WO1998018824A9 - Human tumor necrosis factor receptor-like 2 - Google Patents
Human tumor necrosis factor receptor-like 2Info
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- WO1998018824A9 WO1998018824A9 PCT/US1996/018540 US9618540W WO9818824A9 WO 1998018824 A9 WO1998018824 A9 WO 1998018824A9 US 9618540 W US9618540 W US 9618540W WO 9818824 A9 WO9818824 A9 WO 9818824A9
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- polypeptide
- acid sequence
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to novel members of the Tumor Necrosis Factor (TNF) receptor family. More specifically, isolated nucleic acid molecules are provided encoding a human TNF receptor-related protein, referred to herein as the TR2 receptor of FIG. 1A-1B, having considerable homology to murine CD40. Two different TR2 splice variants, referred to as TR2-SV1 and TR2-SV2, are also provided. TR2 polypeptides are also provided with homology to human type 2 TNF receptor (TNF-RII). Further provided are vectors, host cells and recombinant methods for producing the same. The invention also relates to both the inhibition and enhancement of the activity of TR2 receptor polypeptides and diagnostic methods for detecting TR2 receptor gene expression.
- TNF Tumor Necrosis Factor
- TNF- ⁇ and ⁇ are related members of a broad class of polypeptide mediators, which includes the interferons, interleukins and growth factors, collectively called cytokines (Beutler, B. and Cerami, A., Annu. Rev. Immunol, 7:625-655 (1989)).
- Tumor necrosis factor (TNF- ⁇ and TNF- ⁇ ) was originally discovered as a result of its anti-tumor activity, however, now it is recognized as a pleiotropic cytokine playing important roles in a host of biological processes and pathologies.
- TNF- ⁇ Tumor necrosis factor
- TNF- ⁇ lymphotoxin- ⁇
- LT- ⁇ LT- ⁇
- TRAIL ligands for the Fas receptor
- CD30, CD27, CD40, OX40 and 4- IBB receptors These proteins have conserved C- terminal sequences and variable N-terminal sequences which are often used as membrane anchors, with the exception of TNF- ⁇ . Both TNF- ⁇ and TNF- ⁇ function as homotrimers when they bind to TNF receptors.
- TNF is produced by a number of cell types, including monocytes, fibroblasts, T-cells, natural killer (NK) cells and predominately by activated macrophages.
- TNF- ⁇ has been reported to have a role in the rapid necrosis of tumors, immunostimulation, autoimmune disease, graft rejection, producing an anti-viral response, septic shock, cerebral malaria, cytotoxicity, protection against deleterious effects of ionizing radiation produced during a course of chemotherapy, such as denaturation of enzymes, lipid peroxidation and DNA damage (Nata et al, J. Immunol. 136(7):24 3 (1987)), growth regulation, vascular endothelium effects and metabolic effects.
- TNF- ⁇ also triggers endothelial cells to secrete various factors, including PAI-1, LL-1, GM-CSF and IL-6 to promote cell proliferation.
- TNF- ⁇ up-regulates various cell adhesion molecules such as E-Selectin, ICAM-1 and VCAM-1.
- Fas ligand have also been shown to induce programmed cell death.
- TNF- ⁇ has many activities, including induction of an antiviral state and tumor necrosis, activation of polymorphonuclear leukocytes, induction of class I major histocompatibility complex antigens on endothelial cells, induction of adhesion molecules on endothelium and growth hormone stimulation (Ruddle, N. and Homer, R., Prog. Allergy 40:162-182 (1988)).
- TNF- ⁇ and TNF- ⁇ are involved in growth regulation and interact with hemopoietic cells at several stages of differentiation, inhibiting proliferation of various types of precursor cells, and inducing proliferation of immature myelomonocytic cells.
- mice deficient in TNF- ⁇ production show abnormal development of the peripheral lymphoid organs and morphological changes in spleen architecture (reviewed in Aggarwal et al, Eur Cytokine Netw, 7(2) :93- ⁇ 24 (1996)).
- the lymphoid organs the popliteal, inguinal, para-aortic, mesenteric, axillary and cervical lymph nodes failed to develop in TNF- ⁇ -/- mice.
- peripheral blood from TNF- ⁇ -/- mice contained a three fold reduction in white blood cells as compared to normal mice.
- TNF- ⁇ -/- mice Peripheral blood from TNF- ⁇ -/- mice, however, contained four fold more B cells as compared to their normal counterparts. Further, TNF- ⁇ , in contrast to TNF- ⁇ has been shown to induce proliferation of EBV-infected B cells. These results indicate that TNF- ⁇ is involved in lymphocyte development.
- TNF-RI 55-KDa
- 75-KDa 75-KDa
- TNF-R ⁇ Tumor fibroblasts
- TNF-R ⁇ Tumor fibroblasts
- human and mouse cDNAs corresponding to both receptor types have been isolated and characterized (Loetscher et al, Cell, 67:351 (1990)).
- Both TNF-Rs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions.
- TNF-RI and TNF-RII share 28% identity and are characterized by four repeated cysteine-rich motifs with significant intersubunit sequence homology.
- the majority of cell types and tissues appear to express both TNF receptors and both receptors are active in signal transduction, however, they are able to mediate distinct cellular responses. Further, TNF-RII was shown to exclusively mediate human T-cell proliferation by TNF as shown in PCT WO 94/09137.
- TNF-RI dependent responses include accumulation of C-FOS, LL-6, and manganese superoxide dismutase mRNA, prostaglandin E2 synthesis, IL-2 receptor and MHC class I and II cell surface antigen expression, growth inhibition, and cytotoxicity.
- TNF-RI also triggers second messenger systems such as phospholipase A 2 , protein kinase C, phosphatidylcholine-specific phospholipase
- TNF receptors Several interferons and other agents have been shown to regulate the expression of TNF receptors.
- Retinoic acid for example, has been shown to induce the production of TNF receptors in some cells type while down regulating production in other cells.
- TNF- ⁇ has been shown effect the localization of both types of receptor.
- TNF- ⁇ induces internalization of TNF-RI and secretion of TNF-RII (reviewed in Aggarwal et al, supra).
- TNF-Rs are regulated by a variety of agents.
- the present invention provides isolated nucleic acid molecules comprising polynucleotides encoding a TR2 receptor and splice variants thereof having the amino acid sequences shown in FIG. 1 A-1B (SEQ LD NO:2), FIG. 4A-4B (SEQ
- the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of TR2 polypeptides or peptides by recombinant techniques.
- the invention further provides isolated TR2 polypeptides having amino acid sequences encoded by the polynucleotides described herein.
- the present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by TR2 receptors, which involves contacting cells which express TR2 receptors with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
- a screening assay for agonists and antagonists involves determining the effect a candidate compound has on the binding of cellular ligands to TR2 receptors.
- the method involves contacting TR2 receptors with a ligand polypeptide and a candidate compound and determining whether ligand binding to the TR2 receptors is increased or decreased due to the presence of the candidate compound.
- the invention further provides a diagnostic method useful during diagnosis or prognosis of a disease states resulting from aberrant cell proliferation due to alterations in TR2 receptor expression.
- An additional aspect of the invention is related to a method for treating an individual in need of an increased level of a TR2 receptor activity in the body comprising administering to such an individual a composition comprising a therapeufically effective amount of isolated TR2 polypeptides of the invention or an agonist thereof.
- a still further aspect of the invention is related to a method for treating an individual in need of a decreased level of a TR2 receptor activity in the body comprising, administering to such an individual a composition comprising a therapeufically effective amount of a TR2 receptor antagonist.
- the invention additionally provides soluble forms of the polypeptides of the present invention. Soluble peptides are defined by amino acid sequences wherein the sequence comprises the polypeptide sequences lacking a transmembrane domain. Such soluble forms of the TR2 receptors are useful as antagonists of the membrane bound forms of the receptors.
- FIG. 1A-1B shows the nucleotide (SEQ ID NO: l) and deduced amino acid (SEQ ID NO:2) sequences of a TR2 receptor.
- the protein has a predicted leader sequence of about 36 amino acid residues (underlined) (amino acid residues -36 to -1 in SEQ LD NO:2) and a deduced molecular weight of about 30,417 kDa.
- amino acid residues from about 37 to about 200 constitute the extracellular domain; from about 201 to about 225 (amino acid residues 165 to 189 in SEQ ID NO:2) the transmembrane domain (underlined); and from about 226 to about 283 (amino acid residues 190 to 247 in SEQ LD NO:2) the intracellular domain.
- Two potential asparagine-linked glycosylation sites are located at amino acid positions 110 and 173 (amin ⁇ acid residues 74 to 137 in SEQ LD NO: 2).
- FIG. 2 shows the regions of similarity between the amino acid sequences of the TR2 receptor protein of FIG. 1 A- IB and a murine CD40 protein (SEQ ID NO:3).
- FIG. 3 shows an analysis of the TR2 receptor amino acid sequence of FIG. 1A-1B.
- Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
- amino acid residues 39 to 70, 106 to 120, 142 to 189 and 276 to 283 in FIG. 1 A- IB amino acid residues
- FIG. 4A-4B shows the nucleotide (SEQ LD NO:4) and deduced amino acid (SEQ LD NO:5) sequences of the TR2-SV1 receptor.
- the protein has a predicted leader sequence of about 36 amino acid residues (underlined) (amino acid residues -36 to -1 in SEQ LD NO: 5) and a deduced molecular weight of about 19.5 kDa.
- FIG. 5 shows the regions of similarity between the amino acid sequences of the full-length TR2-SV1 receptor protein and a human type 2 TNF receptor (SEQ ID NO:6).
- FIG. 6 shows an analysis of the TR2-SV1 receptor amino acid sequence.
- Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
- amino acid residues 39 to 70, 99 to 136 and 171 to 185 in FIG. 4A-4B amino acid residues 3 to 34, 63 to 100 and 135 to 149 in SEQ ID NO: 5 correspond to the shown highly antigenic regions of the TR2-SV1 receptor protein.
- FIG. 7A-7B shows the nucleotide (SEQ ID NO: 7) and deduced amino acid (SEQ LD NO: 8) sequences of the TR2-SV2 receptor.
- This protein lacks a putative leader sequence and has a deduced molecular weight of about 14 kDa.
- FIG. 8 shows the regions of similarity between the amino acid sequences of the TR2-SV2 receptor protein and a human type 2 TNF receptor (SEQ LD).
- FIG. 9 shows an analysis of the TR2-SV2 receptor amino acid sequence.
- Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
- FIG. 10 shows the regions of similarity between the amino acid sequences of the TR2 receptor protein of FIG. 1 A- IB and the TR2-SV1 receptor protein of FIG. 4A-4B.
- FIG. 11 shows the regions of similarity between the amino acid sequences of the TR2 receptor protein of FIG. 1 A- IB and the TR2-SV2 receptor protein of FIG. 7A-7B.
- FIG. 12 shows the regions of similarity between the amino acid sequences of the TR2-SV1 and the TR2-SV2 receptor proteins.
- FIG. 13A-13C shows the regions of similarity between the nucleotide sequences encoding the TR2 receptor protein of FIG. 1A-1B and the TR2-SV1 receptor protein of FIG. 4A-4B.
- FIG. 14A-14C shows the regions of similarity between the nucleotide sequences encoding the TR2 receptor protein of FIG. 1A-1B and the TR2-SV2 receptor protein of FIG. 7A-7B.
- FIG. 15A-15E shows the regions of similarity between the nucleotide sequences encoding the TR2-SV1 and the TR2-SV2 receptor proteins.
- FIG. 16 shows an alignment of the amino acid sequence of the TR2 receptor of FIG. 1 A-IB (SEQ ID NO:2) with other TNFR family members.
- the amino acid sequence of TR2 was aligned with those of TNFR-I (SEQ ID NO: 10),
- TNFR- ⁇ SEQ LD NO:l l
- CD40 SEQ LD NO:12
- 4-1BB SEQ ID NO.T3
- the present invention provides isolated nucleic acid molecules comprising polynucleotides encoding a TR2 polypeptide (FIG. 1A-1B (SEQ ID NO:2)) and splice variants thereof, TR2-SV1 (FIG. 4A-4B (SEQ ED NO: 5)) and TR2-SV2 (FIG. 7A-7B (SEQ LD NO: 8)), the amino acid sequences of which were determined by sequencing cloned cDNAs.
- the TR2 protein shown in FIG. 1 A- IB shares sequence homology with the murine CD40 receptor (FIG. 2 (SEQ ED NO:3)).
- a deposit was made at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, and given accession number 97059.
- the nucleotide sequence shown in FIG. 1 A- IB SEQ ID NO:2
- ID NO: l was obtained by sequencing a cDNA clone (Clone ED HLHAB49) containing the same amino acid coding sequences as the clone in ATCC Accession No. 97059 with minor deviation.
- the cDNA sequence shown in FIG. 1A-1B (SEQ LD NO:l) differs from that of the ATCC deposit in the 5 1 and 3' noncoding nucleotide sequences and three nucleotides.
- the TR2 receptor nucleotide sequence shown in FIG. 1A-1B contains an adenine at nucleotide 314, a cytosine at nucleotide 386, and a cytosine at nucleotide 627.
- the clone of ATCC Accession No. 97059 contains a guanine at nucleotide 314, a thymine at nucleotide 386, and a thymine at nucleotide 627.
- the TR2 receptors of the present invention include several allelic variants containing alterations in at least these three nucleotides and two amino acids.
- Nucleotide sequence variants which have been identified include either guanine or adenine at nucleotide 314, thymine or cytosine at nucleotide 386, and thymine or cytosine at nucleotide 627 shown in FIG. 1A-1B (SEQ ID NO:l).
- the alteration at nucleotide 386 results in the codon at nucleotides 385 to 387 encoding either serine or phenylalanine and the alteration at nucleotide 314 results in the codon at nucleotides 313 to 315 encoding either lysine or arginine.
- FIG. 4A-4B The nucleotide sequences shown in FIG. 4A-4B (SEQ ED NO:4) and FIG. 7A-7B (SEQ ED NO:7) were also obtained by sequencing cDNA clones deposited on February 13, 1995 at the American Type Culture Collection and given accession numbers 97058 (TR2-SV1) and 97057 (TR2-SV2), respectively.
- the deposited clones are contained in the pBluescript SK(-) plasmid (Stratagene,
- splice variant refers to cDNA molecules produced from a RNA molecules initially transcribed from the same genomic DNA sequence which have undergone alternative RNA splicing.
- Alternative RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of which may encode different amino acid sequences.
- the term “splice variant” also refers to the proteins encoded by the above cDNA molecules.
- TR2 proteins refer to all proteins resulting from the alternate splicing of the genomic DNA sequences encoding proteins having regions of amino acid sequence identity and receptor activity which correspond to the proteins shown in FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4B (SEQ ID NO:5) or FIG. 7A-7B (SEQ LD NO:8).
- the TR2 protein shown in FIG. 1A-1B, the TR2-SV1 protein shown FIG. 4A-4B and the TR2-SV2 protein shown in FIG. 7A-7B are examples of such receptor proteins.
- nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above.
- any nucleotide sequence determined herein may contain some errors.
- Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.
- the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
- a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
- nucleic acid molecules of the present invention encoding TR2 polypeptides may be obtained using standard cloning and screening procedures, such as those used for cloning cDNAs using mRNA as starting material.
- standard cloning and screening procedures such as those used for cloning cDNAs using mRNA as starting material.
- the nucleic acid molecule described in FIG. 1A-1B (SEQ ID NO: l) was discovered in a cDNA library derived from activated human T-lymphocytes.
- FIG. 4A-4B SEQ ED NO:4
- FIG. 7A-7B SEQ ID NO:7 were discovered in cDNAs library derived from human fetal heart and human stimulated monocytes, respectively.
- TR2 mRNA was detected in numerous tissues including lung, spleen and thymus and may be ubiquitously expressed in human cells. TR2 RNA was also found to be expressed in B lymphocytes (CD19 + ), both CD4 + (T H1 and T H2 clones) and CD8 + T lymphocytes, monocytes and endothelial cells.
- TR2 mRNA was inducible in MG 63 cells by TNF ⁇ . Further, the accumulation of TR2 mRNA was observed in HL60, U937 and THP1 cells upon PMA or DMSO treatment. PMA and DMSO are agents known to induce differentiation of these three cell types.
- the determined nucleotide sequence of the TR2 cDNA of FIG. 1A-1B contains an open reading frame encoding a protein of about 283 amino acid residues, with a predicted leader sequence of about 36 amino acid residues, and a deduced molecular weight of about 30,417 kDa.
- the amino acid sequence of the predicted mature TR2 receptor is shown in FIG. 1A-1B from amino acid residue about 37 to residue about 283 (amino acid residues 1 to 247 in SEQ ID NO:2).
- the location of the leader sequence cleavage site was confirmed for a TR2-Fc fusion protein and found to be between amino acids 36 and 37 shown in FIG.
- FIG. 1 A- IB amino acid residues -1 to 1 in SEQ LD NO:2.
- the TR2 protein shown in FIG. 1 A-IB (SEQ LD NO:2) is about 29% identical and about 47% similar to the murine CD40 protein shown in SEQ ID NO:3 (see FIG. 2).
- the determined cDNA nucleotide sequences of the TR2-SV1 splice variant of TR2 contains an open reading frame encoding a protein of about 185 amino acid residues, with a predicted leader sequence of about 36 a ino acid residues, and a deduced molecular weight of about 19.5 kDa.
- the amino acid sequence of the predicted mature TR2-SV1 receptor is shown in FIG. 4A-4B (SEQ LD NO: 5) from amino acid residue about 37 to residue about 185 (amino acid residues 1 to 149 in (SEQ ID NO:5).
- the TR2-SV1 protein shown in FIG. 4A-4B (SEQ LD NO:5) is about 25% identical and about 48% similar to the human type 2 TNF receptor protein shown in SEQ ID NO:6 (see FIG. 5).
- the determined cDNA nucleotide sequences of the TR2-SV2 splice variant of TR2 contains an open reading frame encoding a protein of about 136 amino acid residues, without a predicted leader sequence, and a deduced molecular weight of about 14 kDa.
- the amino acid sequence of the predicted TR2-SV2 receptor is shown in FIG. 7A-7B (SEQ ID NO: 7)
- TR2-SV2 protein shown in FIG. 7A-7B (SEQ ED NO: 8) is about 27% identical and about 45% similar to the human type 2 TNF receptor protein shown in SEQ ED NO: 9 (see FIG. 8).
- SEQ ED NO: 8 A comparison of both the nucleotide and amino acid sequences of the
- TR2, TR2-SV1 and TR2-SV2 receptor proteins shown in FIG. 1A-1B, FIG. 4A- 4B and FIG. 7A-7B shows several regions of near identity. While the amino acid sequence of the TR2 receptor protein, shown in FIG. 1A-1B (SEQ ID NO: 2), is about 60% identical and about 73% similar to the amino acid sequence of the TR2-SV1 receptor protein, shown in FIG. 4A-4B (SEQ ID NO:5), in approximately the first one hundred amino acids of their respective sequences the two proteins differ in one location (FIG. 10).
- amino acid sequence of the TR2 receptor protein of FIG. 1A-1B (SEQ ED NO:2) is about 60% identical and about 71% similar to the amino acid sequence of the TR2-SV2 receptor protein, shown in FIG. 7A-7B (SEQ LD).
- TR2-SV1 and TR2-SV2 proteins are only about 20% identical and about 38% similar at the amino acid level to each other. Unlike the comparisons of either of these proteins to the TR2 protein shown in FIG. 1A-1B (SEQ ED NO: 2), these proteins share their homology over the entire 136 amino acid sequence of the TR2-SV2 protein (FIG. 12).
- the TR2 cDNAs encoding the disclosed TR2 proteins share large regions of near identity at the nucleic acid level (FIG. 13A- 13C, FIG. 14A-13C and FIG. 15A-15E).
- the cDNA sequences encoding the TR2 and TR2-SV1 proteins for example, share large regions of near identity in their nucleotide sequences which encode both the N termini of the respective proteins and their 5' and 3' noncoding regions (FIG. 13A-13C).
- the nucleotide sequences of the cDNAs encoding the TR2-SV1 and TR2-SV2 proteins share considerable homology but this identity is limited to their 3' regions well beyond their respective coding sequences (FIG. 15A-15E).
- the transcript of the src gene undergoes alternate RNA splicing to produce cell type specific products. Ln most cells the Src protein consists of 533 amino acids while in nerve cells an additional short exon is included in the mRNA resulting in a protein of 539 amino acids. See Alberts, B. et al, MOLECULAR BIOLOGY OF THE CELL (3rd
- Dsx which is approximately 550 amino acids in length in males and 430 amino acids in length in females.
- Dsx is approximately 550 amino acids in length in males and 430 amino acids in length in females.
- RNA (SEQ ED NO:2) is believed to be the full-length polypeptide encoded by the RNA from which the TR2 receptor proteins are translated.
- TR2-SV1 splice variant shown in FIG. 4A-4B (SEQ ED NO:5) is believed to contain an insertion in the region encoding amino acid residue 102 of the amino acid sequence shown in FIG. 1 A- IB and a deletion in the region encoding amino acid residue 184 of the amino acid sequence shown in FIG. 1A-1B.
- the RNA encoding the TR2-SV2 splice variant shown in FIG. 7A-7B is believed to begin with the nucleotide sequence encoding amino acid residue 102 of the amino acid sequence shown in FIG. 1A-1B and contain insertions in the regions encoding amino acid residues 184 and 243 of the amino acid sequence shown in FIG. 1A- 1B.
- the present invention also provides the mature forms of the TR2 receptors of the present invention.
- proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
- Most mammalian cells and even insect cells cleave secreted proteins with the same specificity.
- cleavage of a secreted protein is not entirely uniform, which results in two or more mature species on the protein.
- the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
- the present invention provides nucleotide sequences encoding mature TR2 polypeptides having the amino acid sequences encoded by the cDNA clones contained in the host identified as ATCC Deposit Numbers 97059 and 97058 and as shown in FIG. 1A-1B (SEQ ID NO:2) and FIG. 4A-4B (SEQ ED NO:5).
- the mature TR2 polypeptides having the amino acid sequences encoded by the cDNA clones contained in the host identified as ATCC Deposit Numbers 97059 and 97058 is meant the mature form(s) of the
- TR2 receptors produced by expression in a mammalian cell (e.g., COS cells, as described below) of the complete open reading frame encoded by the human DNA sequence of the clone contained in the vector in the deposited host.
- a mammalian cell e.g., COS cells, as described below
- the invention also provides nucleic acid sequences encoding the TR2-SV2 receptor protein of FIG. 7A-7B (SEQ ED NO: 8), having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit Number 97057, which does not contain a secretory leader sequence.
- Kanehisa Genomics 74:897-911 (1992)
- the analysis by the PSORT program predicted the cleavage sites between amino acids -1 and 1 in SEQ ED NO:2 and SEQ LD NO:5. Thereafter, the complete amino acid sequences were further analyzed by visual inspection, applying a simple form of the (-1,-3) rule of von Heine, von Heinje, supra.
- the leader sequences for the TR2 protein shown in SEQ ED NO:2 and the TR2-SV1 protein are predicted to consist of amino acid residues -36 to -1 in both SEQ ED NO:2 and SEQ ID NO:5, while the predicted mature TR2 proteins consist of amino acid residues 1 to 247 for the TR2 protein shown in SEQ ID NO:2 and residues 1 to 149 for the TR2-SV1 protein shown in SEQ ED NO: 5.
- Example 6 the cleavage site of the leader sequence of a TR2- Fc fusion protein was confirmed using amino acid analysis of the expressed fusion protein. This fusion protein was found to begin at amino acid 37, which corresponds to amino acid 1 in SEQ ID NO:2 and SEQ LD NO:5, indicating that the cleavage site of the leader sequence is between amino acids 36 and 37 in this protein (corresponding to amino acid residues -1 to 1 in SEQ ED NO: 2 and SEQ LD NO:5).
- the TR2 receptor polypeptide encoded by the cDNA of ATCC Deposit Number 97059 comprises about 283 amino acids, but may be anywhere in the range of 250 to 316 amino acids; and the leader sequence of this protein is about 36 amino acids, but may be anywhere in the range of about 30 to about 42 amino acids.
- the TR2-SV1 receptor polypeptide encoded by the cDNA of ATCC Deposit Number 97058 comprises about 185 amino acids, but may be anywhere in the range of 163-207 amino acids; and the leader sequence of this protein is about 36 amino acids, but may be anywhere in the range of about 30 to about 42 amino acids.
- the TR2-SV2 receptor polypeptide encoded by the cDNA of ATCC Deposit Number 97057 comprises about 136 amino acids, but may be anywhere in the range of 120-152 amino acids
- nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
- RNA such as mRNA
- DNA including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
- DNA may be double-stranded or single-stranded.
- Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
- isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment
- recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
- Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
- Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
- Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in FIG. 1A-1B (SEQ ID NO: 1); DNA molecules comprising the coding sequence for the mature TR2 receptor shown in FIG. 1 A- IB (SEQ LD NO:2) (last 247 amino acids); and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TR2 receptor protein shown in FIG. 1A-1B (SEQ ID NO:2).
- ORF open reading frame
- SEQ ID NO: 1 DNA molecules comprising the coding sequence for the mature TR2 receptor shown in FIG. 1 A- IB (SEQ LD NO:2) (last 247 amino acids)
- the genetic code is well known in the art. Thus, it would be routine for one
- isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in FIG. 4A-4B (SEQ LD NO:4); DNA molecules comprising the coding sequence for the mature
- TR2-SV1 receptor shown in FIG. 4A-4B SEQ ID NO:5 (last 149 amino acids); and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TR2-SV1 receptor.
- isolated nucleic acid molecules of the present invention include
- DNA molecules comprising an open reading frame (ORF) shown in FIG. 7A-7B (SEQ ID NO: 7) and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TR2-SV2 receptor.
- the invention provides isolated nucleic acid molecules encoding the TR2, TR2-SV1 and TR2-SV2 polypeptides having the amino acid sequences encoded by the cDNA clones contained in the plasmid deposited as ATCC Deposit No. 97059, 97058 and 97057, respectively, on February 13, 1995.
- these nucleic acid molecules will encode a mature polypeptide or the full-length polypeptide lacking the N-terminal methionine.
- the invention further provides isolated nucleic acid molecules having the nucleotide sequences shown in FIG. 1A-1B (SEQ LD NO:l), FIG. 4A-4B (SEQ ID NO:4), and FIG. 7A-7B (SEQ ED NO: 7); the nucleotide sequences of the cDNAs contained in the above-described deposited clones; or nucleic acid molecules having a sequence complementary to one of the above sequences.
- Such isolated molecules, particularly DNA molecules are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the TR2 receptor genes of the present invention in human tissue, for instance, by Northern blot analysis.
- the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
- FIG. 1A-1B SEQ LD NOT
- FIG. 4A-4B SEQ LD NO:4
- FIG. 7A-7B SEQ LD NO:7
- fragments 50-400 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequences of the deposited cDNAs or as shown in FIG. 1A-1B (SEQ ED NO: 1), FIG. 4A-4B (SEQ ED NO:4), or FIG. 7A-7B (SEQ ED NO:7).
- a fragment at least 20 nt in length for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequences of the deposited cDNAs or the nucleotide sequences as shown in FIG. 1A-1B (SEQ ID NO: l), FIG. 4A-4B (SEQ LD
- FIG. 7A-7B SEQ ID NO:7.
- Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising the TR2 receptor protein of FIG. 1A-1B (SEQ ED NO:2) extracellular domain (predicted to constitute amino acid residues from about 37 to about 200 in FIG. 1 A- IB (amino acid residues 1 to 164 in SEQ ED NO:2)); a polypeptide comprising the TR2 receptor transmembrane domain (amino acid residues 201 to 225 in FIG. 1 A- IB (amino acid residues 165 to 189 in SEQ LD NO:2)); a polypeptide comprising the TR2 receptor intracellular domain (predicted to constitute amino acid residues from about 226 to about 283 in FIG. 1A-1B (amino acid residues 190 to 247 in SEQ
- nucleic acid fragments of the present invention also include nucleic acid molecules encoding polypeptides comprising the mature TR2-SV1 receptor (predicted to constitute amino acid residues from about 37 to about 185 in FIG 4A-4B (amino acid residues 1 to 149 in SEQ ID NO 5)) and the complete TR2-SV2 receptor (predicted to constitute amino acid residues from about 1 to about 136 in FIG 7A-7B (SEQ ID NO 8))
- amino acid residues constituting the extracellular, transmembrane and intracellular domains have been predicted by computer analysis
- the amino acid residues constituting these domains may vary slightly (e g , by about 1 to about 15 amino acid residues) depending on the criteria used to define each domain
- nucleic acid fragments of the present invention also include nucleic acid molecules encoding epitope-bearing portions of the TR2 receptor proteins
- nucleic acid fragments of the present invention include nucleic acid molecules encoding a polypeptide comprising amino acid residues from about 39 to about 70 in FIG 1 A- IB (amino acid residues 3 to 34 in SEQ LD NO 2), a polypeptide comprising amino acid residues from about 106 to about 120 in FIG 1 (amino acid residues 70 to 84 in SEQ ID NO 2), a polypeptide comprising amino acid residues from about 142 to about 189 in FIG 1A-1B (amino acid residues 106 to 153 in SEQ ID NO 2), a polypeptide comprising amino acid residues from about 276 to about 283 in FIG 1A-1B (amino acid residues 240 to 247 in SEQ ID NO 2), a polypeptide comprising amino acid residues from about 39 to about 70 in FIG 4A-4B (amino
- the invention provides isolated nucleic acid molecules comprising polynucleotides which hybridizes under stringent hybridization conditions to a portion of the polynucleotide of one of the nucleic acid molecules of the invention described above, for instance, the cDNA clones contained in ATCC Deposits 97059, 97058 and 97057.
- stringent hybridization conditions is intended overnight incubation at 42 °C in a solution comprising: 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. lx SSC at about 65 °C.
- polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about
- nucleotides and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide.
- a portion of a polynucleotide of "at least 20 nt in length,” for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., the deposited cDNAs or the nucleotide sequences as shown in FIG. 1 A-IB (SEQ ED NO: 1), FIG. 4A-4B (SEQ ED NO:4), or FIG. 7A-7B (SEQ LD NO:7)).
- SEQ ED NO: 1 A-IB SEQ ED NO: 1
- FIG. 4A-4B SEQ ED NO:4
- FIG. 7A-7B SEQ LD NO:7
- nucleic acid molecules of the present invention which encode TR2 polypeptides may include, but are not limited to those encoding the amino acid sequences of the mature polypeptides, by itself; the coding sequence for the mature polypeptides and additional sequences, such as those encoding the about 36 amino acid leader or secretory sequences, such as pre-, or pro- or prepro- protein sequences; the coding sequence of the mature polypeptides, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5 ' and 3 ' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
- the sequence encoding the polypeptides may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
- the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
- hexa-histidine provides for convenient purification of the fusion protein.
- the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37: 767 (1984).
- other such fusion proteins include the TR2 receptors fused to IgG-Fc at the N- or C-terminus.
- the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the TR2 receptors Variants may occur naturally, such as a natural allelic variant
- allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism Genes II, Lewin, B , ed , John Wiley & Sons, New York (1985)
- Non-naturally occurring variants may be produced using art-known mutagenesis techniques
- variants include those produced by nucleotide substitutions, deletions or additions, which may involve one or more nucleotides
- the variants may be altered in coding regions, non-coding regions, or both Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the TR2 receptors or portions thereof Also especially preferred in this regard are conservative substitutions
- nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the TR2 polypeptide having the complete amino acid sequence shown in FIG 1A-1B (amino acid residues -36 to 247 in SEQ ED NO 2), FIG 4A-4B (amino acid residues -36 to 149 in SEQ ED NO 5), or FIG 7A-7B (amino acid residues 1 to 136 in SEQ ID NO 8), (b) a nucleotide encoding the complete amino sequence shown in FIG 1A-1B (amino acid residues -35 to 247 in SEQ LD NO 2), FIG 4A-4B (amino acid residues -35 to 149 in SEQ ID NO 5), or FIG 7A-7B (amino acid residues 2 to 136 in SEQ ID
- nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the TR2 receptors.
- a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
- These mutations of the reference sequence may occur at the 5 ' or 3 ' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
- nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in FIG. 1A-1B (SEQ LD NO:l) or to the nucleotides sequence of the deposited cDNA clone encoding that protein can be determined conventionally using known computer programs such as the Bestfit program
- the present application is directed to nucleic acid molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences shown in FIG. 1A-1B (SEQ ED NOT), FIG. 4A-4B (SEQ ED NO:4), or FIG. 7A-7B (SEQ LD NO: 7) or to the nucleic acid sequence of the deposited cDNAs, irrespective of whether they encode a polypeptide having TR2 receptor activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having TR2 receptor activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
- PCR polymerase chain reaction
- nucleic acid molecules of the present invention that do not encode a polypeptide having TR2 receptor activity include, wter alia, (1) isolating a TR2 receptor gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of a TR2 receptor gene, as described in Verma et al, Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting TR2 receptor mRNA expression in specific tissues.
- FISH in situ hybridization
- nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in FIG. 1A-1B (SEQ ID NO: l), FIG. 4A-4B (SEQ ED NO:4), or FIG. 7A-7B
- TR2 receptor activity polypeptide having TR2 receptor activity
- polypeptide having TR2 receptor activity polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the TR2 receptors of the present invention (either the full-length protein, the splice variants, or, preferably, the mature protein), as measured in a particular biological assay.
- TR2 receptor activity can be measured by determining the ability of a polypeptide-Fc fusion protein to inhibit lymphocyte proliferation as described below in Example 6.
- TR2 receptor activity may also be measured by determining the ability of a polypeptide, such as cognate ligand which is free or expressed on a cell surface, to confer proliferatory activity in intact cells expressing the receptor.
- nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of the deposited cDNAs or the nucleic acid sequences shown in FIG. 1A-1B (SEQ ED NO:l), FIG. 4A-4B (SEQ LD NO:4), or FIG.
- the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of TR2 polypeptides or fragments thereof by recombinant techniques.
- the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
- a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
- the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retro viral LTRs, to name a few.
- an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retro viral LTRs, to name a few.
- the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
- the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or
- the expression vectors will preferably include at least one selectable marker.
- markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
- appropriate heterologous hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as
- CHO, COS and Bowes melanoma cells CHO, COS and Bowes melanoma cells; and plant cells.
- Appropriate culture mediums and conditions for the above-described host cells are known in the art.
- vectors preferred for use in bacteria include pQ ⁇ 70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
- preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
- Other suitable vectors will be readily apparent to the skilled artisan.
- Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986).
- the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
- a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins.
- EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof.
- the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
- Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations.
- human proteins such as, human hIL-5 receptor has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D.
- TR2 receptors can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
- Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
- polypeptides of the present invention may be glycosylated or may be non-glycosylated.
- polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
- the invention further provides isolated TR2 polypeptides having the amino acid sequence encoded by the deposited cDNAs, or the amino acid sequence in FIG. 1 A- IB (SEQ LD NO:2), FIG. 4A-4B (SEQ ID NO: 5), or FIG. 7A-7B (SEQ ID NO: 8), or a peptide or polypeptide comprising a portion of the above polypeptides.
- the polypeptides of this invention may be membrane bound or may be in a soluble circulating form.
- Soluble peptides are defined by amino acid sequence wherein the sequence comprises the polypeptide sequence lacking the transmembrane domain.
- One example of such a soluble form of the TR2 receptor is the TR2-SV1 splice variant which has a secretory leader sequence but lacks both the intracellular and transmembrane domains.
- the TR2-SV1 receptor protein appears to be secreted in a soluble form from cells which express this protein.
- the polypeptides of the present invention may exist as a membrane bound receptor having a transmembrane region and an intra- and extracellular region or they may exist in soluble form wherein the transmembrane domain is lacking.
- a form of the TR2 receptor is the TR2 receptor shown in FIG. 1A-1B (SEQ ID NO:2) which contains, in addition to a leader sequence, transmembrane, intracellular and extracellular domains.
- the invention further includes variations of the TR2 receptors which show substantial TR2 receptor activity or which include regions of TR2 proteins such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and type substitutions. As indicated above, guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J. U., et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990).
- the fragment, derivative or analog of the polypeptides of FIG. 1A- 1B (SEQ ID NO:2), FIG. 4A-4B (SEQ LD NO:5), and FIG. 7A-7B (SEQ ID NO: 8), or that encoded by the deposited cDNAs, 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 substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is
- the replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al, Nature 3(57:266-268 (1993) describes certain mutations resulting in selective binding of TNF- ⁇ to only one of the two known types of TNF receptors.
- the TR2 receptors of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.
- changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1).
- Amino acids in the TR2 proteins of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine- scanning mutagenesis (Cunningham and Wells, Science 244 1081-1085 (1989)) The latter procedure introduces single alanine mutations at every residue in the molecule The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro, or in vitro proliferative activity Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol Biol. 224 899-904 (1992) and de Vos et al. Science 255 306-312 (1992))
- polypeptides of the present invention are preferably provided in an isolated form
- isolated polypeptide is intended a polypeptide removed from its native environment
- a polypeptide produced and contained within a recombinant host cell would be considered “isolated” for purposes of the present invention.
- isolated polypeptide are polypeptides that have been purified, partially or substantially, from a recombinant host.
- recombinantly produced versions of the TR2 receptors can be substantially purified by the one-step method described in Smith and Johnson, Gene 67: 1-40
- the polypeptides of the present invention also include the polypeptide encoded by the deposited cDNAs including the leader; the polypeptide encoded by the deposited the cDNAs minus the leader (i.e., the mature protein); the polypeptides of FIG. 1A-1B (SEQ ED NO:2) or FIG. 4A-4B (SEQ ID NO:5) including the leader; the polypeptides of FIG. 1 A-IB (SEQ ID NO:2) or FIG. 4A- 4B (SEQ LD NO:5) including the leader but minus the N-terminal methionine; the polypeptides of FIG. 1A-1B (SEQ ID NO:2) or FIG.
- polypeptide 4A-4B (SEQ LD NO:5) minus the leader; the polypeptide of FIG. 7A-7B (SEQ ED NO:8); the extracellular domain, the transmembrane domain, and the intracellular domain of the TR2 receptor shown in FIG. 1A-1B (SEQ ED NO:2); and polypeptides which are at least 80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptides described above, and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
- polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a TR2 polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of a
- TR2 receptor to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
- These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
- any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in FIG. 1 A-IB (SEQ ED NO:2), FIG. 4A-4B (SEQ LD NO:5), or FIG. 7A- 7B (SEQ LD NO: 8) or to the amino acid sequence encoded by one of the deposited cDNA clones can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance,
- the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
- polypeptides of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
- the invention provides peptides or polypeptides comprising epitope-bearing portions of the polypeptides of the invention.
- the epitopes of these polypeptide portions are an immunogenic or antigenic epitopes of the polypeptides described herein.
- An "immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen.
- a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
- the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al, Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983).
- Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
- Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al, Cell 37161-11% (1984) at 777.
- Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
- Non-limiting examples of antigenic polypeptides or peptides that can be used to generate TR2 receptor-specific antibodies include: a polypeptide comprising amino acid residues from about 39 to about 70 in FIG. 1 (amino acid residues 3 to 34 in SEQ ED NO:2); a polypeptide comprising amino acid residues from about 106 to about 120 in FIG. 1 A- IB (amino acid residues 70 to 84 in SEQ ED NO:2); a polypeptide comprising amino acid residues from about 142 to about 189 in FIG. 1A-1B (amino acid residues 106 to 153 in SEQ ID NO:2); a polypeptide comprising amino acid residues from about 276 to about 283 in FIG.
- amino acid residues 240 to 247 in SEQ ID NO:2 amino acid residues 240 to 247 in SEQ ID NO:2
- polypeptide comprising amino acid residues from about 39 to about 70 in FIG. 4A-4B amino acid residues 3 to 34 in SEQ ID NO:5
- polypeptide comprising amino acid residues from about 99 to about 136 in FIG. 4A-4B amino acid residues 63 to
- polypeptide comprising amino acid residues from about 171 to about 185 in FIG. 4A-4B (amino acid residues 135 to 149 in SEQ ID NO:5); a polypeptide comprising amino acid residues from about 56 to about 68 in FIG. 7A-7B (SEQ ED NO: 8); and a polypeptide comprising amino acid residues from about 93 to about 136 in FIG. 7A-7B (SEQ LD NO:8).
- the inventors have determined that the above polypeptide fragments are antigenic regions of the TR2 receptor proteins.
- the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 52:5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten et al (1986).
- SMPS Simultaneous Multiple Peptide Synthesis
- TR2 polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
- IgG immunoglobulins
- fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al, Nature 337:84-86 (1988)).
- Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric TR2 receptor proteins or protein fragments alone (Fountoulakis et al, J. Biochem 270:3958-3964 (1995)).
- TNF-family ligands induce various cellular responses by binding to TNF-family receptors, including the TR2 receptors of the present invention.
- TNF- ⁇ a potent ligand of the TNF receptor proteins, is known to be involved in a number of biological processes including lymphocyte development, tumor necrosis, induction of an antiviral state, activation of polymorphonuclear leukocytes, induction of class I major histocompatibility complex antigens on endothelial cells, induction of adhesion molecules on endothelium and growth hormone stimulation (Ruddle and Homer, Prog. Allergy, 40:162-182 (1988)).
- TNF- ⁇ also a ligand of the TNF receptor proteins
- TNF- ⁇ has been reported to have a role in the rapid necrosis of tumors, immunostimulation, autoimmune disease, graft rejection, producing an anti-viral response, septic shock, cerebral malaria, cytotoxicity, protection against deleterious effects of ionizing radiation produced during a course of chemotherapy, such as denaturation of enzymes, lipid peroxidation and DNA damage (Nata et al, J. Immunol 136(7) :2483 (1987); Porter, Tibtech 9: 158-162 (1991)), growth regulation, vascular endothelium effects and metabolic effects.
- TNF- ⁇ also triggers endothelial cells to secrete various factors, including PAI-1, EL-1, GM-CSF and IL-6 to promote cell proliferation.
- TNF- ⁇ up-regulates various cell adhesion molecules such as E-Selectin, ICAM-1 and VCAM-1.
- TNF- ⁇ and the Fas ligand have also been shown to induce programmed cell death.
- a cellular response to a TNF-family ligand is intended any genotypic, phenotypic, and/or morphologic change to a cell, cell line, tissue, tissue culture or patient that is induced by a TNF-family ligand. As indicated, such cellular responses include not only normal physiological responses to TNF-family ligands, but also diseases associated with increased cell proliferation or the inhibition of increased cell proliferation, such as by the inhibition of apoptosis.
- the invention provides a diagnostic method useful during diagnosis of disease states, which involves assaying the expression level of the gene encoding the TR2 receptor protein in mammalian cells or body fluid and comparing the gene expression level with a standard TR2 receptor gene expression level, whereby an increase or decrease in the gene expression level over the standard is indicative of certain disease states associated with aberrant cell survival.
- the present invention is useful as a prognostic indicator, whereby patients exhibiting significantly aberrant TR2 receptor gene expression will experience a worse clinical outcome relative to patients expressing the gene at a lower level.
- assaying the expression level of the gene encoding the TR2 receptor protein is intended qualitatively or quantitatively measuring or estimating the level of the TR2 receptor protein or the level of the mRNA encoding the TR2 receptor protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the TR2 receptor protein level or mRNA level in a second biological sample).
- the TR2 receptor protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard TR2 receptor protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disease state.
- a standard TR2 receptor protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.
- biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source which contains TR2 receptor protein or mRNA.
- Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted mature TR2 receptor protein, and thymus, prostate, heart, placenta, muscle, liver, spleen, lung, kidney and other tissues. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
- cancers such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors
- autoimmune disorders such as systemic lupus erythematosus and immune-related glomerulonephritis rheumatoid arthritis
- viral infections such as herpes viruses, pox viruses and adenoviruses
- information graft v. host disease such as acute graft rejection, and chronic graft rejection.
- apoptosis Diseases associated with decreased cell survival, or increased apoptosis, include ALDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration); myelodysplastic syndromes (such as aplastic anemia), ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
- neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration
- myelodysplastic syndromes such as aplastic anemia
- ischemic injury such as that caused by myocardial infarction, stroke and reperfusion injury
- toxin-induced liver disease such as that caused by alcohol
- septic shock cachexia and anore
- TR2 receptor-protein specific antibodies can be raised against the intact TR2 receptor protein or an antigenic polypeptide fragment thereof, which may presented together with a carrier protein, such as an albumin, to an animal system
- antibody or “monoclonal antibody” (mAb) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab') 2 fragments) which are capable of specifically binding to
- TR2 receptor protein Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl Med. 24:316-325 (1983)). Thus, these fragments are preferred.
- the antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing the TR2 receptor protein or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of TR2 receptor protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
- the antibodies of the present invention are monoclonal antibodies (or TR2 receptor protein binding fragments thereof).
- monoclonal antibodies can be prepared using hybridoma technology (Kohler et al, Nature 256 495 (1975), Kohler et al, Eur. J. Immunol (5 511 (1976), Kohler et al, Eur. J. Immunol.
- Such procedures involve immunizing an animal (preferably a mouse) with a TR2 receptor protein antigen or, more preferably, with a TR2 receptor protein-expressing cell Suitable cells can be recognized by their capacity to bind anti-TR2 receptor protein antibody Such cells may be cultured in any suitable tissue culture medium, however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56°C), and supplemented with about 10 g/1 of nonessential amino acids, about
- mice 1,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin
- a suitable myeloma cell line Any suitable myeloma cell line may be employed in accordance with the present invention, however, it is preferable to employ the parent myeloma cell line (SP 2 O), available from the American Type Culture Collection, Rockville, Maryland
- SP 2 O parent myeloma cell line
- the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80 225-232 (1981))
- the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the TR2 receptor protein antigen
- the present invention is directed to a method for inhibiting an activity of TR2 induced by a TNF-family ligand (e g , cell proliferation, hematopoietic development), which involves administering to a cell which expresses a TR2 polypeptide an effective amount of a TR2 receptor ligand, analog or an antagonist capable of decreasing TR2, receptor mediated signaling
- TR2 receptor mediated signaling is increased to treat a disease wherein increased cell proliferation is exhibited
- An antagonist can include soluble forms of the TR2 receptors and antibodies directed against the TR2 polypeptides which block TR2 receptor mediated signaling.
- TR2 receptor mediated signaling is decreased to treat a disease.
- the present invention is directed to a method for increasing cell proliferation induced by a TNF-family ligand, which involves administering to a cell which expresses a TR2 polypeptide an effective amount of an agonist capable of increasing TR2 receptor mediated signaling.
- TR2 receptor mediated signaling is increased to treat a disease wherein decreased cell proliferation is exhibited.
- Agonists of the present invention include monoclonal antibodies directed against the TR2 polypeptides which stimulate TR2 receptor mediated signaling.
- TR2 receptor mediated signaling is increased to treat a disease.
- agonist is intended naturally occurring and synthetic compounds capable of enhancing cell proliferation and differentiation mediated by TR2 polypeptides. Such agonists include agents which increase expression of TR2 receptors or increase the sensitivity of the expressed receptor. By “antagonist” is intended naturally occurring and synthetic compounds capable of inhibiting TR2 mediated cell proliferation and differentiation. Such antagonists include agents which decrease expression of TR2 receptors or decrease the sensitivity of the expressed receptor. Whether any candidate "agonist” or “antagonist” of the present invention can enhance or inhibit cell proliferation and differentiation can be determined using art-known TNF-family ligand/receptor cellular response assays, including those described in more detail below.
- One such screening technique involves the use of cells which express the receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science 24(5:181-296 (October 1989).
- compounds may be contacted with a cell which expresses the receptor polypeptide of the present invention and a second messenger response, e.g., signal transduction or pH changes, may be measured to determine whether the potential compound activates or inhibits the receptor.
- Another such screening technique involves introducing RNA encoding the receptor into Xenopus oocytes to transiently express the receptor.
- the receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for compounds which are thought to inhibit activation of the receptor.
- Another method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention antagonists by determining inhibition of binding of labeled ligand to cells which have the receptor on the surface thereof.
- Such a method involves transfecting a eukaryotic cell with DNA encoding the receptor such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand.
- the ligand can be labeled, e.g., by radioactivity.
- the amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors.
- Soluble forms of the polypeptides of the present invention may be utilized in the ligand binding assay described above. These forms of the TR2 receptors are contacted with ligands in the extracellular medium after they are secreted. A determination is then made as to whether the secreted protein will bind to TR2 receptor ligands. Further screening assays for agonist and antagonist of the present invention are described in Tartaglia, L.A., and Goeddel, D.V., J. Biol Chem. 267 (7):4304-4301( ⁇ 992).
- a screening method for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand.
- the method involves contacting cells which express TR2 polypeptides with a candidate compound and a TNF-family ligand, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the ligand/receptor signaling pathway and a decreased cellular response compared to the standard indicates that ' the candidate compound is an antagonist of the ligand/receptor signaling pathway.
- assaying a cellular response is intended qualitatively or quantitatively measuring a cellular response to a candidate compound and/or a TNF-family ligand (e.g., determining or estimating an increase or decrease in T cell proliferation or tritiated thymidine labeling).
- a cell expressing a TR2 polypeptide can be contacted with either an endogenous or exogenously administered TNF-family ligand.
- a thymocyte proliferation assay may be employed to identify both ligands and potential drug candidates. For example, thymus cells are disaggregated from tissue and grown in culture medium.
- Agonists according to the present invention include compounds such as, for example, TNF-family ligand peptide fragments, transforming growth factor ⁇ , and neurotransmitters (such as glutamate, dopamine, N-methyl-D-aspartate).
- Preferred agonist include polyclonal and monoclonal antibodies raised against TR2 polypeptide, or a fragment thereof. Such agonist antibodies raised against a TNF- family receptor are disclosed in Tartaglia, L.A., et al, Proc. Natl Acad. Sci.
- chemotherapeutic drugs such as, for example, cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and vincristine. Others include ethanol and ⁇ -amyloid peptide. (Science 267: 1457-1458 (1995)).
- Antagonist according to the present invention include soluble forms of the TR2 receptors (e.g., fragments of the TR2 receptor shown in FIG. 1A-1B that include the ligand binding domain from the extracellular region of the full length receptor). Such soluble forms of the receptor, which may be naturally occurring or synthetic, antagonize TR2, TR2-SV1 or TR2-SV2 mediated signaling by competing with the cell surface bound forms of the receptor for binding to TNF- family ligands.
- Antagonists of the present invention also include antibodies specific for TNF-family ligands and TR2-Fc fusion proteins such as the one described below in Examples 5 and 6.
- TNF-family ligand is intended naturally occurring, recombinant, and synthetic ligands that are capable of binding to a member of the TNF receptor family and inducing the ligand/receptor signaling pathway.
- TNF ligand family include, but are not limited to, TNF- ⁇ , lymphotoxin- ⁇ (LT- ⁇ , also known as TNF- ⁇ ), LT- ⁇ (found in complex heterotrimer LT- ⁇ 2- ⁇ ), FasL, CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF).
- Example 6 demonstrate that the TR2 receptors of the present invention are capable of inducing the proliferation of lymphocytes. Further, such proliferation can be inhibited by a TR2 protein fragment fused to an Fc antibody fragment.
- TR2 receptors of the present invention include both TR2 amino acid sequences and antibodies capable of preventing TNFR mediated viral entry into cells. Such sequences and antibodies can function by either competing with cell surface localized TNFR for binding to virus or by directly blocking binding of virus to cell surface receptors.
- Antibodies according to the present invention may be prepared by any of a variety of methods using TR2 receptor immunogens of the present invention.
- TR2 receptor immunogens include the TR2 receptor protein shown in FIG. 1A-1B (SEQ ED NO:2) and the TR2-SV1 (FIG. 4A-4B (SEQ ID NO: 5)) and TR2-SV2 (FIG. 7A-7B (SEQ ED NO: 8)) polypeptides (any of which may or may not include a leader sequence) and polypeptide fragments of the receptors comprising the ligand binding, extracellular, transmembrane, the intracellular domains of the TR2 receptors, or any combination thereof.
- mAb monoclonal antibody
- Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl Med. 24:3 X6-325 (1983)).
- antibodies according to the present invention are mAbs.
- Such mAbs can be prepared using hybridoma technology (Kohler and Millstein, Nature 256:495-491 (1975) and U.S. Patent No. 4,376, 110; Hariow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyses, Plenum Press, New York, NY, 1980; Campbell, "Monoclonal Antibody Technology," In: Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon et al, eds.), Elsevier,
- Proteins and other compounds which bind the TR2 receptor domains are also candidate agonist and antagonist according to the present invention.
- Such binding compounds can be "captured” using the yeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989)).
- a modified version of the yeast two- hybrid system has been described by Roger Brent and his colleagues (Gyuris, J. et al, Cell 75:791-803 (1993); Zervos, A.S. et al, Cell 72:223-232 (1993)).
- the yeast two-hybrid system is used according to the present invention to capture compounds which bind to the ligand binding, extracellular, intracellular, and transmembrane domains of the TR2 receptors.
- Such compounds are good candidate agonist and antagonist of the present invention.
- the intracellular domain of the TR2 receptor may be used to identify cellular proteins which interact with the receptor in vivo.
- Such an assay may also be used to identify ligands with potential agonistic or antagonistic activity of TR2 receptor function.
- This screening assay has previously been used to identify protein which interact with the cytoplasmic domain of the murine TNF-RII and led to the identification of two receptor associated proteins. Rothe, M. et al, Cell 75:681 (1994).
- proteins and amino acid sequences which bind to the cytoplasmic domain of the TR2 receptors are good candidate agonist and antagonist of the present invention.
- screening techniques include the use of cells which express the polypeptide of the present invention (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, 24(5: 181-296 (1989).
- potential agonists or antagonists may be contacted with a cell which expresses the polypeptide of the present invention and a second messenger response, e.g., signal transduction may be measured to determine whether the potential antagonist or agonist is effective.
- the TR2 receptor agonists may be employed to stimulate ligand activities, such as inhibition of tumor growth and necrosis of certain transplantable tumors.
- the agonists may also be employed to stimulate cellular differentiation, for example, T-cell, fibroblasts and hemopoietic cell differentiation.
- Agonists to the TR2 receptor may also augment TR2's role in the host's defense against microorganisms and prevent related diseases (infections such as that from Listeria monocytogenes) and Chlamidiae.
- the agonists may also be employed to protect against the deleterious effects of ionizing radiation produced during a course of radiotherapy, such as denaturation of enzymes, lipid peroxidation, and DNA damage.
- Agonists to the receptor polypeptides of the present invention may be used to augment TNF's role in host defenses against microorganisms and prevent related diseases.
- the agonists may also be employed to protect against the deleterious effects of ionizing radiation produced during a course of radiotherapy, such as denaturation of enzymes, lipid peroxidation, and DNA damage.
- the agonists may also be employed to mediate an anti-viral response, to regulate growth, to mediate the immune response and to treat immunodeficiencies related to diseases such as FLTV by increasing the rate of lymphocyte proliferation and differentiation.
- the antagonists to the polypeptides of the present invention may be employed to inhibit ligand activities, such as stimulation of tumor growth and necrosis of certain transplantable tumors.
- the antagonists may also be employed to inhibit cellular differentiation, for example, T-cell, fibroblasts and hemopoietic cell differentiation.
- Antagonists may also be employed to treat autoimmune diseases, for example, graft versus host rejection and allograft rejection, and T-cell mediated autoimmune diseases such as AEDS. It has been shown that T-cell proliferation is stimulated via a type 2 TNF receptor. Accordingly, antagonizing the receptor may prevent the proliferation of T-cells and treat T-cell mediated autoimmune diseases.
- HIV-induced apoptotic cell death has been demonstrated not only in vitro but also, more importantly, in infected individuals (Ameisen, J.C., AIDS 5:1197-1213 (1994) ; Finkel, T.H., and Banda, N.K., Curr. Opin. Immunol (5:605-615(1995); Muro-Cacho, CA. et al, J. Immunol 154:5555-5566 (1995)). Furthermore, apoptosis and CD4 + T-lymphocyte depletion is tightly correlated in different animal models of AIDS (Brunner, T., et al, Nature 373:441-444 (1995); Gougeon, M.L., et al, AIDS Res.
- a method for treating HIV + individuals involves administering an agonist of the present invention to increase the rate of proliferation and differentiation of CD4 + T-lymphocytes.
- Such agonists include agents capable of inducing the expression of TR2 receptors (e.g.,
- TNF ⁇ TNF ⁇ , PMA and DMSO
- Modes of administration and dosages are discussed in detail below.
- the immune system of the recipient animal In rejection of an allograft, the immune system of the recipient animal has not previously been primed to respond because the immune system for the most part is only primed by environmental antigens. Tissues from other members of the same species have not been presented in the same way that, for example, viruses and bacteria have been presented.
- immunosuppressive regimens are designed to prevent the immune system from reaching the effector stage.
- the immune profile of xenograft rejection may resemble disease recurrence more that allograft rejection.
- the immune system In the case of disease recurrence, the immune system has already been activated, as evidenced by destruction of the native islet cells. Therefore, in disease recurrence the immune system is already at the effector stage.
- Antagonists of the present invention are able to suppress the immune response to both allografts and xenografts by decreasing the rate of TR2 mediated lymphocyte proliferation and differentiation.
- Such antagonists include the TR2-Fc fusion protein described in Examples 5 and 6.
- the present invention further provides a method for suppression of immune responses.
- TNF ⁇ has been shown to prevent diabetes in strains of animals which are prone to this affliction resulting from autoimmunity. See Porter, A., Tibtech 9: 158-162 (1991).
- agonists and antagonists of the present invention may be useful in the treatment of autoimmune diseases such as type 1 diabetes.
- TR2 receptors may in some circumstances induce an inflammatory response, and antagonists may be useful reagents for blocking this response.
- TR2 receptor antagonists e.g., soluble forms of the TR2 receptors; neutralizing antibodies
- TR2 receptor antagonists may be useful for treating inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, psoriasis, septicemia, and inflammatory bowel disease.
- Antagonists to the TR2 receptor may also be employed to treat and/or prevent septic shock, which remains a critical clinical condition. Septic shock results from an exaggerated host response, mediated by protein factors such as TNF and LL-1, rather than from a pathogen directly. For example, lipopolysaccharides have been shown to elicit the release of TNF leading to a strong and transient increase of its serum concentration. TNF causes shock and tissue injury when administered in excessive amounts. Accordingly, it is believed that antagonists to the TR2 receptor will block the actions of TNF and treat/prevent septic shock. These antagonists may also be employed to treat meningococcemia in children which correlates with high serum levels of TNF.
- TR2 receptor antagonists there are included, inflammation which is mediated by TNF receptor ligands, and the bacterial infections cachexia and cerebral malaria.
- TR2 receptor antagonists may also be employed to treat inflammation mediated by ligands to the receptor such as TNF.
- the agonist or antagonists described herein can be administered in vitro, ex vivo, or in vivo to cells which express the receptor of the present invention.
- administration of an "effective amount" of an agonist or antagonist is intended an amount of the compound that is sufficient to enhance or inhibit a cellular response to a TNF-family ligand and include polypeptides.
- administration of an "effective amount” of an agonist or antagonists is intended an amount effective to enhance or inhibit TR2 receptor mediated activity.
- an agonist according to the present invention can be co-administered with a TNF-family ligand.
- an agonist or antagonist can be determined empirically and may be employed in pure form or in pharmaceutically acceptable salt, ester or pro-drug form.
- the agonist or antagonist may be administered in compositions in combination with one or more pharmaceutically acceptable excipients.
- the total pharmaceutically effective amount of a TR2 polypeptide administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone.
- the TR2 polypeptide is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed.
- compositions containing the TR2 receptor polypeptides of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
- pharmaceutically acceptable carrier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
- parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
- the bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311). pQE60 encodes ampicillin antibiotic resistance ("Amp r ”) and contains a bacterial origin of replication ("ori”), an IPTG inducible promoter, a ribosome binding site
- RBS nickel-nitrilo-tri-acetic acid
- Ni-NTA nickel-nitrilo-tri-acetic acid
- the polypeptide coding sequence is inserted such that translation of the six His codons is prevented and, therefore, the polypeptide is produced with no 6 X His tag.
- the DNA sequence encoding the desired portion of the TR2 protein lacking the hydrophobic leader sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers which anneal to the amino terminal sequences of the desired portion of the TR2 protein and to sequences in the deposited construct 3' to the cDNA coding sequence. Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively.
- the 5' primer has the sequence:
- 5' CGCCCATGGCCCCAGCTCTGCCGTCCT 3' (SEQ ID NO: 14) containing the underlined Ncol restriction site followed by 18 nucleotides complementary to the amino terminal coding sequence of the mature TR2 sequence in FIG. 1 A- IB.
- the 3' primer has the sequence:
- the amplified TR2 DNA fragments and the vector pQE60 are digested with Ncol and HindLU and the digested DNAs are then ligated together. Insertion of the TR2 DNA into the restricted pQE60 vector places the TR2 protein coding region including its associated stop codon downstream from the EPTG-inducible promoter and in-frame with an initiating AUG. The associated stop codon prevents translation of the six histidine codons downstream of the insertion point.
- E. coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kan r "), is used in carrying out the illustrative example described herein.
- This strain which is only one of many that are suitable for expressing TR2 protein, is available commercially from QIAGEN, Inc., supra. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.
- Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
- the O/N culture is used to inoculate a large culture, at a dilution of approximately 1:25 to 1 :250.
- the cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
- Isopropyl-b-D- thiogalactopyranoside (“LPTG”) is then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter, by inactivating the lad repressor.
- Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation. The cells are then stirred for 3-4 hours at 4° C in 6 M guanidine-HCl, pH
- the cell debris is removed by centrifugation, and the supernatant containing the TR2 is dialyzed against 50 mM Na-acetate buffer pH 6, supplemented with 200 mMNaCl.
- the protein can be successfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH 7.4, containing protease inhibitors.
- the protein can be purified by ion exchange, hydrophobic interaction and size exclusion chromatography.
- an affinity chromatography step such as an antibody column can be used to obtain pure TR2 protein.
- the purified protein is stored at 4°C or frozen at -80°C.
- the plasmid shuttle vector pA2 GP was used to insert the cloned DNA encoding the mature extracellular domain of the TR2 receptor protein shown in FIG. 1A-1B, lacking its naturally associated secretory signal (leader) sequence, into a baculovirus.
- This protein was expressed using a baculovirus leader and standard methods as described in Summers et al, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
- This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the secretory signal peptide (leader) of the baculovirus gp67 protein and convenient restriction sites such as BamHI, Xbal and Asp718.
- the polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation.
- the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene.
- the inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that expresses the cloned polynucleotide.
- baculovirus vectors could be used in place of the vector above, such as pAc373, pVL941 and pAcEMl, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required.
- Such vectors are described, for instance, in Luckow et al, Virology 170:3 ⁇ -39.
- the plasmid was digested with the restriction enzymes BamHI and Asp718 dephosphorylated using calf intestinal phosphatase.
- the DNA was then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA was designated herein "VI”.
- E. coli HBIOI cells were transformed with the ligation mixture and spread on culture plates.
- Other suitable E. coli hosts such as XL-1
- plasmid was designated herein pBacTR2-T.
- pBacTR2-T Five ⁇ g of pBacTR2-T was co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA",
- the transfection mixture was added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate was then incubated for 5 hours at 27° C. After 5 hours the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum was added. The plate was put back into an incubator and cultivation was continued at 27 °C for four days.
- V- TR2-T V- TR2-T
- Sf9 cells were grown in Grace's medium supplemented with 10% heat inactivated FBS
- the cells were infected with the recombinant baculovirus V-TR2-T at a multiplicity of infection ("MOI") of about 2
- MOI multiplicity of infection
- the medium was removed and replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc , Rockville, MD)
- 5 ⁇ Ci of 35 S-methionine and 5 ⁇ Ci 35 S- cysteine available from Amersham
- the cells were further incubated for 16 hours and then they were harvested by centrifugation
- the proteins in the supernatant as well as the intracellular proteins were analyzed by SDS-PAGE followed by autoradiography Microsequencing of the amino acid sequence of the amino terminus of purified protein was used to determine the amino terminal sequence of the mature protein and thus the cleavage point and length of the secretory signal peptide
- the plasmid shuttle vector pA2 was used to insert the cloned DNA encoding the complete protein, including its naturally associated secretary signal (leader) sequence, into a baculovirus to express the mature TR2 protein
- leader naturally associated secretary signal
- Other attributes of the pA2 vector are as described for the pA2 GP vector used in Example 2(a)
- the 5' primer has the sequence:
- 3' primer has the sequence:
- pBacTR2 5 ⁇ g of pBacTR2 was co-transfected with 1 ⁇ g of BaculoGoldTM (Pharmingen) viral DNA and 10 ⁇ l of LipofectinTM (Life Technologies, Inc.) in a total volume of 200 ⁇ l serum free media.
- the primary viruses were harvested at 4-5 days post-infection (pi), and used in plaque assays. Plaque purified viruses were subsequently amplified and frozen, as described in Example 2(a).
- Sf9 cells were seeded in 12 well dishes with 2.0 ml of a cell suspension containing 0.5 x 10 6 cells/ml and allowed to attach for 4 hours. Recombinant baculoviruses were used to infect the cells at an MOI of 1-2. After 4 hours, the media was replaced with 1.0 ml of serum free media depleted for methionine and cysteine (-Met/-Cys). At 3 days pi, the culture media was replaced with 0.5 ml -Met/-Cys containing 2 ⁇ Ci each [ 35 S]-Met and [ 3S S]-Cys.
- Cells were labeled for 16 hours after which the culture media was removed and clarified by centrifugation (Supernatant).
- the cells were lysed in the dish by addition of 0.2 ml lysis buffer (20 mM LTEPES, pH 7.9; 130 mM NaCl; 0.2 mM EDTA; 0.5 mM DTT and 0.5% vol/vol NP-40) and then diluted up to 1.0 ml with dH 2 O (Cell Extract). 30 ⁇ l of each supernatant and cell extract were resolved by 15% SDS-PAGE. Protein gels were stained, destained, amplified, dried and autoradiographed. Labeled bands corresponding to the recombinant proteins were visible after 16-72 hours exposure.
- a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g.,
- Suitable expression vectors for use in practicing the present invention include, for example, vectors such as PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC
- Mammalian host cells that could be used include, human HeLa 293, H9 and Jurkat cells, mouse NEH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
- the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
- a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
- the transfected gene can also be amplified to express large amounts of the encoded protein.
- the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
- Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy etal, Biochem J. 227:211-219 (1991); Bebbington et al,
- the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al, Molecular and Cellular Biology, 438- 447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al, Cell 47:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest.
- the vectors contain in addition the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
- the expression plasmid, pTR2 HA is made by cloning a cDNA encoding TR2 into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.).
- the expression vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate purification) followed by a termination codon and polyadenylation signal arranged so that a cDNA can be conveniently placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker
- the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al, Cell 37:161 (1984) The fusion of the HA tag to the target protein
- a DNA fragment encoding a TR2 is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter
- the plasmid construction strategy is as follows
- the TR2 cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above for construction of vectors for expression of TR2 in E. coh.
- Suitable primers include the following, which are used in this example
- the 5' primer, containing the underlined BamHI site, a Kozak sequence, an AUG start codon and 6 additional codons of the 5' coding region of the complete TR2 has the following sequence
- the 3' primer, containing the underlined Xbal site, a stop codon, HA tag, and 19 bp of 3' coding sequence has the following sequence (at the 3' end) 5' GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGT
- the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with BamHL and Xbal and then ligated
- the ligation mixture is transformed into E. coh strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies Plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other means for the presence of the TR2-encoding fragment
- COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al, Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). Cells are incubated under conditions for expression of TR2 by the vector.
- TR2-HA fusion protein is detected by radiolabeling and immunoprecipitation, using methods described in, for example Hariow et al, Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing 35 S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al cited above.
- Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
- Plasmid pC4 is used for the expression of TR2 protein.
- Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).
- the plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
- Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate.
- the amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F.
- Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al, Molecular and Cellular Biology, March 1985:438-447) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al, Cell 47:521-530 (1985)).
- LTR long terminal repeat
- CMV cytomegalovirus
- BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene.
- Other high efficiency promoters can also be used for the expression, e.g., the human ⁇ -actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
- Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the TR2 protein in a regulated way in mammalian cells (Gossen, M., & Bujard, H.
- telomere sequence For the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well. Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
- the plasmid pC4 is digested with the restriction enzymes BamHI and Asp718 and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated from a 1% agarose gel.
- the DNA sequence encoding the complete TR2 protein including its leader sequence is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
- the 5' primer has the sequence:
- 5' GCGCGGATCCACCATGGAGCCTCCTGGAGACTGG 3' (SEQ ID NO:22) containing the underlined BamHI restriction enzyme site followed by an efficient signal for initiation of translation in eukaryotes, as described by Kozak, M., J. Mol. Biol. 196:941-950 (1987), and 21 bases of the coding sequence of TR2 protein shown in FIG. 1 A-IB (SEQ LD NO: 1).
- the 3' primer has the sequence: 5' GCGCGGTACCTCTACCCCAGCAGGGGCGCCA 3' (SEQ LD NO: 19) containing the underlined Asp718 restriction site followed by 21 nucleotides complementary to the non-translated region of the TR2 gene shown in FIG. 1 A- IB (SEQ ID NO: 1).
- the amplified fragment is digested with the endonucleases BamHI and Asp718 and then purified again on a 1% agarose gel.
- the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
- E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
- Chinese hamster ovary cells lacking an active DHFR gene are used for transfection.
- 5 ⁇ g of the expression plasmid pC4 is cotransfected with 0.5 ⁇ g of the plasmid pSV2-neo using lipofectin (Feigner et al, supra).
- the plasmid pSV2- neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
- the cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
- the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).
- methotrexate 50 nM, 100 nM, 200 nM, 400 nM, 800 nM.
- Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 ⁇ M, 2 ⁇ M, 5 ⁇ M, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 ⁇ M. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.
- Northern blot analysis is carried out to examine TR2 gene expression in human tissues, using methods described by, among others, Sambrook et al, cited above.
- a cDNA probe containing the entire nucleotide sequence of the TR2 protein (SEQ ED NO: 1) is labeled with 32 P using the r ⁇ / ⁇ rimeTM DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN- 100TM column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT 1200- 1. The purified labeled probe is then used to examine various human tissues for TR2 mRNA.
- the putative transmembrane domain of translated TR2 receptor was determined by hydrophobicity using the method of Goldman et al. (Ann. Rev. of
- TR2-Fclink plasmid The PCR product was digested with EcoRI and Asp718I and ligated into the COSFclink plasmid (Johansen, et al, J. Biol. Chem. 270:9459-9471 (1995)) to produce TR2-
- COS cells were transiently transfected with TR2-Fclink and the resulting supernatant was immunoprecipitated with protein A agarose.
- Western blot analysis of the immunoprecipitate using goat anti-human Fc antibodies revealed a strong band consistent with the expected size for glycosylated TR2-Fc (greater than 47.5 kD).
- a 15L transient COS transfection was performed and the resulting supernatant was purified (see below). The purified protein was used to immunize mice following DNA injection for the production of mAbs.
- CHO cells were transfected with TR2-Fclink to produce stable cell lines. Five lines were chosen by dot blot analysis for expansion and were adapted to shaker flasks.
- TR2-Fc protein expression was identified by Western blot analysis. TR2-Fc protein purified from the supernatant of this line was used for cell binding studies by flow cytometry, either as intact protein or after factor Xa cleavage and biotinylation (see below).
- Clone HTXBS40 is an allelic variant of TR2 which differs from the sequence shown in FIG. 1A-1B (SEQ ID NO.T) in that HTXBS40 contains guanine at nucleotide 314, thymine at nucleotide 386 and cytosine at nucleotide 627.
- a plasmid suitable for expression of the extracellular domain of TR2 was constructed as follows to immunize mice for the production of anti-TR2 mAbs.
- the Fc fragment was removed from TR2-Fclink by a Bglll/Xbal digestion, Klenow was used to fill in the overhangs, and the blunt ends of the plasmid were religated.
- the resulting frame shift introduced a stop codon immediately following the amino acids which had originally been introduced into TR2-Fclink by the addition of the Bgi ⁇ site.
- the C terminus of the extracellular domain of TR2 is followed by only 2 amino acids (RS) in this constructed (TR2exlink).
- SDS-PAGE gels run under reducing and non-reducing conditions. Protein concentration was monitored by A 280 assuming an extinction coefficient of 0.7 for the receptor and 1.28 for the chimera, both calculated from the sequence.
- the nonbound fraction from the Protein G column was concentrated in a Centricon 10K cell (Amicon) to about 1 ml to a final concentration of 3.5 mg/ml estimated by A 280 , extinction coefficient 0.7.
- the concentrated sample was diluted to 5 ml with 20 mM sodium phosphate, pH 6 and applied to a 0.5 X 5 cm Mono S column equilibrated in 20 mM sodium phosphate, pH 6 at a linear flow rate of 300 cm/h.
- the column was washed with 20 mM sodium phosphate, pH 6 and eluted with a 20 column volume linear gradient of 20 mM sodium phosphate, pH 6 to 20 mM sodium phosphate,
- the 3 ml nonbound fraction from the Mono S column was concentrated to 1 ml as above using a Centricon 10K cell and dialyze against 20 mM sodium phosphate, 150 mM sodium chloride, pH 7. The concentration following dialysis was 2.1 mg/ml.
- TR2 0.5 mg was dialyzed against 100 mM borate, pH 8.5. A 20-fold molar excess of NHS-LC Biotin was added and the mixture was left on a rotator overnight at 4°C. The biotinylated TR2 was dialyzed against. 20 mM sodium phosphate, 150 mM sodium chloride, pH 7, sterile filtered and stored at -70°C. Biotinylation was demonstrated on a Western blot probed with strepavidin HRP and subsequently developed with ECL reagent.
- the Membrane Bound Form of the TR2 Receptor is a TNFR which Induces Lymphocytes Proliferation and Differentiation
- TNFR tumor necrosis factor
- NGFR nerve growth factor receptor
- receptors contain a hinge-like region immediately adjacent to the transmembrane domain, characterized by a lack of cysteine residues and a high proportion of serine, threonine, and proline, which are likely to be glycosylated with O-linked sugars.
- a cytoplasmic part of these molecules shows limited sequence similarities - a finding which may be the basis for diverse cellular signaling.
- the members identified from human cells include CD40 (Stamenkovic, I. et al, EMBO J. 5: 1403 (1989)), 4-1BB (Kwon, B.S. and Weissman, S.M., Proc. Natl. Acad. Sci.
- EST expressed sequence tag
- cDNA data base obtained over 500 different cDNA libraries (Adams, M.D. et al, Science 252:1651 (1991); Adams, M.D. et al, Nature 355:632 (1992)), was screened for sequence similarity with cysteine-rich motif of the TNFR superfamily, using the blastn and tblastn algorithms (Altschul, S.F. et al, J. Mol. Biol 275:403 (1990)).
- One EST (HT1SB52 - ATCC Accession No. 97059) was identified in a human T cell line library which showed significant identity to TNFR-II at the amino acid level. This sequence was used to clone the missing 5' end by RACE (rapid amplification of cDNA ends) using a 5'-RACE-ready cDNA of human leukocytes (Clontech,
- the myeloid and B-cell lines studied represent cell types at different stages of the differentiation pathway.
- KGla and PLB 985 (Koeffler, H. et al, Blood 56:265 (1980); Tucker, K. et al, Blood 70:312 (1987)) were obtained from Phillip Koeffler (UCLA School of Medicine)
- BJA-B was from Z. Jonak (SmithKline Beecham)
- TF 274 a stromal cell line exhibiting osteoblastic features, was generated from the bone marrow of a healthy male donor (Tan & Jonak, unpublished). All of the other cell lines were obtained from the American Type Culture Collection (Rockville, MD).
- Monocytes were prepared by differential centrifugation of peripheral blood mononuclear cells (PBMC) and adhesion to tissue culture dish.
- PBMC peripheral blood mononuclear cells
- PBMC PBMC by immunomagnetic beads (Dynal, Lake Success, NY). Endothelial cells from human coronary artery were purchased from clonetics (Clonetics, CA).
- RNA of adult tissues was purchases from Clontech (Palo Alto, CA), or extracted from primary cells and cell lines with TriReagent (Molecular
- RNA was fractionated in a 1% agarose gel containing formaldehyde, as described (Sambrook et al, Molecular Cloning, Cold Springs Harbor (1989)) and transferred quantitatively to Zeta-probe nylon membrane (Biorad, Hercules, CA) by vacuum-blotting. The blots were prehybridized, hybridized with 32 P-labeled
- XhoI/EcoRI fragment of TR2 or OX-40 probe washed under stringent conditions and exposed to X-ray films.
- High molecular weight human DNA was digested with various restriction enzymes and fractionated in 0 8% agarose gel The DNA was denatured, neutralized and transferred to nylon membrane and hybridized to 32 P-labeled TR-2 or its variant cDNA
- TR2-Fc Fusion Proteins The 5' portion of the TR2 containing the entire putative open reading frame of extracellular domain was amplified by polymerase chain reaction (Saiki, R K et al, Science 239 487 (1988)) For correctly oriented cloning, a Hindlll site on the 5' end of the forward primer and a Bglll site on the 5' end of the reverse primer were created.
- the Fc portion of human IgG j was PCR-amplified from ARH-77 (ATCC) cell RNA and cloned in Smal site of pGem7 vector
- the Fc fragment including hinge, CH 2 , and CH 3 domain sequences contained a Bglll site at its 5' end and an Xhol site at its 3' end
- the Hindlll- Bglll fragment of TR2 cDNA was inserted into the upstream of human IgG,Fc and an in frame fusion was confirmed by sequencing
- the TR2-Fc fragment was released by digesting the plasmid with Hindlll-Xhol and cloned it into pcDNA3 expression plasmid
- the TR2-Fc plasmid, linearized with Pvul, was transferred into NIH 3T3 by the calcium phosphate co-precipitation method After selection in 400 ⁇ g/ml G418, neomycin-resistant colonies were picked and expanded ELISA with anti- human IgG j and Northern analysis with 32 P-labeled TR2 probe were used to select clones that produce high levels of TR2-Fc in the supernatant
- PBMC peripheral blood mononuclear cells
- TR2 is a New Member of the TNFR Superfamily
- FIG 1A-1B shows the amino acid sequence of TR2 deduced from the longest open reading frame of one of the isolated cDNAs (HLHAB49) Comparison with other sequenced cDNAs and ESTs in the database indicated potential allelic variants which resulted in amino acid changes at positions 17 (either Arg or Lys) and 41 (either Ser or Phe) of the protein sequence shown in FIG 1 A- IB (amino acid residues -20 and 5 in SEQ LD NO 2) The open reading frame encodes 283 amino acids with a calculated molecular weight of 30,417
- the TR2 protein was expected to be a receptor Therefore, the potential signal sequence and transmembrane domain were sought A hydrophobic stretch of 23 amino acids towards the C terminus (amino acids 201-225) (FIG 1 A- IB) was assigned as a transmembrane domain because it made a potentially single helical span, but the signal sequence was less obvious
- the potential ectodomain TR2 was expressed in NTH 3T3 and
- TR2 Using a polyclonal rabbit antibody raised to TR2, the molecular size of natural TR2 was determined to be 38 kD by Western analysis Since the protein backbone of processed TR2 would be composed of 247 amino acids with an Mr of 26,000, the protein must be modified post-translationally Two potential asparagine-linked glycosylation sites are located at amino acid positions 110 and
- TR2 contains the characteristic cysteine-rich motifs which have been shown by X-ray crystallography (Banner et al, Cell 73 431 (1993)) to represent a repetitive structural unit (Banner, D W et al, Cell 73 431 (1993))
- FIG 16 shows the potential TNFR domain aligned among TR2 (SEQ ID NO 2), TNFR-I (SEQ LD
- TR2 contained two perfect TNFR domain and two imperfect ones
- TR2 cytoplasmic tail appears to be more closely related to those of CD40cy and 4-lBBcy, and does not contain the death domain seen in the Fas and TNFR-I intracellular domains. Although the homology is moderate, the Thr 266 ofTR2 is aUgned with Thr 233 of 4- IBB and Thr 254 of CD40. This may be significant because Inui et al, (Inui, S. et al, Eur. J. Immunol. 20 ⁇ 141 (1990)) found that Thr 254 was essential for CD40 signal transduction and when the Thr 254 of CD40 was mutated, the CD40 bd did not bind to the CD40cy (Hu, H.M.
- TR2 RNA was detected in several tissues with a relatively high level in the lung, spleen and thymus (Table 2) but was not detected by this method in the brain, liver or skeletal muscle (Table 2)
- TR-2 was also expressed in monocytes, CD19 + B cells, and resting or PMA plus PHA-treated CD4 + or CD8 ⁇ T cells It was only weakly expressed in bone marrow and endothelial cells (Tables 2 and 3), although expression was observed in the hematopoietic cell line KGla (Table 2)
- OX-40 another member of the TNFR superfamily, was examined (Table 2) Unlike TR2, OX-40 was not detected in any tissues examined, and was detected only in activated T- cells and KGla
- Several cell lines were negative for TR2 expression, including TF 274 (bone marrow stromal), MG 63 and TE 85 (osteosarcoma), RL 95-2 (
- the FISH mapping procedure was applied to localize the TR2 gene to a specific human chromosomal region.
- the assignment of a hybridization signal to the short arm of chromosome 1 was obtained with the aid of DAPI banding.
- the TR2 position is in close proximity with CD30 (Smith, C A et al, Cell 73 1349-1360 (1993), 4-lBB (Kwon, B S et al, J. Immunol 152 2256-2262 (1994), Goodwin, R G et al, Eur. J Immunol.
- TR2 TNF receptor superfamily
- ADDRESSEE Sterne, Kessler, Goldstein & Fox, P.L.L.C.
- MOLECULE TYPE DNA (genomic)
- TTC TCT CCC AAT GGG ACC CTG GAG GAA TGT CAG CAC CAG ACC AAG TGC 819 Phe Ser Pro Asn Gly Thr Leu Glu Glu Cys Gin His Gin Thr Lys Cys 135 140 145
- Gly Thr Tyr lie Ala His Leu Asn Gly Leu Ser Lys Cys Leu Gin Cys 45 50 55 60
- MOLECULE TYPE DNA (genomic)
- CCCCCTTCTA CAGGAAACCC GGAGTGGACT GGAACGGTGC AGGGGGAGAA CTCGCCCCTC 60
- AGA TCC ACC CCC AGA ACC GAC GTC TTG AGG CTG GTG CTG TAT CTC ACC 456 Arg Ser Thr Pro Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr -20 -15 -10
- MOLECULE TYPE protein
- SEQUENCE DESCRIPTION SEQ ID NO : 5 :
- MOLECULE TYPE DNA (genomic)
- CTGCAAGCCC TCGTCCCACA CGCAGCTCTG CCGTCCCTTG GTGTCCCTCC CGGCCTCAGG 240
- CTTCTCTCCC CTCTCCCTCT GCCGTCCTGT CTCCTGTGGC CAGTCTCTCC TTGTTTCTCT 1394
- CTCTCCCACG TCCTCGGCCC
- TTGTTTGCTC CACAGTTGGC CTAATCATAT GTGTGAAAAG AAGAAAGCCA AGGGGTGATG 1754
- MOLECULE TYPE protein
- MOLECULE TYPE protein
- MOLECULE TYPE cDNA
- MOLECULE TYPE cDNA
- the applicant hereby request that, until the application has been laid open to public inspection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.
- the request to this effect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I of the PCT Applicant's Guide). If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Swedish Patent office or any person approved by the applicant in the individual case.
- the applicant hereby request that, until the application has been laid open to public inspection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.
- the request to this effect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I of the PCT Applicant's Guide). If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Swedish Patent office or any person approved by the applicant in the individual case.
- the applicant hereby request that, until the application has been laid open to public inspection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.
- the request to this effect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I of the PCT Applicant's Guide). If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Swedish Patent office or any person approved by the applicant in the individual case.
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Abstract
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JP10520413A JP2001502912A (en) | 1996-10-30 | 1996-10-30 | Human tumor necrosis factor receptor-like 2 |
EP96942776A EP0961782A4 (en) | 1996-10-30 | 1996-10-30 | Human tumor necrosis factor receptor-like 2 |
PCT/US1996/018540 WO1998018824A1 (en) | 1996-10-30 | 1996-10-30 | Human tumor necrosis factor receptor-like 2 |
CA2270913A CA2270913C (en) | 1996-10-30 | 1996-10-30 | Human tumor necrosis factor receptor-like 2 |
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PCT/US1996/018540 WO1998018824A1 (en) | 1996-10-30 | 1996-10-30 | Human tumor necrosis factor receptor-like 2 |
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WO1998018824A9 true WO1998018824A9 (en) | 1999-09-30 |
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EP (1) | EP0961782A4 (en) |
JP (1) | JP2001502912A (en) |
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US7427492B1 (en) | 1995-06-05 | 2008-09-23 | Human Genome Sciences, Inc. | Polynucleotides encoding human tumor necrosis factor receptor-like2 |
US6291207B1 (en) | 1995-07-28 | 2001-09-18 | Northwestern University | Herpes virus entry receptor protein |
US7118742B2 (en) | 1997-07-07 | 2006-10-10 | La Jolla Institute For Allergy And Immunology | Ligand for herpes simplex virus entry mediator and methods of use |
US6140467A (en) * | 1997-07-07 | 2000-10-31 | La Jolla Institute For Allergy And Immunology | Ligand for herpes simplex virus entry mediator and methods of use |
US6998108B1 (en) | 1997-07-07 | 2006-02-14 | La Jolla Institute For Allergy And Immunology | Antibodies to p30 polypeptides and methods making and using same |
US6346388B1 (en) | 1997-08-13 | 2002-02-12 | Smithkline Beecham Corporation | Method of identifying agonist and antagonists for tumor necrosis related receptors TR1 and TR2 |
CA2340686A1 (en) * | 1998-09-03 | 2000-03-16 | Samantha J. Busfield | Novel molecules of the herpes virus-entry-mediator-related protein family and uses thereof |
US6287808B1 (en) | 1998-09-03 | 2001-09-11 | Millennium Pharmaceuticals, Inc. | Molecules of the herpesvirus-entry-mediator-related protein family and uses thereof |
TR200504220T2 (en) | 1998-12-17 | 2007-04-24 | Biogen Idec Ma Inc. | Active lymphotoxin-beta receptor immunoglobulin chime A method for high level expression and purification of purified protein proteins and a method for purification of active lymphotoxin-beta receptor immunoglobulin chimeric proteins. |
CA2399388A1 (en) | 2000-02-11 | 2001-08-16 | Michael J. Lenardo | Identification of a domain in the tumor necrosis factor receptor family that mediates pre-ligand receptor assembly and function |
WO2001079496A2 (en) * | 2000-03-13 | 2001-10-25 | La Jolla Institute For Allergy And Immunology | Ligand for herpes simplex virus entry mediator and methods of use |
EP1274840B1 (en) * | 2000-04-12 | 2007-06-20 | La Jolla Institute For Allergy And Immunology | Ligand for herpes simplex virus entry mediator and methods of use |
US20040235173A1 (en) * | 2000-07-03 | 2004-11-25 | Gala Design, Inc. | Production of host cells containing multiple integrating vectors by serial transduction |
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EP0393438B1 (en) * | 1989-04-21 | 2005-02-16 | Amgen Inc. | TNF-receptor, TNF-binding protein and DNA coding therefor |
US5395760A (en) * | 1989-09-05 | 1995-03-07 | Immunex Corporation | DNA encoding tumor necrosis factor-α and -β receptors |
US5464938A (en) * | 1990-04-09 | 1995-11-07 | Immunex Corporation | Isolated viral protein TNF antagonists |
JPH11507205A (en) * | 1995-04-27 | 1999-06-29 | ヒューマン・ジェノム・サイエンシズ・インコーポレイテッド | Human tumor necrosis factor receptor |
-
1996
- 1996-10-30 WO PCT/US1996/018540 patent/WO1998018824A1/en not_active Application Discontinuation
- 1996-10-30 JP JP10520413A patent/JP2001502912A/en not_active Ceased
- 1996-10-30 EP EP96942776A patent/EP0961782A4/en not_active Withdrawn
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EP0961782A4 (en) | 2003-07-16 |
CA2270913A1 (en) | 1998-05-07 |
JP2001502912A (en) | 2001-03-06 |
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