EP1135412A2

EP1135412A2 - Tumor necrosis factor receptor homologue-1 (trh1)

Info

Publication number: EP1135412A2
Application number: EP99968873A
Authority: EP
Inventors: Michael A. Bowen; Nathan Siemers
Original assignee: Bristol Myers Squibb Co
Current assignee: Bristol Myers Squibb Co
Priority date: 1998-12-11
Filing date: 1999-12-10
Publication date: 2001-09-26
Also published as: CA2354612A1; AU2708000A; WO2000034294A3; JP2002531112A; WO2000034294A2

Abstract

The present invention discloses the identification of the new human tumor necrosis factor receptor homologue ('TRH1'), as identified by its nucleic acid and amino acid sequences disclosed herein. The invention also includes methods of using the nucleic acid sequence, the TRH1 protein, a monoclonal antibody specific for the novel tumor necrosis factor receptor, a ligand for the novel tumor necrosis factor receptor, and fusion proteins comprising all or a portion of the TRH1 disclosed herein.

Description

TUMOR NECROSIS FACTOR RECEPTOR HOMOLOGUE-1 ("TRHl")

Background of the Invention

The tumor necrosis factor receptor ("TNFr") superfamily consists primarily of transmembrane proteins that elicit signal transduction in a variety of cells. The TNFr superfamily of cell surface receptors includes TNFrl, TNFr2, Fas, 4- IBB, OX-40, DR4, DR5 and others. These molecules contain extracellular cysteine-rich TNFr domains, a transmembrane domain, and an intracellular cytoplasmic tail which mediates receptor interactions with intracellular signaling molecules. Members of this family mediate diverse biological events including cell activation, proliferation, differentiation, and apoptosis. (See, e.g., Smith, C.A., et al, (1994) Cell, 76:959-962, incorporated herein by reference).

Recently, a novel soluble member was described, called osteoprotegrin, which contains four cysteine-rich TNFr domains but lacks transmembrane and cytoplasmic domains. This molecule has been shown to regulate osteoclast differentiation and therefore may play a role in regulating bone mass during development and in the mature animal. (Simonet, W.S., et al., (1997) Cell, 89:309-319, incorporated herein by reference).

Because of the diverse biological activities of members of the TNFr superfamily and their close relationship to immune cell function (see, e.g., US Patent 5,670,319 to Goeddel et al., incorporated herein by reference), those skilled in the art are interested in identifying novel members of this family to: (1) better understand the mechanisms underlying immune cell function; (2) regulate or control immune reactions or disease; and (3) screen for compounds effective in modulating the activity of TNFr' s.

Summary of the Invention

The present invention includes a novel TNFr homologue 1 ("TRHl") and the cDNA encoding said TRHl. The nucleotide sequence of the isolated cDNA is disclosed herein along with the deduced amino acid sequence. The cDNA gene has been deposited with the American Type Culture Collection and given the Accession Number ATCC .

The present inventors sequenced the cDNA encoding the novel TRHl and determined the primary sequence of the deduced protein. The novel TRHl has homology to known TNFr sequences.

The TRHl of the present invention can be produced by: (1) inserting the cDNA of the disclosed TRHl into an appropriate expression vector; (2) transfecting the expression vector into an appropriate transfection host(s); (3) growing the transfected host(s) in appropriate culture media; and (4) purifying the receptor protein from the culture media.

The present invention therefore provides a purified and isolated nucleic acid molecule, preferably a DNA molecule, having a sequence which codes for a TRHl, or an oligonucleotide fragment of the nucleic acid molecule which is unique to the TRHl of the invention. In a preferred embodiment of the invention, the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO: 1. In another preferred embodiment, the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:2. In still another preferred embodiment the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:3. In still another preferred embodiment of the present invention the purified and isolated nucleic acid molecule has the nucleotide sequence as shown in SEQ ID NO:4.

The invention also contemplates a double stranded nucleic acid molecule comprising a nucleic acid molecule of the invention or an oligonucleotide fragment thereof hydrogen bonded to a complementary nucleotide base sequence.

The terms "isolated and purified nucleic acid" and "substantially pure nucleic acid", e.g., substantially pure DNA, refer to a nucleic acid molecule which is one or both of the following: (1) not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (i.e., one at the 5' end and one at the 3 'end) in the naturally occurring genome of the organism from which the nucleic acid is derived; or (2) which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure or isolated and purified DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional TRHl /TNFr sequence.

The present invention provides in one embodiment: (a) an isolated and purified nucleic acid molecule comprising a sequence encoding all or a portion of a protein having the amino acid sequence as shown in SEQ ID NO:5; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which exhibit at least

80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions. In a particular embodiment, the fragment is a sequence encoding a TRHl having the amino acid sequence as shown in SEQ ID NO:7 (encompassing from amino acid number 42 to 655 of Figure 2) and sequences having at least 80%), more preferably at least 90%, more preferably at least 95%>, and most preferably at least 98%> sequence identity thereto.

The degree of homology (percent identity) between a native and a mutant sequence may be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose. One suitable program is the GAP computer program described by Devereux et al., (1984) Nucl. Acids Res. 12:387. The GAP program utilizes the alignment method of Needleman and Wunsch (1970) J. Mol. Biol. 48:433, as revised by Smith and Waterman (1981) Adv. Appl. Math. 2:482. Briefly, the GAP program defines percent identity as the number of aligned symbols (i.e., nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter of the two sequences.

As used herein the term "stringent conditions" encompasses conditions known in the art under which a nucleotide sequence will hybridize to an isolated and purified nucleic acid molecule comprising a sequence encoding a protein having the amino acid sequence as shown herein, or to (b) a nucleic acid sequence complementary to (a). Screening polynucleotides under stringent conditions may be carried out according to the method described in Nature, 313:402-404 (1985). Polynucleotide sequences capable of hybridizing under stringent conditions with the polynucleotides of the present invention may be, for example, allelic variants of the disclosed DNA sequences, or may be derived from other sources. General techniques of nucleic acid hybridization are disclosed by Sambrook et al., "Molecular Cloning: A Laboratory Manual", 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1984); and by Haymes et al., "Nucleic Acid Hybridization: A Practical Approach", IRL Press, Washington, D.C. (1985), which references are incorporated herein by reference.

The present invention provides in another embodiment: (a) an isolated and purified nucleic acid molecule comprising a sequence encoding all or a portion of a protein having the amino acid sequence as shown in SEQ ID NO:6; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are at least 80%>, more preferably at least 90%>, more preferably at least 95%>, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions.

The present invention provides in another embodiment: (a) an isolated and purified nucleic acid molecule comprising a sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO:8 (Gln42 through Pro351 of Figure 2); (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are at least 80%o, more preferably at least 90%o, more preferably at least 95%, and most preferably at least 98%> sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions. The present invention also provides: (a) a purified and isolated nucleic acid molecule comprising a sequence as shown in SEQ ID NO:l; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 80%, more preferably at least 90%>, more preferably at least 95%, and most preferably at least 98%o sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions. The present invention further provides: (a) a purified and isolated nucleic acid molecule comprising a sequence as shown in SEQ ID NO:2; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 80%>, more preferably at least 90%o, more preferably at least 95%>, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions.

The present invention further provides: (a) a purified and isolated nucleic acid molecule comprising a sequence as shown in SEQ ID NO:3; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 80%>, more preferably at least 90%>, more preferably at least 95%, and most preferably at least 98%o sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions.

The present invention further provides: (a) a purified and isolated nucleic acid molecule comprising a sequence as shown in SEQ ID NO:4; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 80%>, more preferably at least 90%>, more preferably at least 95%>, and most preferably at least 98%o sequence identity to (a); or (d) a fragment of (a) or (b) that is at least 18 bases and which will hybridize to (a) or (b) under stringent conditions.

The present invention additionally covers nucleic acid molecules of the present invention having one or more structural mutations including replacement, deletion or insertion mutations. For example, a signal peptide may be deleted, or conservative amino acid substitutions may be made to generate a protein that is still biologically competent or active.

The invention further contemplates a recombinant molecule comprising a nucleic acid molecule of the present invention or an oligonucleotide fragment thereof and an expression control sequence operatively linked to the nucleic acid molecule or oligonucleotide fragment. A transformant host cell including a recombinant molecule of the invention is also provided.

In another aspect, the invention features a cell or purified preparation of cells which include a novel gene encoding a TRHl of the present invention, or which otherwise misexpresses a gene encoding a TRHl of the present invention. The cell preparation can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a TRHl transgene, e.g., a heterologous form of a TRHl gene, e.g., a gene derived from humans (in the case of a non-human cell). The TRHl transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpresses an endogenous TRHl gene, e.g., a gene that expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or misexpressed TRHl alleles for use in drug screening. Still further, the invention provides plasmids which comprise the nucleic acid molecules of the invention.

The present invention also includes a novel TRHl of the present invention, or an active part thereof. A biologically competent or active form of the protein or part thereof is also referred to herein as an "active TRHl or part thereof. The invention further contemplates antibodies having specificity against an epitope of the TRHl of the present invention or part of the protein. These antibodies may be polyclonal or monoclonal. The antibodies may be labeled with a detectable substance and they may be used, for example, to detect the novel TRHl of the invention in tissue and cells. Additionally, the antibodies of the present invention, or portions thereof, may be used to make targeted antibodies that destroy TRHl expressing cells (e.g., antibody-toxin fusion proteins, or radiolabelled antibodies).

The invention also permits the construction of nucleotide probes which encode part or all of the novel TRHl protein of the invention or a part of the protein. Thus, the invention also relates to a probe comprising a nucleotide sequence coding for a protein, which displays the properties of the novel TRHl of the invention or a peptide unique to the protein. The probe may be labeled, for example, with a detectable (e.g., radioactive) substance and it may be used to select from a mixture of nucleotide sequences a nucleotide sequence coding for a protein which displays the properties of the novel TRHl of the invention. The present invention also provides a transgenic non-human animal (e.g., a rodent, e.g., a mouse or a rat, a rabbit or a pig) or embryo all of whose germ cells and somatic cells contain a recombinant molecule of the invention, preferably a recombinant molecule comprising a nucleic acid molecule of the present invention encoding the TRHl of the invention or part thereof. The recombinant molecule may comprise a nucleic acid sequence encoding the TRHl of the present invention with a structural mutation, or may comprise a nucleic acid sequence encoding the TRHl of the invention or part thereof and one or more regulatory elements which differ from the regulatory elements that drive expression of the native protein. In another preferred embodiment, the animal has a TRHl gene which is misexpressed or not expressed, e.g., a knockout. Such transgenic animals can serve as a model for studying disorders which are related to mutated or misexpressed TRHl of the present invention.

The invention still further provides a method for identifying a substance which is capable of binding the novel TRHl of the invention, comprising reacting the novel TRHl of the invention or part of the protein under conditions which permit the formation of a complex between the substance and the novel TRHl protein or part of the protein, and assaying for substance-TRHl complexes, for free substance, for non- complexed TRHl, or for activation of the TRHl.

An embodiment of the invention provides a method for identifying ligands which are capable of binding to the novel TRHl protein of the invention, isoforms thereof, or part of the protein, said method comprising reacting the novel TRHl protein of the invention, isoforms thereof, or part of the protein, with at least one ligand which potentially is capable of binding to the protein, isoform, or part of the protein, under conditions which permit the formation of ligand-receptor protein complexes, and assaying for ligand-receptor protein complexes, for free ligand, for non-complexed TRHl protein, or for activation of the TRHl . In a preferred embodiment of the method, ligands are identified which are capable of binding to and activating the novel TRHl protein of the invention, isoforms thereof, or part of the protein.

The invention also relates to a method for assaying a medium for the presence of an agonist or antagonist of the interaction of the novel TRHl protein and a substance which is capable of binding the TRHl, said method comprising providing a known concentration of the TRHl, reacting the TRHl with a substance which is capable of binding to the TRHl and a suspected agonist or antagonist under conditions which permit the formation of substance-TRHl complexes, and assaying for substance-TRHl complexes, for free substance, for non-complexed TRHl, or for activation of the TRH 1.

The invention further provides a method for identifying a substance which is capable of binding to an activated TRHl protein of the present invention or an isoform or a part of the protein, said method comprising reacting an activated TRHl of the present invention, or an isoform or part of the protein, with at least one substance which potentially can bind with the TRHl , isoform or part of the protein, under conditions which permit the formation of substance-activated TRHl complexes, and assaying for substance-TRHl complexes, for free substance, or for non- complexed TRHl . The method may be used to identify intracellular ligands containing proteins which bind to an activated TRHl protein of the present invention or parts thereof, or intracellular ligands which may be affected in other ways by the activated TRHl of the invention.

Also included within the scope of the present invention is a composition which includes the TRHl of the present invention, a fragment thereof (or a nucleic acid encoding said TRHl or fragment thereof) and one or more additional components, e.g., a earner, diluent or solvent. The additional component can be one which renders the composition useful for in vitro, in vivo, pharmaceutical or veterinary use.

In another aspect, the present invention relates to a method of treating a mammal, e.g., a human, at risk for a disorder, e.g., a disorder characterized by aberrant or unwanted level or biological activity of the TRHl of the present invention, or characterized by an aberrant or unwanted level of a ligand that specifically binds to the TRHl of the present invention. For example, the TRHl of the present invention may be useful to leach out or block a ligand which is found to bind to the TRHl of the present invention. Encompassed within the scope of the invention is a soluble form of the TRHl of the present invention, e.g., a fragment of the receptor, that may be used to inhibit activation of the receptor by binding to the ligand a polypeptide of the present invention and preventing the ligand from interacting with membrane bound TRHl.

Also within the scope of the present invention are fusion proteins comprising all or a portion of the TRHl of the present invention. Preferably, the fusion protein comprises all or a portion of the extracellular region of the TRHl of the present invention as shown in SEQ ID NO: 8. All or a portion of the extracellular portion of the TRHl of the present invention may be attached to another molecule or polypeptide, e.g., a hinge and/or constant region of an immunoglobulin ("Ig") protein.

The primary object of the present invention is the identification of the new human TRHl, as identified by its sequence disclosed herein. Additional objects of the invention are the methods of using the cDNA, the TRHl protein, the monoclonal antibody specific for the novel TRHl, fusion proteins comprising a portion of the TRHl protein of the present invention, and a ligand for the novel TRHl as described above.

Brief Description of the Figures

Figure 1 shows the DNA sequences of TRHl cDNA clones 2733717 (Figure 1A; SEQ ID NO:l) and 2098183 (Figure IB; SEQ ID NO:l 1). These represent two of the cDNA clones that were identified by searches of the Incyte LifeSeq database. Figure 2A-2D gives the deduced amino acid sequence of TRHl (SEQ ID NO:5).

Figure 3 shows the results of Northern blot analysis of the tissue distribution of TRHl mRNA. Filters containing polyA enriched RNA from multiple tissues (A, B, C, D) were hybridized with radiolabeled TRHl cDNA probe. The sources of

RNAs are listed across the tops of each panel and the sizes of RNA size standards in kb are shown by arrows on the left side of each panel. A mRNA of approximately 4.2 kb (3.5 kb in testis) hybridized with the TRHl probe in many tissues.

Figure 4 demonstrates the production of TRHl Ig fusion proteins. COS cells were transiently transfected with plasmids encoding the extracellular region of TRHl fused to the hinge, CH2, and CH3 domains of mouse IgG2a (mlg) or human IgGl (hlg). Fusion proteins from the supernatants of transfected cells that were pulsed with ³⁵S-methionine were purified by protein A affinity chromatography and subjected to SDS-PAGE analysis and autoradiography. The apparent masses of molecular weight standards in kDa are shown to the left of the panel. Figure 5 shows the alignment of the peptide sequences of TRHl with osteoprotegerin ("OPG") and TNF receptor type II ("TNFR2"). The extracellular region of TRHl is aligned with the cysteine rich motifs of OPG and TNFR2 that are characteristic of TNFR family members. Aligned residues are: TRHl 50-213 (SEQ ID NO:12); OPG 24-187 (SEQ ID NO:13) from GenPept accession number 2072185; and TNFR2 38-202 (SEQ ID NO: 14) from GenPept accession number 1469541. Residues that are strictly conserved between the three proteins are in bold lettering and all cysteine residues are underlined. TRHl shares 34% identity with OPG and 39%o identity with TNFR2 in the aligned extracellular regions.

Detailed Description of the Invention

In order to identify novel members of the tumor necrosis factor receptor ("TNFR") superfamily, BLAST (basic local alignment sequence tool) searches of EST (expressed sequence tag) databases were performed with peptide sequences of known TNFR family members. Two cDNA clones in the Incyte LifeSeq database were identified. One clone was derived from a kidney tumor cDNA library while the other was isolated from a teratocarcinoma cell line that had neural differentiation characteristics. They were obtained from Incyte and sequenced.

Since they did not contain the complete coding region, they were used in further BLAST searches to identify two additional clones, one from a brain tumor cDNA library and the other from an ovarian tumor library. These two cDNA clones of TNFR homologue 1 ("TRHl") were sequenced in entirety on both strands by the fluorescent dideoxy chain termination method. INCYTE cDNA clones 2733717 and 2098183 (Figure 1) both contain the complete peptide encoding sequences for TRHl (Figure 2). Although both clones encode identical proteins, their 5' and 3' untranslated regions are not identical. There are at least two possible translation start sites in these clones (Met-1 and Met-25) (Figure 2). The predicted mature peptide sequence begins at Gln42. Overall, the cDNA's encode for a 655 residue type 1 membrane protein. After cleavage of the predicted signal peptide, the 614 amino acid mature protein contains a 310 residue extracellular region (SEQ ID NO: 8), a transmembrane spanning segment, and a 285 amino acid cytoplasmic region. The predicted molecular mass of the mature protein before posttranslational modification is approximately 68 kDa. Comparison of the extracellular region of TRHl to other members of the TNFR superfamily reveals that it is most closely related to osteoprotegerin (Simonet, supra) and TNFr2 (see Figure 5).

A preferred nucleic acid sequence encoding a TRHl of the present invention comprises nucleotides 52 through 2016 (SEQ ID NO:2) of the following SEQ ID

NO:l:

ATTCGGCTGC CCCGCGCGCC CCGGGCGCCC CTGCGAGTCC CCGGTTCAGC C

51

ATG GGG ACC TCT CCG AGC AGC AGC ACC GCC CTC GCC TCC TGC 93

AGC CGC ATC GCC CGC CGA GCC ACA GCC ACG ATG ATC GCG GGC

135

TCC CTT CTC CTG CTT GGA TTC CTT AGC ACC ACC ACA GCT CAG

177 CCA GAA CAG AAG GCC TCG AAT CTC ATT GGC ACA TAC CGC CAT

219

GTT GAC CGT GCC ACC GGC CAG GTG CTA ACC TGT GAC AAG TGT

261

CCA GCA GGA ACC TAT GTC TCT GAG CAT TGT ACC AAC ACA AGC 303

CTG CGC GTC TGC AGC AGT TGC CCT GTG GGG ACC TTT ACC AGG

345

CAT GAG AAT GGC ATA GAG AAA TGC CAT GAC TGT AGT CAG CCA

387 TGC CCA TGG CCA ATG ATT GAG AAA TTA CCT TGT GCT GCC TTG

429

ACT GAC CGA GAA TGC ACT TGC CCA CCT GGC ATG TTC CAG TCT

471

AAC GCT ACC TGT GCC CCC CAT ACG GTG TGT CCT GTG GGT TGG 513 GGT GTG CGG AAG AAA GGG ACA GAG ACT GAG GAT GTG CGG TGT

555

AAG CAG TGT GCT CGG GGT ACC TTC TCA GAT GTG CCT TCT AGT

597 GTG ATG AAA TGC AAA GCA TAC ACA GAC TGT CTG AGT CAG AAC

639

CTG GTG GTG ATC AAG CCG GGG ACC AAG GAG ACA GAC AAC GTC

681

TGT GGC ACA CTC CCG TCC TTC TCC AGC TCC ACC TCA CCT TCC 723

CCT GGC ACA GCC ATC TTT CCA CGC CCT GAG CAC ATG GAA ACC

765

CAT GAA GTC CCT TCC TCC ACT TAT GTT CCC AAA GGC ATG AAC

807 TCA ACA GAA TCC AAC TCT TCT GCC TCT GTT AGA CCA AAG GTA

849

CTG AGT AGC ATC CAG GAA GGG ACA GTC CCT GAC AAC ACA AGC

891

TCA GCA AGG GGG AAG GAA GAC GTG AAC AAG ACC CTC CCA AAC 933

CTT CAG GTA GTC AAC CAC CAG CAA GGC CCC CAC CAC AGA CAC

975

ATC CTG AAG CTG CTG CCG TCC ATG GAG GCC ACT GGG GGC GAG

1017 AAG TCC AGC ACG CCC ATC AAG GGC CCC AAG AGG GGA CAT CCT

1059

AGA CAG AAC CTA CAC AAG CAT TTT GAC ATC AAT GAG CAT TTG

1101

CCC TGG ATG ATT GTG CTT TTC CTG CTG CTG GTG CTT GTG GTG 1143

ATT GTG GTG TGC AGT ATC CGG AAA AGC TCG AGG ACT CTG AAA

1185

AAG GGG CCC CGG CAG GAT CCC AGT GCC ATT GTG GAA AAG GCA

1227 GGG CTG AAG AAA TCC ATG ACT CCA ACC CAG AAC CGG GAG AAA TGG ATC TAC TAC TGC AAT GGC CAT GGT ATC GAT ATC CTG AAG

1311

CTT GTA GCA GCC CAA GTG GGA AGC CAG TGG AAA GAT ATC TAT

1353 CAG TTT CTT TGC AAT GCC AGT GAG AGG GAG GTT GCT GCT TTC

1395

TCC AAT GGG TAC ACA GCC GAC CAC GAG CGG GCC TAC GCA GCT

1437

CTG CAG CAC TGG ACC ATC CGG GGC CCC GAG GCC AGC CTC GCC 1479

CAG CTA ATT AGC GCC CTG CGC CAG CAC CGG AGA AAC GAT GTT

1521

GTG GAG AAG ATT CGT GGG CTG ATG GAA GAC ACC ACC CAG CTG

1563 GAA ACT GAC AAA CTA GCT CTC CCG ATG AGC CCC AGC CCG CTT

1605

AGC CCG AGC CCC ATC CCC AGC CCC AAC GCG AAA CTT GAG AAT

1647

TCC GCT CTC CTG ACG GTG GAG CCT TCC CCA CAG GAC AAG AAC 1689

AAG GGC TTC TTC GTG GAT GAG TCG GAG CCC CTT CTC CGC TGT

1731

GAC TCT ACA TCC AGC GGC TCC TCC GCG CTG AGC AGG AAC GGT

1773 TCC TTT ATT ACC AAA GAA AAG AAG GAC ACA GTG TTG CGG CAG

1815

GTA CGC CTG GAC CCC TGT GAC TTG CAG CCT ATC TTT GAT GAC

1857

ATG CTC CAC TTT CTA AAT CCT GAG GAG CTG CGG GTG ATT GAA 1899

GAG ATT CCC CAG GCT GAG GAC AAA CTA GAC CGG CTA TTC GAA

1941

ATT ATT GGA GTC AAG AGC CAG GAA GCC AGC CAG ACC CTC CTG

1983 GAC TCT GTT TAT AGC CAT CTT CCT GAC CTG CTG TAGAACATAG

2026 GGATACTGCA TTCTGGAAAT TACTCAATTT AGTGGCAGGG TGGTTTTTTA 2076 ATTTTCTTCT GTTTCTGATT TTTGTTGTTT GGGGTGTGTG TGTGTGTTTG 2126 TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TTTAACAGAG AATATGGCCA 2176 GTGCTTGAGT TCTTTCTCCT TCTCTCTCTC TCTTTTTTTT TTAAATAACT 2226 CTTCTGGGAA GTTGGTTTAT AAGCCTTTGC CAGGTGTAAC TGTTGTGAAA 2276 TACCCACCAC TAAAGTTTTT TAAGTTCCAT ATTTTCTCCA TTTTGCCTTC 2326 TTATGTATTT TCAAGATTAT TCTGTGCACT TTAAATTTAC TTAACTTACC 2376 ATAAATGCAG TGTGACTTTT CCCACACACT GGATTGTGAG GCTCTTAACT 2426 TCTTAAAAGT ATAATGGCAT CTTGTGAATC CTATAAGCAG TCTTTATGTC 2476 TCTTAACATT CACACCTACT TTTTAAAAAC AAATATTATT ACTATTTTTA 2526 TTATTGTTTG TCCTTTATAA ATTTTCTTAA AGATTAAGAA AATTTAAGAC 2576 CCCATTGAGT TACTGTAATG CAATTCAACT TTGAGTTATC TTTTAAATAT 2626 GTCTTGTATA GTTCATATTC ATGGCTGAAA CTTGACCACA CTATTGCTGA 2676 TTGTATGGTT TTCACCTGGA CACCGTGTAG AATGCTTGAT TACTTGTACT 2726

CTTCTTATGC TAATATGCTC TGGGCTGGAG AAATGAAATC CTCAAGCCAT

2776 CAGGATTTGC TATTTAAGTG GCTTGACAAC TGGGCCACCA AAGAACTTGA 2826 ACTTCACCTT TTAGGATTTG AGCTGTTCTG GAACACATTG CTGCACTTTG 2876 GAAAGTCAAA ATCAAGTGCC AGTGGCGCCC TTTCCATAGA GAATTTGCCC 2926 AGCTTTGCTT TAAAAGATGT CTTGTTTTTT ATATACACAT AATCAATAGG 2976 TCCAATCTGC TCTCAAGGCC TTGGTCCTGG TGGGATTCCT TCACCAATTA

3026 CTTTAATTAA AAATGGCTGC AACTGTAAGA ACCCTTGTCT GATATATTTG 3076 CAACTATGCT CCCATTTACA AATGTACCTT CTAATGCTCA GTTGCCAGGT 3126 TCCAATGCAA AGGTGGCGTG GACTCCCTTT GTGTGGGTGG GGTTTGTGGG 3176 TAGTGGTGAA GGACCGATAT CAGAAAAATG CCTTCAAGTG TACTAATTTA 3226 TTAATAAACA TTAGGTGTTT GTTAAAAAAA AAATGGTTAA TAAAAAAAAG 3276 G

3277

The deduced amino acid sequence of a TRHl of the present invention comprises the following SEQ ID NO:5: MGTSPSSSTA LASCSRIARR ATATMIAGSL LL GFLSTTT AQPEQKASN

50 IGTYRHVDRA TGQVLTCDKC PAGTYVSEHC TNTSLRVCSS CPVGTFTRHE 100 NGIEKCHDCS QPCP PMIEK LPCAALTDRE CTCPPGMFQS NATCAPHTVC 150 PVG GVRKKG TETEDVRCKQ ^"CARGTFSDVP SSVMKCKAYT DCLSQNLWI 200 KPGTKETDNV CGTLPSFSSS TSPSPGTAIF PRPEHMETHE VPSSTYVPKG 250 MNSTESNSSA SVRPKVLSSI QEGTVPDNTS SARGKEDVNK TLPNLQWNH 300 QQGPHHRHIL KLLPSMEATG GEKSSTPIKG PKRGHPRQNL HKHFDINEHL 350 PWMTVT.FT.T.T. VT.WTWCST RKRSPTT.KKG PRQDPSAIVE KAGLKKSMTP 400 TQNREK IYY CNGHGIDILK LVAAQVGSQW KDIYQFLCNA SEREVAAFSN 450 GYTADHERAY AALQH TIRG PEASLAQLIS ALRQHRRNDV VEKIRGLMED 500 TTQLETDKLA LPMSPSPLSP SPIPSPNAKL ENSALLTVEP SPQDK KGFF 550

VDESEPLLRC DSTSSGSSAL SRNGSFITKE KKDTVLRQVR LDPCDLQPIF 600 DDMLHFLNPE ELRVIEEIPQ AEDKLDRLFE IIGVKSQEAS QTLLDSVYSH 650 LPDLL* 655

The predicted signal peptide of the above TRHl protein spans residues 1-41. The two potential translational start sites are Metl and Met25 (bold). After cleavage of the predicted signal peptide, the mature protein (SEQ ID NO: 7) begins at Gln42 (bold and underlined). The predicted membrane spanning region is underlined. The present invention relates to the nucleic acid sequence or a fragment thereof (referred to herein as a "polynucleotide") of the novel TRHl as shown above (SEQ ID NO:2), as well as to the amino acid sequence of the TNFr (SEQ ID NO:5), and biologically active portions thereof. SEQ ID NO:3 shows the nucleic acid sequence of a portion (Met25 through Leu655) of the TRHl receptor of the present invention; SEQ ID NO: 6 shows the amino acid sequence of a portion of the TRHl receptor sequence. SEQ ID NO: 8 shows the amino acid sequence of the extracellular portion of the TRHl protein of the present invention.

The present invention further relates to variants of the hereinabove described nucleic acid sequence which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of SEQ ID NO:5 or the polypeptide encoded by the cDNA of the deposited clones. The variants of the nucleic acid sequence may be naturally occurring variants of the nucleic acid sequence or non-naturally occurring variants of the nucleic acid sequence. Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in SEQ ID NO:5, or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of SEQ ID NO:5 or the polypeptide encoded by the cDNA of the deposited clones. Specifically included within the scope of the present invention is a polynucleotide as shown in SEQ ID NO:3, encoding a 631 amino acid protein as shown in SEQ ID NO:6 (Met25 through Leu655); and a polynucleotide as shown in SEQ ID NO:4, encoding a 614 amino acid protein as shown in SEQ ID NO:7 (Gln42 through Leu655). Such nucleotide variants include deletion variants, substitution variants and addition or insertion (splice) variants.

The polynucleotides may also encode for a soluble form of the TRHl receptor polypeptide of the present invention which is the extracellular portion of the polypeptide which has been cleaved from the transmembrane and intracellular domain of the full length polypeptide of the present invention. A preferred polynucleotide encodes the polypeptide of SEQ ID NO:8, which represents the extracellular portion of the TRHl of the present invention.

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be used as hybridization probes for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to a gene of the present invention or similar biological activity. Probes of this type preferably have at least between 20 and 30 bases, and may contain, for example, 50 or more bases. The probes may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene of the present invention including regulatory and promoter regions, exons, and introns. The present invention further relates to polynucleotides that hybridize to the polynucleotide sequences disclosed herein, if there is at least 80%, preferably at least 90%), and more preferably at least 95%> identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides described herein.

Alternatively the polynucleotide may have at least 20 bases, preferably at least 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, for example for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.

Thus the present invention is directed to polynucleotides having at least 80%> identity, preferably at least 90%> and more preferably at least 95 % identity to a polynucleotide of the present invention, including polynucleotides encoding the polypeptide of SEQ ID NO:5, as well as fragments thereof, which fragments have at least 20 or 30 bases, and preferably at least 50 bases, and to polypeptides encoded by such polynucleotides.

The present invention further relates to a tumor necrosis factor receptor homologue polypeptide, TRHl, which has the deduced amino acid sequence as shown in SEQ ID NO:5 (Figure 2), or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide. Encompassed within the scope of the present invention are polypeptides as shown in SEQ ID NO:6, SEQ ID NO:7 (the mature protein) and SEQ ID NO:8 (the extracellular portion of the TRHl protein).

The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-Organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence(s) of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with

Other analogs within the invention are those with modifications which increase protein or peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the protein or peptide sequence. Also included are analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.

In terms of general utility of the novel TRHl protein of the present invention, the expression pattern of the protein and homology to members of the TΝFr superfamily indicate that this novel TΝFr homologue plays a role in cellular function, including but not limited to cell activation, proliferation, differentiation, and apoptosis. Similar to other receptor systems being investigated (e.g., CD40/CD40L, 4-1BB/4-1BBL, Fas/FasL) it is contemplated by the present invention that the interaction between the novel TΝFr protein of the present invention and intracellular signaling molecules and/or its potential co-receptor may serve as a novel target for immunosuppressive, antiinflammatory and/or immunostimulatory drug development.

Gene constructs of the present invention can also be used as part of a gene therapy protocol to deliver nucleic acids encoding the TRHl of the present invention, or an agonist or antagonist form of a TRHl protein or peptide. The invention features expression vectors for in vivo transfection and expression of a TRHl . Expression constructs of the TRHl of the present invention, may be administered in any biologically effective carrier, e.g., any formulation or composition capable of effectively delivering the TRHl gene to cells in vivo. Approaches include insertion of any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

Analogs of the novel TRHl of the present invention are also within the scope of the present invention. Analogs can differ from the naturally occurring TRHl of the present invention in amino acid sequence or in ways that do not involve sequence, or both. Non-sequence modifications include in vivo or in vitro chemical derivitization of the TRHl of the present invention. Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation. Preferred analogs include the novel TRHl of the present invention (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions or insertions which do not abolish the biological activity of the TRHl of the present invention. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative amino acid substitutions can be taken from the table below.

Table 1 Conservative amino acid replacements

the subject gene in viral vectors including recombinant retro viruses, adenoviruses, adeno-associated viruses, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; an advantage of infection of cells with a viral vector is that a large proportion of the targeted cells can receive the nucleic acid. Several viral delivery systems are known in the art and can be utilized by one practicing the present invention.

In addition to viral transfer methods, non-viral methods may also be employed to cause expression of the TRHl in the tissue of an animal. Most non-viral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes. DNA of the present invention may also be introduced to cell(s) by direct injection of the gene construct or electroporation.

In clinical settings, the gene delivery systems for the therapeutic TRHl gene can be introduced into a patient by any of a number of methods, each of which is known in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.

The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.

Another aspect of the invention relates to the use of an isolated nucleic acid in "antisense" therapy. As used herein, "antisense" therapy refers to administration or in situ generation of ohgonucleotides or their derivatives which specifically hybridize under cellular conditions, with the cellular mRNA and/or genomic DNA encoding the TRHl of the present invention so as to inhibit expression of the encoded protein, e.g., by inhibiting transcription and/or translation. In general, "antisense" therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences. Fragments of the TNFr of the present invention are also within the scope of

Applicant's invention. Fragments of the protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Digestion with "end- nibbling" endonucleases can thus generate DNA's which encode an array of fragments. DNA's which encode fragments of the TRHl protein can also be generated by random shearing, restriction digestion or a combination of the above- discussed methods. Fragments can also be chemcially synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.

Amino acid sequence variants of the TRHl protein of the present invention can be prepared by random mutagenesis of DNA which encodes a protein or a particular domain or region of the protein. Useful methods are known in the art, e.g., PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate ohgonucleotides sequences, a process known and practiced by those skilled in the art.

Non-random or directed mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions of residues of the known amino acid sequence of the TRHl protein of the present invention. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids then with more radical choices depending upon results achieved; (2) deleting the target residue; or (3) inserting residues of the same or a different class (e.g., hydrophobic or hydrophihc) adjacent to the located site, or a combination of options (l)-(3). Alanine scanning mutagenesis is a useful method for identification of certain functional residues or regions of a desired protein that are preferred locations or domains for mutagenesis. Oligonucleotide-mediated mutagenesis, cassette mutagenesis, and combinatorial mutagenesis are useful methods known to those skilled in the art for preparing substitution, deletion, and insertion variants of DNA.

The present invention also relates to methods of screening. Various techniques are known in the art for screening generated mutant gene products. Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, e.g., in this case binding of a ligand to the TRHl of the present invention. Techniques known in the art are amenable to high through-put analysis for screening large numbers of sequences created, e.g., by random mutagenesis techniques. Two hybrid assays can be used to identify fragments or analogs of a protein or peptide which bind to the TRHl of the present invention. These may include agonists or antagonists. In one approach to screening assays, the candidate protein or peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay". In a similar fashion, a detectably labeled ligand can be used to score for potentially functional peptide homologues. Fluorescently labeled ligands, e.g., receptors, can be used to detect homologue which retain ligand-binding activity. The use of fluorescently labeled ligand allows cells to be visually inspected and separated under fluorescence microscope or to be separated by a fluorescence- activated cell sorter.

High through-put assays can be followed by secondary screens in order to identify further biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists. The type of a secondary screen used will depend on the desired activity that needs to be tested. For example, an assay can be developed in which the ability to inhibit an interaction between the TRHl of the present invention and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated through one of the primary screens. Therefore, methods for generating fragments and analogs and testing them for activity are known in the art. Once a sequence of interest is identified, it is routine for one skilled in the art to obtain agonistic or antagonistic analogs, fragments, and/or ligands. Drug screening assays are also provided in the present invention. By producing purified and recombinant TRHl of the present invention, or fragments thereof, one skilled in the art can use these to screen for drugs which are either agonists or antagonists of the normal cellular function or their role in cellular signaling. In one embodiment, the assay evaluates the ability of a compound to modulate binding between the TRHl of the present invention and a naturally occurring ligand. The term "modulating" encompasses enhancement, diminishment, activation or inactivation of the TRHl receptor. Assays useful to identify ligands to the TRHl receptor of the present invention, including peptides, proteins, small molecules, and antibodies that are capable of binding to the TRHl receptor are encompassed herein. A variety of assay formats will suffice and are known by those skilled in the art.

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as primary screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound.

Also within the scope of the present invention is a process for modulating the TRHl of the present invention. The term "modulating" encompasses enhancement, diminishment, activation or inactivation of the TRHl receptor. Ligands to the TRHl receptor of the present invention, including peptides, proteins, small molecules, and antibodies, that are capable of binding to the TRHl receptor and modulating its activity are encompasses herein. These compounds are useful in modulating the activity of the TRHl receptor and in treating TRHl -associated disorders. "TRH1- associated disorders" refers to any disorder or disease state in which the TRHl protein plays a regulatory role in the metabolic pathway of that disorder or disease. Such disorders or diseases may include rheumatoid arthritis and transplant rejection. As used herein the term "treating" refers to the alleviation of symptoms of a particular disorder in a patient, the improvement of an ascertainable measurement associated with a particular disorder, or the prevention of a particular immune, inflammatory or cellular response (such as transplant rejection).

The invention also includes antibodies specifically reactive with the TRHl of the present invention, or a portion thereof. Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard known procedures. A mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques known in the art. An immunogenic portion of the TRHl of the present invention can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum.

The term "antibody" as used herein is intended to include fragments thereof which are also specifically reactive with the TRHl of the present invention. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. The antibody of the present invention is further intended to include chimeric and humanized molecules that recognize and bind to the TRHl of the present invention. Both monoclonal and polyclonal antibodies directed against the TRHl of the present invention, and antibody fragments such as Fab', sFv and F(ab')2, can be used to block the action of the TRHl of the present invention and allow study of the role of a particular TRHl of the present invention. Alternatively, such antibodies can be used therapeutically to block the TRHl of the present invention in a subject mammal, e.g., a human. In a preferred embodiment a therapeutic compositions comprising an antibody of the present invention can also comprise a pharmaceutically acceptable carrier, solvent or diluent, and be administered by systems known in the art. Antibodies of the present invention may also be useful as potential agonists of the TRHl of the present invention. Such agonistic antibodies tend to aggregate and crosslink the receptor, which induces signaling, proliferation, differentiation and/or cell death (apoptosis). Antibodies which specifically bind to the TRHl of the present invention, or fragments thereof, can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern expression of the TRHl of the present invention. Antibodies can be used diagnostically in immunoprecipitation, immunoblotting, and enzyme linked immunosorbent assay (ELISA) to detect and evaluate levels of the TRHl of the present invention in tissue or bodily fluid.

EXAMPLES

The following examples are included for understanding the present invention and are not intended to limit the scope of Applicants invention.

Example 1 : Northern blot analysis In order to determine the tissue distribution of TRHl mRNA, Northern blot analyses of filters containing polyA enriched RNA from multiple tissues and cells lines was performed. Multiple tissue Northern blots were obtained commercially (Clontech, Palo Alto, C A). A fragment of clone 2098183 was labeled with P³²-dCTP using the random prime labeling kit (Boerhinger) and then hybridized to filters that had been prehybridized for 3 hours at 65° with prehybridization/hybridization solution (0.5 M NaHPO₄ pH 7.2, 1 mM EDTA, 1% bovine serum albumin, 7% sodium dodecyl sulfate). Hybridization was performed overnight at 65°C. The filters were subsequently washed with low stringency wash buffer (0.5%> bovine serum albumin, 1 mM EDTA, 40 mM NaHPO₄ pH 7.2, 5% sodium dodecyl sulfate) for 30 minutes at room temperature and 30 minutes at 42°C. The filters were then washed with high stringency wash buffer (40 mM NaHPO₄ pH 7.2, 1 mM EDTA, 1% sodium dodecyl sulfate) for 30 minutes at 42°C and 30 minutes 55°C. The filters were then exposed to X-ray film at -70°C with intensifying screens and then developed. From Northern analysis, an approximately 4.2 kb mRNA is expressed in multiple tissues and cell lines. TRHl is most highly expressed in thymus, prostate, small intestine (Fig 3A), lymph node (Fig 3B), HeLa (cervical adenocarcinoma), Molt4 (T cell lymphoma), SW480 (colorectal carcinoma), A549 (lung carcinoma), and G361 (melanoma) (Fig 3C), and heart, brain, placenta, kidney, and pancreas (Fig 3D). Messenger RNAs of approximately 4.2 and 3.5 kb also hybridized in the testis (Fig 3A).

Example 2: Production of TRHl immunoglobulin (Ig) fusion proteins Complementary DNA fragments encoding the extracellular region of TRHl

(Metl to Pro351) (although the mature protein would be expected to not have the signal peptide encompassing amino acids 1-41, it was encoded in the fragment used to construct the fusion protein) were produced by polymerase chain reaction (PCR) methodology using cDNA clone 2098183 as a template with ohgonucleotides containing the appropriate endonuclease restriction sites to mediate fusion with human IgGl or mouse IgG2a expression cassettes as already described (Aruffo et al., (1990) Cell 61 :1303-1313; Bowen et al., (1996) JBC 271(29):17390-17396). The sense oligonucletide was (GCC AAG CTT CGA GTC CCC GGT TCA GCC ATG (SEQ ID NO:9)) and encoded a Hindlll restriction site and bases 34 to 54 of clone 2733717. The antisense oligonucletide (GCG TGG ATC CGG ATG TCC CCT CTT GGG GCC (SEQ ID NO: 10)) contained a BamHI restriction site and encoded the reverse complement of bases 1039 to 1058 of clone 2733717. These ohgonucleotides were used to amplify cDNA sequences that encode amino acids Metl through Pro336 of TRHl (Fig 2). The PCR was performed with high fidelity Taq polymerase (Boerhinger) with the following cycling conditions: 1 cycle: 94°C 3 min; 40 cycles: 94° 1 min., 60° 1 min., 72° 2.5 min.; 1 cycle: 72° 5 min.

Proteins were produced by transient transfection of COS cells that had been pulsed 16h with ³⁵S-Methionine (Amersham Corp.) 24h after transfection. The fusion proteins were purified with Protein A Sepharose and subsequently analyzed by SDS- PAGE and autoradiography (Fig 4). Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

We claim:

I . A purified and isolated nucleic acid sequence encoding all or a portion of a tumor necrosis factor receptor homologue, said tumor necrosis factor receptor homologue comprising the amino acid sequence as shown in SEQ ID NO:5. 2. The nucleic acid sequence of claim 1 comprising the nucleic acid sequence as shown in SEQ ID NO:2.

3. A purified and isolated nucleic acid sequence encoding all or a portion of a tumor necrosis factor receptor homologue, said tumor necrosis factor receptor homologue comprising the amino acid sequence as shown in SEQ ID NO:6. 4. The nucleic acid sequence of claim 3 comprising the nucleic acid sequence as shown in SEQ ID NO:3.

5. A purified and isolated nucleic acid sequence encoding all or a portion of a tumor necrosis factor receptor homologue, said tumor necrosis factor receptor homologue comprising the amino acid sequence as shown in SEQ ID NO:7. 6. The nucleic acid sequence of claim 5 comprising the nucleic acid sequence as shown in SEQ ID NO:4.

7. An expression vector comprising a nucleic acid molecule as claimed in claim 1, 2, 3, 4, 5, or 6 and an expression control sequence operatively linked to the nucleic acid molecule. 8. A transformant host cell including an expression vector comprising a nucleic acid molecule as claimed in claim 1, 2, 3, 4, 5 or 6 and an expression control sequence operatively linked to the nucleic acid molecule.

9. A purified and isolated nucleic acid sequence comprising the complement of the nucleic acid sequence of claim 1, 2, 3, 4, 5 or 6. 10. A tumor necrosis factor receptor homologue comprising the amino acid sequence as shown in SEQ ID NO:5.

I I. A tumor necrosis factor receptor homologue comprising the amino acid sequence as shown in SEQ ID NO:6.

12. A tumor necrosis factor receptor homologue comprising the amino acid sequence as shown in SEQ ID NO:7.

13. A method of producing a tumor necrosis factor receptor homologue, said method comprising the steps of: a) inserting a nucleic acid sequence according to claim 1, 2, 3, 4, 5 or 6 encoding said tumor necrosis factor receptor homologue into an appropriate expression vector, b) transfecting said expression vector into an appropriate transfection host cell, c) growing said transfected host cells in an appropriate culture media, and d) purifying the tumor necrosis factor receptor from said culture media.

14. An isolated nucleic acid sequence which hybridizes under stringent conditions to the nucleic acid sequence of claim 2, 4 or 6.

15. An antibody specific for the tumor necrosis factor receptor homologue as claimed in claim 10.

16. The antibody of claim 15 wherein said antibody is a monoclonal antibody.

17. An antibody specific for the tumor necrosis factor receptor homologue as claimed in claim 11. 18. The antibody of claim 17 wherein said antibody is a monoclonal antibody.

19. An antibody specific for the tumor necrosis factor receptor homologue as claimed in claim 12.

20. The antibody of claim 19 wherein said antibody is a monoclonal antibody.

21. The tumor necrosis factor receptor homologue of claim 10, produced by: a) inserting a nucleic acid sequence encoding said tumor necrosis factor receptor homologue into an appropriate expression vector, b) transfecting said expression vector into an appropriate transfection host cell, c) growing said transfected host cells in an appropriate culture media, and d) purifying the tumor necrosis factor receptor from said culture media. 22. The tumor necrosis factor receptor homologue of claim 11 , produced by: a) inserting a nucleic acid sequence encoding said tumor necrosis factor receptor homologue into an appropriate expression vector, b) transfecting said expression vector into an appropriate transfection host cell, c) growing said transfected host cells in an appropriate culture media, and d) purifying the tumor necrosis factor receptor from said culture media. 23. A method for identifying a ligand which is capable of binding to the tumor necrosis factor receptor homologue of claim 10, or to a part of said tumor necrosis factor receptor homologue, said method comprising the steps of:

(a) reacting said tumor necrosis factor receptor homologue, or part of said tumor necrosis factor receptor homologue, with said ligand which potentially is capable of binding to the tumor necrosis factor receptor homologue or part of said tumor necrosis factor receptor homologue, under conditions which permit the formation of ligand-tumor necrosis factor receptor homologue complexes; and

(b) assaying for ligand-tumor necrosis factor receptor homologue complexes, for free ligand, for non-complexed tumor necrosis factor receptor homologue, or for activation of the tumor necrosis factor receptor homologue.

24. A method for identifying a ligand which is capable of binding to the tumor necrosis factor receptor homologue of claim 11, or to a part of said tumor necrosis factor homologue, said method comprising the steps of:

25. A method for identifying a ligand which is capable of binding to the tumor necrosis factor receptor homologue of claim 12, or to a part of said tumor necrosis factor receptor homologue, said method comprising the steps of:

26. A fusion protein comprising all or a portion of the tumor necrosis factor receptor homologue as shown in SEQ ID NO:5, attached to a second polypeptide. 27. The fusion protein of claim 26 comprising the extracellular portion of the tumor necrosis factor receptor homologue as shown in SEQ ID NO:5 attached to all or a portion of the hinge and/or constant region of a human IgG molecule.

28. A fusion protein comprising all or a portion of the polypeptide as shown in SEQ ID NO:8.