein fragments, as well as the protein fragments comprising each domain separately or in various combinations. The invention provides nucleic acids containing nucleotides 43-88, 43-91 and 43-99, which encode the ss4623 signal peptides residing at amino acids 1-15, 1-16 and 1-19 of SEQ ID NO:4; nucleotides 89-1443, 92-1443 and 100-1443, which encode mature ss4623 polypeptides comprising, respectively, amino acids 16-467, 17-467 and 20-467 of SEQ ID NO:4; nucleotides 1108-1170, encoding a transmembrane region comprising amino acids 356-376; nucleotides 89-1107, 92-1107 and 100-1107, encoding extracellular portions of the ss4623 protein; and nucleotides 1171-1443, encoding a cytoplasmic domain comprising amino acids 377-467. Also contemplated is an ss4623 polypeptide comprising the first two Ig-like domains (encoded by nucleotides 209-714 of SEQ ID NO:3) in which sialic acid binding capacity may reside.

The invention further contemplates ss4623 or ss5846 polypeptides comprising more than one but less than all of the above described protein fragments, such as for example, an otherwise full-length ss4623 molecule from which the transmembrane region, the cytoplasmic region, the extracellular region, and/or the signal peptide is deleted.

The discovery of the nucleic acids of the invention enables the construction of expression vectors comprising nucleic acid sequences encoding polypeptides; host cells transfected or transformed with the expression vectors; isolated and purified biologically active polypeptides and fragments thereof; the use of the nucleic acids or oligonucleotides thereof as probes to identify nucleic acids encoding related siglec family proteins; the use of the nucleic acids or oligonucleotides thereof to correlate the location of the ss4623 or ss5846 genes with chromosome regions associated with human diseases, the use of the nucleic acid or oligonucleotides thereof to identify genes associated with tumors, immune disorders, syndromes or other human conditions; the administration of the disclosed proteins or fragments thereof for the treatment of disorders characterized by a mutation in the ss4623 or ss5846 gene or by an excess or a deficit of an ss4623 or ss5846 protein; the use of single-stranded sense or antisense oligonucleotides to inhibit expression of polynucleotides encoded by the ss4623, ss5846 or closely related siglecs; the use of the disclosed polypeptides and soluble fragments thereof as competitive inhibitors of the binding of native ss4623 or ss5846 polypeptides to their ligands or counter-structure binding partners; the use of ss4623 and ss5846 polypeptides and peptide fragments as unique molecular weight markers or as controls for peptide fragmentation, and kits comprising these reagents; the use of such polypeptides and fragments thereof to generate antibodies; and the use of such antibodies to purify the ss4623 and ss5846 polypeptides, as affinity reagents for the separation of hematopoietic cells expressing the proteins.

As used herein, an "isolated nucleic acid molecule" refers to a nucleic acid molecule (DNA or RNA) in the form of a separate fragment or as a component of a larger nucleic acid construct that is substantially free of proteins and lipids. Expression of the subject nucleic acid molecules generally involves using a form of the molecule wherein it contain an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA may be present 5' or 3' from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.

NUCLEIC ACID MOLECULES

Nucleic acid molecules of the invention include DNA and RNA in both single- stranded and double-stranded form, as well as their corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA may be isolated by conventional techniques, e.g., using the sequences of SEQ ID NOS:l, 3, 5, 7, 9 or 11, or suitable fragments thereof, as probes.

The DNA molecules of the invention include full length genes encoding the polypeptides of ss5846 and ss4623, as well as polynucleotides and fragments thereof. Other embodiments include DNA encoding soluble forms of the proteins, e.g., fragments comprising the extracellular domains. The polypeptide encoded by ss5846-c appears to be a soluble protein, and may be secreted in vivo. Based on comparison with known members of the siglec family, amino acids 16-355 of the ss4623 polypeptide correspond to the extracellular portion of this polypeptide. In other embodiments, the extracellular domain of ss4623 comprises amino acids 17-355 or 20-355. The nucleic acids of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well. Due to the known degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, a DNA sequence can vary from that shown in SEQ ID NOS:l, 3, 5, 7, 9 or 11 and still encode polypeptides having the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 10 or 12, respectively. Such variant DNA sequences are encompassed by the invention, and can result from silent mutations (e.g., occurring during PCR amplification), or can be the product of deliberate mutagenesis of a native sequence.

The invention thus provides isolated nucleic acids molecules encoding polypeptides of the invention, selected from: (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9 or 11; (b) a nucleic acid molecule encoding a polypeptide as shown in SEQ ID NOS:2, 4, 6, 8, 10 or 12; (c) a nucleic acid molecule capable of hybridization to a nucleic acid molecule of (a) or (b) under highly stringent or moderately stringent conditions and that encodes a polypeptide of the invention; and (d) a nucleic acid molecule that encodes a polypeptide that is encoded by a nucleic acid of (a), (b), (c), but that differs in codon usage from the nucleic acid of (a), (b) or (c) due to the degeneracy of the genetic code. Also provided are nucleic acid molecules encoding functional fragments of the proteins encoded by (a), (b), (c) and (d), as well as polypeptides that differ in that they comprise an inactivated N-glycosylation site(s), an inactivated protease processing site(s), or a conservative amino acid substitution(s), as described below. In addition, the invention encompasses the polypeptides encoded by the aforementioned nucleic acid sequences, and fragments thereof.

The nucleic acid molecules of the invention also include DNAs and RNAs that are at least 85% identical to one of the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9 and 11. Also contemplated are nucleic acid molecules whose sequence is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to the disclosed siglec nucleotide sequences.

The percent identity for nucleic acids may be determined by visual inspection and mathematical calculation, or alternatively, by comparing the sequences using a computer program. An example of a suitable computer program is GAP, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984), which is available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities), and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6145, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Equivalent comparisons may also be performed using other computer programs known to those skilled in the art.

The invention also provides isolated nucleic acids useful in the production of polypeptides. Such polypeptides may be prepared by any of a number of conventional techniques. A DNA sequence encoding the polypeptides of the invention, or desired fragments thereof, may be subcloned into an expression vector for production of the polypeptide or fragment. The DNA sequence advantageously is fused to a sequence encoding a suitable leader or signal peptide. Alternatively, the desired fragment may be chemically synthesized using known techniques. DNA fragments encoding desired polypeptide fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels, or by PCR amplification of desired regions of DNA. If necessary, oligonucleotides that reconstruct the 5' or 3' terminus to a desired point may be ligated to a DNA fragment generated by restriction enzyme digestion. Such oligonucleotides may additionally contain a restriction endonuclease cleavage site upstream of the desired coding sequence, and position an initiation codon (ATG) at the N-terminus of the coding sequence. The well-known polymerase chain reaction (PCR) procedure also may be employed to isolate and amplify a DNA sequence encoding a desired protein fragment. Oligonucleotides that define the desired termini of the DNA fragment are employed as 5' and 3' primers. The oligonucleotides may additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified DNA fragment into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990). Provided herein are nucleic acid molecules that hybridize to a denatured, double- stranded DNA comprising all or selected portions of SEQ ID NO:l and SEQ ID NO:3. Also encompassed are isolated nucleic acid molecules that are derived by in vitro mutagenesis of nucleic acid molecules comprising sequences of SEQ ID NOS:l or 3 that are degenerate from nucleic acid molecules comprising sequences of SEQ ID NOS:l or 3, and that are naturally occuring allelic variants of these nucleic acids. Further encompassed by the invention are mRNA splicing variants that may be derived from the nucleic acids that encode ss5846 or ss4623, as well as the polypeptides translated from such variants. In addition, the invention encompasses methods of using the nucleic acid molecules noted above to identify nucleic acids encoding additional novel members of the siglec family. Moreover, the nucleic acids of the present invention are useful for chromosome identification, as they map to chromosome 19, and moreover probes unique to ss5846 are useful for mapping to a position 276.25 cR from the top of chromosome 19. The disclosed nucleic acids hybridize to a particular location on an individual human chromosome, and therefore can be correlated with diseases that are associated with that same chromosome locus (see, e.g., V. McKusick, Mendelian Inheritance in Man, available at the Johns Hopkins University Medical Library website).

POLYPEPTIDES AND FRAGMENTS THEREOF

Polypeptides encompassed by the invention include the ss4623 polypeptide that is shown in SEQ ID NO:4, and which is encoded by the nucleotide sequence of SEQ ID NO:3. as well as the ss5846 polypeptides whose sequences are shown in SEQ ID NOS:2, 6, 8, 10 and 12 and which are encoded, respectively, by the open reading frames shown in SEQ ID NOS: l, 5, 7, 9 and 11. The invention encompasses purified polypeptides and fragments thereof in various forms, including those that are naturally occurring or produced through various techniques, such as procedures involving recombinant DNA technology. Such forms include, but are not limited to, derivatives, variants, and oligomers, as well as fusion proteins or fragments thereof.

The polypeptides of the invention include full length proteins encoded by the nucleic acid sequences set forth above. Such polypeptides comprise the amino acid sequence of SEQ ID NOS:2, 4, 6, 8, 10 and 12. The subject polypeptides include fragments comprising amino acids corresponding to identifiable domains of the ss4623 polypeptide. Referring to SEQ ID NO:4, the signal peptide may be cleaved to produce signal peptides comprising amino acids 1-15, 1-16 or 1-19; the sialic acid binding domain comprises at least amino acids 23-114; the extracellular portion of the molecule comprises amino acids 16-355 or 17-355 or 20-355, depending on the cleavage site for the signal peptide; the transmembrane region comprises amino acids 356-376; and the cytoplasmic region of the protein, comprises amino acids 377-467. The skilled artisan will recognize that the above- described boundaries of such regions of the polypeptide are approximate and that the boundaries of the transmembrane region (which may be predicted by using computer programs available for that purpose) may differ from those described above.

The polypeptides of the invention may be membrane-bound or they may be secreted and thus soluble. Soluble polypeptides are capable of being secreted from the cells in which they are expressed. In general, soluble polypeptides may be identified (and distinguished from non-soluble membrane-bound counterparts) by separating intact cells which express the desired polypeptide from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired polypeptide. The presence of polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus is a soluble form of the protein. In one embodiment, the soluble polypeptides and fragments thereof comprise all or part of the extracellular domain, but lack the transmembrane region that would cause retention of the polypeptide in a cell membrane. A soluble polypeptide according to the invention may include the cytoplasmic domain, or a portion thereof, as long as the polypeptide is secreted from the cell in which it is produced. In general, the use of soluble forms is advantageous for certain applications.

Purification of the polypeptides from recombinant host cells is facilitated, since the soluble polypeptides are secreted from the cells. Further, soluble polypeptides are generally more suitable for intravenous administration.

The invention also provides polypeptides and fragments of the extracellular domain that retain the capacity to bind sialic acid. Such a fragment may be a soluble polypeptide, as described above.

Also provided herein are polypeptide fragments comprising at least 20, or at least 30, contiguous amino acids of the sequence of SEQ ID NOS:2, 4, 6, 8, 10 or 12. Fragments derived from the cytoplasmic domain find use in studies of signal transduction, and in regulating cellular processes associated with transduction of biological signals, such as inhibitory signals, and in identifying small molecule mimics or inhibitors of receptor interaction with signaling molecules. Polypeptide fragments comprising at least 8-11, or more preferably 10-30, contiguous amino acids of SEQ ID

NOS:2, 4, 6, 8, 10 or 12 also may be employed as immunogens for generating antibodies, as well as larger polypeptides.

Variants

Naturally occurring variants as well as derived variants of the disclosed polypeptides and fragments are provided herein. Variants may exhibit amino acid sequences that are at least 80% identical to the disclosed polypeptides and fragments. Also provided are polypeptides or fragments comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to the amino acid sequences disclosed herein. Percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined by comparing sequence information using the a computer program, such as the GAP program, based on the algorithm of Needleman and Wunsch (J. Mol. Bio. 48:443, 1970) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a scoring matrix, blosum62, as described by Henikoff and Henikoff (Proc. Natl. Acad. Sci. USA 89:10915, 1992); (2) a gap weight of 12; (3) a gap length weight of 4; and (4) no penalty for end gaps. Similar comparison parameters can be implemented using other computer programs. The variants of the invention include, for example, those that result from alternate mRNA splicing events or from proteolytic cleavage. Alternate splicing of mRNA may, for example, yield a truncated but biologically active protein, such as a naturally occurring soluble form of the protein. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the protein (generally from 1-5 terminal amino acids). Proteins in which differences in amino acid sequence are attributable to genetic polymorphism (allelic variation among individuals producing the protein) are also contemplated herein. Additional variants within the scope of the invention include polypeptides that may be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives may be prepared by linking the chemical moieties to functional groups on amino acid side chains or at the N-terminus or C- terminus of a polypeptide. Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein, as discussed in more detail below.

Other derivatives include covalent or aggregative conjugates of the polypeptides with other proteins or polypeptides, such as by synthesis in recombinant culture as N- terminal or C-terminal fusions. Examples of fusion proteins are discussed below in connection with oligomers. Further, fusion proteins can comprise peptides added to facilitate purification and identification. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988. One such peptide is the FLAG^® peptide, Asp-Tyr- Lys-Asp-Asp-Asp-Asp-Lys, which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. A murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAG^® peptide in the presence of certain divalent metal cations, as described in U.S. Patent 5,011,912, hereby incoφorated by reference. The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259. Monoclonal antibodies that bind the FLAG^® peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Connecticut.

Among the variant polypeptides provided herein are variants of native polypeptides that retain the native binding properties of ss5846 or ss4623 or the substantial equivalent thereof. One example is a variant that binds its binding partner with essentially the same binding affinity as does the native form. Binding affinity can be measured by conventional procedures, e.g., as described in U.S. Patent No. 5,512,457 and as set forth below. Variants include polypeptides that are substantially homologous to the native form, but which have an amino acid sequence different from that of the native form because of one or more deletions, insertions or substitutions. Particular embodiments include, but are not limited to, polypeptides that comprise from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a native sequence.

A given amino acid may be replaced, for example, by a residue having similar physiochemical characteristics. Examples of such conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one another; substitutions of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gin and Asn; or substitutions of one aromatic residue for another, such as Phe, Trp, or Tyr for one another. Other conservative substitutions, e.g., involving substitutions of entire regions having similar hydrophobicity characteristics, are well known.

Similarly, the DNAs of the invention include variants that differ from a native DNA sequence because of one or more deletions, insertions or substitutions, but that encode a biologically active polypeptide, e.g., variants that exhibit inhibitory activity.

Sialoadhesins contain a number of potential glycosylation sites. The invention further includes polypeptides of the invention with or without associated native-pattern glycosylation. Polypeptides expressed in yeast or mammalian expression systems (e.g.,

COS-1 or COS-7 cells) can be similar to or significantly different from a native polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of any of the polypeptides of the invention in bacterial expression systems, such as E. coli, provides non-glycosylated forms of the polypeptides.

Further, a given preparation may include multiple differentially glycosylated species of the protein. Glycosyl groups can be removed through conventional methods, in particular those utilizing glycopeptidase. In general, glycosylated polypeptides of the invention can have their carbohydrate moieties removed by being incubated with a molar excess of glycopeptidase (Boehringer Mannheim).

Correspondingly, similar DNA constructs that encode various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences are encompassed by the invention. For example, N-glycosylation sites in the polypeptide extracellular domain can be modified to preclude glycosylation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Appropriate substitutions, additions, or deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain. Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an N-glycosylation site. Alternatively, the Ser or Thr can by replaced with another amino acid, such as Ala. Known procedures for inactivating N-glycosylation sites in proteins include those described in U.S. Patent 5,071,972 and EP 276,846.

In another example of variants, sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon folding or renaturation.

Other variants are prepared by modification of adjacent dibasic amino acid residues, to enhance expression in yeast systems in which KEX2 protease activity is present. EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein. KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites. Oligomers Encompassed by the invention are oligomers or fusion proteins that contain ss4623 polypeptides or one of the disclosed ss5846 polypeptides. In a preferred embodiment, the fusion partners are linked to the C-terminus of the ss4623 or ss5846 polypeptide or fragment. Such oligomers may be in the form of covalently-linked or non- covalently-linked multimers, including dimers, trimers, or higher oligomers. As noted above, preferred polypeptides are soluble and thus these oligomers may comprise soluble polypeptides. In one aspect of the invention, the oligomers maintain the binding ability of the polypeptide components and provide therefor, bivalent, trivalent, etc., binding sites.

One embodiment of the invention is directed to oligomers comprising multiple polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the polypeptides. Such peptide moieties may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of the polypeptides attached thereto, as described in more detail below. Immunoglobulin-based Oligomers

As one alternative, an oligomer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1 - 10.19.11, 1992).

One embodiment of the present invention is directed to a dimer comprising two fusion proteins created by fusing a polypeptide of the invention to an Fc polypeptide derived from an antibody. A gene fusion encoding the polypeptide/Fc fusion protein is inserted into an appropriate expression vector. Polypeptide/Fc fusion proteins are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent molecules.

The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides made up of the Fc region of an antibody comprising any or all of the CH domains of the Fc region. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. Preferred polypeptides comprise an Fc polypeptide derived from a human IgGl antibody. The Fc polypeptides preferably are linked to the COOH-terminus of a polypeptide of the invention.

One suitable Fc polypeptide, described in PCT application WO 93/10151 (hereby incorporated by reference), is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgGl antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Patent 5,457,035 and in Baum et al., (EMBO J. 13:3992-4001, 1994) incoφorated herein by reference. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

The above-described fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns. In other embodiments, the polypeptides of the invention may be substituted for the variable portion of an antibody heavy or light chain. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form an oligomer with as many as four ss5846 or ss4623 extracellular regions.

Pepti de-linker Based Oligomers

Alternatively, the oligomer is a fusion protein comprising multiple polypeptides, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233, which are hereby incoφorated by reference. A DNA sequence encoding a desired peptide linker may be inserted between, and in the same reading frame as, the DNA sequences of the invention, using any suitable conventional technique. For example, a chemically synthesized oligonucleotide encoding the linker may be ligated between the sequences. In particular embodiments, a fusion protein comprises from two to four soluble ss5846 or ss4623 polypeptides, separated by peptide linkers. Leucine-Zippers

Another method for preparing the oligomers of the invention involves use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.

The zipper domain (also referred to herein as an oligomerizing, or oligomer- forming, domain) comprises a repetitive heptad repeat, often with four or five leucine residues interspersed with other amino acids. Examples of zipper domains are those found in the yeast transcription factor GCN4 and a heat-stable DNA-binding protein found in rat liver (C/EBP; Landschulz et al., Science 243:1681, 1989). Two nuclear transforming proteins, fos and jun, also exhibit zipper domains, as does the gene product of the murine proto-oncogene, c-myc (Landschulz et al., Science 240: 1759, 1988). The products of the nuclear oncogenes/os andjun comprise zipper domains that preferentially form heterodimer (O'Shea et al., Science 245:646, 1989, Turner and Tjian, Science 243:1689, 1989). The zipper domain is necessary for biological activity (DNA binding) in these proteins. The fusogenic proteins of several different viruses, including paramyxovirus, coronavirus, measles virus and many retroviruses, also possess zipper domains (Buckland and Wild, Nature 338:547,1989; Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS Research and Human Retroviruses 6:703, 1990). The zipper domains in these fusogenic viral proteins are near the transmembrane region of the proteins; it has been suggested that the zipper domains could contribute to the oligomeric structure of the fusogenic proteins. Oligomerization of fusogenic viral proteins is involved in fusion pore formation (Spruce et al, Proc. Natl. Acad. Sci. U.S.A. 88:3523, 1991). Zipper domains have also been recently reported to play a role in oligomerization of heat-shock transcription factors (Rabindran et al., Science 259:230, 1993).

Zipper domains fold as short, parallel coiled coils (O'Shea et al., Science 254:539, 1991). The general architecture of the parallel coiled coil has been well characterized, with a "knobs-into-holes" packing as proposed by Crick in 1953 (Ada Crystallogr. 6:689). The dimer formed by a zipper domain is stabilized by the heptad repeat, designated (abcdefg)_n according to the notation of McLachlan and Stewart (J. Mol. Biol. 98:293; 1975), in which residues a and d are generally hydrophobic residues, with d being a leucine, which line up on the same face of a helix. Oppositely-charged residues commonly occur at positions g and e. Thus, in a parallel coiled coil formed from two helical zipper domains, the "knobs" formed by the hydrophobic side chains of the first helix are packed into the "holes" formed between the side chains of the second helix.

The residues at position d (often leucine) contribute large hydrophobic stabilization energies, and are important for oligomer formation (Krystek: et al., Int. J. Peptide Res. 38:229, 1991). Lovejoy et al. (Science 259:1288, 1993) recently reported the synthesis of a triple-stranded • -helical bundle in which the helices run up-up-down. Their studies confirmed that hydrophobic stabilization energy provides the main driving force for the formation of coiled coils from helical monomers. These studies also indicate that electrostatic interactions contribute to the stoichiometry and geometry of coiled coils. Further discussion of the structure of leucine zippers is found in Harbury et al (Science 262:1401, 26 November 1993). Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308, as well as the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe et al. (FEBS Letters 344:191, 1994), hereby incoφorated by reference. The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow et al. (Semin. Immunol. 6:267-278, 1994). Recombinant fusion proteins comprising a soluble polypeptide fused to a leucine zipper peptide are expressed in suitable host cells, and the soluble oligomer that forms is recovered from the culture supernatant.

Certain leucine zipper moieties preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD) noted above, as described in Hoppe et al. (EE5S Letters 344: 191, 1994) and in U.S. Patent 5,716,805, hereby incoφorated by reference in their entirety. This lung SPD-derived leucine zipper peptide comprises the amino acid sequence Pro Asp Val Ala Ser Leu Arg Gin Gin Val Glu Ala Leu Gin Gly Gin Val Gin His Leu Gin Ala Ala Phe Ser Gin Tyr.

Another example of a leucine zipper that promotes trimerization is a peptide comprising the amino acid sequence Arg Met Lys Gin He Glu Asp Lys He Glu Glu He Leu Ser Lys He Tyr His He Glu Asn Glu He Ala Arg He Lys Lys Leu He Gly Glu Arg, as described in U.S. Patent 5,716,805. In one alternative embodiment, an N-terminal Asp residue is added; in another, the peptide lacks the N-terminal Arg residue.

Fragments of the foregoing zipper peptides that retain the property of promoting oligomerization may be employed as well. Examples of such fragments include, but are not limited to, peptides lacking one or two of the N-terminal or C-terminal residues presented in the foregoing amino acid sequences. Leucine zippers may be derived from naturally occurring leucine zipper peptides, e.g., via conservative substitution(s) in the native amino acid sequence, wherein the peptide 's ability to promote oligomerization is retained.

Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric ss5846 or ss4623 polypeptides. Alternatively, synthetic peptides that promote oligomerization may be employed. In particular embodiments, leucine residues in a leucine zipper moiety are replaced by isoleucine residues. Such peptides comprising isoleucine may be referred to as isoleucine zippers, but are encompassed by the term "leucine zippers" as employed herein. PRODUCTION OF POLYPEPTIDES AND FRAGMENTS THEREOF

Expression, isolation and purification of the polypeptides and fragments of the invention may be accomplished by any suitable technique, including but not limited to the following:

Expression Systems

Provided herein are recombinant cloning and expression vectors containing DNA, as well as host cell containing the recombinant vectors. Expression vectors comprising DNA may be used to prepare the polypeptides or fragments of the invention encoded by the DNA. A method for producing polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding the polypeptide, under conditions that promote expression of the polypeptide, then recovering the expressed polypeptides from the cells or from culture medium in which the host cell is grown. The procedure for purifying the expressed polypeptides will vary according to the type of host cells employed, and whether the polypeptide is membrane-bound or is a secreted soluble form of the protein.

Suitable expression vectors include a DNA encoding a polypeptide or fragment of the invention, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a DNA sequence if the promoter nucleotide sequence controls the transcription of the DNA sequence. An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incoφorated into the expression vector.

In addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incoφorated into expression vectors. A DNA sequence for a signal peptide may be fused in frame to the nucleic acid sequence of the invention so that the DNA is initially transcribed, and the mRNA translated, into a fusion protein comprising the signal peptide. Signal peptides may be employed that direct transmembrane proteins to the cell surface, or different signal peptides may be used that promote the secretion of a soluble form of the protein. Generally, the signal peptide is cleaved during maturation of the protein.

The skilled artisan will also recognize that the position(s) at which the signal peptide is cleaved may differ from that predicted by computer program, and may vary according to such factors as the type of host cells employed in expressing a recombinant polypeptide. A protein preparation may include a mixture of protein molecules having different N-terminal amino acids, resulting from cleavage of the signal peptide at more than one site. Particular embodiments of mature ss4623 proteins provided herein include, but are not limited to, proteins wherein the N-terminus is cleaved at amino acid 14, 15, 16, 17, 18, 19 or 20 of SEQ ID NO:4.

Suitable host cells for expression of polypeptides include prokaryotes, yeast, plant cells, insect or higher eukaryotic cells. Most typically, yeast or mammalian cells are used. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from DNA constructs disclosed herein. Prokaryotic Systems Suitable prokaryotic host cells for transformation may be gram-negativr or gram- positive, and include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, a polypeptide may include an N-terminal methionine (met) residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant polypeptide.

Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes, which may include, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Useful prokaryotic expression vectors include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017), which ampicillin and tetracycline resistance genes. Other suitable vectors include, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, Wl, USA). An appropriate promoter and a DNA sequence encoding the desired polypeptide may be inserted into the vector.

Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include • -lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., NαtMre 281:544, 1979), tryptophan (tφ) promoter system (Goeddel et al., Nucl. Acids Res. 5:4057, 1980; and EP-A-36776) and tac promoter (Maniatis et al., Molecular Cloning: A Laboratory Manual, first ed., Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic host cell expression system employs a phage ^»P_L promoter and a cI857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incoφorate derivatives of the ^»P_L promoter include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).

Yeast Systems Alternatively, the polypeptides may be expressed in yeast host cells, such as from the Saccharomyces genus (e.g., S. cerevisiae). Alternatively, Pichia, Kluyveromyces, or other yeast genera may be employed. Yeast vectors will often contain an origin of replication sequence from a 2^» yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences include those derived from the yeast metallothionein or 3-phosphoglycerate kinase genes (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other genes encoding glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 77:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho-glucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are known in the art (e.g., see in Hitzeman, ΕPA-73,657; Russell et al., J. Biol. Chem. 258:2614, 1982; and Beier et al., Nature 300:124, 1982). The yeast • -factor leader sequence may be employed to direct secretion of the polypeptide, and often is inserted between the promoter sequence and the structural gene sequence (e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984). Yeast transformation protocols are known to those of skill in the art, including a proticol involving selection for Tφ⁺ transformants in a medium containing yeast nitrogen base, casamino acids, glucose, 10 mg/ml adenine and 20 mg/ml uracil (e.g., Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978. In other protocols, yeast cells transformed by vectors containing an ADH2 promoter sequence may be grown for inducing expression in a "rich" medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.

Mammalian or Insect Systems

Mammalian or insect host cell culture systems also may be employed to express recombinant polypeptides, such as the bacculovirus systems reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Established cell lines of mammalian origin also may be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:115, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991). Established methods for introducing DNA into mammalian cells have been described (Kaufman, R.J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69). Additional protocols using commercially available reagents, such as Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine-Plus lipid reagent, can be used to transfect cells (Feigner et al., Proc. Natl. Acad. Sci. USA 84:1413-1411, 1987). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al., 1989. Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cy to toxic drugs. Kaufman et al., Meth. in Enzymology iS5:487-511, 1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable host strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980). A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media. Other examples of selectable markers include cDNAs conferring resistance to antibiotics, such as G418 and hygromycin B, which permit selection of cells harboring the vector on the basis of resistance to these compounds.

Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment, which can also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. in Enzymology, 1990). Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.

Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors include such elements as the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al., Animal Cell Technology, 1997, pp. 529-534 and PCT Application WO 97/25420) and the tripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem. 257:13475-13491, 1982). The internal ribosome entry site (IRES) sequences of viral origin allows dicistronic mRNAs to be translated efficiently (Oh and Sarnow, Current Opinion in Genetics and Development 5:295-300, 1993; Ramesh et al., Nucleic Acids Research 24:2691-2100, 1996). Expression of a heterologous cDNA as part of a dicistronic mRNA followed by the gene for a selectable marker (e.g. DHFR) has been shown to improve transfectability of the host and expression of the heterologous cDNA (Kaufman, Meth. in Enzymology, 1990). Exemplary expression vectors that employ dicistronic mRNAs are pTR-DC/GFP described by Mosser et al., Biotechniques 22:150- 161, 1997, and p2A5I described by Morris et al., Animal Cell Technology, 1997, pp. 529- 534.

A useful high expression vector, pCAVNOT, has been described by Mosley et al., Cell 59:335-348, 1989. Other expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable high level expression of mammalian cDNAs in C 127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 25:935, 1986). A useful high expression vector, PMLSV N1/N4, described by Cosman et al., Nature 312:168, 1984, has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, and in WO 91/18982, incoφorated by reference herein. In yet another alternative, the vectors can be derived from retroviruses.

Additional useful expression vectors, pFLAG^® and pDC311, can also be used. FLAG^® technology is centered on the fusion of a low molecular weight (lkD), hydrophilic, FLAG^® marker peptide to the N-terminus of a recombinant protein expressed by pFLAG" expression vectors. pDC311 is another specialized vector used for expressing proteins in CHO cells. pDC311 is characterized by a bicistronic sequence containing the gene of interest and a dihydrofolate reductase (DHFR) gene with an internal ribosome binding site for DHFR translation, an expression augmenting sequence element (EASE), the human CMV promoter, a tripartite leader sequence, and a polyadenylation site.

Signal peptides may be employed to direct secretion of recombinant proteins. If desired, the native signal peptide may be replaced by a heterologous signal peptide or leader sequence, or proteins not normally secreted may be prepared for secretion by excision of nucleotides encoding a transmembrane region, and fusion of a signal peptide capable of directing secretion of the protein. The choice of signal peptide or leader may depend on factors such as the type of host cells in which the recombinant polypeptide is to be produced. To illustrate, examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in United States Patent 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Patent 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846. Isolation and Purification

The invention also includes methods of isolating and purifying the polypeptides and fragments thereof. The "isolated" polypeptides or fragments thereof encompassed by this invention are polypeptides or fragments that are not in an environment identical to an environment in which it or they can be found in nature. The "purified" polypeptides or fragments thereof encompassed by this invention are essentially free of association with other proteins or polypeptides, for example, as a purification product of recombinant expression systems such as those described above or as a purified product from a non- recombinant source such as naturally occurring cells and/or tissues.

In one preferred embodiment, the purification of recombinant polypeptides or fragments can be accomplished using fusions of polypeptides or fragments of the invention to another polypeptide to aid in the purification of polypeptides or fragments of the invention. Such fusion partners can include the poly-His or other antigenic identification peptides described above as well as the Fc moieties described previously. Procedures for purifying a recombinant polypeptide or fragment will vary according to such factors as the type of host cells employed and whether or not the recombinant polypeptide or fragment is secreted into the culture medium.

In general, the recombinant polypeptide or fragment can be isolated from the host cells if not secreted, or from the medium or supernatant if soluble and secreted, followed by one or more concentration, salting-out, ion exchange, hydrophobic interaction, affinity purification or size exclusion chromatography steps. If desired, the culture medium first can be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed, including various insoluble matrices comprising sulfopropyl or carboxymethyl groups. In addition, a chromatofocusing step or, alternatively, a hydrophobic interaction chromatography step can be employed. Suitable matrices can be phenyl or octyl moieties bound to resins. In addition, affinity chromatography with a matrix which selectively binds the recombinant protein can be employed. Examples of such resins employed are lectin columns, dye columns, and metal-chelating columns. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gel or polymer resin having pendant methyl, octyl, octyldecyl or other aliphatic groups) can be employed to further purify the polypeptides. Some or all of the foregoing purification steps, in various combinations, are well known and can be employed to provide an isolated and purified recombinant protein.

It is also possible to utilize an affinity column comprising a polypeptide-binding protein of the invention, such as a monoclonal antibody generated against polypeptides of the invention, to affinity-purify expressed polypeptides. These polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the invention.

In this aspect of the invention, polypeptide-binding proteins, such as the anti- polypeptide antibodies of the invention or other proteins that may interact with the polypeptide of the invention, can be bound to a solid phase support such as a column chromatography matrix or a similar substrate suitable for identifying, separating, or purifying cells that express polypeptides of the invention on their surface. Adherence of polypeptide-binding proteins of the invention to a solid phase contacting surface can be accomplished by any means, for example, magnetic microspheres can be coated with these polypeptide-binding proteins and held in the incubation vessel through a magnetic field. Suspensions of cell mixtures are contacted with the solid phase that has such polypeptide-binding proteins thereon. Cells having polypeptides of the invention on their surface bind to the fixed polypeptide-binding protein and unbound cells then are washed away. This affinity-binding method is useful for purifying, screening, or separating such polypeptide-expressing cells from solution. The cells can be released, for example, by using a preferably non-toxic enzyme that cleaves the cell-surface binding partner, or by effecting such release by modifying the compostion of the buffer.

Alternatively, mixtures of cells suspected of containing polypeptide-expressing cells of the invention first can be incubated with a biotinylated polypeptide-binding protein, such as an anti-siglec antibody. Sufficient binding usually occurs within about one hour, after which the mixture then is passed through a column packed with avidin- coated beads, to which the biotin moiety will bind with high affinity (see Berenson, et al. J. Cell. Biochem., 10D:239, 1986). Unbound cells are washed free of the column, and bound cells are eluted according to conventional methods. This method can be used to isolate cells expressing membrane-bound ss4623 or ss5846. When purifying isolated siglec proteins, the desired degree of purity will depend on the intended use of the protein. A relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example. In such a case, the polypeptides typically are purified such that no protein bands corresponding to other proteins are detectable by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). One skilled in the art will understand that multiple bands corresponding to the polypeptide may be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptide of the invention is purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDS- PAGE. The protein band may be visualized by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography. Assays

The purified polypeptides of the invention (including proteins, polypeptides, fragments, variants, oligomers, and other forms) may be tested for the ability to bind a ss5846 or a ss4623 binding partners, such as sialic acid-containing proteins, in any suitable assay, such as a conventional binding assay. To illustrate, the polypeptide may be labeled with a detectable reagent (e.g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like), and then contacted with cells expressing sialic acid-containing surface proteins. The cells are washed to remove unbound labeled polypeptide, and the presence of cell-bound label is determined by a suitable technique.

One example of such a binding assay, a recombinant expression vector is constructed containing cDNA corresponding to a ss5846 or ss4623 protein (or fragments thereof) fused to an Fc region according to methods well known in the art. The cDNA may encode, for example, soluble ss4623 or ss5846, comprising the extracellular portions of such proteins, or may include the extracellular domain and a cytoplasmic domain with the transmembrane region removed. CVl-EBNA-1 cells (ATCC CRL 10478), which constitutively express EBV nuclear antigen- 1, are transfected with the recombinant expression vector. The derivation of this cell line is described by McMahan et al. (EMBO J. 10:2821, 1991). The transfected cells are cultured for 24 hours, and the cells in each dish then are split into a 24-well plate. After culturing an additional 48 hours, medium containing the soluble ss4623 or other soluble polypeptide of the invention is collected from the transfected cells (about 4 x 10⁴ cells per well), and the amount of the polypeptide is quantified using standard methods.

Cells expressing the binding partner are cultured as above, and washed with BM- NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50 mg/ml nonfat dry milk has been added. The cells then are incubated for 1 hour at 37 »C with various concentrations of, for example, a soluble polypeptide/Fc fusion protein made as expressed in a vector as set forth above. Cells then are washed and incubated with a constant saturating concentration of a ¹²⁵I-mouse anti-human IgG in binding medium, with gentle agitation for 1 hour at 37* C. After extensive washing, cells are released via trypsinization.

The mouse anti-human IgG employed above is directed against the Fc region of human IgG and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, PA. The antibody is radioiodinated using the standard chloramine-T method. The antibody will bind to the Fc portion of any polypeptide/Fc protein that has bound to the

1 cells. In all assays, non-specific binding of I-antibody is assayed in the absence of the Fc fusion protein/Fc, as well as in the presence of the Fc fusion protein and a 200-fold molar excess of unlabeled mouse anti-human IgG antibody.

Cell-bound ¹²⁵I-antibody is quantified on a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. N.Y. Acad. Sci. 51:660, 1949) are generated on RS/1 (BBN Software, Boston, MA) run on a Microvax computer.

Another type of suitable binding assay is a competitive binding assay. To illustrate, biological activity of a variant may be determined by assaying for the variant's ability to compete with the native proteins for binding to its binding partner. Competitive binding assays can be performed by conventional methodology.

Reagents that may be employed in competitive binding assays include a radiolabeled soluble ss5846 or ss4623 polypeptide or intact cells expressing these same polypeptides (endogenous or recombinant) on the cell surface. For example, a radiolabeled soluble ss5846 or ss4623 fragment can be used to compete with a soluble variant for binding to a cell surface binding partner. Instead of intact cells, one could substitute a soluble ss5846 or ss4623/Fc fusion protein bound to a solid phase through the interaction of Protein A or Protein G (on the solid phase) with the Fc moiety. Chromatography columns that contain Protein A and Protein G include those available from Pharmacia Biotech, Inc., Piscataway, NJ.

Another type of competitive binding assay utilizes a radiolabeled soluble ss5846 or ss4623, such as a soluble ss5846 or ss4623/Fc fusion protein, and intact cells expressing sialic acid-containing binding partners. Qualitative results can be obtained by competitive autoradiographic plate binding assays, while Scatchard plots (Scatchard, Ann.

N.Y. Acad. Sci. 51:660, 1949) may be utilized to generate quantitative results.

USE OF ss5846 or ss4623 NUCLEIC ACIDS

In addition to being used to express polypeptides as described above, the nucleic acids of the invention, including DNA, and oligonucleotides thereof can be used: as probes to identify nucleic acid encoding proteins having the ability to bind with sialic acid-containing proteins; to identify genes associated with certain diseases, syndromes, or other conditions associated with the human chromosomes to which the genes of the invention map; as single-stranded sense or antisense oligonucleotides, to inhibit expression of polypeptides encoded by the ss5846 or ss4623 genes; to identify disease states in which expession of ss5846 or ss4623 is abnormal or perturbed; - to help detect defective genes in an individual; and for gene therapy. Probes

Among the uses of nucleic acids of the invention is the use of fragments as probes or PCR primers. Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence. In other embodiments, a DNA fragment comprises at least 30, or at least 60, contiguous nucleotides of a DNA sequence.

Because homologs of SEQ ID NOS:l, 3, 5, 7, 9 and 11 from other mammalian species are contemplated herein, probes based on the disclosed human DNA sequences may be used to screen cDNA libraries derived from other mammalian species, using conventional cross-species hybridization techniques, e.g., hybridization under conditions of moderate to low stringency. Using knowledge of the genetic code in combination with the amino acid sequences set forth above, sets of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful as primers, e.g., in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified. Identifying Associated Diseases

Nucleic acid molecules of the invention may be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of, the genes corresponding to the nucleic acids of the invention. Disclosure herein of native nucleotide sequences permits the detection of defective genes, and the replacement thereof with normal genes. Defective genes may be detected in in vitro diagnostic assays, and by comparison of a native nucleotide sequence disclosed herein with that of a gene derived from a person suspected of harboring a defect in either of these genes. Sense-Antisense Other useful fragments of the disclosed nucleic acids include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise fragments of the nucleic acid molecules of SEQ ID NOS:l, 3, 5, 7, 9, and 11. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and

Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block or inhibit protein expression by one of several means, including enhanced degradation of the mRNA by RNAse H, inhibition of splicing, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of proteins. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar- phosphodiester backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences. Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.

Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, lipofection, CaPO -mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus or adenovirus.

Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.

USE OF ss5846 and ss4623 POLYPEPTIDES AND FRAGMENTED POLYPEPTIDES

Uses include, but are not limited to, the following:

Purifying proteins and measuring activity thereof

Delivery agents

Therapeutic reagents

Reasearch reagents - Molecular weight and isoelectric focusing markers

Controls for peptide fragmentation

Identification of unknown proteins Preparation of polyclonal and monoclonal antibodies Purification Reagents

The polypeptides of the invention find use as protein purification reagents. The polypeptides may be attached to a solid support material and used to purify sialic acid- containing binding partners by affinity chromatography. In particular embodiments, a polypeptide (in any form described herein that is capable of binding to the binding partners of ss5846 or ss4623) is attached to a solid support by conventional procedures. As one example, chromatography columns containing functional groups that will react with functional groups on amino acid side chains of proteins are available (Pharmacia Biotech, Inc., Piscataway, NJ). In an alternative, a polypeptide/Fc protein (as discussed above) is attached to Protein A- or Protein G-containing chromatography columns through interaction with the Fc moiety.

The polypeptides of the invention also find use in purifying or identifying cells that express sialic acid-containing binding partners on the cell surface. The polypeptides are bound to a solid phase such as a column chromatography matrix or a similar suitable substrate. For example, magnetic microspheres can be coated with the polypeptides and held in an incubation vessel through a magnetic field. Suspensions of cell mixtures containing binding partner-expressing cells are contacted with the solid phase having the polypeptides thereon. Cells expressing the binding partner on the cell surface bind to the fixed polypeptides, and unbound cells then are washed away. Thus, for example, this procedure provides for the separation from cell mixtures of dendritic cells or T cells, which are cells expressing ss5846.

Alternatively, the polypeptides can be conjugated to a detectable moiety, then incubated with cells to be tested for ss5846 or ss4623 binding partner expression. After incubation, unbound labeled matter is removed and the presence or absence of the detectable moiety on the cells is determined.

In a further alternative, mixtures of cells suspected of containing ss5846 or ss4623 binding partner-expressing cells are incubated with biotinylated ss5846 or ss4623 polypeptides. Incubation periods are typically at least one hour in duration to ensure sufficient binding. The resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides binding of the desired cells to the beads. Procedures for using avidin-coated beads are known (see Berenson, et al. J. Cell. Biochem., 10D:239, 1986). Washing to remove unbound material, and the release of the bound cells, are performed using conventional methods. Measuring Activity

Polypeptides also find use in measuring the biological activity of sialic acid moities in terms of their binding affinity. The polypeptides thus may be employed by those conducting "quality assurance" studies, e.g., to monitor shelf life and stability of protein under different conditions. For example, the polypeptides may be employed in a binding affinity study to measure the biological activity of a sialic acid-containing binding partner protein that has been stored at different temperatures, or produced in different cell types. The proteins also may be used to determine whether biological activity is retained after modification of a binding partner protein (e.g., chemical modification, truncation, mutation, etc.). The binding affinity of the modified binding partner protein is compared to that of an unmodified binding partner protein to detect any adverse impact of the modifications on biological activity of the binding partner. The biological activity of a binding partner protein thus can be ascertained before it is used in a research study, for example.

Delivery Agents

The polypeptides also find use as carriers for delivering agents attached thereto to cells expressing specific binding proteins bearing sialic acid moieties. The polypeptides thus can be used to deliver diagnostic or therapeutic agents to such cells, or to other cell types found to express the ss5846 or ss4623 binding partners on the cell surface in in vitro or in vivo procedures.

Antibodies against ss5846 or ss4623 polypeptides also may be used as vehicles to deliver diagnostic or therapeutic agents to cells expressing ss5846 or ss4623. Detectable (diagnostic) and therapeutic agents that may be attached to such antibodies include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction, and the like, with the particular agent being chosen according to the intended application. Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins such as trichothecenes, and derivatives and fragments (e.g., single chains) thereof. Radionuclides suitable for diagnostic use include, but are not limited to, ¹²³I, ¹³¹I, ⁹⁹Tc, ^{ι n}In, and ⁷⁶Br. Examples of radionuclides suitable for therapeutic use are ¹³¹I, ²¹¹At, ⁷⁷Br, ¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu. Such agents may be attached to the antibodies by any suitable conventional procedure. The antibodies comprise functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example. Alternatively, the protein or agent may be derivatized to generate or attach a desired reactive functional group. The derivatization may involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to proteins (Pierce Chemical Company, Rockford, Illinois). A number of techniques for radiolabeling proteins are known. Radionuclide metals may be attached to antibodies by using a suitable bifunctional chelating agent, for example. Conjugates comprising antibodies and a suitable diagnostic or therapeutic agent

(preferably covalently linked) are thus prepared. The conjugates are administered or otherwise employed in an amount appropriate for the particular application. Similarly, conjugates may be made of soluble forms of ss5846 or ss4623 for delivery of the diagnostic or therapeutic agent to cells expressing receptors for ss5846 or ss4623 proteins.

Therapeutic Agents

As discussed below, the subject polypeptides may be used in treating any disorder mediated (directly or indirectly) by defective, or insufficient amounts of the subject polypeptides. Research Agents

Another use of the polypeptide of the present invention is as a research tool for studying the biological effects that result from inhibiting interactions of ss5846 or ss4623 polypeptides in different cell types. Polypeptides also may be employed in in vitro assays for detecting ss5846 or ss4623 or interactions with their binding partners. Another embodiment of the invention relates to uses of the polypeptides of the invention to study cell signal transduction. SS4623, and the membrane forms of ss5846, like other cell surface proteins, could play a central role in immune responses which includes cellular signal transduction, antigen uptake and/or presentation, cell migration, cell-cell interactions, and cell adhesions to other cells or to the extracellular matrix. As such, alterations in the expression and/or activation of the polypeptides of the invention can have profound effects on a plethora of cellular processes. Expression of ss5846 or ss4623, functionally inactive mutants of these polypeptides, or the soluble extracellular forms of these proteins can be used to identify the role a particular protein plays in mediating specific signaling events.

Cellular signaling often involves a molecular activation cascade, during which a receptor propagates a ligand-receptor mediated signal by specifically activating intracellular kinases which phosphorylate target substrates. These substrates can themselves be kinases which become activated following phosphorylation. Alternatively, they can be adaptor molecules that facilitate down stream signaling through protein- protein interaction following phosphorylation. Regardless of the nature of the substrate molecule(s), expressed functionally active versions of ss4623 and ss5846, can be used in assays such as the yeast 2-hybrid assay to identify what substrate(s) were recognized and activated by these polypeptides' binding partners. As such, these novel siglec family members can be used as reagents to identify novel molecules involved in signal transduction pathways. Antibodies Antibodies that are specifically immunoreactive with the polypeptides of the invention are provided herein. By "specific" is meant that such antibodies do not bind under standard antibody-antigen binding conditions with any siglecs other than a ss4623 polypeptide or fragment thereof, or a ss5846 polypeptide or fragment thereof, or a polypeptide or fragment thereof that has at least 80% amino acid sequence homology with a ss4623 or a ss5846 polypeptide. Immunogenic peptides providing the desired specificity may be derived, for example, from portions of the ss5846 or ss4623 polypeptides that have no known counteφarts, such as those encoded by the aforementioned highly unique nucleotide sequences. Alternatively, specific antibodies may be derived from complex epitopes involving more than one contiguous amio acid sequence (see below). Screening procedures by which such antibodies may be identified are well known, and for example may involve immunoaffinity chromatography.

The polypeptides, fragments, variants, fusion proteins, etc., as set forth above may be employed as "immunogens" in producing antibodies immunoreactive therewith. More specifically, the polypeptides, fragment, variants, fusion proteins, etc. contain antigenic determinants or epitopes that elicit the formation of antibodies. Suitable antigenic determinants or epitopes may be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon protein folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded proteins have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes may be identified by any of the methods known in the art.

The epitopes derived from the disclosed polypeptides are useful for raising antibodies, including monoclonal antibodies, and can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology. The polyclonal and monoclonal antibodies elicited by the disclosed polypeptides, whether the epitopes have been isolated or remain part of the polypeptides, may be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein, and may be produced and identified by conventional techniques. One method for producing such a hybridoma cell line comprises immunizing an animal with a polypeptide; harvesting spleen cells from the immunized animal; fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds the polypeptide. The monoclonal antibodies may be recovered by conventional techniques.

The monoclonal antibodies of the present invention include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies may be prepared by known techniques and offer the advantage of reduced immunogenicity when the antibodies are administered to humans, such as for therapeutic puφoses. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winter and Harris (77RS 14:139, May, 1993). Procedures to generate antibodies transgenically can be found in GB 2,272,440, US Patent Nos. 5,569,825 and 5,545,806 and related patents claiming priority therefrom, all of which are incoφorated by reference herein.

Antigen-binding fragments of the antibodies, which may be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab')₂ fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided. Uses Thereof

The antibodies of the invention can be used in assays to detect the presence of the polypeptides or fragments of the invention, either in vitro or in vivo. The antibodies also may be employed in purifying polypeptides or fragments of the invention by immunoaffinity chromatography. Those antibodies that additionally can block binding of the polypeptides of the invention to their binding partners may be used to inhibit a biological activity that results from such binding. Such blocking antibodies may be identified using any suitable assay procedure, such as by testing antibodies for the ability to inhibit binding of ss5846 or ss4623 polypeptides to certain cells expressing the binding partners of these polypeptides. Alternatively, blocking antibodies may be identified in assays for the ability to inhibit a biological effect that results from binding of the polypeptides of the invention to target cells. Antibodies may be assayed for the ability to inhibit ss5846 of ss4623-mediated cellular activities, for example.

Such antibodies may be employed in in vitro procedures, or administered in vivo to inhibit a biological activity mediated by the entity that generated the antibody. Disorders caused or exacerbated (directly or indirectly) by the interaction of the polypeptides of the invention with cell surface (binding partner) receptor thus may be treated. A therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective in inhibiting an ss5846 or ss4623-mediated biological activity. Monoclonal antibodies are generally preferred for use in such therapeutic methods. In one embodiment, an antigen-binding antibody fragment is employed.

Antibodies may be screened for agonistic (i.e., ligand-mimicking) properties. Such antibodies, upon binding to cell surface sialic acid containing proteins, induce biological effects (e.g., transduction of biological signals) similar to the biological effects induced when ss5846 or ss4623 binds to cell surface ligands. Agonistic antibodies may be used to antagonize such responses .

Compositions comprising an antibody that is directed against ss5846 or ss4623 and a physiologically acceptable diluent, excipient, or carrier, are provided herein. Suitable components of such compositions are as described above for compositions containing ss5846 or ss4623 proteins.

Also provided herein are conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to the antibody. Examples of such agents are presented above. The conjugates find use in in vitro or in vivo procedures. Diagnostic Assays

The nucleic acids and polypeptides provided herein are useful as diagnostic reagents in assays to detect malfunctioning ss4623 or ss5846 genes. Samples for diagnostic reagents may be obtained from a patient's tissues, for example, throat swab, blood sample, tissue biopsy, urine, saliva and so on. Similar samples are taken from normal individuals, i.e., from persons not suffering from the disorder in question, and these normal samples may provide a basis for comparison. Once normal levels are established, purified reagents (e.g., siglec nucleic acids, proteins and antibodies) may be used as standards for the diagnostic assays. In some embodiments, the subject nucleic acids are used as probes for Northern or Southern blots or as PCR primers to detect mutated forms of ss4623 or ss5846 that are associated with a malfunctioning ss4623 or ss5846 protein.

Conditions that may be diagnosed include those characterized by an excess or deficit of an ss4623 or ss5846 polypeptide, or that are characterized by a mutated form of such a polypeptide. Such conditions include, but are not limited to, absence of the protein in a cell that requires its expression, altered enzymatic activity, altered signalling ability, overexpression or underexpression. Particular conditions that may be diagnosed using these assays include but are not limited to: rheumatologic diseases (e.g., rheumatoid arthritis, psoriatic arthritis, seronegative spondyloarthropathies), inflammatatory conditions, bone marrow or solid organ transplantation, graft-versus-host disease, allergies (e.g., asthma, allergic rhinitis), neurologic disorders (e.g., Alzheimer's, Parkinson's, dementia, brain cancer, Bell's palsy, post-heφetic neuralgia), cancers (e.g., lymphoma, B-cell, T-cell and myeloid cell leukemias), infections (e.g., bacterial, parasitic, protozoal and viral infections, including AIDS), chemotherapy or radiation-induced toxicity, cachexia, cardiovascular disorders (e.g., congestive heart failure, myocardial infarction, ischemia/reperfusion injury, arteritis, stroke), gastrointestinal disorders (e.g., inflammatory bowel disease, Crohn's disease, celiac disease), diabetes mellitus, skin diseases (e.g., psoriasis, scleroderma, dermatomyositis), hematologic disorders (e.g., myelodysplastic syndromes, acquired or Fanconi's aplastic anemia), septic shock, liver diseases (e.g., viral hepatitis or alcohol- associated), bone disorders (e.g., osteoporosis, osteopetrosis). In some embodiments of the invention, the condition being diagnosed is a hematologic disorder, and the tissue sample is blood or a lymph node biopsy. Screening Assays for Agonists and Antagonists of ss4623 and ss5846 The ss4623 and ss5846 nucleic acids and proteins disclosed herein find use in screening assays for identifying agonists and antagonists of the subject proteins. Molecules that bind specifically to the subject siglecs may act either as an agonist or as an antagonist, depending on the effect this binding exerts on the biological activity of the bound siglec. Once identified, such antagonists may be administered, for example, to suppress siglec expression in conditions characterized by oveφroduction of these or other siglecs. Agonists identified in these assays may be used to stimulate the biological activity of the subject proteins in cultured cells or in patients suffering from diseases characterized by a deficit of the normal endogenous activator of these siglecs. Molecules that bind specifically with the subject siglecs are expected to exhibit either an agonistic or antagonistic effect on siglec activity.

Methods of screening a test molecule for its capacity to bind specifically with ss4623 or ss5846 may involve: i) contacting an ss4623 or ss5846 polypeptide with a specific antibody in the presence or absence of the test molecule under conditions that specific binding of the antibody and polypeptide, ii) detecting the extent of binding between polypeptide and antibodyin the presence and absence of the test molecule, and iii) determining that the test molecule binds specifically to the polypeptide if the extent of antibody binding is decreased when the test molecule is present. One means of determining the extent of antibody binding is to measure the capacity of the polypeptide to bind to a solid phase medium to which the antibody is anchored. For this approach, the polypeptide may be pre-incubated with the test molecule, or may be mixed with a solution containing the test molecule during the incubation of the polypeptide with the solid phase medium. In one embodiment, the polypeptide is present on the surface of a cell expressing the polypeptide, or alternatively, a purified soluble form of the polypeptide is used. The amount of antibody binding to the siglec is measured by standard procedures.

Provided also are screening assays for identifying agonists or antagonists that may affect siglec activity directly or indirectly, such as by targetting another molecule that indirectly affects siglec activity. Target molecules having an indirect effect include, for example, ligands that bind specifically with a ss4623 or ss5846 polypeptide, or other molecules capable of forming functional heteromers with the siglec.

To screen a molecule for its capacity to agonize or antagonize the secretion of a soluble siglec polypeptide, cells expressing the polypeptide are incubated in the presence or absence of the test molecule, then the amount of secreted polypeptide is measured by any convenient method, and the test molecule is recognized to be an agonist of polypeptide secretion if the amount secreted is increased in the presence of the test molecule. The test molecule is identified as an antagonist if the amount of polypeptide secreted is reduced in the presence of the test molecule. Secreted polypeptides thus assayed include soluble forms of ss4623 and ss5846, including a protein having amino acids 1-257 of SEQ ID NO: 12, or a fragment thereof. In one such assay, the ITIM-mediated inhibitory activity of ss4623, ss5846-t2, or

-t3 is triggered in a cell expressing the siglec, wherein during the triggering the cells are exposed to the test molecule. The effect of the test molecule is then determined by comparing the extent of ITIM-mediated inhibitory activity in exposed and unexposed cells, then determining that the test molecule is an antagonist if the degree of inhibitory activity detected is decreased when the molecule is present, or that the molecule is an agonist if its presence results in increased inhibitory activity.

Cells used for these screening assays may include cells that naturally express ss4623 or ss5846, such as glial cells, T-cells, myeloid cells and other hematopoietic cells, or any convenient cell type that has been transformed with a vector that directs the expression of the ss4623 or ss5846 polypeptide. For this latter puφose, any suitable mammalian cell may be used that is capable of expressing the transfected gene. Any transmembrand form of the ss4623 or ss5846 proteins described herein are suitable for expression in cells to be used for these screening assays.

In other assays, cells expressing a soluble form of ss5846 may be cultured with the test molecule to determine whether the molecule has the capacity to agonize or antagonize the amount of soluble ss5846 produced by the cells. The amount of soluble ss5846 produced may be measured by any suitable method, including enzyme-linked immunosorbent assay (ELISA), dot blot employing an antibody that binds soluble ss5846, or aa solid phase binding assay. Methods of Therapy

The polypeptides and nucleic acids of the invention may be administered therapeutically to a mammalian patient (human or animal) having a disorder involving a malfunctioning ss4623 or ss5846 gene or polypeptide, including an excess or a deficit of such a polypeptide, or expression of a deleterious mutant form of the polypeptide. Such disorders include conditions caused (directly or indirectly) or exacerbated by such forms of the polypeptides.

For these therapeutic methods, agonists and antagonists of ss4623 or ss5846 may be employed. Such agonists or antagonists are identified by screening, such as by employing the screening methods disclosed herein, and molecules with therapeutic activity include the disclosed polypeptides, small molecules that display agonistic or antagonistic activity, and antibodies specific for a ss4623 or ss5846 polypeptide or a naturally-occuring ligand of such proteins, as well as ligands and fragments thereof, and antisense oligonucleotides. Antibodies that bind specifically with the siglec or its ligand may agonize or antagonize the biologic activity of the ss4623 oar ss5846. Agonistic antibodies are capable of triggering the activity of the membrane-bound forms of the proteins.

Disorders treatable by the aforementioned polypeptides include but are not limited to: rheumatologic disorders (e.g., rheumatoid arthritis, psoriatic arthritis, seronegative spondyloarthropathies), bone marrow or solid organ transplant, graft-versus-host reaction, inflammatory conditions, autoimmune disorders (e.g., systemic lupus erythematosus, Hashimoto's thyroiditis, Sjogren's syndrome), allergies (e.g., asthma, allergic rhinitis), neurologic disorders (e.g., Alzheimer's, Parkinson's, dementia, brain cancer, Bell's palsy, post-heφetic neuralgia), cancers (e.g., lymphoma, B-cell, T-cell and myeloid cell leukemias), infections (e.g., bacterial, parasitic, protozoal and viral infections, including AIDS), chemotherapy or radiation-induced toxicity, cachexia, cardiovascular disorders (e.g., congestive heart failure, myocardial infarction, ischemia/reperfusion injury, arteritis, stroke), diabetes mellitus, skin diseases (e.g., psoriasis, scleroderma, dermatomyositis), hematologic disorders (e.g., myelodysplastic syndromes, acquired or Fanconi's aplastic anemia), septic shock, liver diseases (e.g., viral hepatitis or alcohol- associated), bone disorders (e.g., osteoporosis, osteopetrosis). For treating the above disorders, the therapeutic agent,may be administered in an amount effective to measurably reduce one or more signs or symptoms of the disorder being treated. In addition, such disorders may be treated by administration in vivo or ex vivo of a vector or liposome that delivers a non-defective form of the malfunctioning gene to the cell type in which the malfunction is present. Therapeutic compositions may comprise an isolated ss4623 or ss5846 polypeptide in any form described herein, such as native proteins, variants, derivatives, oligomers, fusion proteins and biologically active protein fragments. The composition may comprise a soluble polypeptide or an oligomer comprising soluble ss5846 or ss4623 polypeptides, and in other embodiments, comprises an antibody directed against at least one ss4623 or ss5846 epitope.

Combination therapies also are envisioned, in which another pharmacologically active compound is co-administered with the therapeutic agents of the present invention. Compounds suitable for co-administration include but are not limited to cytokines, lymphokines, chemokines, chemotherapy agents, anti-inflammatories, DMARDs, or any other compound effective in treating the target disease.

Pharmaceutical compositions of the present invention furthermore may comprise other components such as a physiologically acceptable diluent, carrier, or excipient, are provided herein, and are formulated according to known methods. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphate buffered solutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton, PA.

In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incoφorated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incoφorated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application. The compositions of the invention can be administered in any suitable manner, e.g., orally, topically, parenterally, or by inhalation. The term "parenteral" includes injection, e.g., by subcutaneous, intravenous, or intramuscular routes, also including localized administration, e.g., at a site of disease or injury. Sustained release from implants is also contemplated. Suitable dosages will vary, depending upon such factors as the nature of the disorder to be treated, the patient's body weight, age, and general condition, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices.

The dose, route of administration, frequency of administration and duration of an effective regimen of treatment will vary, depending factors such as the particular condition being treated, the severity of the condition, the age of the patient, and so on, and may be adjusted accordingly by the patient's physician.

In one method of treatment, the active agent is a polypeptide, and is administered by injection one to three times a week at a dose ranging from 0.1-100 mg/kg, or more preferably at a dose of 0.4-50 mg/kg. Treatment is continued until a measurable improvement in the patient's condition has been ascertained, which in most cases will require at least two to eight weeks or more of treatment. Maintenance doses may be administered thereafter, and treatment may be resumed if evidence of disease should reappear. Suitable regimens for other routes of administration may be determined according to methods known in the art. Similarly, suitable regimens for administering antibodies, small molecules, antisense or gene therapy reagents may be determined according to methods known in the art. Included within the scope of the invention are pharmacologically acceptable compositions comprising the aforedescribed therapeutic agents, including compositions suitable for administration by each of the aforedesribed routes. Such compositions are forumlated in accord with standard practices. The following examples are provided to further illustrate particular embodiments of the invention, and are not to be construed as limiting the scope of the present invention.

Example 1. Isolation of Nucleic Acids A cDNA library was prepared using as a template messenger RNA from CD34+ dendritic cells derived from human bone marrow, and individual cDNA clones from this library were sequenced by high-throughput sequencing . Two of the clones sequenced from this library were ss5846-p (SEQ ID NO: l) and ss4623 (SEQ ID NO:3), whose predicted open reading frames are highly similar. The polypeptides encoded by these two clones are shown, respectively, in SEQ ID NO:2 and NO:4. Amino acid sequence comparisons indicated that the proteins predicted to be encoded by the open reading frames of ss5846 and ss4623 are similar to members of the sialic acid-binding Ig-like lectin (siglec) family of proteins.

Specific oligonucleotide primers based on the ss5846-p nucleotide sequence were used in RT-PCR reactions to identify additional sources of ss5846 cDNA. Templates for the RT-PCR reactions included mRNA from a human T cell clone ("clone 22") and a human glioma cell line A-172 (ATCC #CRL 1620). Both of these mRNAs sources yielded PCR products of the size predicted for a PCR product primed by these primers from an ss5846 mRNA. Thus, it is apparent that both T cells and glioma cells express the ss5846 gene.

A random-primed DNA probe prepared from the ss5846-p insert was used in hybridizations to screen the clone 22 cDNA library. Several positive clones were identified and purified. The inserts from the positive clones were amplified by PCR and their nucleotide sequences determined. The sequence thus obtained indicated that there were three closely-related variants of ss5846. Because these clones appear to encode transmembrane proteins, they were named ss5846-tl, ss5846-t2 and ss5846-t3. The nucleotide sequences of these clones are shown, respectively, in SEQ ID NOS:5, 7 and 9, and the polypeptides they encode are shown in SEQ ID NOS:6, 8 and 10. In comparing the 5 '-terminal nucleotide sequences ss5846-tl, ss5846-t2 and ss5846-t3 with sequences of other siglecs, it was noted that the 5' ends of all three ss5846 variants appear to contain a one nucleotide deletion in the region encoding the protein leader sequence. If the 5' nucleotide sequences of the ss5846 variants are translated with this deletion, the predicted amino acid sequence of the resulting signal peptide is quite different from the highly conserved signal sequence found in all of the other known siglecs. Since it appears in all three variants, it is assumed that this one nucleotide deletion probably resulted from an error that occurred during transcription in the cell. Accordingly, the nucleotide sequences depicted in SEQ ID NOS:5, 7, 9 and 11 have been adjusted by insertion of a "G" residue at the position of the deletion, thus giving rise to a leader whose amino acid sequence conforms with those found in other siglecs. The predicted signal peptide cleavage site for ss5846 is not affected by the addition of this nucleotide, i.e., the amino acid sequences of the bodies of the predicted ss5846 proteins is exactly the same regardless of whether or not this correction is made. This inserted "G" corresponds to nucleotide positions 39, 30, 48 and 40, respectively, in SEQ ID NOS:5, 7, 9 and 11.

The amino acid sequence of the ss5846-p polypeptide (SEQ ID NO:2) shares extensive similarity with the polypeptides encoded by ss5846-tl, ss5846-t2 and ss5846-t3 (SEQ ID NOS:6, 8 and 10). However, the ss5846-p sequence diverges at its carboxyl terminus from the proteins encoded by the three T-cell-derived cDNAs. A sequence of about 25 amino acids at the -COOH terminus of ss5846-p (amino acid residues 148-172 of SEQ ID NO:2) lacks any appreciable homology with the corresponding -COOH termini of the ss5846-tl, ss5846-t2 or ss5846-t3 proteins, i.e., with amino acids 250-295 of SEQ ID NO:6; 247-298 of SEQ ID NO:8; or 235-286 of SEQ ID NO: 10. The position at which ss5846-p diverges from ss5846-t-l, -2 and -t3 is located upstream from the transmembrane domains of the latter group of proteins. Thus, ss5846-p lacks any identifiable transmembrane region, and appears to be derived from a mRNA encoding a soluble or secreted form of the protein. The predicted full-length nucleotide sequence for the soluble form of ss5846 is shown in SEQ ID NO:l 1, and its predicted protein is shown in SEQ ID NO: 12.

Using the UWGG program TRANSMEMBRANE and by comparison with other transmembrane proteins, the transmembrane regions of ss5846-tl, ss5846-t2 and ss5846-t3 are predicted to be located at amino acids 257-279, 254-276, and 242-264, respectively, of SEQ ID NOS:6, 8 and 10.

Computerized analysis of the transmembrane variants of ss5846 also revealed a modified ITIM motif in the cytoplasmic domains of two of these transmembrane proteins, ss5846-t2 and -t3. This ITIM motif is located at amino acids 292-297 and 280-285, respectively, of SEQ ID NOS: 8 and 10. The ss5846 ITIM motif has the sequence NVYAVM (SEQ ID NO: 15), which deviates somewhat from the ITIM consensus sequence. However, since ITIMs are present in the cytoplasmic regions of other siglecs, it is likely that the ss5846-t2 and -t3 ITIM motifs function to transmit an inhibitory signal to the cell whenever ss5846-t2 or -t3 is triggered, which could occur, for example, by cross-linking of cell surface proteins, stimulation by a cytokine, binding to an agonistic antibody, or binding to a naturally-occuring ligand.

Example 2. Preparation of Monoclonal Antibodies This example illustrates a method for preparing monoclonal antibodies (MABs) that bind the polypeptides of the invention. Suitable immunogens that may be employed in generating such antibodies include, but are not limited to, purified recombinant ss5846 or ss4623, or fragments thereof. Such fragments may be generated by digestion with proteases such as trypsin, papain or other proteases. Other suitable immunogens include the extracellular or cytoplasmic domains of ss5846 or ss4623, or fusion proteins comprising ss5846 or ss4623 or a subportion thereof, e.g., a soluble ss5846 or ss4623/Fc fusion protein. To obtain purified ss5846 or ss4623 polypeptides for use in raising antibodies (or for use in binding assays or therapeutic puφoses), ss5846 or ss4623 nucleic acid molecules, or portions thereof, are expressed using recombinant DNA techniques.

Alternatively, portions of the predicted ss5846 or ss4623 polypeptides that are unique can be used to design synthetic peptides that can be used as immunogens. After immunizing with synthetic peptides, the immune response is boosted by administering a recombinant Fc fusion protein comprising the extracellular portion of the target siglec, an approach that gives high titres of antibodies specific for the target siglec protein.

Expression of these polypeptides may be accomplished by using the expression vector designated pDC409, which is a mammalian expression vector derived from the pDC406 vector described in McMahan et al. (EMBO J. 10:2821-2832, 1991; hereby incoφorated by reference). Features added to pDC409 (compared to pDC406) include additional unique restriction sites in the multiple cloning site (mcs); three stop codons (one in each reading frame) positioned downstream of the mcs; and a T7 polymerase promoter, downstream of the mcs, that facilitates sequencing of DNA inserted into the mcs.

For expression of full length human ss5846 or ss4623 proteins, the entire coding region (i.e., the DNA sequences presented in SEQ ID NOS:3, 9 or 11) are amplified by polymerase chain reaction (PCR). The 5' coding sequences of SEQ ID NOS: 5 and 7 can be incoφorated into the 5' PCR promers and thus added to the amplified DNA molecules during the PCR reaction. The template employed in the PCR are the cDNA clones isolated from a human dendritic cell cDNA library or T-cell clone cDNA library, as described in Example 1. The isolated and amplified DNA is inserted into the expression vector pDC409, to yield a constructs designated as pDC409-ss5846 and -ss4623. Proteins expressed from these constructs are purified by standard purification techniques for use in the assays. The purified ss5846 and 4623 polypeptides are employed to isolate heamatopoietic cells, as assay reagents, as molecular weight markers, or for other puφoses, as discussed above.

The purified recombinant polypeptides, fragments, or fusion proteins as described above can be used to generate MABs using conventional techniques, such as those described in U.S. Patent 4,411,993. Briefly, mice are immunized with the ss5846 or ss4623 immunogen emulsified in complete Freund's adjuvant, and injected in amounts ranging from 10-100 μg subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals are boosted with additional immunogen emulsified in incomplete Freund's adjuvant. Mice are periodically boosted thereafter on a weekly to bi-weekly immunization schedule. Serum samples are periodically taken to test for ss5846 or ss4623 antibodies by dot blot assay, enzyme-linked immunosorbent assay (ELISA; see below) or inhibition of ss5846 or ss4623 binding to polysaccharide.

Following detection of an appropriate antibody titer, positive animals are provided one last intravenous injection of ss5846 or ss4623 in saline. Three to four days later, the animals are sacrificed, spleen cells harvested and fused to a murine myeloma cell line, e.g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). These cell fusions generate hybridoma cells, which are plated in multiple microtiter plates in a selective medium containing hypoxanthine, aminopterin and thymidine (HAT) to inhibit proliferation of non-fused cells, myeloma cell hybrids and spleen cell hybrids. For characterizing individual hybridoma clones, ELISA may be performed as in Engvall et al. (Immunochem. 8:871, 1971) and in U.S. 4,703,004. Screening may be performed by the antibody capture technique described in Beckmann et al. (J. Immunol. 144:4212, 1990), or by other known methods. To perform ELISA, serial dilutions of ss5846 or ss4823-containing samples (in 50 mM NaHCO₃, brought to pH 9 with NaOH) are coated onto Linbro/Titertek 96 well flat bottom E.I. A. microtitration plates (ICN Biomedicals Inc., Aurora, OH) at 100:l/well. After incubation at 4 ^»C for 16 hours, the wells are washed six times with 200:1 PBS containing 0.05% Tween-20 (PBS-Tween). The wells are then incubated with FLAG®-ss5846 (or -ss4623) at 1 mg/ml in PBS-Tween with 5% fetal calf serum (FCS) for 90 minutes (100:1 per well), followed by washing as above. Next, each well is incubated with the anti-FLAG® (monoclonal antibody M2 at 1 mg/ml in PBS-Tween containing 5% FCS for 90 minutes (100:1 per well), followed by washing as above. Subsequently, wells are incubated with a polyclonal goat anti-mlgGl-specific horseradish peroxidase-conjugated antibody (a 1:5000 dilution of the commercial stock in PBS- Tween containing 5% FCS) for 90 minutes (100:1 per well). The HRP-conjugated antibody is obtained from Southern Biotechnology Associates, Inc., Birmingham, Alabama. Wells then are washed six times, as above.

For development of the ELISA, a substrate mix [100:1 per well of a 1:1 premix of the TMB Peroxidase Substrate and Peroxidase Solution B (Kirkegaard Perry Laboratories, Gaithersburg, Maryland)] is added to the wells. After sufficient color reaction, the enzymatic reaction is terminated by addition of 2 N H₂SO (50 :1 per well). Color intensity (indicating ss5846- (or ss4623-) binding activity) is determined by measuring extinction at 450 nm on a V Max plate reader (Molecular Devices, Sunnyvale, CA).

Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/C mice to produce ascites containing high concentrations of anti-ss5846 or ss4623 MABs. Alternatively, hybridoma cells can be grown in vitro in flasks or roller bottles with appropriate media. Monoclonal antibodies produced in mouse ascites can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to Protein A or Protein G can also be used to purify the MABs, as can affinity chromatography based upon binding to ss5846 or ss4623 polypeptides. Example 3. Tissues Expressing ss5846 and ss4623

Expression of ss5846 occurs in brain cells and T-lymphocytes, as well as in dendritic cells. Expression of this gene in brain and T-cells was determined by PCR- priming of first strand cDNA derived from mRNA extracted from these two cell types, using PCR primers derived from unique regions of nucleotide sequence of SEQ ID NO: 1.

The tissue distribution of ss5846 and 4623 mRNA is investigated by Northern blot analysis, as follows. An aliquot of a radiolabeled probe derived from one of the unique portions of ss5846 or ss4623 DNA is added to two different human multiple tissue

Northern blots (Clontech, Palo Alto, CA; Biochain, Palo Alto, CA). Hybridization is conducted overnight under stringent conditions, which can be achieved, for example, by using suitable salt and/or formamide concentrations in the hybridization buffer, or by varying the incubation temperature during the hybridization reaction (see Sambrook et al.,

1989). Stringent hybridization for mRNA detection can be performed by hybridizing labeled oligonucleotide probes to filter-bound target nucleic acids overnight at 50-55° C in aqueous buffer containing 5 x SSC (1 x SSC=0.15 M NaCl, 0.015 M sodium citrate), followed by washes in 6 x SSC at 50-55° C, though these conditions may be adjusted to take into account the length and base composition of the particular probe being used. A glycerol-aldehyde-phosphate dehydrogenase (GAPDH) specific probe is used to standardize for RNA loadings on the gel. Example 4. Binding Assay ss5846 and ss4623 polypeptides or fragments thereof are expressed by recombinant DNA techniques, purified and tested for the ability to bind with various cells of the hematopoietic lineage. The binding assays employ ss5846 or ss4623 polypeptides, including soluble forms of these proteins, and oligomers formed as described below. Oligomers for assays are prepared as follows. Fusion proteins comprising a leucine zipper peptide fused to the COOH-terminus of an ss5846 or ss4623 polypeptide are constructed as described above. Preferably, the polypeptide comprises a soluble form of ss5846 or ss4523, such as the extracellular region of ss4623 (amino acids 18-275 in SEQ ID NO: 12), or an extracellular region of an ss5846 polypeptide (e.g., amino acids 14-253 of SEQ ID NO:8, or amino acids 20-241 of SEQ ID NO: 10). An expression construct is prepared, essentially as described in Baum et al. (EMBO J. 13:3992-4001, 1994). The construct, in expression vector pDC409, encodes a leader sequence derived from human cytomegalovirus, followed by the leucine zipper moiety fused to the C- terminus of a soluble ss5846 or ss4623 polypeptide. Alternatively, a gene fusion encoding an ss5846 or ss4623 polypeptide/Fc fusion protein is inserted into an appropriate expression vector. Polypeptide/Fc fusion proteins are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble by the formation of interchain disulfide bonds between the Fc moieties, thus yielding dimeric molecules.

Example 5. Chromosome Mapping

This example describes the chromosomal mapping of the ss5846 gene using PCR- based mapping strategies. Initial human chromosomal assignments were made using ss5846-specific PCR primers and a BIOS Somatic Cell Hybrid PCRable DNA kit from BIOS Laboratories (New Haven, CT), following the manufacturer's instructions. ss5846 mapped to human chromosome 19. More detailed mapping was performed using a Genebridge 4 Radiation Hybrid Panel (Research Genetics, Huntsville, AL; described in Walter, MA et al., Nature Genetics 7:22-28, 1994). Data from this analysis was then submitted electronically to the MIT Radiation Hybrid Mapper (URL: http://www- genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) following the instructions contained therein. This analysis yielded specific genetic marker names which, when submitted electronically to the NCBI Genemap browser http://www.ncbi .nlm.nih . gov/genemap/map.cgi ?CHR= 19), yielded the specific chromosome 19 interval. This analysis showed that ss5846 mapped 3.46 cR distal to marker NIB 1805, which is located at 272.79 cR from the top of chromosome 19 on the Genebridge 4 map. ss5846 thus mapped to the position 276.25 cR from the top of chromosome 19, which is very close to the previously mapped position (275.75 cR from the top) of CD33.

The embodiments within the specification provide representative examples of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan will readily recognize that many other embodiments are encompassed by the invention.

Claims

What is claimed is:

1. An isolated nucleic acid molecule selected from the group consisting of:

(a) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9 and 11; (b) a nucleic acid molecule capable of encoding an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10 and 12;

(c) a nucleic acid molecule capable of hybridizing under moderately stringent conditions to a DNA that is complementary to a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 5, 7, 9 and 11; (d) a nucleic acid molecule capable of hybridizing under highly stringent conditions to a DNA that is complementary to a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 5, 7, 9 and 11;

(e) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of nucleotides 76-138 of SEQ ID NO:3, 181-270 of SEQ ID NO:3, and 766-825 of SEQ ID NO:3;

(f) a nucleic acid molecule derived by in vitro mutagenesis from a nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9 and 11; and

(g) a nucleic acid molecule having at least 85% identity with a nucleic acid molecule capable of encoding an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 6, 8, 10 and 12.

2. A recombinant vector that directs the expression of a polypeptide encoded by a nucleic acid molecule according to Claim 1.

3. A host cell transfected or transformed with a vector according to claim 2.

4. A nucleic acid molecule capable of hybridizing under highly stringent conditions with a nucleotide sequence selected from the group consisting of: (a) nucleotides 50-94 of SEQ ID NO: 1 ;

(b) nucleotides 395-520 of SEQ ID NO:l;

(c) nucleotides 142-167 of SEQ ID NO:9;

(d) nucleotides 737-764 of SEQ ID NO:9; and (e) nucleotides 775-1392 of SEQ ID NO:9.

5. An isolated polypeptide encoded by a nucleic acid molecule according to Claim 1, or a fragment thereof.

6. An isolated polypeptide according to claim 5 in non-glycosylated form.

7. An isolated polypeptide having an at least 80% identity to a polypeptide according to claim 5.

8. An isolated antibody capable of binding specifically to a polypeptide of claim 6.

9. An antibody according to claim 8, wherein the antibody is a monoclonal antibody.

10. A host cell according to claim 3, wherein the host cell is selected from the group consisting of a bacterial cell, a yeast cell, a plant cell, an insect cell, and a mammalian cell.

11. A method for producing a ss5846 or ss4623 polypeptide comprising culturing a host cell according to claim 3 in a culture medium under conditions promoting expression of a recombinant protein, and recovering the polypeptide from the host cell or from the culture medium.

12. A polypeptide consisting of an amino acid sequence selected from the group consisting of:

(a) amino acids 1-172 of SEQ ID NO:2;

(b) amino acids 1-295 of SEQ ID NO:6; (c) amino acids 1-298 of SEQ ID NO:8;

(d) amino acids 1-286 of SEQ ID NO: 10;

(e) amino acids 1-275 of SEQ ID NO: 12;

(f) amino acids 1-467 of SEQ ID NO:4; and (g) a peptide fragment of (a)-(f), said fragment being capable of eliciting an antibody that binds specifically with a polypeptide of (a)-(f).

13. A siglec polypeptide having an at least 80% identity to a polypeptide according to claim 12(a), 12(b), 12(c), 12(d) or 12(e).

14. A pharmaceutically acceptable composition comprising a polypeptide selected from the group consisting of:

(a) amino acids 1-172 of SEQ ID NO: 2 or fragments thereof; (b) amino acids 1-295 of SEQ ID NO:6 or fragments thereof;

(c) amino acids 1-298 of SEQ ID NO:8 or fragments thereof;

(d) amino acids 1-286 of SEQ ID NO: 10 or fragments thereof;

(e) amino acids 1-275 of SEQ ID NO: 12 or fragments thereof; and

(f) amino acids 1-467 of SEQ ID NO:4.

15. A pharmaceutical composition according to claim 14, further comprising a physiologically acceptable carrier, excipient or diluent.

16. A method comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition according to claim 14.

17. A method comprising administering to a patient in need thereof an effective amount of an antagonist of ss4623 or ss5846, wherein the antagonist comprises a molecule selected from the group consisting of a soluble ss4623 or ss5846 polypeptide or fragment thereof, a fusion protein comprising a soluble ss4623 or ss5846 polypeptide or fragment thereof, an antibody that specifically binds ss4623 or ss5846, and an antisense oligonucleotide.

18. A method comprising administering to a patient in need thereof an effective amount of an agonist of ss4623 or ss5846, wherein the agonist comprises a molecule selected from the group consisting of a soluble ligand of ss4623 or ss5846 and an antibody that specifically binds ss4623 or ss5846.

19. A method according to claim 16, wherein the polypeptide is administered by bolus injection, intracerebral injection, orally, aerosol, continuous infusion or sustained release from an implant.

20. A method according to claim 17, wherein the molecule is administered by bolus injection, intracerebral injection, orally, aerosol, continuous infusion or sustained release from an implant.

21. A method according to claim 18, wherein the molecule is administered by bolus injection, intracerebral injection, orally, aerosol, continuous infusion or sustained release from an implant.

22. A method according to claim 16, wherein the patient suffers from a condition selected from the group consisting of: an rheumatologic disease; an inflammatory condition; an allergy; a bone marrow or solid organ transplant; a graft-versus-host reaction; a neurologic disorder; a hematologic disorder; a cancer; an infection; septic shock; a gastrointestinal disorder; chemotherapy or radiation-induced toxicity; cachexia; a cardiovascular disorder; diabetes mellitus; a liver disease; a kidney disease; a skin disease; a hematologic disorder; and a bone disorder.

23. A method according to claim 17, wherein the patient suffers from a condition selected from the group consisting of: an rheumatologic disease; an inflammatory condition; an allergy; a bone marrow or solid organ transplant; a graft-versus-host reaction; a neurologic disorder; a hematologic disorder; a cancer; an infection; septic shock; a gastrointestinal disorder; chemotherapy or radiation-induced toxicity; cachexia; a cardiovascular disorder; diabetes mellitus; a liver disease; a kidney disease; a skin disease; a hematologic disorder; and a bone disorder.

24. A method according to claim 18, wherein the patient suffers from a condition selected from the group consisting of: an rheumatologic disease; an inflammatory condition; an allergy; a bone marrow or solid organ transplant; a graft-versus-host reaction; a neurologic disorder; a hematologic disorder; a cancer; an infection; septic shock; a gastrointestinal disorder; chemotherapy or radiation-induced toxicity; cachexia; a cardiovascular disorder; diabetes mellitus; a liver disease; a kidney disease; a skin disease; a hematologic disorder; and a bone disorder.

25. A method of screening a test molecule for its capacity to bind specifically with a polypeptide according to claim 5, wherein the method comprises: a) contacting the polypeptide with the test molecule prior to or during an incubation of the polypeptide with an antibody that is capable of binding specifically with the polypeptide; b) detecting the extent to which the antibody specifically binds the polypeptide during said incubation; and c) determining that the test molecule binds specifically to the polypeptide if the extent of antibody binding is decreased when the polypeptide is contacted with the test molecule prior to or during said incubation.

26. A method of screening a test molecule for its capacity to agonize or antagonize the secretion of a soluble siglec polypeptide comprising amino acids 1-275 of SEQ ID NO: 12 or a fragment therof , wherein said method comprises: a) incubating a culture of cells expressing said polypeptide in the presence and absence of the test molecule; b) detecting the amount of the polypeptide secreted; c) determining that the test molecule agonizes the secretion of the polypeptide if the amount of polypeptide secreted is increased in the presence of the test molecule; and d) determining that the test molecule antagonizes the secretion of the polypeptide if the amount of polypeptide secreted is decreased in the presence of the test molecule.

27. A method for diagnosing a condition characterized by an excess or a deficit of a siglec polypeptide according to claim 6, wherein the method comprises: a) detecting the amount of said polypeptide present in sample of a tissue from a patient suspected of suffering from a condition characterized by an excess or a deficit of the polypeptide; b) determining that the patient suffers from a condition characterized by an excess of the polypeptide if the amount of polypeptide detected in the tissue sample from the patient exceeds the amount of the polypeptide in a sample of said tissue from a normal individual; and c) determining that the patient suffers from a condition characterized by a deficit of the polypeptide if the amount of polypeptide detected in the tissue sample is less than the amount of the polypeptide in a sample of said tissue from a normal individual.

28. A method according to claim 27, wherein the condition characterized by an excess or deficit of the polypeptide is a hematologic disorder, and further wherein the tissue sample is blood or a lymph node biopsy.