EP1088095A1 - Novel molecules of the t110-related protein family and uses thereof - Google Patents

Abstract

Novel T110 polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated, full-length T110 proteins, the invention further provides isolated T110 fusion proteins, antigenic peptides and anti-T110 antibodies. The invention also provides T110 nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which a T110 gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

Description

NOVEL MOLECULES OF THE TllO-RELATED PROTEIN FAMILY

AND USES THEREOF

Cross Reference To Related Applications

This application is a continuation-in-part of application serial number 09/102,705, filed June 22, 1998.

Background of the Invention The invention relates to a novel cell surface or secreted protein and the gene encoding it .

Many membrane-associated and secreted proteins, for example, cytokines, play a vital role in the regulation of cell growth, cell differentiation, and a variety of specific cellular responses. A number of medically useful proteins, including erythropoietin, granulocyte-macrophage colony stimulating factor, human growth hormone, and various interleukins, are secreted proteins. Thus, an important goal in the design and development of new therapies is the identification and characterization of membrane-associated and secreted proteins and the genes which encode them.

Many membrane-associated proteins are receptors which bind a ligand and transduce an intracellular signal, leading to a variety of cellular responses. The identification and characterization of such a receptor enables one to identify both the ligands which bind to the receptor and the intracellular molecules and signal transduction pathways associated with the receptor, permitting one to identify or design modulators of receptor activity, e.g., receptor agonists or antagonists and modulators of signal transduction. Summary of the Invention The present invention is based, at least in part, on the discovery of a gene encoding TllO. TllO protein is related to four-jointed (fj) protein of Drosophila Melanogaster. TllO is predicted to be a member of the type-II membrane protein superfamily. Such proteins usually employ a transmembrane domain as the internal signal sequence. The amino terminal end of such proteins is normally intracellular, and the carboxy terminal end is normally extracellular. However, some type II membrane proteins are secreted from the cell while others are initially expressed on the surface of the cell and are subsequently processed to release a soluble fragment . The human TllO cDNA described below (SEQ ID NO:l) has a 1311 nucleotide open reading frame (nucleotides 131 to 1441 of SEQ ID NO:l; SEQ ID NO:3) which encodes a 437 amino acid protein (SEQ ID NO: 2) . Figure 8 depicts a potential alternative translation product (SEQ ID NO:4) for the above-described human TllO cNDA. It is possible that this alternative translation product is not full length. Those skilled in the art can isolate full-length clones having additional 5' coding sequence using the methods described below.

The mouse TllO cDNA described below (SEQ ID NO: 5) has a 1350 nucleotide open reading frame (nucleotides 103 to 1452 of SEQ ID NO : 5 ; SEQ ID NO: 7) which encodes a 450 amino acid protein (SEQ ID NO: 6) . Figure 6 depicts a potential alternative translation product (SEQ ID NO: 8) for the above-described murine TllO cDNA. It is possible that this alternative translation product is not full length. Those skilled in the art can isolate full-length clones having additional 5' coding sequence using the methods described below.

A partial rat TllO cDNA is also described below (SEQ ID NO: 9) . It has a 507 nucleotide open reading frame (nucleotides 1 to 507 of SEQ ID NO: 9) which encodes a 169 amino acid peptide (SEQ ID NO: 10) . Those skilled in the art can isolate full-length clones having additional 5' sequence using the methods described below. The TllO molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes, e.g., cell proliferation and/or cell differentiation. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding TllO proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of TllO-encoding nucleic acids.

The invention features a nucleic acid molecule which includes a fragment of at least 400 (450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1420) nucleotides of the nucleotide sequence shown in SEQ ID NO:l or a complement thereof; or a fragment of at least 200 (250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1110, 1200, 1300, 1400, or 1420) nucleotides of the nucleotide sequence shown in SEQ ID NO: 3 or a complement thereof; or a fragment of at least 450 (500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1450) nucleotides of the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7, or a complement thereof .

In a preferred embodiment, a TllO nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:l, or SEQ ID NO: 3, or SEQ ID NO: 5, or SEQ ID NO : 7. Also within the invention is a nucleic acid molecule which encodes a fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:4, or SEQ ID NO: 6 or SEQ ID NO : 8 , the fragment including at least 70 (80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 480) contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4; or the fragment including at least 150 (160, 170, 180, 200, 250, 300, 350, 400, 450, or 480) contiguous amino acids of SEQ ID NO: 6 or SEQ ID NO: 8. The invention includes a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or SEQ ID NO : 6 or SEQ ID NO : 8 , wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:l or SEQ ID NO: 3, or SEQ ID NO: 5 or SEQ ID NO : 7 under stringent conditions. Also within the invention is an isolated TllO protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 9.

Also within the invention is a polypeptide which is a naturally occurring allelic variant of a polypeptide that includes the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:4, or SEQ ID NO : 6 or SEQ ID NO : 8 , wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:l or SEQ ID NO: 3, or SEQ ID NO : 5 or SEQ ID NO : 7 under stringent conditions; Another embodiment of the invention features TllO nucleic acid molecules which specifically detect TllO nucleic acid molecules. For example, in one embodiment, a TllO nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID

NO: 5, or SEQ ID NO: 7, or a complement thereof. In another embodiment, the TllO nucleic acid molecule is at least 440 (450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1420) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence as shown in SEQ ID NO:l or a complement thereof; or a fragment of at least 220 (250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1420) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence as shown in SEQ ID NO: 3 or a complement thereof; or a fragment of at least 450 (500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1420) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 7, or a complement thereof. In another embodiment, the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a TllO nucleic acid.

Another aspect of the invention provides a vector, e.g., a recombinant expression vector, comprising a TllO nucleic acid molecule of the invention. In another embodiment the invention provides a host cell containing such a vector. The invention also provides a method for producing TllO protein by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a TllO protein is produced.

Another aspect of this invention features isolated or recombinant TllO proteins and polypeptides. Preferred TllO proteins and polypeptides possess at least one biological activity possessed by naturally occurring human TllO, e.g., modulation of cellular proliferation. In one embodiment, an isolated TllO protein has an extracellular domain and lacks both a transmembrane and a cytoplasmic domain. In another embodiment, an isolated TllO protein is soluble under physiological conditions. The TllO proteins of the present invention, or biologically active portions thereof, can be operably linked to a non-THO polypeptide (e.g., heterologous amino acid sequences) to form TllO fusion proteins. The invention further features antibodies that specifically bind TllO proteins, such as monoclonal or polyclonal antibodies. In addition, the TllO proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detecting the presence of TllO activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of TllO activity such that the presence of TllO activity is detected in the biological sample.

In another aspect, the invention provides a method for modulating TllO activity comprising contacting a cell with an agent that modulates (inhibits or stimulates) TllO activity or expression such that TllO activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to TllO protein. In another embodiment, the agent modulates expression of TllO by modulating transcription of a TllO gene, splicing of a TllO mRNA, or translation of a TllO mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the TllO mRNA or the TllO gene. In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant TllO protein activity or nucleic acid expression by administering an agent which is a TllO modulator to the subject. In one embodiment, the TllO modulator is a TllO protein. In another embodiment the TllO modulator is a TllO nucleic acid molecule. In other embodiments, the TllO modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant TllO protein or nucleic acid expression is neoplasia, inappropriate angiogenesis, or inappropriate tissue regeneration after injury.

The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: (i) aberrant modification or mutation (including partial or complete deletion or amplification) of a gene encoding a TllO protein; (ii) mis-regulation of a gene encoding a TllO protein; and (iii) aberrant post- translational modification of a TllO protein, wherein a wild-type form of the gene encodes a protein with a TllO activity.

In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of a TllO protein. In general, such methods entail measuring a biological activity of a TllO protein in the presence and absence of a test compound and identifying those compounds which alter the activity of the TllO protein. The invention also features methods for identifying a compound which modulates the expression of TllO by measuring the expression of TllO in the presence and absence of a compound.

A plasmid containing DNA encoding human TllO and a plasmid containing DNA encoding murine TllO were deposited with the American Type Culture Collection (ATCC) , 10801 University Boulevard, Manassas, Virginia, 20110-2209, on June 22, 1998, and have been assigned ATCC

Accession Nos. and , respectively. The deposits were made according to the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The plasmid containing human DNA was deposited in E. coli (strain designation Epfthb llOd) , which contains a human TllO DNA in the plasmid vector pZLl . The plasmid containing murine DNA was also deposited in E. coli (strain designation Epftmb llOg) , which contains a murine TllO DNA in the plasmid vector pZLl . The deposits were made merely as a convenience for those of skill in the art and are not an admission that deposits are required under 35 U.S. C. § 112.

The invention includes a nucleic acid molecule that contains the nucleotide sequence of the cDNA having ATCC Accession No. , the coding sequence of that cDNA (i.e., the cDNA having ATCC Accession No. ), or complements thereof. Similarly, the invention includes a nucleic acid molecule that contains the nucleotide sequence of the cDNA having ATCC Accession No. , the coding sequence of that cDNA (i.e., the cDNA having ATCC Accession No. ) , or complements thereof .

The invention includes polypeptides encoded by the coding sequence of the nucleic acid molecules described above, i.e., sequence contained within the nucleic acid molecules deposited with the ATCC and assigned ATCC

Accession Nos. and , and biologically active fragments thereof. Moreover, those of ordinary skill in the art will recognize that many, if not all, of the methods described herein can be practiced with the nucleic acid molecules (or complements or fragments thereof) deposited with the ATCC, as described above, and/or the polypeptides (or fragments thereof) encoded by those molecules, just as they can be practiced as described herein by reference to a given SEQ ID No. Other features and advantages of the invention will be apparent from the following detailed description and claims .

Brief Description of the Drawings Figure 1 depicts the cDNA sequence (SEQ ID N0:1) and predicted amino acid sequence (SEQ ID NO: 2) of human TllO. The open reading frame of SEQ ID N0:1 extends from nucleotide 131 to nucleotide 1441 (SEQ ID NO: 3) .

Figure 2 is a hydropathy plot of human TllO. The location of the predicted transmembrane (TM) , and extracellular (OUT) domains are indicated as are the position of cysteines (cys; vertical bars) and potential glycosylation sites (Ngly; vertical bars) . Relative hydrophobicity is shown above the dotted line, and relative hydrophilicity is shown below the dotted line. Figure 3 depicts the cDNA sequence (SEQ ID NO: 5) and predicted amino acid sequence (SEQ ID NO: 6) of mouse TllO. The open reading frame of SEQ ID NO: 5 extends from nucleotide 103 to nucleotide 1452 (SEQ ID NO: 7) . Figure 4 is a hydropathy plot of mouse TllO. The location of the predicted transmembrane (TM) , and extracellular (OUT) domains are indicated as are the position of cysteines (cys; vertical bars) and potential glycosylatin sites (Ngly; vertical bars) . Relative hydrophobicity is shown above the dotted line, and relative hydrophilicity is shown below the dotted line.

Figure 5A depicts the partial cDNA sequence of rat TllO (SEQ ID NO: 9) .

Figure 5B depicts the predicted amino acid sequence (SEQ ID NO: 10) of rat TllO. The coding region of SEQ ID NO: 10 extends from nucleotide 1 to nucleotide 507 of SEQ ID NO : 9.

Figure 6 depicts the cDNA sequence (SEQ ID NO:l) and predicted amino acid sequence (SEQ ID NO: 4) of a potential alternative human TllO translation product. The open reading frame extends from nucleotide 2 to 1411 of SEQ ID NO:l (SEQ ID NO:12).

Figure 7 is a hydropathy plot of a potential alternative human TllO translation product. The location of the predicted transmembrane (TM) , and extracellular (OUT) domains are indicated as are the position of cysteines (cys; vertical bars) and potential glycosylation sites (Ngly; vertical bars) . Relative hydrophobicity is shown above the dotted line, and relative hydrophilicity is shown below the dotted line. Figure 8 depicts the cDNA sequence (SEQ ID NO: 5) and predicted amino acid sequence (SEQ ID NO: 8) of a potential alternative murine TllO translation product. The open reading frame extends from nucleotide 1 to 1452 of SEQ ID NO: 5 (SEQ ID NO: 13) .

Figure 9 is a hydropathy plot of a potential alternative murine TllO translation product. The location of the predicted transmembrane (TM) , and extracellular (OUT) domains are indicated as are the position of cysteines (cys; vertical bars) and potential glycosylation sites (Ngly; vertical bars) . Relative hydrophobicity is shown above the dotted line, and relative hydrophilicity is shown below the dotted line. Figure 10 depicts the sequence alignment of

D. melanogaster four jointed protein (SEQ ID NO: 11) with human TllO protein (SEQ ID NO:2) .

Figure 11 is a plot showing predicted structural features of a potential alternative human TllO protein.

Detailed Description of the Invention

The present invention is based on the discovery of a cDNA molecule encoding human TllO, a member of the type- II membrane protein superfamily. A nucleotide sequence encoding a human TllO protein is shown in Figure 1 (SEQ ID NO:l; SEQ ID NO : 3 includes the open reading frame only) . A predicted amino acid sequence of TllO protein is also shown in Figure 1 (SEQ ID NO: 2) . This protein includes a predicted signal peptide of about 28 amino acids (from amino acid 1 to about amino acid 28 of SEQ ID NO: 2) . The predicted mature protein extends from about amino acid 29 to amino acid 437 of SEQ ID NO: 2 (SEQ ID NO: 14) .

The human TllO cDNA of Figure 1 (SEQ ID N0:1), which is approximately 2401 nucleotides long including untranslated regions, encodes a protein amino acid having a molecular weight of approximately 48 kDa (excluding post-translational modifications) .

Human TllO protein and D. melanogaster four- jointed (fj) protein share many primary features. They are proteins of similar size and both contain a single predicted hydrophobic region near the N-terminus that may be a transmembrane domain rather than a signal sequence. Thus, the hydrophobic region from amino acids 1-28 (or 7- 30) might be a transmembrane domain that acts as an internal signal sequence. Each protein contain two pairs of conserved cysteine residues, one pair near the center of the molecule (cys₁₆₁ and cys₁₇₈) , the other pair near the C-terminus of the molecule (cys₃₆₅ and cys₄₂₇) . Regions of highest identity between the two proteins surround the two pairs of cysteines in the extracellular domains. Each protein also contains putative N-glycosylation sites, two of which are in approximately the same position, i.e., between the two pairs of cysteines (amino acid residuess 248 to 251 and amino acid residues 277 to

280) . A sequence alignment of human TllO protein and D. melanogaster fj protein is depicted in Figure 6. In this alignment, human TllO protein and D. melanogaster fj protein display about 30% identity and about 36% similarity. An approximately 2.4 kb human TllO mRNA transcript is expressed at the highest level in brain, heart, placenta, and pancreas. Low levels of this transcript have been observed in liver, skeletal muscle, and kidney. No detectable message is seen in lung. Embryonic expression is seen in week 8-9 fetus and week 20 liver and spleen mixed tissues. Embryonic expression is also observed in neuronal tissue.

Human TllO is one member of a family of molecules (the "TllO family") having certain conserved structural and functional features. The present invention provides detailed description of various members of the "TllO family", e.g., human TllO, mouse TllO, and rat TllO. The term "family" when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain and having sufficient amino acid or nucleotide sequence identity as defined herein. Such family members can be naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin and a homologue of that protein of murine origin, as well as a second, distinct protein of human origin and a murine homologue of that protein. Members of a family may also have common functional characteristics.

Preferred TllO polypeptides of the present invention have an amino acid sequence sufficiently identical to the consensus amino acid sequence of human TllO protein. As used herein, the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences which contain a common structural domain having about 65% identity, preferably 75% identity, more preferably 85%, 95%, or 98% identity are defined herein as sufficiently identical.

As used interchangeably herein a "TllO activity", "biological activity of TllO" or "functional activity of TllO", refers to an activity exerted by a TllO protein, polypeptide or nucleic acid molecule on a TllO responsive cell as determined in vivo, or in vi tro, according to standard techniques. A TllO activity can be a direct activity, such as an association with or an enzymatic activity on a second protein or an indirect activity, such as a cellular signaling activity mediated by interaction of the TllO protein with a second protein. In a preferred embodiment, a TllO activity includes at least one or more of the following activities: (i) the ability to interact with proteins in the TllO signalling pathway (ii) the ability to interact with a TllO ligand or receptor (iii) the ability to interact with an intracellular target protein; and (iv) the ability to interact with proteins involved in cellular proliferation or differentiation. Accordingly, another embodiment of the invention features isolated TllO proteins and polypeptides having a TllO activity.

Various aspects of the invention are described in further detail in the following subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid molecules that encode TllO proteins or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify TllO-encoding nucleic acids (e.g., TllO mRNA) and fragments for use as PCR primers for the amplification or mutation of TllO nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated TllO nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO : 5 , SEQ ID NO: 7, or a complement of any of these nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of SEQ ID N0:1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 as a hybridization probe, TllO nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al . , eds., Molecular Cloning: A Laboratory Manual . 2nd, ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) .

A nucleic acid of the invention can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to TllO nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO: 3,

SEQ ID NO: 5, SEQ ID NO: 7, or a portion thereof. A nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.

Moreover, the nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding TllO, for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of TllO. The nucleotide sequence determined from the cloning of the human TllO gene allows for the generation of probes and primers designed for use in identifying and/or cloning TllO homologues in other cell types, e.g., from other tissues, as well as TllO homologues from other mammals. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of the sense or anti-sense sequence of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO : 5 , SEQ ID NO : 7 or of a naturally occurring mutant of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO : 7.

Probes based on the human TllO nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or identical proteins. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express a TllO protein, such as by measuring a level of a TllO-encoding nucleic acid in a sample of cells from a subject, e.g., detecting TllO mRNA levels or determining whether a genomic TllO gene has been mutated or deleted.

A nucleic acid fragment encoding a "biologically active portion of TllO" can be prepared by isolating a portion of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO : 5 , SEQ ID NO: 7 which encodes a polypeptide having a TllO biological activity, expressing the encoded portion of TllO protein (e.g., by recombinant expression in vi tro) and assessing the activity of the encoded portion of TllO.

The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ ID NO:l, SEQ ID NO : 3 , SEQ ID NO: 5, or SEQ ID NO : 7 due to degeneracy of the genetic code and thus encode the same TllO protein as that encoded by the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.

In addition to the human TllO nucleotide sequence shown in SEQ ID NO : 1 and SEQ ID NO: 3, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of TllO may exist within a population (e.g., the human population) . Such genetic polymorphism in the TllO gene may exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a TllO protein, preferably a mammalian TllO protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the TllO gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily accomplished using hybridization probes recognizing TllO sequences to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms in TllO that are the result of natural allelic variation and that do not alter the functional activity of TllO are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding TllO proteins from other species (TllO homologues) , which have a nucleotide sequence which differs from that of a human

TllO, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the TllO cDNA of the invention can be isolated based on their identity to the human TllO nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. For example, a soluble human TllO cDNA can be isolated based on its identity to human membrane-bound TllO. Likewise, a cDNA encoding a membrane-bound form of human TllO can be isolated based on its identity to soluble human TllO.

Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 440 (450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1420) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule having the nucleotide sequence as shown in SEQ ID NO:l or a complement thereof; or the isolated nucleic acid molecule is at least 220 (250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1420) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule having the nucleotide sequence as shown in SEQ ID NO: 3 or a complement thereof; or the isolated nucleic acid molecule is at least 450 (500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1450) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 7, or a complement thereof .

As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, preferably 75%, 85%, or 95%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 corresponds to a naturally- occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the TllO sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, thereby leading to changes in the amino acid sequence of the encoded TllO protein, without altering the functional ability of the TllO protein. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of TllO (e.g., the sequence of SEQ ID NO:2 or SEQ ID NO: 6) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the TllO proteins of various species are predicted to be particularly unamenable to alteration.

For example, preferred TllO proteins of the present invention contain at least two pairs of conserved cysteines in the extracellular domain. Such conserved amino acids are less likely to be amenable to mutation. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved among TllO of various species) may not be essential for activity and thus are likely to be amenable to alteration.

In another example, preferred TllO proteins of the present invention contain residues that are identical between human TllO and mouse TllO. Such conserved amino acids between human TllO and mouse TllO are likely to be structurally or functionally significant. Accordingly, it is preferable to preserve these conserved residues unless one wishes to decrease protein stability or activity.

Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding TllO proteins that contain changes in amino acid residues that are not essential for activity. Such TllO proteins differ in amino acid sequence from SEQ ID NO: 2, 4, 6 or 8 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an amino acid sequence that is at least about 65% identical, 75%, 85%,

95%, or 98% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8.

An isolated nucleic acid molecule encoding a TllO protein having a sequence which differs from that of SEQ ID NO:2, 4, 6, or 8 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO : 7 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in TllO is preferably replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of a TllO coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for TllO biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

In a preferred embodiment, a mutant TllO protein can be assayed for: (1) the ability to form protein:protein interactions with proteins in a TllO signalling pathway; (2) the ability to bind a TllO ligand or receptor (3) the ability to bind to an intracellular target protein; or (4) the ability to interact with a protein involved in cellular proliferation or differentiation. In yet another preferred embodiment, a mutant TllO can be assayed for the ability to modulate cellular proliferation or cellular differentiation. The present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a protein, e.g., complementary to the coding strand of a double- stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire TllO coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame) . An antisense nucleic acid molecule can be antisense to a noncoding region of the coding strand of a nucleotide sequence encoding TllO. The noncoding regions ("5' and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids. Given the coding strand sequences encoding TllO disclosed herein (e.g., SEQ ID NO:l or SEQ ID NO: 3), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of TllO mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of TllO mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of TllO mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxyτnethyl-aminomethyl-2-1hiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosme, inosine, N6-iso-pentenyladenine, 1- methylguanine , 1-methylinosine, 2 , 2-di-methylguanine, 2- methyladenine, 2 -methylguanine, 3 -methyl-cytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2- thiouracil, beta-D-mannosylqueosine, 5 ' -methoxycarboxy- methyluracil, 5-methoxyuracil , 2-methyl-thio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v) , wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyl-uracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v) , 5-methyl-2-thiouracil , 3- (3- amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6- diaminopurine . Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection) . The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in si tu such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a TllO protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomeric nucleic acid molecule. An ce-anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual /3-units, the strands run parallel to each other (Gaultier et al . (1987) Nucleic Acids . Res . 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2 ' -o-methylribonucleotide (Inoue et al . (1987) Nucleic Acids Res . 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al . (1987) FEBS Lett . 215:327- 330) .

The invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave TllO mRΝA transcripts to thereby inhibit translation of TllO mRΝA. A ribozyme having specificity for a T110- encoding nucleic acid can be designed based upon the nucleotide sequence of a TllO cDΝA disclosed herein (e.g., SEQ ID ΝO:l, SEQ ID NO:3). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a TllO-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al . U.S. Patent No. 5,116,742. Alternatively, TllO mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418. The invention also encompasses nucleic acid molecules which form triple helical structures. For example, TllO gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the TllO (e.g., the TllO promoter and/or enhancers) to form triple helical structures that prevent transcription of the TllO gene in target cells. See generally, Helene (1991) Anticancer Drug Des . 6(6):569-84; Helene (1992) Ann. N. Y. Acad. Sci . 660:27- 36; and Maher (1992) Bioassays 14 (12) : 807-15. In preferred embodiments, the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al . (1996) Bioorganic & Medicinal Chemistry 4(1) : 5-23) . As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al . (1996) supra; Perry-0' Keefe et al . (1996) Proc . Natl . Acad . Sci . USA 93: 14670-675.

PNAs of TllO can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of TllO can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1996) supra ; or as probes or primers for DNA sequence and hybridization (Hyrup (1996) supra; Perry-O' Keefe et al . (1996) Proc . Natl . Acad . Sci . USA 93: 14670-675).

In another embodiment, PNAs of TllO can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of TllO can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAse H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) supra) . The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) supra and Finn et al. (1996) Nucleic Acids Research 24 (17) : 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5'- (4-meth-oxytrityl) amino-5 ' -deoxy-thymidine phosphoramidite can be used as a link between the PNA and the 5' end of DNA (Mag et al . (1989) Nucleic Acid Res . 17:5973-88) . PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al . (1996) Nucleic Acids Research 24 (17) :3357-63) . Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et al . (1975) Bioorganic Med. Chem. Lett . 5:1119-11124).

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo) , or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al . (1989) Proc . Natl . Acad. Sci . USA 86:6553-6556; Lemaitre et al . (1987) Proc . Natl . Acad . Sci . USA 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W0 89/10134) . In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al . (1988) Bio/Techniques 6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm . Res . 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

II. Isolated TllO Proteins and Anti-TllO Antibodies

One aspect of the invention pertains to isolated TllO proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-TllO antibodies. In one embodiment, native TllO proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, TllO proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a TllO protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the TllO protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of TllO protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, TllO protein that is substantially free of cellular material includes preparations of TllO protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-THO protein (also referred to herein as a "contaminating protein") . When the TllO protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When TllO protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.

Accordingly such preparations of TllO protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or non-THO chemicals.

Biologically active portions of a TllO protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the TllO protein (e.g., the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, or SEQ ID NO: 8), which include fewer amino acids than the full length TllO proteins, and exhibit at least one activity of a TllO protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the TllO protein. A biologically active portion of a TllO protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. Preferred biologically active polypeptides include one or more identified TllO structural domains, e.g., the extracellular domain (SEQ ID NO:4 and SEQ ID NO : 8) . Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native TllO protein. Preferred TllO protein has the amino acid sequence shown of SEQ ID NO: 2 or SEQ ID NO: 6. Other useful TllO proteins are substantially identical to SEQ ID NO: 2 or SEQ ID NO: 6 and retain the functional activity of the protein of SEQ ID NO: 2 or SEQ ID NO: 6 yet differ in amino acid sequence due to natural allelic variation or mutagenesis. Accordingly, a useful TllO protein is a protein which includes an amino acid sequence at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6 and retains the functional activity of the TllO proteins of SEQ ID NO:2. In other instances, the TllO protein is a protein having an amino acid sequence 55%, 65%, 75%, 85%, 95%, or 98% identical to the TllO extracellular domain (SEQ ID NO: 4 or SEQ ID NO: 8) . In a preferred embodiment, the TllO protein retains a functional activity of the TllO protein of SEQ ID NO: 2 or SEQ ID NO: 6.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of overlapping positions x 100) . Preferably, the two sequences are the same length. The determination of percent homology between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc . Nat 'l Acad . Sci . USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc . Nat 'l Acad. Sci . USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al . (1990) J. Mol . Biol . 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to TllO nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to TllO protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res . 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989) . Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.

The invention also provides TllO chimeric or fusion proteins. As used herein, a TllO "chimeric protein" or "fusion protein" comprises a TllO polypeptide operably linked to a non-THO polypeptide. A "TllO polypeptide" refers to a polypeptide having an amino acid sequence corresponding to TllO, whereas a "non-THO polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially identical to the TllO protein, e.g., a protein which is different from the TllO protein and which is derived from the same or a different organism. Within a TllO fusion protein the TllO polypeptide can correspond to all or a portion of a TllO protein, preferably at least one biologically active portion of a TllO protein. Within the fusion protein, the term "operably linked" is intended to indicate that the TllO polypeptide and the non-THO polypeptide are fused in- frame to each other. The non-THO polypeptide can be fused to the N-terminus or C-terminus of the TllO polypeptide .

One useful fusion protein is a GST-T110 fusion protein in which the TllO sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant TllO.

In yet another embodiment, the fusion protein is an TllO-immunoglobulin fusion protein in which all or part of TllO is fused to sequences derived from a member of the immunoglobulin protein family. The T110- immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a TllO ligand and a TllO protein on the surface of a cell, to thereby suppress TllO-mediated signal transduction in vivo. The TllO-immunoglobulin fusion proteins can be used to affect the bioavailability of a TllO cognate ligand. Inhibition of the TllO ligand/THO interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the TllO-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-TllO antibodies in a subject, to purify TllO ligands and in screening assays to identify molecules which inhibit the interaction of TllO with a TllO ligand.

Preferably, a TllO chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in- frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Current Protocols in Molecular Biology, Ausubel et al . eds . , John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide) . An TllO-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the TllO protein.

The present invention also pertains to variants of the TllO proteins (i.e., proteins having a sequence which differs from that of a naturally occurring TllO) which function as either TllO agonists (mimetics) or as TllO antagonists. Variants of the TllO protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the TllO protein. An agonist of the TllO protein can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the TllO protein. An antagonist of the TllO protein can inhibit one or more of the activities of the naturally occurring form of the TllO protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the TllO protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the TllO proteins.

Variants of the TllO protein which function as either TllO agonists (mimetics) or as TllO antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the TllO protein for TllO protein agonist or antagonist activity. In one embodiment, a variegated library of TllO variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.

A variegated library of TllO variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential TllO sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of TllO sequences therein. There are a variety of methods which can be used to produce libraries of potential TllO variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential TllO sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al . (1984) Annu. Rev. Biochem. 53:323; Itakura et al . (1984) Science 198:1056; Ike et al . (1983) Nucleic Acid Res . 11:477) .

In addition, libraries of fragments of the TllO protein coding sequence can be used to generate a variegated population of TllO fragments for screening and subsequent selection of variants of a TllO protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a TllO coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the TllO protein.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of TllO proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM) , a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify TllO variants (Arkin and Yourvan (1992) Proc . Natl . Acad. Sci . USA 89:7811-7815; Delgrave et al . (1993) Protein Engineering 6 (3) : 327-331) . An isolated TllO protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind TllO using standard techniques for polyclonal and monoclonal antibody preparation. The full-length TllO protein can be used or, alternatively, the invention provides antigenic peptide fragments of TllO for use as immunogens. The antigenic peptide of TllO comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO : 6 and encompasses an epitope of TllO such that an antibody raised against the peptide forms a specific immune complex with TllO.

Preferred epitopes encompassed by the antigenic peptide are regions of TllO that are located on the surface of the protein, e.g., hydrophilic regions. Other important criteria include a preference for a terminal sequence, high antigenic index (e.g., as predicted by the Jameson-Wolf algorithm), ease of peptide synthesis (e.g., avoidance of prolines) , and high surface probability (e.g., as predicted by the Emini algorithm) .

A TllO immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed TllO protein or a chemically synthesized TllO polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic TllO preparation induces a polyclonal anti-TllO antibody response. Accordingly, another aspect of the invention pertains to anti-TllO antibodies. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as TllO. A molecule which specifically binds to TllO is a molecule which binds TllO, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains TllO. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')₂ fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind TllO. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of TllO. A monoclonal antibody composition thus typically displays a single binding affinity for a particular TllO protein with which it immunoreacts .

Polyclonal anti-TllO antibodies can be prepared as described above by immunizing a suitable subject with a TllO immunogen. The anti-TllO antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized TllO. If desired, the antibody molecules directed against TllO can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti- T110 antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al . (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al . (1985) , Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing various antibodies and monoclonal antibody hybridomas is well known ( see generally Current Protocols in Immunology (1994) Coligan et al . (eds.) John Wiley & Sons, Inc., New York, NY) . Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a TllO immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds TllO.

Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-TllO monoclonal antibody (see, e.g., Current Protocols in

Immunology, supra; Galfre et al . (1977) Nature 266:55052; R.H. Kenneth, in Monoclonal Antibodies : A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); and Lerner (1981) Yale J. Biol . Med . , 54:387-402. Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line, e.g., a myeloma cell line that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium") . Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3- NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed) . Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind TllO, e.g., using a standard ELISA assay. Alternative to preparing monoclonal antibody- secreting hybridomas, a monoclonal anti-TllO antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with TllO to thereby isolate immunoglobulin library members that bind TllO. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene Surf ZAP™ Phage Display Ki t, Catalog No. 240612) . Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al . (1991) Bio/Technology 9:1370-1372; Hay et al . (1992) Hum. Anti od. Hybridomas 3:81-85; Huse et al . (1989) Science 246:1275-1281; Griffiths et al . (1993) EMBO J 12:725-734.

Additionally, recombinant anti-TllO antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al . (1987) Proc . Natl . Acad. Sci . USA 84:3439-3443; Liu et al . (1987) J. Immunol . 139:3521-3526; Sun et al . (1987) Proc . Natl . Acad. Sci . USA 84:214-218; Νishimura et al . (1987) Cane . Res . 47:999-1005; Wood et al . (1985) Nature 314:446-449; and Shaw et al . (1988) J^". Natl . Cancer Inst . 80:1553- 1559); Morrison, (1985) Science 229:1202-1207; Oi et al . (1986) Bio/Techniques 4:214; U.S. Patent 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al . (1988) Science 239:1534; and Beidler et al . (1988) J^". Immunol . 141:4053-4060. Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of TllO. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Jnt. .Rev. Immunol . 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016; and U.S. Patent 5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, CA) , can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above .

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.

First, a non-human monoclonal antibody which binds a selected antigen (epitope), e.g., an antibody which inhibits TllO activity, is identified. The heavy chain and the light chain of the non-human antibody are cloned and used to create phage display Fab fragments . For example, the heavy chain gene can be cloned into a plasmid vector so that the heavy chain can be secreted from bacteria. The light chain gene can be cloned into a phage coat protein gene so that the light chain can be expressed on the surface of phage. A repertoire (random collection) of human light chains fused to phage is used to infect the bacteria which express the non-human heavy chain. The resulting progeny phage display hybrid antibodies (human light chain/non-human heavy chain) . The selected antigen is used in a panning screen to select phage which bind the selected antigen. Several rounds of selection may be required to identify such phage. Next, human light chain genes are isolated from the selected phage which bind the selected antigen. These selected human light chain genes are then used to guide the selection of human heavy chain genes as follows. The selected human light chain genes are inserted into vectors for expression by bacteria.

Bacteria expressing the selected human light chains are infected with a repertoire of human heavy chains fused to phage. The resulting progeny phage display human antibodies (human light chain/human heavy chain) . Next, the selected antigen is used in a panning screen to select phage which bind the selected antigen. The phage selected in this step display completely human antibody which recognize the same epitope recognized by the original selected, non-human monoclonal antibody. The genes encoding both the heavy and light chains are readily isolated and can be further manipulated for production of human antibody. This technology is described by Jespers et al . (1994, Bio/technology 12:899-903) . An anti-TllO antibody (e.g., monoclonal antibody) can be used to isolate TllO by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-TllO antibody can facilitate the purification of natural TllO from cells and of recombinantly produced TllO expressed in host cells. Moreover, an anti-TllO antibody can be used to detect TllO protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the TllO protein. Anti-TllO antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance . Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, jβ-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol ; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding TllO (or a portion thereof) . As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid" , which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) . Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors) . However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) , which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence (s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vi tro transcription/translation system or in a host cell when the vector is introduced into the host cell) . The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) . Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) . It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., TllO proteins, mutant forms of TllO, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed for expression of TllO in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli , insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) . Alternatively, the recombinant expression vector can be transcribed and translated in vi tro, for example using T7 promoter regulatory sequences and T7 polymerase .

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST) , maltose E binding protein, or protein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al . , (1988) Gene 69:301-315) and pET lid (Studier et al . , Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89) . Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET lid vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl) . This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV5 promoter.

One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave, the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128) . Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al . (1992) Nucleic Acids Res . 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be accomplished by standard DNA synthesis techniques.

In another embodiment, the TllO expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari et al . (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA) , and picZ (Invitrogen Corp, San Diego, CA) .

Alternatively, TllO can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al . (1983) Mol . Cell Biol . 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31- 39) .

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al . (1987) EMBO J. 6:187- 195) . When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al . ( supra) . In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid) . Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al . (1987) Genes

Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol . 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc . Natl . Acad . Sci . USA 86:5473-5477), pancreas-specific promoters (Edlund et al . (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally- regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to TllO mRNA. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al . {Reviews - Trends in Genetics, Vol. 1(1) 1986) . Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, TllO protein can be expressed in bacterial cells such as E. coli , insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells

(CHO) or COS cells) . Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al . ( supra) , and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding TllO or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die) .

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) TllO protein. Accordingly, the invention further provides methods for producing TllO protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding TllO has been introduced) in a suitable medium such that TllO protein is produced. In another embodiment, the method further comprises isolating TllO from the medium or the host cell.

The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which TllO-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous TllO sequences have been introduced into their genome or homologous recombinant animals in which endogenous TllO sequences have been altered. Such animals are useful for studying the function and/or activity of TllO and for identifying and/or evaluating modulators of TllO activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene . Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal , thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, an "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous TllO gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing TllO-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The TllO cDNA sequence e.g., that of (SEQ ID N0:1, SEQ ID NO : 3 , SEQ ID NO: 5, and SEQ ID NO: 7) can be introduced as a transgene into the genome of a non-human animal . Alternatively, a nonhuman homologue of the human TllO gene, such as a mouse TllO gene can be used as a transgene. A nonhuman homologue of the human TllO can be isolated based on hybridization to the human TllO cDNA. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence (s) can be operably linked to the TllO transgene to direct expression of TllO protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, U.S. Patent No. 4,873,191 and in Hogan, Manipulating the Mouse

Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. , 1986) . Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the TllO transgene in its genome and/or expression of TllO mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding TllO can further be bred to other transgenic animals carrying other transgenes .

To create an homologous recombinant animal , a vector is prepared which contains at least a portion of a TllO gene (e.g., a human or a non-human homolog of the TllO gene, e.g., a murine TllO gene) into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the TllO gene. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous TllO gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector) . Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous TllO gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous TllO protein) . In the homologous recombination vector, the altered portion of the TllO gene is flanked at its 5' and 3' ends by additional nucleic acid of the TllO gene to allow for homologous recombination to occur between the exogenous TllO gene carried by the vector and an endogenous TllO gene in an embryonic stem cell. The additional flanking TllO nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.

Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi (1987) Cell 51:503 for a description of homologous recombination vectors) . The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced TllO gene has homologously recombined with the endogenous TllO gene are selected (see, e.g., Li et al . (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas and Embryonic Stem Cells : A Practical Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113- 152) . A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley

(1991) Current Opinion in Bio/Technology 2:823-829 and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene.

One example of such a system is the cre/loxP recombinase system of bacteriophage PI. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al .

(1992) Proc . Natl . Acad. Sci . USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (0' Gorman et al . (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase .

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al . (1997) Nature 385:810- 813 and PCT Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G₀ phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

IV. Pharmaceutical Compositions

The TllO nucleic acid molecules, TllO proteins, and anti-TllO antibodies (also referred to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal , and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine-tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF; Parsippany, NJ) or phosphate buffered saline (PBS) . In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a TllO protein or anti-TllO antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means . For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors . Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al . (1994) Proc . Natl . Acad . Sci . USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) detection assays (e.g., chromosomal mapping, tissue typing, forensic biology) , c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenomics) ; and d) methods of treatment (e.g., therapeutic and prophylactic). TllO protein interacts with other cellular proteins and can thus be used for (i) regulation of cellular proliferation and (ii) regulation of cellular differentiation. The isolated nucleic acid molecules of the invention can be used to express TllO protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect TllO mRNA (e.g., in a biological sample) or a genetic lesion in a TllO gene, and to modulate TllO activity. In addition, the TllO proteins can be used to screen drugs or compounds which modulate TllO activity or expression as well as to treat disorders characterized by insufficient or excessive production of TllO protein or production of TllO protein forms which have decreased or aberrant activity compared to TllO wild type protein. In addition, the anti-TllO antibodies of the invention can be used to detect and isolate TllO proteins and modulate TllO activity.

This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.

A. Screening Assays The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to TllO proteins or have a stimulatory or inhibitory effect on, for example, TllO expression or TllO activity.

In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a TllO protein or polypeptide or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des . 12:145) . Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al . (1993) Proc . Natl . Acad . Sci . U. S .A . 90:6909; Erb et al . (1994) Proc . Natl . Acad . Sci . USA 91:11422; Zuckermann et al . (1994). J^". Med. Chem.

37:2678; Cho et al . (1993) Science 261:1303; Carrell et al. (1994) Angrew. Chem. Int . Ed. Engl . 33:2059; Carell et al. (1994) Angrew. Chem. Int . Ed. Engl . 33:2061; and Gallop et al . (1994) J^". Med . Chem . 37:1233. Libraries of compounds may be presented in solution (e.g., Houghten (1992) Bio/Techniques 13:412- 421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364 : 555-556) , bacteria (U.S. Patent No. 5,223,409), spores (Patent Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al . (1992) Proc . Natl . Acad. Sci . USA 89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al . (1990) Proc . Natl . Acad . Sci . 87:6378-6382; and Felici (1991) J. Mol . Biol . 222:301- 310) .

In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of TllO protein, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a TllO protein determined. The cell, for example, can be a yeast cell or a cell of mammalian origin. Determining the ability of the test compound to bind to the TllO protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the TllO protein or biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, test compounds can be enzyτnatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In a preferred embodiment, the assay comprises contacting a cell which expresses a membrane- bound form of TllO protein, or a biologically active portion thereof, on the cell surface with a known compound which binds TllO to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a TllO protein, wherein determining the ability of the test compound to interact with a TllO protein comprises determining the ability of the test compound to preferentially bind to TllO or a biologically active portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane- bound form of TllO protein, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the TllO protein or biologically active portion thereof.

Determining the ability of the test compound to modulate the activity of TllO or a biologically active portion thereof can be accomplished, for example, by determining the ability of the TllO protein to bind to or interact with a TllO target molecule.

As used herein, a "target molecule" is a molecule with which a TllO protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a TllO protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A TllO target molecule can be a non-THO molecule or a TllO protein or polypeptide of the present invention. In one embodiment, a TllO target molecule is a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a membrane-bound TllO molecule or by binding of a soluble form of TllO to a cellular receptor) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with TllO. Determining the ability of the TllO protein to bind to or interact with a TllO target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the TllO protein to bind to or interact with a TllO target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca²⁺, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a T110- responsive regulatory element operably linked to a nucleic acid encoding a detectable marker, e.g. luciferase) , or detecting a cellular response, for example, cellular differentiation or cell proliferation.

In yet another embodiment, an assay of the present invention is a cell-free assay comprising contacting a TllO protein or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the TllO protein or biologically active portion thereof (e.g., the extracellular domain of TllO) . Binding of the test compound to the TllO protein can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the TllO protein or biologically active portion thereof with a known compound which binds TllO to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a TllO protein, wherein determining the ability of the test compound to interact with a TllO protein comprises determining the ability of the test compound to preferentially bind to TllO or biologically active portion thereof as compared to the known compound. In another embodiment, an assay is a cell-free assay comprising contacting TllO protein or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the TllO protein or biologically active portion thereof.

Determining the ability of the test compound to modulate the activity of TllO can be accomplished, for example, by determining the ability of the TllO protein to bind to a TllO target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of TllO can be accomplished by determining the ability of the TllO protein to further modulate a TllO target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting the TllO protein or biologically active portion thereof with a known compound which binds TllO to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a TllO protein, wherein determining the ability of the test compound to interact with a TllO protein comprises determining the ability of the TllO protein to preferentially bind to or modulate the activity of a TllO target molecule.

The cell-free assays of the present invention are amenable to use of both the soluble form or the membrane- bound form of TllO. In the case of cell-free assays comprising the membrane-bound form of TllO, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of TllO is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl- N-methylglucamide, Triton^® X-100, Triton^® X-114, Thesit^®, Isotridecypoly (ethylene glycol ether)n, 3- [ (3-cholamidopropyl) dimethylamminio] -1-propane sulfonate (CHAPS), 3- [ (3-cholamidopropyl) dimethylamminio] -2-hydroxy-1-propane sulfonate (CHAPSO) , or N-dodecyl=N,N-dimethyl-3-ammonio- 1-propane sulfonate.

In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either TllO or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. A test compound to TllO, or interaction of TllO with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S- transferase/ TllO fusion proteins or glutathione-S- transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or TllO protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH) . Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of TllO binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either TllO or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated TllO or target molecules can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, IL) , and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical) . Alternatively, antibodies reactive with TllO or target molecules but which do not interfere with binding of the TllO protein to its target molecule can be derivatized to the wells of the plate, and unbound target or TllO trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the TllO or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the TllO or target molecule.

In another embodiment, modulators of TllO expression are identified in a method in which a cell is contacted with a candidate compound and the expression of TllO mRNA or protein in the cell is determined. The level of expression of TllO mRNA or protein in the presence of the candidate compound is compared to the level of expression of TllO mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of TllO expression based on this comparison. For example, when expression of TllO mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of TllO mRNA or protein expression. Alternatively, when expression of TllO mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of TllO mRNA or protein expression. The level of TllO mRNA or protein expression in the cells can be determined by methods described herein for detecting TllO mRNA or protein. In yet another aspect of the invention, the TllO proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al . (1993) Cell 72:223-232; Madura et al. (1993) J. Biol . Chem. 268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al .

(1993) Oncogene 8:1693-1696; and PCT Publication No. WO 94/10300) , to identify other proteins, which bind to or interact with TllO ( "TllO-binding proteins" or "TllO-bp") and modulate TllO activity. Such TllO-binding proteins are also likely to be involved in the propagation of signals by the TllO proteins as, for example, upstream or downstream elements of the TllO pathway.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for TllO is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4) . In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming an TllO-dependent complex, the DNA- binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with TllO. This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing) ; and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. Accordingly, TllO nucleic acid molecules described herein or fragments thereof, can be used to map the location of TllO genes on a chromosome. The mapping of the TllO sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease. Briefly, TllO genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the TllO sequences. Computer analysis of TllO sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes . Only those hybrids containing the human gene corresponding to the TllO sequences will yield an amplified fragment. Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells) . As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow (because they lack a particular enzyme) , but human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio et al . (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the TllO sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a TllO sequence to its chromosome include in si tu hybridization (described in Fan et al . (1990) Proc . Natl . Acad. Sci . USA 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.

Fluorescence in si tu hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical, e.g., colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al . , (Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York, 1988)).

Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping. Once a sequence has 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, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library) .

The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland et al . (1987) Nature, 325:783-787.

Moreover, differences in the DΝA sequences between individuals affected and unaffected with a disease associated with the TllO gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The TllO sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).

Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the TllO sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it .

Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The TllO sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO:l can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000. If a panel of reagents from TllO sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

3. Use of Partial TllO Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual) . As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO:l are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the TllO sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO : 1 or SEQ ID NO: 5 having a length of at least 20 or 30 bases. The TllO sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in si tu hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such TllO probes can be used to identify tissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., TllO primers or probes can be used to screen tissue culture for contamination (i.e., screen for the presence of a mixture of different types of cells in a culture) .

C. Predictive Medicine

The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trails are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining TllO protein and/or nucleic acid expression as well as TllO activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant TllO expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with TllO protein, nucleic acid expression or activity. For example, mutations in a TllO gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with TllO protein, nucleic acid expression or activity.

Another aspect of the invention provides methods for determining TllO protein, nucleic acid expression or TllO activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent . )

Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs or other compounds) on the expression or activity of TllO in clinical trials.

These and other agents are described in further detail in the following sections.

1. Diagnostic Assays An exemplary method for detecting the presence or absence of TllO in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting TllO protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes TllO protein such that the presence of TllO is detected in the biological sample. A preferred agent for detecting TllO mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to TllO mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length TllO nucleic acid, such as the nucleic acid of SEQ ID NO: 1, 3, 5, or 7 , or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to TllO mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

A preferred agent for detecting TllO protein is an antibody capable of binding to TllO protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect TllO mRNA, protein, or genomic DNA in a biological sample in vi tro as well as in vivo. For example, in vi tro techniques for detection of TllO mRNA include Northern hybridizations and in si tu hybridizations. In vi tro techniques for detection of TllO protein include enzyme linked immunosorbent assays (ELISAs) , Western blots, immunoprecipitations and immunofluorescence . In vi tro techniques for detection of TllO genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of TllO protein include introducing into a subject a labeled anti-TllO antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques .

In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting TllO protein, mRNA, or genomic DNA, such that the presence of TllO protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of TllO protein, mRNA or genomic DNA in the control sample with the presence of TllO protein, mRNA or genomic DNA in the test sample. The invention also encompasses kits for detecting the presence of TllO in a biological sample (a test sample) . Such kits can be used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with aberrant expression of TllO (e.g., an immunological disorder). For example, the kit can comprise a labeled compound or agent capable of detecting TllO protein or mRNA in a biological sample and means for determining the amount of TllO in the sample (e.g., an anti-TllO antibody or an oligonucleotide probe which binds to DNA encoding TllO, e.g., SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7) . Kits may also include instruction for observing that the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of TllO if the amount of TllO protein or mRNA is above or below a normal level. For antibody-based kits, the kit may comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to TllO protein; and, optionally, (2) a second, different antibody which binds to TllO protein or the first antibody and is conjugated to a detectable agent .

For oligonucleotide-based kits, the kit may comprise, for example: (1) an oligonucleotide, e.g., a detectably labelled oligonucleotide, which hybridizes to a TllO nucleic acid sequence or (2) a pair of primers useful for amplifying a TllO nucleic acid molecule;

The kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit may also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate) . The kit may also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of TllO.

2. Prognostic Assays

The methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with aberrant TllO expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with TllO protein, nucleic acid expression or activity, e.g., a cell proliferation disorder. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing such a disease or disorder. Thus, the present invention provides a method in which a test sample is obtained from a subject and

TllO protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of TllO protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant TllO expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue. Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant TllO expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease TllO activity) . Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant TllO expression or activity in which a test sample is obtained and TllO protein or nucleic acid is detected (e.g., wherein the presence of TllO protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant TllO expression or activity) .

The methods of the invention can also be used to detect genetic lesions or mutations in a TllO gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant TllO expression or activity, e.g., aberrant cell proliferation and/or differentiation. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding a T110- protein, or the mis-expression of the TllO gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from a TllO gene; 2) an addition of one or more nucleotides to a TllO gene; 3) a substitution of one or more nucleotides of a TllO gene; 4) a chromosomal rearrangement of a TllO gene; 5) an alteration in the level of a messenger RNA transcript of a TllO gene; 6) aberrant modification of a TllO gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a TllO gene, 8) a non-wild type level of a TllO-protein, 9) allelic loss of a TllO gene, 10) inappropriate post-translational modification of a TllO-protein, and 11) amplification of a TllO gene. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a TllO gene.

In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) ( see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) ( see, e.g., Landegran et al . (1988) Science 241:1077-1080; and Nakazawa et al . (1994) Proc . Natl . Acad. Sci . USA 91:360- 364) , the latter of which can be particularly useful for detecting point mutations in the TllO-gene (see, e.g., Abravaya et al . (1995) Nucleic Acids Res . 23:675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a TllO gene under conditions such that hybridization and amplification of the TllO-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequence replication (Guatelli et al . (1990) Proc . Natl . Acad. Sci . USA 87:1874-1878), transcriptional amplification system (Kwoh, et al . (1989) Proc . Natl . Acad. Sci . USA 86:1173-1177), Q-Beta Replicase (Lizardi et al . (1988) Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers .

In an alternative embodiment, mutations in a TllO gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally) , digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyτne cleavage site.

In other embodiments, genetic mutations in TllO can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al . (1996) Human Mutation 7:244-255; Kozal et al . (1996) Nature Medicine 2:753-759). For example, genetic mutations in TllO can be identified in two-dimensional arrays containing light-generated DΝA probes as described in Cronin et al . supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DΝA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene .

In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the TllO gene and detect mutations by comparing the sequence of the sample TllO with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) Proc . Natl . Acad . Sci . USA 74:560) or Sanger ((1977) Proc . Natl . Acad . Sci . USA 74:5463) . It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ( (1995) Bio/Techniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et al . (1996) Adv. Chromatogr. 36:127-162; and

Griffin et al . (1993) Appl . Biochem. Biotechnol . 38:147- 159) .

Other methods for detecting mutations in the TllO gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al . (1985) Science 230:1242). In general, the technique of "mismatch cleavage" entails providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type TllO sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. RNA/DNA duplexes can be treated with RNase to digest mismatched regions, and DNA/DNA hybrids can be treated with SI nuclease to digest mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton et al (1988) Proc . Natl Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol . 217:286- 295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in TllO cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at

G/T mismatches (Hsu et al . (1994) Carcinogenesis 15:1657- 1662) . According to an exemplary embodiment, a probe based on a TllO sequence, e.g., a wild-type TllO sequence, is hybridized to a cDNA or other DNA product from a test cell (s) . The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039. In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in TllO genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al . (1989) Proc . Natl . Acad. Sci USA 86:2766; see also Cotton (1993) Λfutat. .Res. 285:125-144; Hayashi (1992) Genet . Anal . Tech . Appl . 9:73-79). Single-stranded DNA fragments of sample and control TllO nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA) , in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al . (1991) Trends Genet 7:5).

In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al . (1985) Nature 313:495) . When DGGE is used as the method of analysis, DΝA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DΝA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al . (1986) Nature 324:163); Saiki et al . (1989) Proc . Natl Acad. Sci USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention.

Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al . (1989) Nucleic Acids Res . 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent or reduce polymerase extension (Prossner (1993) Tijbtec 11:238) . In addition, it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol . Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc . Natl . Acad . Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification. The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a TllO gene.

Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which TllO is expressed may be utilized in the prognostic assays described herein.

3. Pharmacogenomics

Agents, or modulators which have a stimulatory or inhibitory effect on TllO activity (e.g., TllO gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., a proliferative disorder) associated with aberrant TllO activity. In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual ' s genotype and that individual ' s response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of TllO protein, expression of TllO nucleic acid, or mutation content of TllO genes in an individual can be determined to thereby select appropriate agent (s) for therapeutic or prophylactic treatment of the individual .

Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Linder (1997) Clin . Chem. 43(2) :254- 266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body are referred to as "altered drug action" conditions.

Genetic conditions transmitted as single factors altering the way the body acts on drugs are referred to as "altered drug metabolism" conditions. These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6- phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM) . The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

Thus, the activity of TllO protein, expression of TllO nucleic acid, or mutation content of TllO genes in an individual can be determined to thereby select appropriate agent (s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a TllO modulator, such as a modulator identified by one of the exemplary screening assays described herein.

4. Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of TllO (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent, as determined by a screening assay as described herein to increase TllO gene expression, increase protein levels, or upregulate TllO activity, can be monitored in clinical trials of subjects exhibiting decreased TllO gene expression, decreased protein levels, or downregulated TllO activity. Alternatively, the effectiveness of an agent, as determined by a screening assay, to decrease TllO gene expression, decrease protein levels, or downregulate TllO activity, can be monitored in clinical trials of subjects exhibiting increased TllO gene expression, increased protein levels, or upregulated TllO activity. In such clinical trials, the expression or activity of TllO and, preferably, other genes that have been implicated in, for example, a cellular proliferation disorder can be used as a marker.

For example, and not by way of limitation, genes, including TllO, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates TllO activity (e.g., as identified in a screening assay described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of TllO and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of TllO or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent . In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a TllO protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the TllO protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the TllO protein, mRNA, or genomic DNA in the pre-administration sample with the

TllO protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of TllO to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of TllO to lower levels than detected, i.e., to decrease the effectiveness of the agent .

C. Methods of Treatment The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant TllO expression or activity. Such disorders include neoplasia, inappropriate angiogenesis, or inappropriate tissue regeneration.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant TllO expression or activity, by administering to the subject an agent which modulates TllO expression or at least one TllO activity. Subjects at risk for a disease which is caused or contributed to by aberrant TllO expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the TllO aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of TllO aberrancy, for example, a TllO agonist or TllO antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

2. Therapeutic Methods Another aspect of the invention pertains to methods of modulating TllO expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of TllO protein activity associated with the cell. An agent that modulates TllO protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a TllO protein, a peptide, a TllO peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more of the biological activities of TllO protein. Examples of such stimulatory agents include active TllO protein and a nucleic acid molecule encoding TllO that has been introduced into the cell. In another embodiment, the agent inhibits one or more of the biological activities of TllO protein. Examples of such inhibitory agents include antisense TllO nucleic acid molecules and anti- TllO antibodies. These modulatory methods can be performed in vi tro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject) . As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a TllO protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein) , or combination of agents that modulates (e.g., upregulates or downregulates) TllO expression or activity. In another embodiment, the method involves administering a TllO protein or nucleic acid molecule as therapy to compensate for reduced or aberrant TllO expression or activity. Stimulation of TllO activity is desirable in situations in which TllO is abnormally downregulated and/or in which increased TllO activity is likely to have a beneficial effect. Conversely, inhibition of TllO activity is desirable in situations in which TllO is abnormally upregulated and/or in which decreased TllO activity is likely to have a beneficial effect.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

EXAMPLES Example 1 : Isolation and Characterization of Human TllO cDNAs

A cDNA library was prepared from polyA mRNA isolated from ratPC12 cells (PC6-3 subline) that had been cultured in the absence of neurotrophic factors (NGF) for 12 hours. Random 5' sequencing yielded a single clone with homology to the D. melanogaster fj gene. This partial rat clone was used to screen mouse and human fetal brain cDNA libraries. These screens have yielded clones containing mouse TllO and human TllO.

Complete sequencing of the human TllO clone revealed an approximately 2.4 kb cDNA insert with a 1311 base pair open reading frame predicted to encode a novel secreted protein, i . e . , human TllO. Complete sequencing of the mouse TllO clone revealed an approximately 2.1 kb cDNA insert with a 1350 base pair open reading frame predicted to encode a novel secreted protein, i . e . , mouse TllO. The mouse and human protein sequences are about 85% identical. The major region of divergence is towards the N-terminus.

Figure 6 depicts the cDNA sequence (SEQ ID NO:l) and predicted amino acid sequence (SEQ ID NO: 4) of a potential alternative human TllO translation product. The open reading frame extends from nucleotide 2 to 1411 of SEQ ID NO:l) .

Figure 8 depicts the cDNA sequence (SEQ ID NO: 5) and predicted amino acid sequence (SEQ ID NO: 8) of a potential alternative murine TllO translation product. The open reading frame extends from nucleotide 1 to 1452 of SEQ ID NO: 5. Example 2 : Distribution of TllO mRNA in Human Tissues

The expression of TllO was analyzed using Northern blot hybridization. In rat, the Northern blot analysis of adult tissues showed highest expression in brain and kidney. Expression was also observed in heart and lung.

No mRNA was detected in spleen, liver, skeletal muscle or testis.

To examine the tissue distribution of human TllO, the rat partial cDNA sequence was used as a probe for the Northern blot analysis. The cDNA was radioactively labeled with ³²P-dCTP using the Prime-It kit (Stratagene;

La Jolla, CA) according to the instructions of the supplier. Filters containing human mRNA (MTNI and MTNII:

Clontech; Palo Alto, CA) were probed in ExpressHyb hybridization solution (Clontech, Palo Alto, CA) and washed at high stringency according to manufacturer's recommendations .

These studies revealed that human TllO was expressed as an approximately 2.4 kilobase transcript at highest level in brain, heart, placenta, and pancreas.

Lower levels of transcript were seen in liver, skeletal muscle, and kidney. Transcript was not detected in lung.

Embryonic expression was seen in week 8-9 fetus and week

20 liver and spleen mixed tissue. In si tu expression assays on mouse embryos revealed that TllO is expressed in the nervous system.

In adult mice, in si tu expression assays revealed that

TllO is expressed in discrete regions of the brain, including the cerebellum and olfactory bulb, and in the non-islet cells of the pancreas.

Example 3 : Characterization of TllO Proteins

The human TllO cDNA (Figure 1; SEQ ID NO:l) isolated as described above encodes a 437 amino acid protein (Figure 1; SEQ ID NO: 2) . A hydropathy plot of TllO is presented in Figure 2. This plot shows the presence of a signal sequence (amino acids 1-28) and a hydrophobic region that may indicate a transmembrane domain (amino acid 7-30) that acts as an internal signal sequence .

Figure 7 is a plot showing predicted structural features of a potential alternative human TllO protein (SEQ ID NO: 4) . This figure shows predicted alpha helix regions (Garnier-Robson and Chou-Fasman) , predicted beta sheet regions (Garnier-Robson and Chou-Fasman) , predicted turn regions (Garnier-Robson and Chou-Fasman) , predicted coil regions (Garnier-Robson) , predicted hydrophilicity, predicted alpha amphipathic regions (Eisenberg) predicted beta amphipathic regions (Eisenberg) , predicted flexible regions (Karplus-Schultz) , predicted antigenic index (Jameson-Wolf) , and surface probability (Emini) .

A sequence alignment of human TllO protein and D. melanogaster fj protein, as shown in Figure 6, reveals that both proteins are of similar size, contain a single predicted hydrophobic region as the transmembrane and internal signal sequence, and include a large extracellular domain with two pairs of conserved cysteine residues. In this alignment, which includes gaps, the proteins are 20.7% identical and 35.9% similar. Mature human TllO has a predicted MW of 48 kDa, not including post-translational modifications.

A secretion assay revealed that TllO is a secreted protein. It may be secreted using a signal peptide (amino acids 1-28) or a transmembrane region (amino acids 7-30) that acts as an internal signal sequence.

Example 4 : Preparation of TllO Proteins

Recombinant TllO can be produced in a variety of expression systems. For example, the mature TllO peptide can be expressed as a recombinant glutathione-S- transferase (GST) fusion protein in E. coli and the fusion protein can be isolated and characterized. Specifically, as described above, TllO can be fused to GST and this fusion protein can be expressed in E. coli strain PEB199. Expression of the GST-T110 fusion protein in PEB199 can be induced with IPTG. The recombinant fusion protein can be purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads .

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

What is claimed is:

Claims

1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a fragment of at least 450 nucleotides of the nucleotide sequence of SEQ ID N0:1 or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 220 nucleotides of the nucleotide sequence of SEQ ID NO: 3 or a complement thereof; c) a nucleic acid molecule comprising a fragment of at least 450 nucleotides of the nucleotide sequence of

SEQ ID NO: 5 or SEQ ID NO: 7, or a complement thereof; d) a nucleic acid molecule which encodes a 'polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO : 4 , SEQ ID NO: 6, or SEQ ID NO: 8; e) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein the fragment comprises at least 70 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4; f) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8, wherein the fragment comprises at least 150 contiguous amino acids of SEQ ID NO: 6 or SEQ ID NO: 8; and g) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO : 8 , wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 under stringent conditions.

2. The isolated nucleic acid molecule of claim 1, which is selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or a complement thereof; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO : 4 , SEQ ID NO: 6, or SEQ ID NO : 8.

3. The nucleic acid molecule of claim 1 further comprising vector nucleic acid sequences.

4. The nucleic acid molecule of claim 1 further comprising nucleic acid sequences encoding a heterologous polypeptide .

5. A host cell which contains the nucleic acid molecule of claim 1.

6. The host cell of claim 5 which is a mammalian host cell.

7. A non-human mammalian host cell containing the nucleic acid molecule of claim 1.

8. An isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:4, wherein the fragment comprises at least 70 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO:4; b) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8, wherein the fragment comprises at least 150 contiguous amino acids of SEQ ID NO: 6 or SEQ ID NO: 8; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 under stringent conditions.

9. The isolated polypeptide of claim 8 comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6 or SEQ ID NO: 8.

10. The polypeptide of claim 8 further comprising heterologous amino acid sequences.

11. An antibody which selectively binds to a polypeptide of claim 8.

12. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, or SEQ ID NO: 8; b) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:4, wherein the fragment comprises at least 70 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO:4; c) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8, wherein the fragment comprises at least 150 contiguous amino acids of SEQ ID NO: 6 or SEQ ID NO: 8; and d) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO : 4 , SEQ ID NO: 6, or SEQ ID NO : 8 , wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:l, SEQ ID NO : 3 , SEQ ID NO: 5, or SEQ ID NO: 7 under stringent conditions; comprising culturing the host cell of claim 5 under conditions in which the nucleic acid molecule is expressed.

13. The isolated polypeptide of claim 8 comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, or SEQ ID NO: 8.

14. A method for detecting the presence of a polypeptide of claim 8 in a sample, comprising: a) contacting the sample with a compound which selectively binds to a polypeptide of claim 8; and b) determining whether the compound binds to the polypeptide in the sample.

15. The method of claim 14, wherein the compound which binds to the polypeptide is an antibody.

16. A kit comprising a compound which selectively binds to a polypeptide of claim 8 and instructions for use .

17. A method for detecting the presence of a nucleic acid molecule of claim 1 in a sample, comprising the steps of: a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.

18. The method of claim 17, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.

19. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of claim 1 and instructions for use.

20. A method for identifying a compound which binds to a polypeptide of claim 8 comprising the steps of: a) contacting a polypeptide, or a cell expressing a polypeptide of claim 8 with a test compound; and b) determining whether the polypeptide binds to the test compound.

21. The method of claim 20, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of test compound/polypeptide binding; b) detection of binding using a competition binding assay; c) detection of binding using an assay for T110- mediated signal transduction.

22. A method for modulating the activity of a polypeptide of claim 8 comprising contacting a polypeptide or a cell expressing a polypeptide of claim 8 with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.

23. A method for identifying a compound which modulates the activity of a polypeptide of claim 8, comprising : a) contacting a polypeptide of claim 8 with a test compound; and b) determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.