WO1996011259A1 - TGF-β1, ACTIVIN RECEPTORS 1 AND 3 - Google Patents

Abstract

Polynucleotides encoding TAR-1 and TAR-3 receptor polypeptides, as well as such polypeptides, antibodies and antagonists against such polypeptides are disclosed. Also disclosed is a procedure forproducing such polypeptides by recombinant techniques. Use of agonists and antagonists for therapeutic purposes, for example, to inhibit growth of cells and to treat lung and liver fibrosis is also disclosed.

Description

TGF-01, ACTIVIN RECEPTORS 1 AND 3

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is TGF-01, Activin Receptors 1 and 3, sometimes hereinafter referred to as "TAR- 1 and TAR-3". The invention also relates to inhibiting the action of such polypeptides.

The cloning of TGF-31 and the resultant elucidation of its precursor structure (Derynck, et al., Nature. 316:701-705 (1985)) have led to the identification of at least four other forms of TGF-S and the definition of a larger gene family comprising many other structurally related, but functionally distinct, regulatory proteins.

There are now many polypeptides that belong to the TGF-3 supergene family by virtue of amino acid homologies, particularly with respect to the conservation of seven of the nine cysteine residues of TGF-S among all known family members. These include the mammalian inhibins (Mason, et al., Nature. 318:659-663 (1985)) and activins fLing, et al., Nature. 779-782 (1986)), and ullerian inhibitory substance (Cate, et al., Cell. 45:685-698 (1986)), as well as the predicted products of both a pattern gene in Drosophila (Padgett, et al., Nature. 325:81-84 (1987)), and an amphibian gene expressed in frog oocytes (Weeks and Melton, Cell. 51:861-867 (1987)). Most recently, three new proteins, called bone morphogenetic proteins (BMPε), have been added to the family.

A unifying feature of the biology of these polypeptides is their ability to regulate developmental processes. The inhibins and activins, as discussed further below, regulate reproductive functions and erythropoietic activity. The BMPs are thought to play a role in the formation of cartilage and bone in vivo . TGF-3 itself (Kimelman and Kirschner, Cell. 51:869-877 (1987)) has been shown to augment the ability of fibroblast growth factor to induce esoderm and plays a pivotal role in morphogenesis and organogenesiε in mammalian embryos. In addition, like activin, TGF-/3 is reported to possess follicle-stimulating-hormone (FSH)-releasing activity. (Ying, et al., Biochem. Biophvs. Res. Commun.. 135:950-956 (1986).)

Furthermore, TGF-_/3 has been shown to inhibit the growth of several human cancer cell lines (Roberts et al., PNAS, 82:119-123 (1985)). TGF-/3 has also been described as being able to inhibit production of the HIV virus and decrease syncytia formation, U.S. Pat. No. 5,236,905.

Activins have an extensive anatomical distribution and are implicated in the regulation of many biological processes, including the proliferation of many cell lines (Gonzalez-Manchon, C. and Vale, W., Endocrinology. 125:1666- 1672 (1989)), control of the secretion and expression of the anterior pituitary hormones FSH, GH, and ACTH (Vale, w. , et al., Nature. 321:776-779 (1986)), neuronal survival (Hashimoto, M., et al., Biochem. Biophvs. Res. Commun.. 173:193-200 (1990)), hypothalamic oxytocin secretion (Sawchenko, P.E., et al., Nature. 334:615-617 (1988)), erythropoiesis (Eto, Y., et al., Biochem. Biophvs. Res. Commun.. 142:1095-1103 (1987)), and early embryonic development (Green and Smith, Nature. 347:391-394 (1990)). Activin has also been shown to suppress androgen production (Hsueh et al., PNAS, 84:5082-5086 (1987)), therefore activin decreases follicular size and may lead to female infertility (Woodruff et al., Endocrinol., 127:3196-3205 (1990)).

There are three activins (A, B, and AB), comprising different combinations of two closely-related beta sub-units, (Mason, A.J., et al., Nature. 318:659-663 (1985)). Activins impinge on a much broader spectrum of cells than inhibins, however, in those systems in which both proteins are functional, they have opposing biological effects. The mechanistic basis for this antagonism is unknown.

Cross-linking of labeled TGF-_/5 to membrane receptors has revealed three distinct classes of integral cell membrane components that bind TGF-/S (Massague, J., Biol. Chem. , 260:7059-7066 (1985)). Class I components are 65 Kd in all species, whereas Class II components range from 85 Kd in rodent cells to 95 Kd in monkey and human cells to 110 Kd in chicken cells. Most frequently, all three classes of these binding proteins coexist on cells.

Type I and II TGF-S receptor components, like most growth factor receptors, are glycoproteins. The type III protein is a proteoglycan consisting predominantly of heparin sulfate glycosaminoglycan chains with a smaller amount of chondroitin or der atan sulfate attached to a core protein of about 100-140 Kd. The binding site for TGF-S resides in this core protein (Cheifetz, et al., J. Biol. Chem.. 263:16904- 16991 (1988)).

An isolated TGF-S supergene family receptor polypeptide has been disclosed in U.S. Patent No. 5,216,126. However, this receptor polypeptide has approximately 75% sequence identity with the mature human inhibin/activin receptor sequence. A protein designated as an "activin receptor" has been expression-cloned (Mathews and Vale, Cell. 65:1-20 (1991)), however, it does not appear to be a mammalian receptor in that it is a serine kinase.

In accordance with one aspect of the present invention, there are provided novel polypeptides which have been identified as TAR-1 and TAR-3 receptors, as well as fragments, analogs and derivatives thereof. The receptor polypeptides of the present invention are of human origin.

In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such receptor polypeptides.

In accordance with yet a further aspect of the present invention, there is provided a process for producing such receptor polypeptides by recombinant techniques.

In accordance with yet a further aspect of the present invention, there are provided processes for utilizing such receptor polypeptides, or polynucleotides encoding such receptor polypeptides for therapeutic purposes, for example, to determine the extent of the receptors in the cells of a patient, to measure all bindable forms of activin and TGF-/3 and to screen for receptor antagonists and/or agonists to the TAR-1 and TAR-3 receptors.

In accordance with another aspect of the present invention there is provided a process of using such antagonists to treat liver and lung fibrosis.

In accordance with another aspect of the present invention there is provided a process of using such agonists for inhibiting proliferation of HIV virus and inhibiting the growth of cancer cells.

In accordance with yet a further aspect of the present invention, there are provided antibodies against such receptor polypeptides.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein. The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

Figure 1 is a cDNA sequence and corresponding deduced amino acid sequence of the TAR-1 receptor polypeptide. The signal sequence of the receptor polypeptide is the first 15 amino acids indicated and the transmembrane portion is underlined.

Figure 2 is the cDNA sequence and corresponding deduced amino acid sequence of the TAR-3 receptor polypeptide wherein the first 21 amino acids represent the signal sequence and the transmembrane portion is underlined. The standard one- letter abbreviation for amino acids is used throughout the figures.

Figure 3 illustrates the amino acid sequence homology between TAR-1 and TAR-3 and rat type I TGF-3 receptor. Boxed areas indicate identical amino acids between the different sequences.

Sequencing inaccuracies are a common problem when attempting to determine polynucleotide sequences. Accordingly, the sequences of Figure 1 and 2 are based on several sequencing runs and the sequencing accuracy is considered to be at least 97%.

The receptors of the present invention have been putatively identified as a TAR-1 and TAR-3 receptor. This identification has been made as a result of amino acid sequence homology.

In accordance with an aspect of the present invention, there are provided isolated nucleic acid (polynucleotides) which encode for the mature receptor polypeptides having the deduced amino acid sequence of Figure 1 and Figure 2 or for the mature receptor polypeptides encoded by the cDNA of the clone deposited as ATCC Deposit No. 75843 and ATCC Deposit No. 75842 for TAR-1 and TAR-3, respectively, which were deposited on July 27, 1994. The polynucleotide encoding for TAR-1 was discovered in a cDNA library derived from a human adult spleen. It is structurally related to the TGF-S receptor family. It contains an open reading frame encoding a protein of 509 amino acid residues of which approximately the first 27 amino acids residues are the putative signal sequence such that the mature protein comprises 482 amino acids. TAR-1 exhibits the highest degree of homology to type I TGF-0 receptor with 49% identity and 67% similarity over a 503 amino acid stretch.

The polynucleotide encoding for TAR-3 was discovered in a cDNA library derived from a human placenta. It is also structurally related to the TGF-S receptor family. It contains an open reading frame encoding a protein of about 503 amino acid residues of which approximately the first 21 amino acids residues are the putative signal sequence such that the mature protein comprises 482 amino acids. TAR-3 also exhibits the highest degree of homology to type I TGF-/S receptor with 47% identity and 64% similarity over a 500 amino acid stretch.

The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature receptor polypeptides may be identical to the coding sequence shown in Figure 1 and Figure 2 or that of the deposited clone(ε) or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature receptor polypeptides as the DNA of Figure 1 and Figure 2 or the deposited cDNA.

The polynucleotides which encode for the mature receptor polypeptides of Figure 1 and Figure 2 or for the mature receptor polypeptides encoded by the deposited cDNA(ε) may include: only the coding sequence for the mature receptor polypeptides; the coding sequence for the mature receptor polypeptides and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature receptor polypeptides (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5 ' and/or 3' of the coding sequence for the mature receptor polypeptides.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequence of Figure 1 and Figure 2 or the polypeptide encoded by the cDNA(ε) of the deposited clone(s). The variant of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides.

Thus, the present invention includes polynucleotides encoding the same mature receptor polypeptides as shown in Figure 1 and Figure 2 or the same mature receptor polypeptides encoded by the cDNA(s) of the deposited clone(ε) aε well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the receptor polypeptides of Figure 1 and Figure 2 or the receptor polypeptides encoded by the cDNA(s) of the deposited clone(ε). Such nucleotide variantε include deletion variantε, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotideε may have a coding sequence which is a naturally occurring allelic variant of the coding sequences shown in Figure 1 and Figure 2 or of the coding sequences of the deposited'^"clone(s) . As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the coding sequence for the mature receptor polypeptides may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein pluε additional 5' amino acid residues. A mature protein having a prosequence is a proprotein and iε an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotideε of the present invention may encode or mature receptor proteins, or for a receptor protein having a prosequence or for a receptor protein having both a prosequence and a presequence (leader sequence) .

The polynucleotideε of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the receptor polypeptides of the present invention. The marker sequence may be a hexa- hiεtidine tag εupplied by a pQE-9 vector to provide for purification of the mature receptor polypeptideε fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al.. Cell, 37:767 (1984)).

The present invention further relateε to polynucleotideε which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequenceε. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode receptor polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA(ε) of Figure 1 and Figure 2 or the deposited cDNA(s).

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 purposeε of Patent Procedure. These depositε are provided merely aε convenience to those of skill in the art and are not an admission that a deposit iε required under 35 U.S.C. §112. The sequence of the polynucleotideε contained in the depoεited aterialε, aε 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 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.

The present invention further relates to TAR-1 and TAR-3 receptor polypeptides which have the deduced amino acid sequence of Figure 1 and Figure 2 or which have the amino acid sequences encoded by the deposited cDNA(s), as well as fragments, analogs and derivatives of such polypeptide. The terms "fragment," "derivative" and "analog" when referring to the receptor polypeptides of Figure 1 and Figure 2 or that encoded by the deposited cDNA(s), meanε receptor polypeptides which retain essentially the same biological function or activity aε such polypeptides. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce active mature receptor polypeptides.

The polypeptides of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the receptor polypeptides of Figure 1 and Figure 2 or that encoded by the deposited cDNA(ε) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a εubεtituent group, or (iii) one in which the mature polypeptide is fused with another compound, such aε a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectorε of the invention and the production of polypeptideε of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form cf a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the TAR-1 and TAR-3 genes. The culture conditions, such as temperature, pH and the like, are those previously uεed with the hoεt cell εelected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing receptor polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expresεing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox viruε, and pseudorabies. However, any other vector may be used as long as it iε replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence iε inserted into an appropriate restriction endonuclease site(s) by procedureε known in the art. Such procedureε and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. Aε repreεentative exampleε of εuch promoterε, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P_L promoter and other promoterε known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expresεion vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells εuch aε dihydrofolate reductase or neomycin resiεtance for eukaryotic cell culture, or εuch aε tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA εequence aε hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the proteins.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli. Streptomγces, Salmonella typhimurium: fungal cells, such as yeast; insect cells such as Drosophila and Sf9; animal cellε such as CHO, COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences aε broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoterε are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pOE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNHlβA, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used aε long aε they are replicable and viable in the hoεt.

Promoter regionε can be εelected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markerε. Two appropriate vectorε are PKK232-8 and PCM7. Particular named bacterial promoterε include lad, lacZ, T3, T7, gpt, lambda P„, P_L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRε from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The hoεt cell can be a higher eukaryotic celT, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the hoεt cell can be a prokaryotic cell, εuch as a bacterial cell. Introduction of the construct into the hoεt cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L. , Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide syntheεizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hoεtε are deεcribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the diεcloεure of which iε hereby incorporated by reference.

Transcription of the DNA encoding the receptor polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Exampleε including the SV40 enhancer on the late εide of the replication origin bp 100 to 270, a cytomegaloviruε early promoter ennancer, the polyoma enhancer on the late εide of the replication origin, and adenoviruε enhancers.

Generally, recombinant expreεsion vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such aε 3-phosphoglycerate kinase (PGK), o-factor, acid phosphatase, or heat shock proteins, among others. The heterologouε structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus εubtiliε. Salmonella tvphimurium and variouε εpecieε within the genera Pεeudomonaε, Streptomyceε, and Staphylococcuε, although otherε may alεo be employed aε a matter of choice.

Aε a repreεentative but nonlimiting example, useful expresεion vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sectionε are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expresεion of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Various mammalian cell culture syεtemε can alεo be employed to express recombinant protein. Examples of mammalian expression syεtemε include the COS-7 lines of monkey kidney fibroblastε, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation εite, εplice donor and acceptor siteε, transcriptional termination sequenceε, and 5' flanking nontranscribed εequenceε. DNA εequenceε derived from the SV40 splice, and polyadenylation εiteε may be uεed to provide the required nontranscribed genetic elements.

The TAR-1 and TAR-3 receptor polypeptides can be recovered and purified from recombinant cell cultureε by methodε including ammonium εulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The receptor polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) . Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. The polypeptides may also include an initial methionine residue.

The present invention also relates to detecting altered levels of expression of mRNA encoding TAR-1 and TAR-3 receptors. The overexpression of these genes or loss of expression is indicative of cancer or the risk of cancer. Assays employed to detect the expression of these nucleic acid sequenceε are well-known by those skilled in the art and include, for example, nucleic acid probe hybridization, PCR and Southern blot analysis. In the hybridization technique, a sample is derived from a host and analyzed to determine whether the sample contains altered levels of TAR receptor gene expression.

TAR-1 and TAR-3 receptor polypeptideε are useful in radio-receptor assays to measure all bindable (and active) forms of a TGF supergene family member such as activin. Such an assay would be specific for activin and would be conducted using the naturally purified or recombinant TAR-1 and TAR-3 receptors aε the receptor element.

In addition, TAR-1 and TAR-3 receptor polypeptideε are useful for screening for compoundε that bind to them and have TGF supergene family biological activity as defined above. Preferably, these compounds are small molecules εuch aε organic or peptide moleculeε that exhibit one or more of the desired activities of a TGF supergene family member such aε activin. Screening assays of this kind are conventional in the art, and any such screening procedure may be employed, whereby the test sample iε contacted with the TAR-1 or TAR-3 receptor herein and the extent of binding and biological activity of the bound moleculeε iε determined.

TAR-1 and TAR-3 receptor polypeptides are additionally useful in affinity purification of TGF supergene family memberε that bind TAR-1 and TAR-3 receptorε and in purifying antibodieε thereto. The receptor iε typically coupled to an immobilized resin such as Affi-Gel 10 (Bio- ad, Richmond, CA) or other such resins (support matrices) by means well-known in the art. The resin is equilibrated in a buffer and the preparation to be purified is placed in contact with the resin, whereby the molecules are selectively adsorbed to the receptor on the resin. The resin is then sequentially washed with suitable buffers to remove non-adsorbed material, including unwanted contaminants, from the mixture to be purified, uεing, for an activin mixture, e.g., lOOmM glycine, pH 3, and 0.1% octyl 0-glucoside. The resin is then treated so aε to elute the compound using a buffer that will break the bond between the compound and receptor.

The TAR-1 and TAR-3 receptorε of the present invention may also be employed in a procesε for εcreening for antagoniεtε and/or agonists for the receptors. As an example, mammalian cells or membrane preparations expressing either the TAR-1 or TAR-3 receptor would be incubated with labeled ligand in the presence of compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of ligand and receptor would be measured compared in the presence or absence of the compound. Such second meεεenger systems include but are not limited to, cAMP guanylate cyclaβe, ion channels or phosphoinoεitide hydrolysis.

Other screening techniques include the use of cells which express the receptorε (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, aε described in Science, volume 246, pages 181-296 (October 1989)'. For example, potential agonists or antagonists may be contacted with a cell which expresses the receptors and a second messenger response, e.g. signal transduction or pH changes, may be measured to determine whether the potential agonist or antagoniεt is effective.

Another such screening technique involves introducing RNA encoding the receptors into xenopuε oocyteε to transiently express the receptor. The receptor oocytes may then be contacted in the case of antagonist screening with the receptor ligand and a compound to be screened, followed by detection of inhibition of a calcium signal.

Another screening technique involves expresεing the TAR- 1 or TAR-3 receptor in which the receptor iε linked to a phoεpholipase C or D. As representative examples of such cells, there may be mentioned endothelial cells, smooth muscle cells, embryonic kidney cells, etc. The screening for an antagoniεt or agonist may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal.

A potential antagonist iε an antibody, or in some cases an oligonucleotide, which binds to the TAR-1 and TAR-3 receptors but does not elicit a second messenger response such that the activity of the receptors is prevented.

Potential antagonists also include proteins which are closely related to the ligand of the receptors, i.e. a fragment of the ligand, which have lost biological function and when binding to the TAR-1 and TAR-3 receptorε, elicit no response.

A potential antagonist also includes an antisense construct prepared through the use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide iε designed to be complementary to a region of the gene involved in tranεcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of TAR-1 and TAR- 3 receptors. The antisense RNA oligonucleotide hybridizes to the mRNA n vivo and blocks translation of the mRNA molecule into the receptorε (antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides aε Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotideε deεcribed above can alεo be delivered to cellε such that the antisenεe RNA or DNA may be expreεεed in vivo to inhibit production of the receptorε.

Another potential antagoniεt iε a small molecule which binds to the TAR-1 and/or TAR-3 receptorε, making them inaccessible to ligands such that normal biological activity is prevented. Examples of εmall moleculeε include but are not limited to εmall peptideε or peptide-like moleculeε.

Potential antagonists also include a soluble form of a TAR-1 and TAR-3 receptors, e.g. a fragment of the receptor, which binds to the ligand and prevents the ligand from interacting with membrane bound receptorε. Theεe εoluble fragments bind to TGF-/5" and/or activin and prevent binding to membrane-bound receptorε.

TGF-/S εtimulateε fibroblasts to migrate to a site of injury, proliferate and produce collagen, TGF-/3 also inhibits collagen degradation. These excessive levels of collagen lead to liver and lung fibrosis. Accordingly, antagonists of TAR-1 and TAR-3 receptors will prevent liver and lung fibrosis.

The antagonists may also be employed to treat female sterility, εince activin decreases follicle size and may lead to this disorder. The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.

Agonists to the TAR-1 and TAR-3 receptors may be employed to activate these receptors and upregulate the functional application of TGF-S. Since TGF-_/S has been implicated in suppressing growth of cancerous cells, the activation of the TAR-1 and TAR-3 receptor may be employed to treat cancer. TGF-)8 also inhibits growth and proliferation of the HIV virus and accordingly stimulating the TAR-1 and TAR-3 receptors treats HIV infection.

Agonistε which stimulate these receptors also will upregulate activins and stimulate spermatogonial proliferation to treat male infertility.

The agonistε and antagonists of the present invention may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the agonist or antagonist and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredientε of the pharmaceutical compoεitionε of the invention. Associated with such container(ε) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological productε, which notice reflectε approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositionε may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositionε are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, the pharmaceutical compositions will be administered in an amount of at least about 10 μg/kg body weight and in most cases they will be administered in an amount not in excess of about 3 mg/Kg body weight per day. In most caεeε, the doεage is from about 10 μg/kg to about 1 g/kg body weight daily, taking into account the routes of administration, symptomε, etc.

The TAR-1 and TAR-3 receptor polypeptides and agoniεtε and antagoniεtε which are polypeptideε may alεo be employed in accordance with the invention by expreεsion of the polypeptides in vivo , which is often referred to as "gene therapy" .

For example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding the TAR-1 and TAR-3 polypeptides ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptides. Such methods are well-known in the art. For example, cells may be engineered by procedureε known in the art by uεe of a retroviral particle containing RNA encoding a polypeptide of the preεent invention. Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo . These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there iε a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA(ε). Computer analysis of the cDNA(ε) iε uεed to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, εublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomeε and preεelection by hybridization to construct chromosome specific-cDNA(s) libraries.

Fluorescence in situ hybridization (FISH) of a cDNA(s) cloneε to a metaphaεe chromosomal spread can be uεed to provide a preciεe chromosomal location in one step. Thiε technique can be used with cDNA(s) as short as 500 or 600 bases; however, cloneε larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requirsε use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 iε better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see Verma et al., Human Chromosomeε: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical poεition of the εequence 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 Hopkinε Univerεity Welch Medical Library). The relationεhip between geneε and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

Next, it is necesεary to determine the differenceε in the cDNA(ε) or genomic εequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation iε likely to be the causative agent of the disease.

With current resolution of physical mapping and genetic mapping techniques, a cDNA(s) precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).

The polypeptides, their fragments or other derivativeε, or analogs thereof, or cells expressing them can be used aε an immunogen to produce antibodieε thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well aε Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and ragments.

Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptideε to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptideε itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodieε can then be used to isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodieε, any technique which provideε antibodieε produced by continuouε cell line cultureε can be used. Examples include the hybrido a technique (Kohler and Milεtein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybriBoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Lies, Inc., pp. 77-96).

Techniqueε described for the production of single chain antibodieε (U.S. Patent 4,946,778) can be adapted to produce εingle chain antibodieε to immunogenic polypeptide productε of thiε invention.

The preεent invention will be further deεcribed with reference to the following examples; however, it is to be underεtood that the present invention is not limited to such exampleε. All partε or amounts, unleεε otherwise specified, are by weight.

In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basiε. or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plaεmidε to thoεe deεcribed are known in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequenceε in the DNA. The variouε restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used aε would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and εubstrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37^*C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980).

"Oligonucleotideε" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phoεphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds between two double εtranded nucleic acid fragments (Maniatiε, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed aε deεcribed in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Exam l? 1

Expression of Recombinant TAR-1 in COS cells

The expression of plasmid, TAR-1 HA iε derived from a vector pcDNA(ε)I/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire TAR-1 precursor and a HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to our target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy iε deεcribed as follows:

The DNA sequence encoding TAR-1, ATCC # 75843, was constructed by PCR on the original EST cloned using two primers: the 5' primer 5' GCGCGAAGCTTTACAATGGTAGATGG AGTGAT 3' contains a Hind III site followed by 21 nucleotides of TAR-1 coding sequence including the initiation codon; the 3' sequence 5' GCGCGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGG GTAACAGTCAGTTTTCAATTTGT 3' contains complementary sequences to an Xho site, translation stop codon, HA tag and the last 20 nucleotides of the TAR-1 coding sequence (not including the stop codon). Therefore, the PCR product contains a Hind III site, TAR-1 coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xho site. The PCR amplified DNA fragment and the vector, pcDNA(s)I/Amp, were digeεted with Hind III and Xho I restriction enzyme and ligated. The ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning Systemε, 11099 North Torrey Pineε Road, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and examined by restriction analysiε for the presence of the correct fragment. For expression of the recombinant TAR-1, COS cells were transfeeted with the expression vector by DEAE-DEXTRAN method. (J. Sa brook, E. Fritsch, T. Maniatiε, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the TAR-1 HA protein was detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with ^3SS-cysteine two days post transfection. Culture media were then collected and cells were lyβed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5). (Wilson, I. et al.. Id. 37:767 (1984)). Both cell lysate and culture media were precipitated with a HA specific monoclonal antibody. Proteinε precipitated were analyzed on 15% SDS-PAGE gels.

Example 2 Expresεion of Recombinant TAR-3 in COS cells

The expression of plasmid, TAR-3 HA is derived from a vector pcDNA(ε)I/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire TAR-3 precursor and a HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previouεly described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to our target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows: The DNA εequence encoding TAR-3, ATCC No. 75842, waε constructed by PCR on the Full-length TAR-3 clone using two primers: the 5' primer is 5' GCGCGAAGCTTACCATGACC TTGGGCTCCCCCA 3' contains a Hind III site followed by 22 nucleotideε of TAR-3 coding εequence starting from the initiation codon; the 3' sequence 5' GCGCGTCTAGATCAAGCGTAG TCTGGGACGTCGTATGGGTAGTGAATCACTTTGGGCTTCTC 3' contains complementary sequences to an Xba I site, translation stop codon, HA tag and the last 21 nucleotides of the TAR-3 coding sequence (not including the stop codon). Therefore, the PCR product contains a Hind III site, TAR-3 coding sequence followed by HA tag fused in frame, a translation termination εtop codon next to the HA tag, and an Xba I site. The PCR amplified DNA fragment and the vector, pcDNA(s)I/Amp, were digested with Hind III and Xba I restriction enzyme and ligated. The ligation mixture waε transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and reεiεtant colonies were selected. Plasmid DNA waε isolated from tranεformantε and examined by reεtriction analysis for the presence of the correct fragment. For expresεion of the recombinant TAR-3, COS cellε were tranεfected with the expreεsion vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatiε, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεε, (1989)). The expression of the TAR-3 HA protein was detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with ^3SS-cysteine two days post tranεfection. Culture media were then collected and cells were lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Triε, pH 7.5). (Wilεon, I. et al., Id. 37:767 (1984)). Both cell lyεate and culture media were precipitated with a HA specific monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: HE, ET AL.

(ii) TITLE OF INVENTION: TAR-1 and TAR-3

(ϋi) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART & OLSTEIN

(B) STREET: 6 BECKER FARM ROAD

(C) CITY: ROSELAND

(D) STATE: NEW JERSEY

(E) COUNTRY: USA

(F) ZIP: 07068

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 INCH DISKETTE

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WORD PERFECT 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: Submitted herewith

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA

(A) APPLICATION NUMBER:

(B) FILING DATE:

-32- (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: FERRARO, GREGORY D.

(B) REGISTRATION NUMBER: 36,134

(C) REFERENCE/DOCKET NUMBER: 325800-132

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-994-1700

(B) TELEFAX: 201-994-1744

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1539 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TACAATGGTA GATGGAGTGA TGATTCTTCC TGTGCTTATC ATGATTGCTC TCCCCTCCCC 60

TAGTATGGAA GATGAGAAGC CCAAGGTCAA CCCCAAACTC TACATGTGTG TGTGTGAAGG 120

TCTCTCCTGC GGTAATGAGG ACCACTGTGA AGGCCAGCAG TGCTTTTCCT CACTGAGCAT ISO

CAACGATGGC TTCCACTGCT ACCAGAAAGG CTCCTTCCAG GTTTATGAGC AGGGAAAGAT 240

GACCTGTAAG ACCCCGCCGT CCCCTGGCCA ACCTCTGGAG TGCTCCCAAG GGGACTGGTG 300

TAACAGGAAC ATCACGGCCC AGCTGCCCAC TAAAGGλλλA TCCTTCCCTG GAACACAGAA 360

TTTCCACTTG GAGGTTGGCC TCATTATCCT CTCTGTAGTG TTCGCAGTAT GTCTTTTAGC 420

CTGCCTGCTG GGAGTTGCTC TCCGAAΛATT TAAAAGGCGC AACCAAGAAC GCCTCAATCC 480

CCGAGACGTG GACTATGGCA CTATCGAGGG GCTCATCACC ACCAATGTGG GAGACAGCAC 540

TTTAGCAGAT TTATTGGATC ATTCGTGTAC ATCAGGAAGT GGCTCTGCTC TTCCTTTTCT 600

GGTACAAAGA ACAGTGGCTC GCCAGATTAC ACTGTTGGAG TGTGTCGGGA AAGGCAGGTA 660

TGGTGAGGTG TGGAGGGGCλ GCTGGCAAGG GGAAAATGTT GCCGTGAAGA TCCTCTCCTC 720

CCGTGATGAG AAGTCATGGT TCAGGGAAAC GGAATTGTAC AACACTGTCA TGCTGAGGCA 780

TGAAAATATC TTAGGTTTCA TTGCTTCAGA CATGACATCA AGACACTCCA GTACCCAGCT 840

GTGGTTAATT ACACATTATC ATGAAATGGG ATCGTTGTAC GλCTATCTTC AGCTTACTAC 900

TCTGGATACA GTTAGCTGTC TTCGAATAGT GCTGTCCATA GCTAGTGGTC TTGCACATTT 960

GCACATAGAG ATATTTGGCA CCCAAGGGAA ACCAGCCATT GCCCATCGAG ATTTAAAGAG 1020 CAAAAATACT CTGGTTAAGA AGAATGGACA GTGTTGCATA GCAGATTTGG GCCTGGCAGT 1080 CATGCATTCC CAGAGCACCA ATCAGCTTGA TGTGGGGAAC AATCCCCGTG TGGGCACCAA 1140 GCGCTACATG GCCCCCGAAG TTCTAGATGA AACCATCCAG GTGGATTGTT TCGATTCTTA 1200 TAAAAGGGTC GATATTTGGG CCTTTGGACT TGTTTTGTGG GAAGTGGCCA GGCGGATGGT 1260 GAGCAATGGT ATAGTGGAGG ATTACAAGCC ACCGTTCTAC GATGTGGTTC CCAATAACCC 1320 AAGTTTTGAA GATATGAGGA AGGTAGTCTG TGTGGATCAA CAAAGGCCAA ACATACCCAA 1380 CAGATGGTTC TCAGACCCGA CATTAACCTC TCTGGCCAAG CTAATGAAAG AATGCTGGTA 1440 TCAAAATCCA TCCGCAAGAC TCλCAGCACT GCGTATCAAA AAGACTTTGA CCAAAATTGA 1500 TAATTCCCTC GACAAATTGA AAACTGACTG TTGACATTT 1539

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 509 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: cDNA

Met Val Asp Gly Val Met He Leu Pro Val Leu He Met He Ala

-25 -20 -15

Leu Pro Ser Pro Ser Met Glu Asp Glu Lyε Pro Lys Val Aεn Pro

-10 -5 1

Lys Leu Tyr Met Cys Val Cyε Glu Gly Leu Ser Cys Gly Aεn Glu

5 10 15

Aεp His Cys Glu Gly Gin Gin Cyε Phe Ser Ser Leu Ser He Asn

20 25 30

Aεp Gly Phe Hiε Val Tyr Gin Lyε Gly Cyε Phe Gin Val Tyr Glu

35 40 45

Gin Gly Lyε Met Thr Cys Lyε Thr Pro Pro Ser Pro Gly Gin Ala

50 55 60

Val Glu Cyε Cyε Gin Gly Aεp Trp Cys Asn Arg Asn He Thr Ala

65 70 75

Gin Leu Pro Thr Lyε Gly Lyε Ser Phe Pro Gly Thr Gin Aεn Phe

80 85 90

Hiε Leu Glu Val Gly Leu He He Leu Ser Val Val Phe Ala Val

95 100 105 Cys Leu Leu Ala Cys Leu Leu Gly Val Ala Leu Arg Lys Phe Lys

110 115 120

Arg Arg Asn Gin Glu Arg Leu Asn Pro Arg Asp Val Glu Tyr Gly

125 130 135

Thr He Glu Gly Leu He Thr Thr Asn Val Gly Asp Ser Thr Leu

140 145 150

Ala Asp Leu Leu Asp His Ser Cys Thr Ser Gly Ser Gly Ser Gly

155 160 165

Leu Pro Phe Leu Val Gin Arg Thr Val Ala Arg Gin He Thr Leu

170 175 180

Leu Glu Cys Val Gly Lys Gly Arg Tyr Gly Glu Val Trp Arg Gly

185 ^'-^•■' 190 195

Ser Trp Gin Gly Gly Asn Val Ala Val Lys He Leu Ser Ser Arg

200 205 210

Asp Glu Lys Ser Trp Phe Arg Glu Thr Glu Leu Tyr Asn Thr Val

215 220 225

Met Leu Arg His Glu Asn He Leu Gly Phe He Ala Ser Asp Met

230 235 240

Thr Ser Arg Hiε Ser Ser Thr Gin Leu Trp Leu He Thr His Tyr

245 250 255

His Glu Met Gly Ser Leu Tyr Asp Tyr Leu Gin Leu Thr Thr Leu

260 265 270

Asp Thr Val Ser Cys Leu Arg He Val Leu Ser He Ala Ser Gly

275 280 285

Leu Ala His Leu His He Glu He Phe Gly Thr Gin Gly Lyε Pro

290 295 300

Ala He Ala Hiε Arg Asp Leu Lys Ser Lys Asn Thr Leu Val Lyε

305 310 315

Lys Asn Gly Gin Cys Cys He Ala Aεp Leu Gly Leu Ala Val Met

320 325 330

Hiε Ser Gin Ser Thr Asn Gin Leu Asp Val Gly Asn Asn Pro Arg

335 340 345

Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu Aεp Gly Thr

350 355 360 He Gin Val Asp Cys Phe Asp Ser Tyr Lyε Arg Val Aεp He Trp

365 370 375 Ala Phe Gly Leu Val Leu Trp Glu Val Ala Arg Arg Met Val Ser

380 385 390

Aβn Gly He Val Gly Asp Tyr Lyε Pro Pro Phe Tyr Asp Val Val

395 400 405

Pro Asn Asn Pro Ser Phe Glu Asp Met Arg Lyε Val Val Cyε Val

410 415 420

Asp Gin Gin Arg Pro Asn He Pro Asn Arg Trp Phe Ser Asp Pro

425 430 435

Thr Leu Thr Ser Leu Ala Lyε Leu Met Lyε Glu Cys Trp Tyr Gin

440 445 450

Asn Pro Ser Ala Arg Leu Thr Ala Leu Arg He Lys Lyε Thr Leu

455 460 465

Thr Lys He Asp Asn Ser Leu Asp Lyε Leu Lys Thr Aεp Cys

470 475 480

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1596 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: cDNA

( i) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GCACGAGGAG GGAGCCACGG CCAGCGGCTG TAACACTTCA TGGCTCTTAC TCCACCTCTC 60

TTGCTCCTCT CTGAAGGGAC CATGACCTTG GGCTCCCCCA GGAAAGGCCT TCTGATGCTG 120

CTGATGGCCT TGGTGACCCA GGGAGACCCT GTGAAGCCGT CTCGGGGCCC GCTGGTGACC 180

TGCACGTGTG AGAGCCCACA TTGCAAGGGG CCTACCTGCC GGGGGGCCTG GTGCACAGTA 240

GTGCTGGTGC GGGAGGAGGG GAGGCACCCC CAGGAACATC GGGGCTGCGG GAACTTGCAC 300

AGGGAGCTCT GCAGCGGGCG CCCCACCGAG TTCGTCAACC ACTACTGCTG CGACAACCAC 360

CTCTGCAACC ACAACGTGTC CCTGGTGCTG GAGGCCACCC AACCTCCTTC GGAGCAACCG 420

GGAACAGATG GCCAGCTGGC CCTGATCCTG GGCCCCGTGC TGGCCTTGCT GGCCCTGGTG 480 GCCCTGGGTG TCCTGGGCCT GTGGCATGTC CGACGGAGGC AGGAGAAGCA GCGTGACCTG 540 CACAGCGAGC TGGGAGAGTC CAGTCTCATC CTGAAAGCAT CTGAGCAGGA CGACAGCATG 600 TTGGGGGACC TCCTGGACAG TGACTGCACC ACAGGGAGTG GCTCAGGGCT CCCCTTCCTG 660 GTGCAGAGGA CAGTGGCACG GCAGGTTGCC TTGGTGGAGT GTGTGGGλλλ AGGCCGCTAT 720 GGCGAAGTGT GGCGGGGCTT GTGGCACGGT GAGAGTGTGG CCGTCAAGAT CTTCTCCTCG 780 AGGGATGAAC AGTCCTGGTT CCGGGAGACT GAGATCTATA ACACAGTGTT GCTCAGACAC 840 GACAACATCC TAGGCTTCAT CGCCTCAGAC ATGACCTCCC GCAACTCGAG CACGCAGCTG 900 TGGCTCATCλ CGCACTACCA CGAGCACGGC TCCCTCTACG ACTTTCTGCC GAGACAGACG 960 CTGGAGCCCC ATCTCGCTCT GAGGCTAGCT GTGTCCGCGG CATGCGGCCT GGCGCACCTG 1020 CACGTGGAGA TCTTTGGTAC CACAGGGCAA CCAGCCATTG CCCACCGCGA CTTCAAGΛAC 1080 CGCAATGTGC TGGTCAAGAG CAACCTGCAG TGTTGCATCG CCGACCTGGG CCTGGCTGTG 1140 ATGCACTCAC AGGGCAGCGA TTACCTGGAC ATCGGCAACA ACCCGAGAGT GGGCACCAAG 1200 CGGTACATGG CACCCGAGGT GCTGCACGAG CAGATCCGCA CGGACTGCTT TGAGTCCTAC 1260 AAGTGGACTG ACATCTGGGC CTTTGGCCTG GTGCTGTGGG AGATTGCCCG CCGGACCATC 1320 GTGAATGGCA TCGTGGAGGA CTATAGACCA CCCTTCTATG GTGTGGTGCC CAATGACCCC 1380 AGCTTTGAGG ACATGAAGAA GGTGGTGTGT GTGGATCAGC AGACCCCCAC CATCCCTAAC 1440 CGGCTGGCTG CAGACCCGGT CCTCTCAGGC CTAGCTCAGA TGATGCGGGA GTGCTGGTAC 1500 CCAAACCCCT CTGCCCGACT CAACGCGCTG CGGATCAAGA AGACACTACA AAAAATTAGT 1560 AACAGTCCAG AGAAGCCCAA AGTGATTCAC TAGCCC 1596

(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 503 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS:

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Thr Leu Gly Ser Pro Arg Lys Gly Leu Leu Met Leu Leu Met

-20 -15 -10

Ala Leu Val Thr Gin Gly Aεp Pro Val Lyε Pro Ser Arg Gly Pro

-5 1 5

Leu Val Thr Cyε Thr Cyε Glu Ser Pro Hiε Cyε Lyε Gly Pro Thr

10 15 20 *^•

Cys Arg Gly Ala Trp Cys Thr Val Val Leu Val Arg Glu Glu Gly

25 30 35 Arg Hiε Pro Gin Glu Hiε Arg Gly Cyε Gly Asn Leu Hiε Arg Glu

40 45 50 Leu Cyε Arg Gly Arg Pro Thr Glu Phe Val Asn Hiε Tyr Cyε Cyε

55 60 65

Asp Asn His Leu Cyε Asn Hiε Asn Val Ser Leu Val Leu Glu Ala

70 75 80

Thr Gin Pro Pro Ser Glu Gin Pro Gly Thr Asp Gly Gin Leu Ala

85 90 95

Leu He Leu Gly Pro Val Leu Ala Leu Leu Ala Leu Val Ala Leu

100 105 110

Gly Val Leu Gly Leu Trp His Val Arg Arg Arg Gin Glu Lys Gin

115 120 125

Arg Asp Leu His Ser Glu Leu Gly Glu Ser Ser Leu He Leu Lyε

130 135 140

Ala Ser Glu Gin Aεp Asp Ser Met Leu Gly Asp Leu Leu Asp Ser

145 150 155

Asp Cys Thr Thr Gly Ser Gly Ser Gly Leu Pro Phe Leu Val Gin

160 165 170

Arg Thr Val Ala Arg Gin Val Ala Leu Val Glu Cyε Val Gly Lyε

175 180 185

Gly Arg Tyr Gly Glu Val Trp Arg Gly Leu Trp Hiε Gly Glu Ser

190 195 200

Val Ala Val Lyε He Phe Ser Ser Arg Aεp Glu Gin Ser Trp Phe

205 210 215

Arg Glu Thr Glu He Tyr Aεn Thr Val Leu Leu Arg His Asp Asn

220 225 230

He Leu Gly Phe He Ala Ser Aεp Met Thr Ser Arg Aεn Ser Ser

235 240 245

Thr Gin Leu Trp Leu He Thr Hiε Tyr Hiε Glu His Gly Ser Leu

250 255 260

Tyr Asp Phe Leu Arg Arg Gin Thr Leu Glu Pro His Leu Ala Leu

265 270 275

Arg Leu Ala Val Ser Ala Ala Cys Gly Leu Ala Hiε Leu Hiε Val

280 285 290 Glu He Phe Gly Thr Gin Gly Lys Pro Ala He Ala His Arg Asp

295 300 305

Phe Lys Asn Arg Asn Val Leu Val Lyε Ser Asn Leu Gin Cys Cys

310 315 320

He Ala Asp Leu Gly Leu Ala Val Met His Ser Gin Gly Ser Asp

325 330 335

Tyr Leu Asp He Gly Asn Asn Pro Arg Val Gly Thr Lyε Arg Tyr

340 345 350

Met Ala Pro Glu Val Leu Asp Glu Gin He Arg Thr Asp Cys Phe

355 360 365

Glu Ser Tyr Lys Trp Thr Asp He Trp Ala Phe Gly Leu Val Leu

370 375 380

Trp Glu He Ala Arg Arg Thr He Val Asn Gly He Val Glu Asp

385 390 395

Tyr Arg Pro Pro Phe Tyr Gly Val Val Pro Asn Asp Pro Ser Phe

400 405 410

Glu Asp Met Lys Lys Val Val Cys Val Asp Gin Gin Thr Pro Thr

415 420 425

He Pro Asn Arg Leu Ala Ala Asp Pro Val Leu Ser Gly Leu Ala

430 435 440

Gin Met Met Arg Glu Cys Trp Tyr Pro Asn Pro Ser Ala Arg Leu

445 450 455

Asn Ala Leu Arg He Lys Lys Thr Leu Gin Lys He Ser Asn Ser

460 465 470

Pro Glu Lys Pro Lys Val He His

475 480

Claims

WHAT lg CLftlMEp _Igi

1. An isolated polynucleotide selected from the groups consisting of:

(a) a polynucleotide encoding for the TAR-1 polypeptide having the deduced amino acid sequence of Figure 1 or a fragment, analog or derivative of said polypeptide;

(b) a polynucleotide encoding for the TAR-3 polypeptide having the deduced amino acid sequence of Figure 2 or a fragment, analog or derivative of said polypeptide;

(c) a polynucleotide encoding for the TAR-1 polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 75843 or a fragment, analog or derivative of said polypeptide;

(d) a polynucleotide encoding for the TAR-3 polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 75842 or a fragment, analog or derivative of said polypeptide.

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

3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA. . The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA.

5. The polynucleotide of Claim 2 wherein said polynucleotide encodes for TAR-1 having the deduced amino acid sequence of Figure 1.

6. The polynucleotide of Claim 2 wherein said polynucleotide encodes for TAR-3 having the deduced amino acid sequence of Figure 1.

7. The polynucleotide of Claim 2 wherein said polynucleotide encodes for the TAR-1 polypeptide encoded by the cDNA(s) of ATCC Deposit No. 75843. 8. The polynucleotide of Claim 2 wherein said polynucleotide encodes for the TAR-3 polypeptide encoded by the cDNA(β) of ATCC Deposit No. 75842.

9. The polynucleotide of Claim 1 having the coding sequence for TAR-1 as shown in Figure 1.

10. The polynucleotide of Claim 1 having the coding sequence for TAR-3 as shown in Figure 2.

11. The polynucleotide of Claim 2 having the coding sequence for TAR-1 deposited as ATCC Deposit No. 75843.

12. The polynucleotide of Claim 2 having the coding sequence for TAR-3 deposited as ATCC Deposit No. 75843.

13. A vector containing the DNA of Claim 2.

14. A host cell genetically engineered with the vector of Claim 13.

15. A process for producing a polypeptide comprising: expressing from the host cell of Claim 14 the polypeptide encoded by said DNA.

16. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 13.

17. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having TAR-1 activity.

18. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having TAR-3 activity.

19. A polypeptide selected from the group consiεting of (i) a TAR-1 polypeptide having the deduced amino acid sequence of Figure 1 and fragments, analogs and derivatives thereof and (ii) a TAR-3 polypeptide having the deduced amino acid sequence of Figure 2 and fragments, analogs and derivatives thereof, (iii) a TAR-1 polypeptide encoded by the cDNA of ATCC Deposit No. 75843 and fragments, analogs and derivatives of said polypeptide; and (iv) a TAR-3 polypeptide encoded by the cDNA of ATCC Deposit No. 75842 and fragments, analogs and derivatives of said polypeptide. 20. The polypeptide of Claim 19 wherein the polypeptide is TAR-1 having the deduced amino acid sequence of Figure 1.

21. The polypeptide of Claim 19 wherein the polypeptide is TAR-3 having the deduced amino acid εequence of Figure 2.

22. An antibody against the polypeptide of Claim 19.

23. A compound which inhibits activation of the polypeptide of Claim 19.

24. A compound which activates the polypeptide of claim 19.

25. A method for the treatment of a patient having need to activate TAR-1 comprising: administering to the patient a therapeutically effective amount of the compound of Claim 24.

26. A method for the treatment of a patient having need to activate TAR-3 compriεing: administering to the patient a therapeutically effective amount of the compound of Claim 24.

27. A method for the treatment of a patient having need to inhibit TAR-1 comprising: administering to the patient a therapeutically effective amount of the compound of Claim 23.

28. A method for the treatment of a patient having need to inhibit TAR-3 comprising: administering to the patient a therapeutically effective amount of the compound of Claim 23.

29. The polypeptide of Claim 19 wherein the polypeptide is a εoluble fragment of the TAR-1 receptor and is capable of binding a ligand for the receptor.

30. The polypeptide of Claim 19 wherein the polypeptide is a soluble fragment of the TAR-3 receptor and iε capable of binding a ligand for the receptor. 31. A process for identifying antagonists and agonists to a TAR receptor comprising: preparing cells for expression of the receptor; contacting the cell with a receptor ligand and a compound to be screened; determining the signal generated by the cell in response to binding of the ligand; and identifying antagonists or agonists to the receptor.

32. A process for detecting cancer or the susceptibility to cancer by determining in a sample derived from a host the level of TAR-1 and TAR-3 receptors, whereby an elevated level indicates a cancer or the susceptibility to cancer.