CA2242964A1 - Immune cell cytokine - Google Patents

Abstract

Human Immune Cell Cytokine-like Hormone polypeptide and DNA (RNA) encoding such polypeptide in a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for stimulating the proliferation and differentiation of stem cells of the immune system. Antagonists against such polypeptides are also disclosed. The antagonists include antibodies which may be employed as a therapeutic to treat leukemia and lymphoblastoma, may also be used as imaging agents and diagnostic agents for detecting expression levels of the protein.
Also disclosed are diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences and altered concentrations of the polypeptides.

Description

W O 97/25338 PCTrUS96/00205 T~une Cell Cy~o~;ne This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptide of the present invention has been putatively i-l~nti fied as a cytokine, more particularly, the polypeptide of the present invention has been identified as an i~nn~ cell cytokine-like potential hormone, sometimes hereina~ter re~erred to as "HLHDC84". The invention al~o relates to inhibiting the action of such polypeptides.
The cytokine family of proteins P~h; h; t a wide variety of functions. A h~l lm~rk feature is their ability to elicit chemotactic migration of distinct cell types, including polymorp~nl~clear cells and macrophages. Many cyto~ine~ have pro-inflammatory activity and are involved in multiple steps during inflammatory reactions. In addition to their involvement in inflammation, cytok; nes have been shown to exhibit other activities. For example, interleukin-8 (IL-8) promotes proliferation of keratinocytes.
In light of the diverse biological activities, it is not surprising that cytokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound h~l ing, hematopoietic regulation and logical disorders such as allergy, asthma and arthritis. The protein of the present invention is a secreted protein, similar to cytokine proteins, and is most homologous at the amino acid level to the fringe (~ng) gene of Drosophila.

W O 97/25338 PCTrUS96/00205 The fringe _ (fng) gene, encodes a molecule that mediates signaling between distinct cell populations (Irvine, ~.D. and ~ieschaus, E., Cell, 79:595-506 (1994). The fng gene encodes a putatively secreted protein, and mediates processes that est~hli~h the wing m~rgin and promote wing outgrowth without otherwise affectiny dorsal-ventral wing cell identity.
The fng cDNA includes a 412 codon open reA~; ng frame encoding for a novel protein. Notably, this predicted protein product includes a signal sequence at its amino-terminal end but lacks predicted trAr ~ - dne ~nn~in~, suggesting that it i5 secreted (gyte J. and Doolittle, R.F., J. Mol. Biol., 157:105-132 (1982);
Eisenberg, D., et al., J. Mol. Biol., 179:125-142 (1984); von Hei~ne, G., Nucl. Acids Res. 14:4583-4690 (1986)). fng may have a role in cell-cell interactions promoting wing margin formation and wing growth. The fng gene affects a class of epith~l;Al cells which ultimately fonm the wing. This is done by altering the differentiation state of the cells and ~nhAncing their proliferation.
In accordance with one aspect of the present invention, there are provided novel polypeptides as well as biologically active and diagnostically or therapeutically useful fra~m~nts, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding such polypeptide~, including mRNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically use~ul fr~m~nts, analogs and derivatives thereof.
In accordance with another aspect of the present invention there i8 provided an isolated nucleic acid molecule encoding a mature polypeptide e~pressed by the DNA CQntA ine~l in ATCC Deposit No. 97351.
In accordance with another aspect o~ the present invention there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to sequences of the present invention.
In accordance with yet a ~urther aspect of the present invention, there is provided a process for producing such polypeptides by recomhinAnt technique~ which c~..~Lises culturing rec~m~in~nt prokaryotic and/or eukaryotic host cells, con~A;ning nucleic acid sequence of the present invention, under conditions promoting expression of said protein and subsequent recovery o~
said protein.
In accordance with yet a further aspect of the present invention, there is provided a process ~or utilizing such polypeptides, or polynucleotides encoding such polypeptides for O therapeutic purposes, for example, to st;m~ te the proliferation, mobilization and differentiation of stem cells in the ~mmlln~ system for autologous transplant and for treating auto-~mlmP disorders, to stimulate growth factor activity and neuronal re-growth and to treat inflammatory disorders.
In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides and a method o~ employing such antibodies to limit cellular proliferation or induced differentiation of stem cells for the purpose of treating and/or preventing leukemia and lymphoblastoma and as a diagnostic to detect c~nr~r.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to ~ nh~ h; t the action of such polypeptides, ~or example, in the treatment of lellkPm;~ or lymphohl~toma, arthritis and as adjunct treatment during chemotherapy.
In accordance with another aspect o~ the present invention there is provided a method of diagnosing a disease or a susceptibility to a disease related to a mutation in the nucleic acid se~l~ncPfi and the protein Pnro~P~ by such nucleic acid se~uences.
In accordance with another aspect of the present invention there is provided a method of delivering the polynucleotide of the present invention to cells of a patient, either ex vivo or i~ vivo, such that the gene product of the present invention is A~mtnistered to the patient.
In accordance with yet a further aspect of the present ~ invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, ~or in vitro purposes related to scientific research, synthesis of DNA
and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

W O 97/25338 PCT~U~96/OQ205 The following drawin~s are illustrative of embo~m~nts of the invention and are not meant to limit the scope of the invention as encompassed by the cl ~m~, Figure 1 displays the cDNA sequence and corresponding deduced amino acid sequence of the HLHDC84 polypeptide of the present invention. The underlined portion represents a putative leader sequence of 20 amino acid residues. The st?n~rd one-letter abbreviations for amino acids are used. Se~1~n~ng was perfonmed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).
Figure 2 is an illustration of amino acid sequence homology between the protein polypeptide of the present invention and the Drosophila fringe protein (SEQ ID NO:9).
In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature polypeptides having the deduced amino acid sequences of Figure 1 (SEQ ID No. 2).
The polynucleotide encoding the polypeptide of the present invention was isolated from a hum~n fetal lung cDNA library. ~ased on results of Northern Blot analyses the polypeptide of the presen~
invention is located primarily in tissues of the bloodstream. The polypeptide contains an open reading frame encoding a protein of 327 amino acids wherein the first 20 amino acids are a putative leader sequence such that the mature protein comprises 307 amino acids. The protein P~Ch;h~ ts the highest degree of homology at the amino acid level to the Drosophila fringe protein (SEQ ID NO:9) with 42.222% identity and 61.270~ similarity.
In accordance with another aspect of the present invention there are provided isolated polynucleotides encoding a mature polypeptide expressed by the human cDNA cont~ n~ in ATCC Deposit No. 97351, deposited with the A~erican Type Culture Collection, 12301 Park Lawn Drive, Roc~ville, Maryland 20852, USA, on November 28, 1995. The deposited material is an E. ~oli host harboring a pBluescript SK (-) vector (Stratagene, La Jolla, CA) which contA~n~
the full-length HLHDC84 cDNA.
The deposit(s) will be ~;nt~ne~ under the tenms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. These deposits are provided W O 97125338 PCT~US96/00205 merely as convenience to those of skill in the art and are not an admission that a deposit is requixed under 35 U.S.C. 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence o~ the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event o~ any con~lict with any description of ~ se~lences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
The polynucleotides o~ the present invention may be in the ~orm o~ RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptides may be identical to the coding sequences shown in Figure 1 (SEQ ID NO:1) or may be a different coding sequence which coding sequence, as a result of the re~lln~ncy or degeneracy of the genetic code, encodes the same mature polypeptides as the DNA of Figure 1 (SBQ ID NO:1).
The polynucleotides which encode ~or the mature polypeptides o~ Figure 1 (SEQ ID NO:2) may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence ~or the mature polypeptides.
Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes coding sequence for the polypeptide and may also include additional coding and/or non-coding sequence such as introns.
The present invention further relates to variants of the here~n~hove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequences of Figure 1 (SEQ ID NO:2). The variant of the ~ polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides.

- Thus, the pre~ent invention includes polynucleotides ~nCo~;ng the same mature polypeptides as shown in Figure 1 (SEQ ID NO:2) as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptides o~ Figure 1 (S~Q ID NO:2). Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As here~n~hove indicated, the polynucleotides may have a coding se~uence which is a naturally occurring allelic variant of the coding seguences shown in Figure 1 (SEQ ID NO:1). As known in the art, an allelic variant is an alternate form of a po~ynucleotide se~uence which may have a substitution, deletion or addition o~ one or more nucleotides, which does not su~st~nt~lly alter the ~unction of the encoded polypeptide.
The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptides may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader se~uence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader se~uence cleaved by the host cell to form the mature ~orm o~ the polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein having a prose~uence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein rem~,n~.
Thus, for example, the polynucleotides of the present invention may encode ~or a mature protein, or for a protein having a prosequence or for a protein having both a prose~uence and a presequence ~leader sequence).
The polynucleotides o~ the present invention may also have the coding sequence fused in frame to a m~rker sequence which allows for purification of the polypeptides of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQ~
vector (Qiagen, Inc.) to provide for puri~ication of the mature polypeptides fused to the m~k~r in the case of a bacterial host, or, ~or example, the marker se~uence may be a hemagglll~;n~n ~HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope deri~ed from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The term "gene~l means the segment o~ DNA involved in pro~llr;n~
a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding se~-A~t~ (exons).
Fragments o~ the full length gene of the present invention may be used as a hybridization probe for a cDNA library to isolate the full length cDNA and to isolate other cDNAs which have a high ~equence simil~rity to the gene or stmi ~~r biological activity.
Probes o~ this type have at least 15 bases, preferably at least 30 bases and may cont~n, for example, ~0 or more ~ases. The probe may also be used to i~nt;fy a cDNA clone correspon~ ng to a ~ull length transcript and a genomic clone or clones that cont~; n the complete gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known D~A sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70~, preferably at least 90%, and more preferably at least 95~ identity between the ~equences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the herP~n~hove-described polynucleotides.
As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95~ and preferably at least 97~ identity between the seqll~nc~s. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain subst~nt~lly the same biological function or activity as the mature polypeptide encoded by the cDNA o~ Figure 1 (SEQ ID N0:1).
Alternatively, the polynucleotide may have at least 1~ bases, ~ preferably at least 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as her~in~hove described, and which may or may not retain activity. For example, such polynucleotides .

W O 97/25338 PCTAJS96/'~-2~5 may be employed a~ probes ~or the polynucleotide of SEQ ID NO:l, ~or example, for recovery of the polynucleotide or as a diagnostic probe or a~ a PCR primer.
Thus, the present invention is directed to polynucleotides having at least a 70~ identity, preferably at least 90% and more preferably at least a 95~ identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 and polynucleotides compl~mPnt~ry thereto as well as portions thereof, which portions have at least 15 consecutive bases and preferably at least 30 consecutive bases and more preferably at least 5Q consecutive bases and to polypeptides encoded by such polynucleotide~.
The present invention further relates to polypeptides which have the deduced amino acid se~l~nc~ o~ Figure 1 (SEQ ID NO:2), as well as fragments, analogs and derivatives of such polypeptides.
The terms l'fragment," "derivative" and "analog" when referring to the polypeptides o~ Figure 1 (SEQ ID NO:2) means polypeptides which retain essentially the same biological function or activity as such polypeptides. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptides of the present invention may be recomh~n~nt polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.
The fragment, derivative or analog of the polypeptides of Figure 1 (SEQ ID NO:2) may be (i) one in which one or more o~ the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a con~erved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene ~lycol), or (iv) one in which the additional amino acids are ~used to the mature polypeptide, such as a leader or secretory sequence or a se~uence which is employed ~or puri~ication of the mature polypeptide or a proprotein se~uence. Such ~r~gm~nts, derivatives and analogs are ~m~ to be within the scope of tho~e ~killed in the art ~rom the te~ch;ngs herein.

- The polypeptides and polynucleotides of the present invention are prefera~ly provided in an isolated form, and preferably are purified to homogeneity.
The term 'lisolated" means that the material is removed from it8 original environm~nt (e.g., the natural envilv- - t i~ it i5 naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living ~nt m~ 1 iS not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition i5 not part of its natural enviLo~ ..t.
The polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% s;m;l~rity (pre~erably at least 70~ identity~ to the polypeptide of SEQ ID
NO:2 and more preferably at least 90% stm; l~ity (more preferably at least 90% identity~ to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% 5tm~ 1 ~ity (still more preferably at least 95~ identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally cont~;ning at least 30 amino acids and preferably at least 50 amino acids.
As known in the art '~simi~rityll between two polypeptides is determined by cQrr3ring the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the se~uence o~ a second polypeptide.
Fra~nts or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fr~m~nt~
may be employed as i~termediates for pro~ tng the full-length polypeptides. Fra~ments or portions o~ the polynucleotides o~ the present invention may be used to synthesize full-length polynucleotides o~ the present invention.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the inv~nt~n and the WO 97/25338 PCT~US96/00205 production of polypeptides of the invention by recomh;n~nt techniques.
Host cells are genetically engineered (transduced or transformed or trans~ected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, ~or example, in the fonm of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes. The culture conditions, such as temperature, p~ and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides ~y recQ~in~nt techniques. Thus, for example, the polynucleotide ~ay be included in any one o~ a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonch~omosomal and synthetic DNA sequences, e.g., derivatives o~ SV40; bacterial plasmid~; phage DNA;
baculovirus; yeast pl ~m; ~; vectors derived from comhtn~tions of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, ~owl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.
The a~L~riate DNA se~uence may be inserted into the vector by a variety of procedures. In general, the DNA se~uence is inserted into an a~o~riate restriction en~onllclease site(s) by procedures known in the art. Such procedures and others are ~m~
to be within the scope o~ those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control se~uence(s) (promoter) to direct m~NA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E.
coli. lac or trp, the phage l~nb~l~ PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruRes. The expre~sion vector also contains a ribo~ome binding site for translation initiation and a transcription terminator. The vector may also include a~L~iate sequences ~or ampli~ying expres~ion.
In addition, the expression vectors preferably contain one or more selecta~le marker genes to provide a phenotypic trait ~or W O 97/25338 PCT~US96/OQ?O5 selection of trans~ormed host cells such as dihydrofolate reductase or neomycin resistance ~or eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
The vector contA; n~ ng the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
As representative examples of appropriate hosts, there may be mentioned: bacterial cellsl such as E. coli, Stre~tomyces, SA1mO~e1 la tYPhimurium; ~ungal cells, such as yeast; insect cells such as Drosophila S2 and SpodoPtera Sf9; An~m~1 cells such as CHO, ~OS or Bowes mPl Ano~A; adenoviruses; plant cells, etc. The selection o~ an appropriate host is ~m~ to be within the scope of those skilled in the art from the t~A~h;ngs herein.
More particularly, the present invention also includes re~nmh;nAnt constructs comprising one or more o~ the sequences as ~roadly described above. The constructs c ~-ise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
In a preferred aspect o~ this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those o~ skill in the art, and are comm~cially aVA;l~hle~ The ~ollowing vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pN~16a, pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
Promoter regions can be selected ~rom any desired gene using CAT (chlorAmrh~n~col trans~erase) vectors or other vectors with 8electable mArk~rS. Two a~u~iate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, 1 AmhflA PR~ PL and trp. Eukaryotic promoters include CMV
immediate early, HSV thymidine kinase, early and late SV40, LTRs ~rom retrovirus, and mouse metallothionein-I. Selection o~ the appropriate vectnr and promoter is well within the level o~
ordinary skill in the art.
In a further em~odiment, the present invention relates to host cells cont~;n;ng the above-de~cribed constructs. The host cell can be a higher eukaryotic cell, such as a m~m~ lian cell, or a lower eu~aryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Di~ner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional ~nn~r to produce the gene products encoded by the recombinant sequences. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
~ ature proteins can be expressed in m~m~-l ian cells, yeast, bacteria, or other cells under the control of a~L~riate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described ~y Sambrook, et al., Molecular Cloning: A Laboratory ~nll~l, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure o~ which is here~y incorporated by re~erence.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an ~nh~ncer sequence into the vector. ~nh~ncers are cis-acting el~m~nts of DNA, usually about from 10 to 3~Q bp that act on a promoter to increase its transcription. Examples include the SV40 ~nh~nc~r on the late side of the replication origin bp 100 to 270, a cytomegalovirus early p~omoter ~nh~ncer, the polyoma ~nh~ncer on the late side of the replication origin, and adenovirus Pnh~ncers.
Generally, recombinant expression vectors will include origins of replication and selectable markers pexmitting transformation of the host cell, e.g., the ampicillin resistance gene of ~. coli and S. cexevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ~-W O 97/25338 PCT~US96i'~2Q5 ~actor, acid phosphatase, or heat shock proteins, among others.
The heterologous structural sequence is assembled in d~ru~Liate phase with translation initiation and termination se~l~n~, and preferably, a leader sequence c~pAhle of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can ~ncoA~ a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expres~ed recQ~hin~nt product.
Useful expression vectors for bacterial use are con~tructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable pro~aryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella tyrhim~l~ium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcu~, although others may also be employed as a matter of choice.
As a representative but nnnl;m~ting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such comm~cial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGE~1 (Promega Biotec, Madison, WI, USA). These pBR322 'lba~k~n~ll section~ are combined with an appropriate promoter and the structural sequence to ~e expressed.
Following transformation of a suitable host strain and growth o~ the ho~t strain to an appropriate cell density, the selected promoter is in~llc~A by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harve8ted by centrifugation, disrupted by phy~ical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, -~onication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
Various m~mm~ l; An cell culture systems can also be employed to express re~mh;n~nt protein. ~xamples of mA~m~ n expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines p~hle o~ expres~ing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. ~ An expression vectors will comprise an oxigin of replication, a suitable promoter and ~nh~ncer, and also any necessary ribosome hin~;ng sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5~ fl~nk; ns nontranscribed seqll~n~. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elPm~nts.
The polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation ~chAn~e chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydlo~lapatite chromatography and lectin chromatography. Protein re~olding steps can be used, as neces~ary, in completing configuration of the mature protein. Finally, high performance li~uid chromatography (HPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and ~mmAl;An cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the in~ention may also include an initial methionine amino acid residue.
The HLHDC84 polypeptide may be employed to st;mll-Ate the proli~eration, mobilization and differentiation of stem cells.
Accordingly, it may be employed to treat auto-;~m~ne diseases and for autologous transplant.

The polypeptide of the present invention may al80 be employed to treat and/or prevent inflammation.
The polypeptide of the present invention may also be employed to stimulate neuronal xe-growth for the regeneration of nerves, and as a research reagent.
The polynucleotides and polypeptides encoded by such polynucleotides may also be utilized for in vi~ro purposes related to scientific research, synthesis of DNA and manufacture of DNA
vectors and ~or designing human therapeutics and diagnostics.
Fragments and analogs and derivatives of the polypeptide of the present invention may be i~n~; fied by assays which detect chemotactic activity. One example of such an assay comprises testing such polypeptides for chemotactic activities toward murine polymorphonllclear cells (PMN) or macrophages, human PMN ~isolated on mono-poly~resolving medium; FLOW, McLean, VA). Conditioned medium (diluted and fully supplemented Dulbecco's Modi~ied Eagles medium) and cell lysates (diluted and supplemented Dulbecco's Modi~ied Eagles medium cnnt~n;ng 0.1~ BSA instead of 10% FCS) ~rom transiently transfected CV-1 cells are tested for chemotactic activities toward murine PMNs. Endotoxin content of media and all ~olutions to be tested are measured using a chl~..L~ye~lic limulus amoebocyte lysate assay (Cape Cod Associates, Woods Eole, MA), which was sensitive to 5 pg endotoxin/ml. Chemotactic activity is defined as the mean number of cells migratiny through the pores of the .~.e"~.~ne in 3 to 5 st~nA~rd fields and quantified by image analysis (Wild-Leitz, Rockleigh, New Jersey) using pl ~ni metry measurements (magnification times 100) or by counting normally.
Endotoxin-activated mouse serum (5~) or FMLP
(10-7M) are used as positive controls.
This invention is also related to the use of the HLHDC84 gene as part of a diagnostic assay for detecting diseases or suscept; h;li ty to diseases related to the presence of mutations in the HLHDC84 nucleic acid sequences. Such diseases are related to under-expression of human HLHDC84 polypeptides, ~or example, lack of proliferation and differentiation of stem cells which may lead to ;- n~ disorders.
Individuals carrying mutations in the HLHDC84 gene may be detected at the DN~ level by a variety of techni~ues. Nucleic acids for diagnosis may be obtained from a patient's cells, --1~--_ W O 97/25338 PCT~US~6~ 2~5 including but not_limited to blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA or cDNA
may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding HLHDC84 polypeptide can be used to identify and analyze HLHDC84 mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in c~mr~ison to the normal genotype. Point mutations can be identified by hybridizing amplified DWA to radiolabeled HLHDC84 RNA or alternatively, radiolabeled HLHDC84 antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
Genetic testing based on DNA sequence differences may be achieved by detection o$ alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fra~mPntfi of different sequences may be distinguished on denaturing form~mi~ gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985)).
Sequence changes at speci~ic locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al ., PNAS, USA, 85:4397-4401 ~1985)).
Thus, the detection of a specific DNA sequence may be achieved ~y methods such as hybridization, RNa~e protection, chemical cleavage, direct DNA sequencing or the use of restriction enzyme~, (e.g., Restriction Fragment Length Polymor~h~sm~ (RFLP)) and Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA
seqll~nci n~, mutations can also be detected by in situ analysis.
The present invention also relates to a diagnostic assay for detecting altered levels of H~HDC84 polypeptide in variou~ tissues since an altered expression of the proteins compared to normal control tissue samples may detect the presence of a disease or .

W O 97125338 PCT~US96/002~5 susceptibility to_ a disease, ~or example, malignancies such as cancers and tumors. Assays ùsed to detect levels of HLHDC84 polypeptide in a sample derived from a host are well-known to those of skill in the art and include radio;mml~noassays, competitive-hin~ng assays, Western Blot analysis, ELISA assays and "sandwich"
assay. An ELISA assay (Coligan, et al., Current Protocols in nology, 1(2), Chapter 6, ~1991~) initially comprises preparing an antibody specific to an HLHDC84 antigen, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal ~ntibody~ To the reporter antibody is att~h~ a detectable reagent such as radioactivity, ~luorescence or, in this example, a horseradish peroxidase enzyme. A sample is ~...oved from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein hi n~i ng sites on the dish are then covered by incubating with a non-specific protein like BSA. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal ~ntihodies attach to any HLHDC84 polypeptide attached to the polystyrene dish.
All unbound monoclonal antibody is washed out with bu~er. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in b;n~;n~ of the reporter ~nt~hody to any monoclonal antibody bound to HLHDC84 polypeptide. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of HLHDC84 polypeptide present in a ~iven volume o~ patient sample when compared against a st~n~rd curve.
A competition assay may be employed wherein antibodies specific to HLHDC84 polypeptide are attached to a ~olid support and labeled HLHDC84 polypeptide and a sample derived from the host are passed over the solid s~oLL and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a ~uantity of HLHDC84 polypeptide in the sample.
A "sandwich" assay is sim;l~r to an ELISA assay. In a "sandwich" assay HLHDC84 polypeptide is passed over a solid support and binds to ~ntthody attached to a solid support. A second Ant; hody is then bound to the Hr~DC84 polypeptide. A third ~nt; ho~y wh~ch is labeled and specific to the second ~nt; hody i8 W O 97/2S338 PCT~US~ 0105 then passed over t~e solid support and binds to the second antibody and an amount can then be quantified.
This invention provides a method for identification of the receptors for the human HLHDC84 polypeptides. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand ~nning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, and a cDNA library created from this RNA is divided into pools and used to trans~ect COS cells or other cell~ tha~ are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the labeled polypeptides. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and ;nCllh~tion~ the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and retransfected using an iterative sub-pooling and rescreening process, eventually yielding a single clone that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell ~..E..~ cu-e or extract preparations that express the receptor molecule. Cros6-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex con~n;ng the receptors o~ the polypeptides can be excised, resolved into peptide ~rayments, and subjected to protein microse~l~nci ng . The amino acid sequence obt~i n~A ~rom microse~l~nci n~ would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
This invention also provides a method of screening compounds to i~nti fy antagonists to the HLHDC84 gene and polypeptides of the present invention. AntagoniSts may be identified by the chemotaxis assay described above.
Examples o~ pot~nti~l HLHDC84 polypeptide antagonists include antibodies, or in some cases, oligonucleotides, which bind to the polypeptides. Another example of a potential antagonist is a negative ~omi n~nt mutant o~ the polypeptides. Negative ~mi n~n~

~utants are polypeptides which hind to the receptor of the wild-type polypeptide, but ~ail to retain biological activity.
Antisense constructs prepared using antisense technology are also potential antagonists. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both o~ which methods are based on binding o~ a polynucleotide to DNA or RNA. For example, the 5' coding portion o~ the polynucleotide se~uence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 ba8e pairs in length. A DNA oligonucleotide is designed to be compl~mPnt~y to a region of the gene involved in transcription (triple- helix, see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 2~1: 1360 (1991)), thereby preventing transcription and the production of human HLHDC84. The antisense RNA oligonucleotide hybridizes to the m~RNA
in vivo and blocks translation of the mRNA molecule into the polypeptides (antisense - Okano, J. Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Tnh; h; tors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA
or DN~ may be expressed in vi~o to ; nh; h; t production Of hu~m~l HLHDC84.
Another potPnt; A 1 human HLHDC84 antagonist is a peptide derivative of the polypeptides which are naturally or synthetically modified analogs of the polypeptides that have lost biological function yet still recognize and bind to the receptors of the polypeptides to thereby effectively block the receptors. Examples of peptide derivatives include, but are not limited to, small peptides or peptide-like molecules.
The antagonists specific to HLHDC84 may be employed to ;nh;h~t and/or limit cellular proliferation or induced differentiation of stem cells of the ;mm~n~ system and may therefore be employed to treat and/or ~-~vel~t cancers such as lellkem; A and ly~rhobl~toma.
- The antagonists may be employed to control the proliferation and differentiation of stem cells, particularly in the l~lnP
system This activity may be employed as adjunct protective treatment during cancer chemotherapy by 1nhihiting bone marrow stem cell colony ~ormation.

W O 97n533~ PCT~US96/00205 - The antagoni,sts may also be employed to ~nh;b; t the differentiatlon and proliferation of cells of the lm~llne system to treat and/or prevent arthritis.
The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
The human HLHDC84 polypeptides and antagonists may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically ef~ective amount of the polypeptide or antagonist, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and comh~n~tions thereof. The formulation should suit the mode of ~mtn; stration.
The invention also provides a pharmaceutical pack or kit comprising one or more co~t~n~s filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
Associated with such con~in~r(s) can be a notice in the form prescribed by a governm~nt~l agency regulating the manu~acture, use or sale o~ pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for hum~n ~ n; stration. In addition, the polypeptides and antagonists may be employed in con~unction with other therapeutic compounds.
The pharmaceutical compositions may be ~m~n~ stered in a convenient m~nn~r such as by the topical, intravenous, intraperitoneal, intramuscular, intratumor, subcllt~n~ous, intr~n~Al or intradermal routes. The pharmaceutical compositions are ~d~tni stered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, the polypeptides will be ~min~ stered in an amount of at least about 10 ~g/kg body weight and in mo~t cases they will be ~m~ n~ stered in an amount not in excess of about 8 mg/Kg body weight per day.
In most cases, the dosage is from about 10 ~g/kg to about 1 mg/kg body weight daily, taking into account the routes of A~; n; ~tration, symptoms, etc.
The HLHDC84 polypeptides and antagonists which are polypeptides, may be employed in accordance with the present -~invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy."
Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle cont~;n~ng RNA encoding a polypeptide of the pre~ent invention.
S;m;l~ly, cells may be engineered in vi~o for expression of a polypeptide in vivo by, for example, procedures known in the art.
As known in the art, a producer cell for producing a retroviral particle cont~i ni ng RNA encoding the polypeptide o~ the present invention may be ~m;ni fitered to a patient for engineering cells in vivo and expres~ion of the polypeptide in vivo. These and other methods for ~mi nt stering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the tPA~h;ngs of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after comh;nAtion with a suitable delivery vehicle.
Retroviruses from which the retroviral plasmid vectors her~;n~hove mentioned may he derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape lellkPmi~ virus, human ~ noAeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one emho~;mpnt~ the retroviral plasmid vector is derived from Moloney Murine LellkPm;~ Virus.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniaues, Vol. 7, No. 9, 980-990 (1989~, or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ~-actin promoters). Other viral ~Lul--uLers which may be employed include, but are not limited to, adenovirus promoters, thymidine ~inase (TK) ~L~IlLoLers, and B19 W O 97/25338 PCT~US96/00205 parvovirus promot~rs. The selection of a suitable promoter will ~e apparent to those skilled in the art from the t~chin~8 cont~;neA herein.
The nucleic acid se~uence encoding the polypeptide of the present invention is under the control of a suitable promoter.
Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter;
~n~l~c;hle promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI
promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the ~-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the gene encoding the polypeptide.
The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Bxamples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-17-H2, ~CRE, ~CRIP, GP+E-86, GP~envAml2, and DAN cell lines as described in Miller, Human Gene Thera~y, Vol. 1, pgs. 5-14 ~1990), which is incorporated herein by reference in its entirety. The vector may transduce the packa~ing cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then ~m;n~stered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in YiVo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, ~ ol~ic stem cells, e"~ly~lic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, W O 97ns338 PCTAUS96/00205 endothPl;Al cells,_and bronchial epithelial cells.The sequences of the present invention are also valuable for chromosome iAF~nt; fication. The sequence is specifically targeted to and can hybridize with a particular location on an individual human ~ chromosome. Moreover, there is a current need for iAPntifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorrh;~) are presently available for marking chromosom~l location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disea8e.
The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with ~ particular location on an individual human chromosome. Moreover, there is a current need for iApntifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorph;l:m~:) are presently available for marking chromo~omal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes as~ociated with disease.
Briefly, seqll~ncp~ can be mapped to chromosomes by preparing PCR primers (pre~erably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids c~nt~;n;ng individual human chromosomes. Only those hybrids cont~ n; ng ~he human gene corrP~L~ .lin~ to the primPr will yield an ~rl;f;PA fr~l ~t, PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular cl~ ome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fra~ ~ from specific chromosomes or pools o~ large genomic clones in an analogous m~nn~r, Other mapping strategies that can s;m;l~rly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.

Fluore~cence in situ hybridization (FISH) of a cDNA clone to a metAph~se chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases. For a review of this technigue, see Verma et al., Human Chromosomes: a ~Anll~l of Basic Techniques, Pel~",oll Press, New York (1988).
Once a sequence ha6 been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, M~nA~liAn Inheritance in Man (available on line through Johns Hopkins Uni~ersity Welch Medical Library).
The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified t}~ouyh linkage analy~is (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA
or genomic sequence between affected and unaffected individuals.
If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expres~ing them can be used as an im~llnogen to produce antibodies thereto. These AntihoA;es can be, for example, polyclonal or monoclonal antibodies. The present invention also includes ch~m~ric, single chain, and h~ n~zed Ant;hoA; es, as well as Fab fra~r~nt~, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fra~ment~.
~ ntihodies generated against the polypeptides correspon~in~
to a sequence of the present invention can be obtA~nP~ by direct injection of the polypeptides into an ~n;mAl or by A~m;ni~tering the polypeptides to an An;m~l, preferably a nonhll~-n The Ant;hody so obtAine~ will then bind the polypeptides itself. In this m~nner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies b; n~; ng the whole W O 97/25338 PCT~US96/00205 native polypeptides. Such ;~nt; hodies can then be used to isolate the polypeptide from tissuè expressing that polypeptide.
Antibodies specific to the polypeptide of the present invention may be employed as a diagnostic to determine altered levels of the polypeptide in a sample derived from a host by techni~ues known in the art. Altered levels are indicative of certain disorders, for example, cancer. The expression level of the protein is altered in cancerous cells and an ~nt; hody which detects the level of this protein is thus useful as a diagnostic for the state of the disease, prognosis, or as an imaging agent for identifying c~nrProus cells, wherein the antibody is labeled with a marker r~p~le of detection by a medical imaging machine.
For preparation of monoclonal antibodies, any technique which provides ~nt t ho~; es produced by continuous cell line cultures can be used. ~xamples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, T~lmology Today 4:72), and the EBV-hybridoma techni~ue to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal ~nt1hodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain t;h~dies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to imml-nogenic polypeptide products o~ this invention. Also, transgenic mice may be used to express hl-m~n;zed antibodies to immunogenic polypeptide products of this invention.
The above-described antibodies may be employed to isolate the polypeptide of the present invention by atta~hmPnt of the ~nt t hody to a solid support and performing affinity chromatography by passing the polypeptide desired to be purified o~er the column and recovering the purified polypeptide.
The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate underst~n~iny of the following examples certain frequently occurring methods and/or term~ will be described.
~ pl;~m;~l~" are desiynated by a lower case p preceded and/or followed by c~pital letters and/or numbers. The starting plasmids W O 97/25338 PCT~US96/00205 herein are ei~her_commercially av~ hl e, publicly available on an unrestricted basis, or can be constructed from aVAi l ~hl e plasmids in accord with pllhl;sh~tl procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage o~ the DNA
with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 ~g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ~1 of buf~er solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~g of DNA are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a ~ingle stranded polydeoxynucleotide or two complPm~nt~ry polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without ~; n~ a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accompl~ ~h~r~ using known buffers and conditions with ~0 units to T4 DNA ligase ("ligase") per O.5 ~g of approximately equimolar amounts of the DNA fragments to be ligated.

W O 97125338 PCT~US96/00205 ~ Unless other~ise stated, transformation was perfonmed as described in the method o~ Graham, F. and Van der Eb, A., Virology, ~2:456-457 (1973).

ExamPle 1 Expression of Recomh;n~nt HLHDC84 in COS cells The expression o~ plasmid, HLHDC84 is derived from a vector pcDNAI/Amp (Invitrogen) cnnt~in;ng: 1) SV40 origin o~ replication, 2) ampic;ll~n resistance gene, 3) E.coli replication origin, 4) CMV
promoter ~ollowed by a polyl;nk~ region, a SV40 intron and polyadenylation site. A DNA ~ragment encoding the entire HLHDC84 precursor is cloned into the polylinker region of the vector, there~ore, the reC~mh;n~nt protein expression is directed under the CMV promoter.
The plasmid construction strategy is described as follows:
The DNA sequence encoding ~or ~LHDC84, ATCC # 97351, is constructed by PCR using two primers: the 5' primer 5~
GTCTGGGATCCCAGG&CGAAGCCATGCAGTGC 3' ~SEQ ID N0:5) cont~;n~ a BamHI
site (in bold) ~ollowed by 9 nucleotides o~ ~T.Rnc84 coding sequence starting ~rom the minus 3 position relative to initiation codon;
the 3' se~uence ~ ~l~A~ GCCCAGC 3' (SEQ ID N0:6) cont~n~
complem~nt~ry sequences to a BamHI site. Therefore, the PCR
product cnnt~;n~ a BamHI site, HLHDC84 coding sequence and a second BamHI site. The PCR ampli~ied DNA ~ragment and the vector, pcDNAI/Amp, are digested with BamHI restriction enzyme and ligated.
The ligation mixture i5 transformed into E. coli strain SURE
(Stratagene Cloning Systems, La Jolla, CA) the transformed culture i8 plated on ampic;ll; n media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and ~m;ne~
by restriction analysis for the presence o~ the correct ~r~m~nt.
For expression of the recombinant HLHDC84, COS cells are transfected with the expression vector by DEAE-DEXTRAN method (J.
Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory nll~l, Cold Spring Laboratory Press, (1989)). The expression of the HhHDC84 HA protein is detected by radiolabelling and t~lln~precipitation method (E. Harlow, D. Lane, ~nt;hodies: A
Laboratory ~nll~l, Cold Spriny Harbor Laboratory Press, (1988)).
Cells are labelled for 8 hours with 35S-cysteine two days post W O 97/25338 PCT~US96/00205 transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1~ NP-40, 0.1% SDS, 1~
NP-40, 0.~ DOC, 50mM Tris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media are precipitated with a HA specific monoclonal antibody. Proteins precipitated are analyzed by SDS-PAGE.

Example 2 Cloninq and expression of HLHDC84 usinq the baculovirus exPression system The DNA sequence encoding the full length HLHDC84 protein, ATCC # 97351, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the se~uence 5' TGGGATCCCAGGCCGC
CATCATGCAGTGCCGGCTCC 3' (SEQ ID NO:7) and cont~n~ a Bar.~I
restriction enzyme site (in bold) followed by 16 nucleotides of coding sequence.
The 3' primer has the sequence 5' GGCTCT~-ACCC
AGCAGTTCAGGATTCATCG 3' (SEQ ID NO:8) and cnnt~in~ the cleavage site for the restriction Pn~nnllclease XbaI and nucleotides compl~m-~nt~ry to the 3' translated sequence of the HLHDC84 gene and stop codon.
The amplified sequences are isolated ~rom a 1~ agarose gel using a com~cially available kit (IlGeneclean," BIO 101 Inc., La ~olla, Ca.). The fragment is then digested with the ~n~nllrleases BamXI
and XbaI, then purified again on a 1~ agarose gel. This ~ragment is designated F2.
The vector pA2 (modification of pVL941 vector, discussed below) is used for the expression of the Hr-~DC84 protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A m~nll~l of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Exper~m~nt~l Station Bulletin No. 1555). This expression vector cont~;n~ the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction ~n~nnllCl eases BamHI and Asp781. The polyadenylation site of the simian virus SV40 is used for efficient polyadenylation. For an easy selection of reco~hin~nt viruses the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the W O 97/25338 PCTrUS96/00205 polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by ~iral sequences ~or the cell-mediated homologous recomh;n~tion of cotransfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pA2 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
- The plasmid is digested with the restriction enzymes BamHI and XbaI and then depho~phorylated using cal~ intestinal phosrh~t~e by procedures known in the art. The DNA is then isolated from a 1~6 agarose gel using the commercially a~ hl e kit ("Geneclean"
BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated ~2.
Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase. E. coli ~M101 cells are then transformed and bacteria identified that cont~;ne~ the plasmid (pBac-HIHDC84) with the HLHDC84 gene using the enzymes BamHI and Asp781. The se~uence o$ the cloned fragment is confirmed by DNA ~e~l~nc~ng.
5 ~g of the pla~mid pBac-HLHDC84 is cotransfected with 1.0 ~g of a c~ -rcially av~ hl e l~nP~rized baculovirus ("BaculoGold~
baculovirus DNA", Phanmingen, San Diego, CA.) using the lipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
l~g o~ BaculoGold~ virus DNA and 5 ~g of the plasmid pBac-HLHDC84 are mixed in a sterile well o~ a microtiter plate cont~n~ng 50 ~1 of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~l Lipofectin plus 90 ~l Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added dropwise to the Sf9 (Spodoptera frugiperda) insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with lml Grace~s medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate i~ then incubated for 5 hours at 27~C.
After 5 hours the transfection solution is ~ ved from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal cal~
serum is added. The plate i8 put back into an incubator and cultivation cnnt;nlle~ at 27~C for four days.
After ~our days the sUpern~t~nt is collected and a pla~ue assay performed similar as described by Summers and Smith (supra).
As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue stained plaques. _(A detailed description of a "plaque assay~ can also be ~ound in the user's guide for insect cell culture and baculovirology distributed by ~ife Technologies Inc., Gaithersburg, page 9-10~.
Four days a~ter the serial dilution,'the viruses are added to the cells and blue stained plaques are picked with the tip of an Eppendorf pipette. The agar cont~;n;ng the reCo~h~n~nt viruses is then resuspended in an ~ppendor~ tube cont~l n; ng 2 0 0 ~1 of Grace's medium. The agar is removed by a brief centrifugation and the supernatant cont~;n;ng the recombinant baculovirus is used to infect S~9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture ~ hF~ are harvested and then stored at 4~C.
Sf9 cells are grown in Grace's medium supplemented with 10~
heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-HLHDC84 at a multiplicity of infection ~MOI) of 2.
Six hours later the medium is L~-,wved and replaced with SF900 II
medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 ~Ci of 35S-methionine and 5 ~Ci 35S
cysteine (Amersham) are added. The cells are ~urther incubated for 16 hours.
The growth medium was harvested on day 4 post-in~ection.
After the removal of baculovirus cells by continuous centrifugation, the supernatant was applied to a cation P~rhAn~e column (pros HS 50 resin from PerSeptive Biosystem) pre-eqll;l;h~ated with buffer A (40 mM NaAcetate, 50 mM NaCl, pH 5.5).
The column was eluted stepwise with increasing NaCl concentration in the ~ame buf~er. The ~ractions contA;n;ng HLHDC84 were pooled, diluted with buffer A and applied to another cation ~h~nge column (CM20), followed by a NaCl gradient elution. The combined fractions were ~urther purified by gel ~iltration chromatography (Superdex 200 or Superdex 75 ~rom Pharmacia Biotech). Hum~n HLHDC84 was finally puri~ied to homogeneity (95%) and concentrated by cation ex~h~n~e chromatography (CM20) after the gel filtration fractionation.

Exam~le 3 ExPressiPn via Gene Thera~v W 097~5338 PCT~US96/00205 Fibroblasts ~re obt~; n~-l from a su}~ject by s3~in biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chllnk~ o~ the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. A~ter 24 hours at room temperature, the flask is inverted and the chunks o~ tis~ue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10~ FBS, penicillin and streptomycin, is added. This is then incu~ated at 37~C for approximately one week.
~t this time, ~resh media i8 added and ~ubsequently changed e~rery several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is tryp8inized and scaled into larger ~lask~.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, i8 digested with EcoRI and HindIII and subse~uently treated with cal~ intestinal phosphatase. The linear vector is ~ractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequences respectively. The 5' primer c~ntA;nin~ an EcoRI site and the 3' primer further includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus l;nP~r ~ackhon~ and the amplified EcoRI and HindIII ~ragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments.
The ligation mixture is used to transfonm bacteria HB101, which are then plated onto agar-cont~;n;ng kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modi~ied Eagles Medium (DMEM) with 10~ calf serum (CS), penicillin and streptomycin. The MSV vector co~t~;n;ng the gene is then added to the media and the packaging cells are transduced with the vector.
The packaging cells now produce infectious viral particles cont~n;ng the gene (the packaging cells are now referred to as producer cells).

W O 97/25338 PCTAUS~G;~05 ~ resh media ~s added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, cnnt~;n;n~ the infectious ~iral particles, is ~iltered through a millipore ~ilter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed ~rom a sub-confluent plate of ~ibroblasts and quickly replaced with the media ~rom the producer cells. This media is removed and replaced with fresh media. I~ the titer o~ virus is high, then virtually all ~ibroblasts will be infected and no selection is required. I~ the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.
The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The ~ibroblasts now produce the protein product.
Numerous modi~ications and variations of the present inv~nt~on are possible in light o~ the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

CA 02242964 l998-07-l0 ~ 96J ~05 iP~ AUG 1997 SEQUENCE LISTING

(1) GENERAh INFORMATION:
(i) APPLICANT: SOPPET, DANIEL R
LI, YI
(ii) TITLE OF INVENTION: IMMUNE CELL CYTOKINE
(iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN
'B STREET: 6 BECKER FARM ROAD
~C CITY: ROSELAND
D~ STATE: NJ
E COUN1~Y: US
,F, ZIP: 07068 - (v) COMPUTER READABLE FORM:
~A'~ MEDIUM TYPE: Floppy disk ~B: COMPUTER: IBM PC compatible C OPERATING SYSTEM: PC-DOS/MS-DOS
~D, SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US96/00205 (B) FILING DATE: 11-JAN-1996 (C) CLASSIFICATIoN:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: MULLINS, ~.G.
(B) REGISTRATION NUMBER: 33,073 (C) REFEREN OE /DOCKET NUMBER: 325800-511 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (201) 994-1700 (B) TELEFAX: (201) 994-1744 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUEN OE CHARACTERISTICS:
,A'~ LENGTH: 2225 base pairs ~:B TYPE: nucleic acid ~C~ STRANDEDNESS: single ~D, TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 368..1330 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

CTTCCTCCCA CCGCAGCAGC TGGCAGAGCG GGGCTGCCCT TTCTGCTGGT GC~-lC~ lC 60 AGCCGCCTCT GTCTGGGGTT CC~-llCGl~l GGCCCCGCCC CTCGGGCTAA GCGGGGCGGG 120 AMEND~D SHEET

,CA 02242964 l998 PCT/~ 9 6 1 0 0 2 IP~'~ 1 3. AUG 1997 ACCAGC-rrCC CCTCCCTGTC TGGTTGGGAT TTGGGGGCTG AGCTGTCTGG GGTCCCAGGG 360 Met Gln Cy9 Arg Leu Pro Arg Gly Leu Ala Gly Ala Leu Leu Thr Leu Leu Cys Met Gly Leu Leu CyS Leu Arg Tyr HiS Leu Asn Leu Ser Pro Gln Arg Val Gln Gly Thr Pro Glu Leu Ser Gln Pro Asn Pro Gly Pro Pro Lys Leu Gln Leu His Asp Val Phe Ile Ala Val Lys Thr Thr Arg Ala Phe His Arg Leu Arg Leu Glu Leu Leu Leu Asp Thr Trp Val Ser Arg Thr Arg Glu Gln Thr Phe Val Phe Thr Asp Ser Pro Asp Lys Gly Leu Gln Glu Arg Leu Gly Ser His Leu Val Val Thr Asn Cys Ser Ala Glu His Ser His Pro Ala Leu Ser Cys Lys Met Ala Ala Glu Phe Asp Thr Phe Leu Ala Ser Gly Leu Arg Trp Phe Cys His Val Asp Asp Asp Asn Tyr Val Asn Pro Arg Ala Leu Leu Gln Leu Leu Arg Ala Phe Pro Leu Ala Arg Asp Val Tyr Val Gly Arg Pro Ser Leu Asn Arg Pro Ile His Ala Ser Glu Pro Gln Pro His Asn Arg Thr Arg Leu Val 175 180 185 l90 Gln Phe Trp Phe Ala Thr Gly Gly Ala Gly Phe Cys Ile Asn Arg Lys - AMENDED SHEET

-CA 02242964 1998-07-10 p ~ 9 6 l 00 20 IP~ AUG 1997 Leu Ala Leu Lys Met Ala Pro Trp Ala Ser Gly Ser Arg Phe Met Asp Thr Ser Ala Leu Ile Arg Leu Pro Asp Asp Cys Thr Met Gly Tyr Ile Ile Glu Cy5 Lys Leu Gly Gly Arg Leu Gln Pro Ser Pro Leu Phe His Ser His Leu Glu Thr Leu Gln Leu Leu Arg Thr Ala Gln Leu Pro Glu CAG GTC ACC CTC AGC TAC GGT GTC TTT GAG GGG AAA CTC A~C GTC ATT 1225 Gln Val Thr Leu Ser Tyr Gly Val Phe Glu Gly Lys Leu Asn Val Ile Lys Leu Gln Gly Pro Phe Ser Pro Glu Glu Asp ,Pro Ser Arg Phe Arg Ser Leu His Cys Leu Leu Tyr Pro Asp Thr Pro Trp Cys Pro Gln Leu Gly Ala Arg TGGGTGGAAT GATGGCAGAA TCCAGGGTCT GCAGCACCTG ~-l~rl~l~lGC CAACCAGTCT 1850 CCCAAAGCTC CTTGCTCCCC ACCCCTTGCG AACAGGACCA GA~ Gl-l-l GGAGCCTCAG 1910 CATGCCGGGC CCAGATGATG GAGCATAACG GGTCCCAGCC AATTGTGATG ANC~ l-lG 1970 cTCATTTCCC AGC~-l-l-l~-l-l GCTGTTAGGG GCTACCATGG GACCAGCTCT GGCCAGAGGG 2030 AACTAAGCAA ATCCAATAGA GAl~l-l-l~lG GGGAAGGTTT TGCAGCCCAC TCCCCATCTT 2090 AMENDED S~IEEJ

~ CA 02242964 l998-07-lO
P~ - 96/ 002(~5 IPEA~ AUG 1997 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 321 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 2:
Met Gln Cys Arg Leu Pro Arg Gly Leu Ala Gly Ala Leu Leu Thr Leu lS
Leu CyS Met Gly Leu Leu Cys Leu Arg Tyr His Leu Asn Leu Ser Pro Gln Arg Val Gln Gly Thr Pro Glu Leu Ser Gln Pro Asn Pro Gly Pro Pro Lys Leu Gln Leu His Asp Val Phe Ile Ala Val Lys Thr Thr Arg Ala Phe His Arg Leu Arg Leu Glu Leu Leu Leu Asp Thr Trp Val Ser 75' 80 Arg Thr Arg Glu Gln Thr Phe Val Phe Thr Asp Ser Pro Asp Lys Gly Leu Gln Glu Arg Leu Gly Ser His Leu Val Val Thr Asn Cys Ser Ala Glu His Ser His Pro Ala Leu Ser Cys Lys Met Ala Ala Glu Phe Asp Thr Phe Leu Ala Ser Gly Leu Arg Trp Phe Cys His Val Asp Asp Asp Asn Tyr Val Asn Pro Arg Ala Leu Leu Gln Leu Leu Arg Ala Phe Pro Leu Ala Arg Asp Val Tyr Val Gly Arg Pro Ser Leu Asn Arg Pro Ile His Ala Ser Glu Pro Gln Pro His Asn Arg Thr Arg Leu Val Gln Phe Trp Phe Ala Thr Gly Gly Ala Gly Phe Cys Ile Asn Arg Lys Leu Ala Leu Lys Met Ala Pro Trp Ala Ser Gly Ser Arg Phe Met Asp Thr Ser Ala Leu Ile Arg Leu Pro Asp Asp Cys Thr Met Gly Tyr Ile Ile Glu 225 230 235 ~ 240 Cys Lys Leu Gly Gly Arg Leu Gln Pro Ser Pro Leu Phe His Ser His Leu Glu Thr Leu Gln Leu Leu Arg Thr Ala Gln Leu Pro Glu Gln Val Thr Leu Ser Tyr Gly Val Phe Glu Gly Lys Leu Asn Val Ile Lys Leu -AMENGE~ S~EET

CA 02242964 l998-07-l0 PCT/US 9 6/ 002Q~
IPE~j 1 1 AUG 1997 Gln Gly Pro Phe Ser Pro Glu Glu Asp Pro Ser Arg Phe Arg Ser Leu His Cys Leu Leu Tyr Pro Asp Thr Pro Trp Cys Pro Gln Leu Gly Ala Arg (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 335 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Gln Pro Gly Ala Gly Ala Pro Ala Ala Ser Pro Thr Thr Val Ile Ile Arg Lys Asp Ile Arg Ser Phe Asn Phe Ser Asp Ile Glu Val Ser Glu Arg Pro Thr Ala Thr Leu Leu Thr Glu Leu Ala Arg Arg Ser Arg Asn Gly Glu Leu Leu Arg Asp Leu Ser Gln Arg Ala Val Thr Ala Thr Pro Gln Pro Pro Val Thr Glu Leu Asp Asp Ile Phe Ile Ser Val Lys Thr 7~ 75 80 ~hr Lys Asn Tyr His Asp Thr Arg Leu Ala Leu Ile Ile Lys Thr Trp ~he Gln Leu Ala Arg Asp Gln Thr Trp Phe~Phe Thr Asp Thr Asp Asp His Tyr Tyr Gln Glu Lys Thr Lys Gly His Leu Ile Asn Thr Lys Cys Ser Gln Gly His Phe Arg Lys Ala Leu Cys Cys Lys Met Ser Ala Glu Leu Asp Val Phe Leu Glu Ser Gly Lys Lys Trp Phe Cys His Phe Asp 145 150 155 . 160 ~sp Asp Asn Tyr Val Asn Val Pro Arg Leu Val Lys Leu Leu Asp Glu ~yr Ser Pro Ser Val Asp Trp Tyr Leu Gly Lys Pro Ser Ile Ser Ser Pro Leu Glu Ile His Leu Asp Ser Lys Asn Thr Thr Thr Asn Lys Lys Ile Thr Phe Trp Phe Ala Thr Gly Gly Ala Gly Phe Cys Leu Ser Arg Ala Leu Thr Leu Lys Met Leu Pro Ile Ala Gly Gly Gly Lys Phe Ile AMEN~E3:) SHFF~

CA 02242964 1998-07-10 PCT/~S 96/ 002 t ~E~ I 1 AUG ~gg~
Ser Ile Gly Asp Lys Ile Arg Phe Pro Asp Asp Val Thr Met Gly Phe Ile Ile Glu His Leu Leu Lys Val Pro Leu Thr Val Val Asp Asn Phe His Ser His Leu Glu Pro Met Glu Phe Ile Arg Gln Asp Thr Phe Gln Asp Gln Val Ser Phe Ser Tyr Ala His Met Lys Asn Gln Trp Asn Val Ile Lys Val Asp Gly Phe Asp Met Lys Thr Asp Pro Lys Arg Phe Tyr Ser Leu His Cys Gln Leu Phe Pro Tyr Phe Ser Phe Cys Pro Pro (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE- DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single AI\/lEN~rD SHEE:r ~ ,CA 02242964 1998-07-10 p ~ 9 6 / O 0 2 0 5 IP~ lAUG1997 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

AMENDcD Sl-'EET

Claims (25)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising a polynucleotide having at least 70% identity to a member selected from the group consisting of:
(a) a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
(b) a polynucleotide encoding a polypeptide comprising amino acids 21 to amino acid 337 of SEQ NO ID:2;
(c) a polynucleotide which is complementary to the polynucleotide of (a); and (d) a polynucleotide comprising at least 15 consecutive bases of the polynucleotide of (a), (b) or (c).

2. The isolated polynucleotide of Claim 1 wherein the polynucleotide is DNA.

3. The isolated polynucleotide of Claim 1 wherein the polynucleotide is RNA.

4. The isolated polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA.

5. The isolated polynucleotide of Claim 2 comprising nucleotide 1 to 2225 set forth in SEQ ID NO:1.

6. The isolated polynucleotide of Claim 2 comprising nucleotide 368 to 1330 set forth in SEQ ID NO:1.

7. The isolated polynucleotide of Claim 2 comprising nucleotide 428 to 1330 set forth in SEQ ID NO:1.

8. The isolated polynucleotide of Claim 2 which encodes a polypeptide comprising amino acid 21 to 337 of SEQ ID NO:2.

9. An isolated polynucleotide comprising a polynucleotide having at least a 70% identity to a member selected from the group consisting of:

(a) a polynucleotide encoding a mature HLHDC84 polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97351;
(b) a polynucleotide complementary to the polynucleotide of (a); and (c) a polynucleotide comprising at least 15 bases of the polynucleotide of (a) or (b).

10. A vector comprising the DNA of Claim 2.

11. A host cell comprising the vector of Claim 9.

12. A process for producing a polypeptide comprising:
expressing from the host cell of claim 10 the polypeptide encoded by said human cDNA.

13. A process for producing a cell comprising: transforming or transfecting the cell with the vector of Claim 9 to thereby express the polypeptide encoded by the human cDNA contained in the vector.

14. A polypeptide comprising a member selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2;
(b) a polypeptide comprising amino acid 21 to 337 of SEQ
ID NO:2;
(c) a polypeptide which is at least 70% identical to the polypeptide of (a) or (b); and (d) a polypeptide having at least 15 amino acid residues of the polypeptide of (a), (b) or (c).

15. A polypeptide comprising amino acid 21 to amino acid 337 of SEQ ID NO:2.

16. An antibody against the polypeptide of Claim 14.

17. A process of isolating an HLHDC84 polypeptide comprising:
attaching the antibody of claim 16 to a substrate and contacting a sample containing the polypeptide with the antibody.

18. An antagonist against the polypeptide of Claim 14.

19. A method for the treatment of a patient having need of HLHDC84 comprising: administering to the patient a therapeutically effective amount of the polypeptide of Claim 14.

20. The method of Claim 19 wherein said therapeutically effective amount of the polypeptide is administered by providing to the patient DNA encoding said polypeptide and expressing said polypeptide in vivo.

21. A method for the treatment of a patient having need to inhibit HLHDC84 comprising:
administering to the patient a therapeutically effective amount of the antagonist of Claim 18.

22. A method for the treatment of leukemia comprising:
administering a therapeutically effective amount of the antibody of Claim 16 to a patient in need thereof.

23. A method for the treatment of lymphoblastoma comprising:
administering a therapeutically effective amount of the antibody of Claim 16 to a patient in need thereof.

24. A diagnostic process comprising:
analyzing for the presence of the polypeptide of Claim 14 in a sample derived from a host.

25. A method for identifying compounds which bind to and inhibit a receptor for the polypeptide of Claim 14 comprising:
contacting a cell expressing on the surface thereof a receptor for the polypeptide, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor, with a compound to be screened under conditions to permit binding to the receptor; and determining whether the compound binds to and inhibits the receptor by detecting the absence of a signal generated from the interaction of the compound with the receptor.