JP2002511268A - U4, a member of the hematopoietin receptor superfamily - Google Patents

Abstract

  (57) [Summary] Disclosed are polynucleotides encoding U4 hematopoietin receptor superfamily chains and fragments thereof. Also disclosed are U4 proteins and methods for their production.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION This invention relates to new members of the mammalian hematopoietin superfamily, including but not limited to human and murine receptor proteins, among other proteins, fragments and recombinant polynucleotides thereof. And cells useful for expression of such proteins.

BACKGROUND OF THE INVENTION Various regulatory molecules known as hematopoietin have been identified that are involved in the development and proliferation of various populations of hematopoiesis or blood cells. Most hematopoietins display specific biological activities by interacting with receptors on the target cell ice surface. Usually, cytokine receptors are composed of one, two or three chains. Many cytokine receptors and some cytokines, such as IL-12 p40, are members of the hematopoietin receptor superfamily of proteins. The identification of new members of the hematopoietin receptor superfamily may be useful in regulating hematopoiesis, regulating immune responses, and identifying other members of the hematopoietin superfamily, including cytokines and receptors. It is desirable to identify and determine DNA and protein sequences for previously unknown members of the hematopoietin receptor superfamily.

SUMMARY OF THE INVENTION According to the present invention, polynucleotides encoding the U4 hematopoietin receptor superfamily chain are disclosed, including but not limited to those of murine and human origin. In certain embodiments, the present invention relates to: (a) a nucleotide sequence from nucleotide 242 to nucleotide 1396 of SEQ ID NO: 4; (b) a nucleotide sequence from nucleotide 71 to nucleotide 1225 of SEQ ID NO: 6; A nucleotide sequence from nucleotide 136 to nucleotide 1401 of No. 9; (d) a nucleotide sequence altered from the sequence of the nucleotide sequence shown in (a), (b) or (c) as a result of the degeneracy of the genetic code (E) a nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide shown in (a) or (b); (f) a species homolog of the sequence shown in (a), (b) or (c); (G) the nucleoside shown in (a), (b) or (c), Provided is an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of allelic variants of the tide sequence.

[0004] Preferably, the nucleotide sequence encodes a protein having the biological activity of the U4 hematopoietin receptor superfamily chain. The nucleotide sequence may be operably linked to an expression control sequence. In a preferred embodiment, the polynucleotide is a nucleotide sequence from nucleotide 242 to nucleotide 1396 of SEQ ID NO: 4; nucleotide 122 to nucleotide 139 of SEQ ID NO: 4.
A nucleotide sequence from nucleotide 71 to nucleotide 1225 of SEQ ID NO: 6; or a nucleotide sequence from nucleotide 11 to nucleotide 1225 of SEQ ID NO: 6; or a nucleotide sequence from nucleotide 136 to nucleotide 1401 of SEQ ID NO: 9 Or a nucleotide sequence from nucleotide 247 to nucleotide 1401 of SEQ ID NO: 9.

[0005] The present invention also provides: (a) an amino acid sequence of SEQ ID NO: 5; (b) an amino acid sequence of amino acids 41 to 425 of SEQ ID NO: 5; (c) an amino acid sequence of SEQ ID NO: 7; (E) amino acid sequence of SEQ ID NO: 10; (f) amino acid sequence of amino acids 38 to 421 of SEQ ID NO: 10; and (g) U4 hematopoietin receptor Also provided is an isolated polynucleotide comprising a nucleotide sequence encoding a peptide or protein comprising an amino acid sequence selected from the group consisting of fragments of (a) to (f) having the biological activity of a superfamily chain. Other preferred embodiments include the amino acid sequence of SEQ ID NO: 5; amino acids 41 to 425 of SEQ ID NO: 5; the amino acid sequence of SEQ ID NO: 7; the amino acid sequence of amino acids 24 to 408 of SEQ ID NO: 7; : 10 amino acid sequence; and encodes the amino acid sequence of amino acids 38 to 421 of SEQ ID NO: 10.

[0006] Also provided is a host cell, preferably a mammalian cell, transformed with the polynucleotide of the present invention.

In another embodiment, the present invention provides a method for producing a U4 protein. The method comprises: (a) growing a culture of a host cell of the invention in a suitable medium; and (b) purifying human U4 protein from the culture. Also provided are proteins produced by these methods.

The present invention also provides: (a) the amino acid sequence of SEQ ID NO: 5; (b) the amino acid sequence of amino acids 41 to 425 of SEQ ID NO: 5; (c) the amino acid sequence of SEQ ID NO: 7; An amino acid sequence from amino acids 24 to 408 of SEQ ID NO: 7; and (e) an amino acid sequence selected from the group consisting of fragments of (a) to (d) having biological activity of the U4 hematopoietin receptor superfamily chain. Also provided is an isolated U4 protein comprising: Preferably, the protein is an amino acid sequence of SEQ ID NO: 5; an amino acid sequence of amino acids 41 to 425 of SEQ ID NO: 5; an amino acid sequence of SEQ ID NO: 7; or SEQ ID NO: 7.
The amino acid sequence from amino acids 24 to 408 of In another preferred embodiment, the specified amino acid sequence is part of a fusion protein (with an additional amino acid sequence not derived from U4). Preferred fusion proteins include an antibody fragment, such as an Fc fragment. Also provided is a pharmaceutical composition comprising a protein of the invention and a pharmaceutically acceptable carrier. Further, the present invention provides a composition comprising an antibody that specifically reacts with the protein of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present inventors have identified and provided for the first time a polynucleotide encoding a U4 hematopoietin receptor superfamily chain (not limited to a polynucleotide encoding murine and human U4). (Hereinafter “U4” or “U4
Protein)). A 79 amino acid region of the human IL-5 receptor (LMTNAFISIIDDLSKYDVQVRAAVSS
Using MCREAGLWSEWSQPIYVGNDEHKPLREWFVIVIMATICFILLIL, SEQ ID NO: 1), TBLA
The GenBank EST database was searched using the STN algorithm. EST W66776 with homology to this region was identified, suggesting that it may encode a novel hematopoietin receptor. Translation of the reverse complement of this EST using the GCG map program revealed a protein sequence in the second reading frame that contained a conserved WSXWS motif found in the hematopoietin receptor. However, a stop codon was also present at nucleotide 227 of the reading frame, indicating that the EST was not a novel hematopoietin receptor or that the DNA sequence in the EST was incorrect. To determine whether this EST sequence may be related to the hematopoietin receptor, we constructed a murine embryo library using an oligonucleotide probe whose sequence was CTTGGCTTGG AAGAGGAAAT CCTTGAGAGC (SEQ ID NO: 2). Screened. The full length cDNA clone U6-3 (1A) was identified and the complete sequence was obtained. The DNA sequence and deduced amino acid sequence for the murine protein are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively. The murine protein encodes a novel member of the hematopoietin receptor family. It contains a leader sequence, and conserved cysteine pairs characteristic of each family, PP, and WSXWS.
Has a motif. This clone has no transmembrane or cytoplasmic domain. Comparison of this clone with ESTs in GenBank juxtaposed revealed that the ESTs had a frameshift mutation. SEQ ID NO: 4 provides the nucleotide sequence of the cDNA encoding murine U4. SEQ ID NO: 5 provides the deduced amino acid sequence of the receptor chain, including the deduced signal sequence of amino acids 1-40. Mature mouse U4 has amino acids 41-38 of SEQ ID NO: 5.
It is thought to have three sequences.

To identify additional related sequences in GenBanbk, the W66776 sequence was used,
GenBank was searched using the BLASTN algorithm. A closely related EST from human genomic DNA, H14009, was identified. Next, a cDNA clone was isolated from a human cDNA library using the EST-derived oligonucleotide CTGAGCGTGC GCTGGGTGTC GCCAC (SEQ ID NO: 3). A cDNA clone (HU4-3B) encoding the full-length mature protein homolog was completely sequenced. This clone does not have a complete signal sequence, but encodes the entire putative full-length mature protein. The human clone has a DNA level of 85 relative to the mouse clone.
% Homology. The deduced amino acid sequence is 95% identical between human and rat. The nucleotide and amino acid sequences of human U4 are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively. SEQ ID NO: 6 provides the nucleotide sequence of the cDNA encoding human U4. SEQ ID NO: 7 provides the predicted amino acid sequence for the receptor chain, including the predicted signal sequence for amino acids 1-23. Mature human U4 has amino acids 24-38 of SEQ ID NO: 7.
It is believed to have an array of zeros. Murine and human clones were isolated from the American Type Cult on January 15, 1997.
ure Collection, accession numbers ATCC 98305 and ATC, respectively.
C 98036.

[0011] By replacing the human leader sequence with a murine leader sequence, or by extending the human leader sequence using amino acids 1-14 of the murine leader sequence (MPAGRPGPVA QSAR, SEQ ID NO: 8), the human U4 protein is Can be expressed. Furthermore, longer cDNA or genomic clones encoding the actual human leader can be isolated using the sequences disclosed herein. Using the human U4 cDNA sequence described above, a longer cDNA encoding the human U4 protein was isolated. The nucleotide sequence of this long clone is shown in SEQ ID NO: 9. The deduced amino acid sequence encoded thereby is shown in SEQ ID NO: 10. The long cDNA clone was transferred to the American Type Culture Coll.
and deposited with Accession No. ATCC 98688. Any form of the U4 protein that is less than full length is encompassed by the present invention and is referred to herein as "U4" or "U4 protein" together with the full length and mature form. Polynucleotide encoding full-length U4 protein (SEQ ID NO: 4 and SEQ ID NO: 6)
By expressing the corresponding fragment of the above, the U4 protein shorter than the full length may be produced. These corresponding polynucleotide fragments are also part of the present invention. Construction of suitable desired deletion mutants, site-directed mutagenesis The modified polynucleotides described above may be produced by standard molecular biology techniques or the polymerase chain reaction using appropriate oligonucleotide primers. For the purposes of the present invention, a protein has "the biological activity of the U4 hematopoietin receptor superfamily chain" if the protein has one or more of the biological activities of the corresponding mature U4 proteins. U4 or an active fragment thereof (U4 protein) may be fused to a carrier molecule such as an immunoglobulin. For example, the soluble form of U4 may be fused to the Fc portion of an immunoglobulin via a "linker" sequence. Other fusion proteins such as GSTLex-A or MBP may be used.

The present invention also provides an allelic variant of the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 6, ie, a U4 protein, preferably a protein having U4 biological activity, SEQ ID NO: 4 or Includes naturally occurring alternative forms of the isolated polynucleotide of SEQ ID NO: 6. Also encompassed by the present invention are isolated polynucleotides that hybridize under very stringent conditions (eg, 0.1 × SSC, 65 ° C.) to the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 6. Isolated polynucleotides that encode the U4 protein but differ from the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 6 due to the degeneracy of the genetic code are also encompassed by the present invention. Changes in the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 6 caused by point mutations or induced modifications are also included in the invention.

[0013] The present invention relates to murine and human U from other animal species, in particular from other mammalian species.
Polynucleotides encoding the four homologs are also provided. Probes or primers are generated from the murine or human sequences disclosed herein, eg, from a PBM of the corresponding species.
Species homologs can be identified and isolated by screening libraries from appropriate species, such as libraries constructed from Cs, thymus or testis.

[0014] pMT disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991)
The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as a 2 or pED expression vector to obtain the U4 protein by recombinant methods. Many suitable control sequences are known in the art. General methods for expressing recombinant proteins are also known and are described in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). "Operably linked," as defined herein, refers to the isolation of the present invention such that the U4 protein is expressed by a host cell transformed (transfected) with the linked polynucleotide / expression control sequence. It means that the polynucleotide and the expression control sequence are arranged in a vector or a cell. Many types of cells can serve as suitable host cells for U4 protein expression. Host cells include, for example, monkey COS cells, Chinese hamster ovary (CHO) cells, human kidney 293 cells, human epithelial A431 cells, human Colo205 cells,
T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells,
Cell lines, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK, Rat2, BaF derived from in vitro culture of primary tissues
3, 32D, FDCP-1, PC12, M1x or C2C12 cells. The U4 protein can also be produced using an insect expression system by operably linking the isolated polynucleotides of the present invention to appropriate control sequences in one or more insect expression vectors. Materials and methods for use in the baculovirus / insect cell expression system are commercially available, for example, in kit form from Invitrogen. San Diego, California, USA (MaxBac ^R kit), and Summers and Smith, Texas Agricultural.
Such methods as described in Experiment Station Bulletin No. 1555 (1987), which is deemed to be described herein by reference, are well known in the art. A soluble form of the U4 protein may be produced in insect cells using the appropriate isolated polynucleotide described above. Alternatively, the U4 protein can be produced in lower eukaryotes, such as yeast, or in prokaryotes, such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces, Candida, or yeast strains capable of expressing heterologous proteins. Include. Suitable bacterial strains include Escherichia coli, Bacillus subtilis (Ba
cillus subtilis), Salmonella typhimurium
Or a bacterial strain capable of expressing a heterologous protein. Expression in bacteria may form inclusion bodies containing the recombinant protein. Thus, regeneration of the recombinant protein may be necessary to obtain an active or more active substance. Several methods for obtaining correctly folded heterologous proteins from bacterial inclusions are known in the art. In general, these methods involve solubilizing the protein from the inclusion bodies and then completely denaturing the protein using a chaotropic agent. If a cysteine residue is present in the primary amino acid sequence of the protein, it must be regenerated in an environment (redox system) that correctly produces disulfide bonds. The general method of regeneration is Kohno, Meth. Enzym.,
185: 187-195 (1990). EP 0433225 and co-pending application USSN 0
8/163877 describes other suitable methods. Express the U4 protein of the invention as a product of a transgenic animal, for example, as a component of transgenic bovine, goat, pig or goat milk characterized by somatic or germ cells containing a nucleotide sequence encoding the U4 protein May be.

[0015] The U4 protein of the present invention may be produced by culturing the transformed host cell under culture conditions necessary for the expression of the desired protein. The expressed protein can then be purified from the medium or cell extract. The soluble form of the U4 protein of the invention may be purified from the conditioned medium. The U4 protein of the present invention in membrane-bound form can be purified by preparing a whole membrane fraction from the expressing cells and then extracting the membrane using a non-ionic masking agent such as Triton X-100. U4 protein can be purified using methods known to those skilled in the art. For example, a U4 protein of the present invention can be purified using a commercially available protein concentration filter such as Amicon or Millipore Pe.
It can be concentrated using an llicon ultrafiltration unit. After performing the concentration step,
The concentrate can be applied to a purification matrix, such as a gel filtration medium. Alternatively, an anion exchange resin such as suspended diethylaminoethyl (DEAE)
) Or a matrix or substrate having polyethyleneimine (PEI) groups. The matrix may be acrylamide, agarose, dextran, cellulose or any other type commonly used for protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred (eg, S-Sepharose ^R columns). Purification of the U4 protein from the culture supernatant includes, for example, one or more column steps with an affinity resin such as Concanavalin A-agarose, Heparin-Toyopearl ^R or Cibachrome Blue 3GA Sepharose ^R ; phenyl ether, butyl ether or propyl One or more steps involving hydrophobic interaction chromatography using a resin such as an ether; or immunoaffinity chromatography may be used. Finally, the U4 protein is purified using one or more reverse phase high quality liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, for example, silica gel with pendant methyl or other aliphatic groups. It can be further purified. Affinity columns containing antibodies to the U4 protein may be used for purification by known methods. Some or all of the above purification steps may be used in various combinations to obtain a substantially homogeneous isolated recombinant protein. Preferably, the isolated U is substantially free of other mammalian proteins.
4 proteins are purified. An agent capable of binding to the U4 protein may be screened using the U4 protein of the present invention. Binding assays using the desired binding protein, either immobilized or not, are well known in the art and can be used for this purpose with the U4 protein of the invention. Such agents may be identified using purified cell-based assays or purified protein-based (cell-free) assays. For example, the U4 protein may be immobilized on a carrier in a purified form, and binding or potential ligand for the purified U4 protein may be determined.

When mixed with a pharmaceutically acceptable carrier, U4 protein purified from cells or produced by recombinant methods can be used as a pharmaceutical composition. Such compositions may contain, in addition to the U4 protein and carrier, diluents, fillers, salts, buffers, stabilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic substance that does not interfere with the effectiveness of the biological activity of the active ingredient. The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the present invention comprises cytokine, lymphokine or M-CSF, GM-C
SF, TNF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7
, IL-8, IL-9, G-CSF, Meg-CSF; other hematopoietic factors such as stem cell factor and erythropoietin. The pharmaceutical composition may further contain other anti-inflammatory agents. Such additional factors and / or agents may be included in the pharmaceutical composition to achieve interaction with the isolated U4 protein, or may minimize side effects caused by the isolated U4 protein. Conversely, the isolated U4 protein is converted to specific cytokines, lymphokines, other hematopoietic factors, thrombolytic or antithrombotic factors,
Alternatively, side effects of cytokines, lymphokines, other hematopoietic factors, thrombolytic or antithrombotic factors, or anti-inflammatory agents may be included in the formulation of the anti-inflammatory agent. The pharmaceutical composition of the invention may be in the form of liposomes, in which, in addition to other pharmaceutically acceptable carriers, the isolated U4 protein is mixed with an amphipathic agent such as a lipid, The amphiphile is present as micelles, insoluble monolayers, liquid crystals or lamellar layers in aqueous solution. Lipids suitable for liposome formulations include monoglycerides, diglycerides, sulfatides, lysolectins, phospholipids, saponins, bile acids, and the like,
Not limited to these. Preparation of such liposome formulations is within the level of ordinary skill in the art, for example, US Pat. No. 4,235,871, US Pat. No. 4,501,728,
As disclosed in U.S. Patent Nos. 4,837,028 and 4,737,323, all of which are hereby incorporated by reference.

As used herein, the term “therapeutically effective amount” indicates a significant benefit to a patient, ie,
It refers to the total amount of each active ingredient or method of the pharmaceutical composition that exhibits treatment, cure, prevention or amelioration of an inflammatory response or condition, or that increases the rate of treatment, cure, prevention or amelioration of such condition. When referring to an individual active ingredient administered alone, the term is used for that ingredient only. When referring to a combination of active ingredients, the term is used for the total amount of active ingredients that produces a therapeutic effect, whether mixed, sequentially or simultaneously. In practicing the treatment method of the present invention, a therapeutically effective amount of the isolated U4 protein is administered to a mammal having the condition to be treated. The isolated U4 protein can be administered according to the methods of the present invention alone, or in combination with other therapies, such as treatment with cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, the isolated U4 protein can be administered simultaneously or sequentially with cytokines, lymphokines, other hematopoietic factors, thrombolytic or anti-thrombotic factors. . In the case of sequential administration, the attending physician
The appropriate order of administration of the isolated U4 protein in combination with lymphokines, other hematopoietic factors, thrombolytic or anti-thrombotic factors will be determined.

The administration of the isolated U4 protein used in the pharmaceutical composition or used in practicing the method of the present invention is performed by various conventional routes, such as oral feeding, inhalation, or dermal, subcutaneous, or intravenous injection. be able to. Intravenous administration to a patient is preferred. When a therapeutically effective amount of the isolated U4 protein is administered orally, the isolated U4 protein may be in a capsule,
It may be in the form of a powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may further contain a solid carrier such as gelatin or an adjuvant. Tablets, capsules, and powders contain about 5 to 95% isolated U4 protein, and preferably about 25 to 90% isolated U4 protein. When administered in liquid form, oils of animal or plant origin, such as water, petroleum, peanut oil, mineral oil, soybean oil or sesame oil, or synthetic oils and fats may be added. Liquid form pharmaceutical compositions may also contain physiological saline solution, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains about 0.5 to 90% by weight of isolated U4 protein, preferably about 1 to 5%.
Contains 0% isolated U4 protein.

When a therapeutically effective amount of the isolated U4 protein is administered by intravenous, dermal or subcutaneous injection, the isolated U4 protein will be in the form of a pyrogen-free parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions is within the skill of the art in regard to pH, isotonicity, stability and the like. Preferred pharmaceutical compositions for intravenous, dermal or subcutaneous injection are, in addition to isolated U4 protein, sodium chloride for injection, dextrose and sodium chloride for injection, Ringer's solution for injection, dextrose for injection and Ringer's lactate for injection (Lactated). It should contain an isotonic carrier such as Ringer's) or other carriers known in the art. The pharmaceutical compositions of the present invention may contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art. The amount of isolated U4 protein in the pharmaceutical composition of the invention will depend on the nature and severity of the condition to be treated and the nature of the treatment that the patient has already received. Ultimately, the attending physician will determine the amount of isolated U4 protein to treat each patient. Initially, the attending physician will administer a low dose of isolated U4 protein and observe the patient's response. Higher doses of the isolated U4 protein can be administered to achieve the optimal effect on the patient, at which point the dose is not further increased. It is believed that various pharmaceutical compositions used in practicing the methods of the present invention should contain from about 0.1 μg to about 100 mg of isolated U4 protein per kg body weight. The duration of intravenous therapy using the pharmaceutical composition of the present invention will depend on the severity of the disease and condition to be treated and the potential unique response of each patient. Each application period of the isolated U4 protein will be a continuous intravenous dose ranging from 12 to 24 hours. Ultimately, the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.

The polynucleotides and proteins of the present invention will exhibit one or more of the following uses or biological activities, including those associated with the assays cited herein. The uses or activities described with respect to the proteins of the present invention may be due to the administration or use of such proteins, or the administration or use of polynucleotides encoding such proteins (eg, such as a vector suitable for gene therapy or DNA transfer). Can be provided.

Cytokines and Cell Proliferation / Differentiation Activity The proteins of the present invention may exhibit cytokine activity, cell proliferation activity (induction or inhibition) or cell differentiation activity (induction or inhibition), or produce other cytokines in certain cell populations. Can be induced. Many proteinaceous factors (including all known cytokines) that have been found to date have demonstrated activity in one or more factor-dependent cell proliferation assays, thus making the assay a convenient method of confirming cytokine activity. Serve as. The activity of the protein of the present invention is determined in cell lines (32D, DA
2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (pr
eB M +), 2E8, RB5, DA1, 123, T1165, HT2, CTL
(Including, but not limited to, L2, TF-1, Mo7e and CMK) are confirmed by any of a number of conventional factor-dependent cell proliferation assays. The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for T cell or thymocyte proliferation are described in Current Protocols in I.
mmunology, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM She
vach, W Strober, Pub.Green Publishing Associates and Wiley-Interscienc
e (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Ch
apter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137: 3
494-3500, 1986; Bertagnolli et al., J. Immunol. 145: 1706-1712, 1990; Ber
tagnolli et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli, et
al., J. Immunol. 149: 3778-3783, 1992; Bowman et al., J. Immunol. 152: 17
56-1761, 1994, including, but not limited to. Assays for cytokine production and / or proliferation of spleen cells, lymph node cells or thymocytes are described in Polyclonal T cell stimulation, Kruisbeek, AM
d Shevach, EM In Current Protocols in Immunology. JEea Coligan eds
Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Meas
urement of mouse and human Interferon g, Schreiber, RD In Current Pro
tocols in Immunology. JEea Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John
Includes, but is not limited to, those described in Wiley and Sons, Toronto. 1994. Assays for proliferation and differentiation of hematopoietic and lymphogenic cells are described in the Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottoml
y, K., Davis, LS and Lipsky, PE In Current Protocols in Immunology.
JEea Coligan eds.Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toron
to. 1991; deVries et al., J. Exp.Med. 173: 1205-1211, 1991; Moreau et al.
., Nature 336: 690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci.
USA 80: 2931-2938, 1983; Measurement of mouse and human interleukin 6
-Nordan, R. In Current Protocols in Immunology. JEea Coligan eds. V
ol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.1991; Smith et al.,
Proc. Natl. Acad. Sci. USA 83: 1857-1861, 1986; Measurement of human I
nterleukin 11-Bennett, F., Giannotti, J., Clark, SC and Turner, K. J
. In Current Protocols in Immunology. JEea Coligan eds. Vol 1 pp.
6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and hum
an Interleukin 9-Ciarletta, A., Giannotti, J., Clark, SC and Turner,
KJ In Current Protocols in Immunology. JEea Coligan eds. Vol 1 p
p. 6.13.1, including but not limited to those described in John Wiley and Sons, Toronto. 1991. Assays for the response of T cell clones to antigens, specifically identifying APC-T cell interactions as well as direct T cell effects by measuring proliferation and cytokine production, are described in Current Protocols in Immunology, Ed by JE.
Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W Strober, Pub.
Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro
assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their ce
llular receptors; Chapter 7, Immunologic studies in Humans); Weinberger
Natl. Acad. Sci. USA 77: 6091-6095, 1980; Weinberger et al.
, Eur. J. Immun. 11: 405-411, 1981; Takai et al., J. Immunol. 137: 3494-35.
00, 1986; Takai et al., J. Immunol. 140: 508-512, 1988, but are not limited thereto.

Immunostimulatory or Suppressive Activity The protein of the present invention may also exhibit immunostimulatory or immunosuppressive activity, including but not limited to the activity in the assays described herein. Protein is
It may be useful in a variety of deficiencies and disorders, including severe immunodeficiency complications (SCID), for example, in up-regulating or down-regulating T and / or B lymphocyte proliferation, and in N
It may be useful in activating the cytolytic activity of K cells and other cell populations. These immunodeficiencies can be genetic and can include viral (
For example, it may be caused by HIV) and bacterial or fungal infections, or may be caused by an autoimmune disease. More specifically, infectious diseases caused by viruses, bacteria, fungi or other infectious agents, for example, various fungal infections such as HIV, hepatitis virus, herpes virus, mycobacteria, Leishmania, malaria and Candida species, It can be treated using the protein of the present invention. Of course, in this regard, the proteins of the invention may be used where a boost to the immune system is needed, ie, in the treatment of cancer. Autoimmune diseases that may be treated using the protein of the present invention include, for example, multiple sclerosis,
Systemic deep elitomades, rheumatoid arthritis, autoimmune pneumonia, Guillain-Barre syndrome, autoimmune thyroiditis, insulin-dependent diabetes mellitus, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory Eye diseases. Such proteins of the invention may be used in the treatment of allergic reactions and conditions, such as asthma or other respiratory diseases. Other conditions in which immunosuppression is desired, including, for example, asthma (particularly allergic asthma) and related respiratory problems, may be treated with the proteins of the invention. Other conditions for which immunosuppression is desired (including, for example, organ transplantation)
May be treated using the protein of the present invention. The proteins of the present invention can also be used to enable an immune response in a number of ways. Down-regulation may be in the form of inhibiting or blocking an immune response already in progress, or may involve interfering with the induction of an immune response. The function of activated T cells may be inhibited by suppressing T cell responses, or by inducing specific resistance in T cells, or both. In general, immunosuppression of a T cell response is an active, non-antigen-specific process that requires continuous exposure of T cells to an inhibitor. Tolerance means non-responsiveness or anergy in T cells, and differs from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent is stopped.
Operationally, resistance may be indicated by a lack of a T cell response when exposed to a specific antigen in the absence of a tolerizing agent. Down-regulating or preventing one or more antigen functions, including but not limited to B-lymphocyte antigen functions (eg, such as B7), eg, high levels of lymphokines by activated T cells Interference with synthesis may be useful in tissue, skin and organ transplantation and graft versus host disease (GVHD). For example, blocking T cell function should reduce tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the graft is initiated by the recognition of the tissue piece as foreign by T cells, followed by an immune response that destroys the graft. A monomeric form having the activity of a molecule that inhibits or blocks the interaction of a B7 lymphocyte antigen with its native ligand on immune cells (another B lymphocyte antigen (eg, B7-1, B7-3)) Mixed with peptides,
Alternatively, pre-transplant administration of a soluble, monomeric form of a peptide with B7-2 activity alone, induces binding of the molecule to its native ligand on immune cells without transmitting a responsive costimulatory signal. there is a possibility. Blocking B lymphocyte antigen function in this regard prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may be sufficient to cause a loss of T cell response. The induction of long-term tolerance by B lymphocyte antigen blocking reagents may obviate the need for repeated administration of these blocking reagents. To achieve sufficient immunosuppression or resistance in a subject, it may also be necessary to block the function of the B lymphocyte antigen combination. The efficacy of individual blocking reagents in preventing organ transplant rejection or GVHD can be evaluated using animal models that can predict efficacy in humans. Examples of suitable systems that can be used include allogeneic heart grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which are used to test the immunosuppressive effects of CTLA4Ig fusion proteins in vivo. (Lensch
ow et al., Science 257: 789-792 (1992) and Turka et al., Proc Natl. Acad.
Sci. USA, 89: 11102-11105 (1992)). Furthermore, a rat model of GVHD (Paul ed., Fundamental Immunology, Ravan Press, New York, 1
989, pp. 846-847) can be used to determine the blocking effect of B lymphocyte antigen function on the progression of the disease in vivo. Also, blocking antigen function can be therapeutically useful for treating autoimmune diseases. Many autoimmune diseases are the result of inappropriate activation of T cells that are reactive to self tissue and promote the production of cytokines and autoantibodies that are involved in the pathology of the disease. Interfering with the activation of autoreactive T cells may reduce or eliminate the symptoms of the disease. Receptors for B lymphocyte antigens: autoantibodies or T cell-derived that inhibit T cell activation using administration of reagents that block T cell costimulation by disrupting ligand interactions and may be involved in the disease process Can interfere with the production of cytokines. In addition, blocking reagents can induce antigen-specific resistance of autoreactive T cells that can lead to long-term remission of the disease. The effectiveness of a blocking reagent in preventing or ameliorating an autoimmune disease can be determined using a number of well-characterized animal models for human autoimmune diseases. Examples are murine experimental autoimmune encephalitis, MRLlpr / lpr mice or N
Systemic deep erythematosus, murine autoimmune collagen arthritis in ZB hybrid mice, diabetes in NOD mice and BB rats, and experimental myasthenia gravis in rats (Paul ed., Fundamental Immunology, Raven Press,
New York, 1989, pp. 840-856). Up-regulation of antigen function (preferably B-lymphocyte antigen function) as a means to up-regulate the immune response is also therapeutically useful.
Up-regulation of the immune response may be in the form of promoting an existing immune response or eliminating the initial immune response. For example, enhancing an immune response by stimulating B lymphocyte antigen function may be useful in the case of a viral infection.
In addition, systemic viral diseases such as influenza, common cold, and encephalitis may be ameliorated by systemic administration of stimulatory forms of B lymphocyte antigen. Alternatively, the T cells are taken from a patient and co-stimulated in vitro with the viral antigen to which the ACPs expressing the peptide of the invention have been added, or combined with a stimulatory form of the soluble peptide of the invention and then in vitro. By reintroducing the T cells activated in the above into the patient, the antiviral immune response in the infected patient may be promoted. Another method of promoting an antiviral immune response is
The infected cells are removed from the patient and transfected with them a nucleic acid encoding a protein of the invention as described herein so that the cells express all or part of the protein on their surface. Will be re-introduced. The infected cells would then be able to transmit the costimulatory signal to the T cells in vivo and thereby activate the T cells. In another application, up-regulation or promotion of antigen function (preferably B lymphocyte antigen function) may be useful in inducing tumor immunity. Administering a tumor cell (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention to a tumor-specific tumor in the subject Can overcome resistance. If desired, tumor cells can be transfected to express the peptide combination. For example, an expression vector that directs the expression of tumor cells obtained from a patient in combination with a peptide having B7-2-like activity alone or with a peptide having B7-1-like activity and / or B7-3-like activity, It can be transfected ex vivo. The transfected tumor cells are returned to the patient and the peptide is expressed on the surface of the transfected cells. Alternatively, tumor cells can be targeted for transfection in vivo using gene therapy. The presence of a peptide of the invention having the activity of a B lymphocyte antigen on the surface of tumor cells provides the necessary costimulatory signals for T cells to induce an immune response to transfected tumor cells mediated by T cells I do. In addition, MHC
Tumor cells lacking class I or MHC class II molecules, or sufficient MHC
Class I or tumor cells can not be re-expressed MHC class II molecules, MHC class Iα chain protein and beta ₂ microglobulin protein or an MHC class IIα chain and an MHC class IIβ chain all or part of the protein (e.g., cytoplasmic truncations protein Domain) can be transfected, whereby the class I or class II MHC protein can be expressed on the cell surface. Expression of appropriate Class I or Class II HMC in combination with a peptide having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-3) is a T cell-mediated transfected tumor Induces an immune response against the cells. If desired, a gene encoding an antisense construct that blocks expression of an MHC class II binding protein, such as an invariant chain, can be co-transfected with a DNA encoding a peptide having B lymphocyte antigen activity. It can also enhance the presentation of tumor-associated antigens and induce tumor-specific immunity. Thus, induction of an immune response mediated by T cells in a human subject may be sufficient to overcome tumor-specific resistance in the subject.

The activity of a protein of the invention may be measured, inter alia, by the following method: Suitable assays for thymocyte or spleen cell cytotoxicity are described in Current Protocols
in Immunology, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM
Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Intersc
ience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19
; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl
Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J. Immunol. 128:
1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai e
t al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140:
508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78: 2488-2492.
Herrmann et al., J. Immunol. 128: 1968-1974, 1982; Handa et al., 1981;
J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 137: 3494-3
500, 1986; Bowmanet al., J. Virology 61: 1992-1998; Takai et al., J. Imm
unol. 140: 508-512, 1988; Bertagnolli et al., Cellular Immunology 133: 3
27-341, 1991; including but not limited to the assays described in Brown et al., J. Immunol. 153: 3079-3092, 1994. Assays for T cell-dependent immunoglobulin responses and isotype switching, specifically to modulate T cell-dependent antibody responses and identify proteins that affect Th1 / Th2 profiles, are described in Maliazewski, J. Immunol. 144: 3028-3033, 1990, including but not limited to assays for B cell function as described in In vitro antibody production, Mond, JJ and Brunswick, M. In Current.
Protocols in Immunology JEColigan eds.Val 1 pp.3.8.1-3.8.16, John Wile
y and Sons, Tronto 1994. Mixed lymphocyte reaction (MLR) assays, which identify proteins that primarily produce Th1 and CTL responses, are described in Current Protocols in Immunology, Ed by
JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W Strober,
Pub.Green Publishing Associates and Wiley-Interscience (Chapter 3, In
Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunol
ogic studies in Humans); Takai et al., J. Immunol. 137: 3494-3500, 1986;
Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., J. Im.
munol. 149: 3778-3783, 1992, but is not limited thereto. Dendritic cell-dependent assays, specifically identifying proteins expressed by dendritic cells that activate intact T cells, are described by Guery et al., J. Immunol. 134: 536-544.
, 1995; Inaba et al., Journal of Experimental Medicine 173: 549-559, 199
1; Macatonia et al., Journal of Immunology 154: 5071-5079, 1995; Porgado
r et al., Journal of Experimental Medicine 182: 255-260, 1995; Nair et a
l., Journal of Virology 67: 4062-4069, 1993; Huang et al., Science 264:
961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:
1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:
797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:
631-640, 1990, including, but not limited to. Assays for lymphocyte survival / apoptosis, specifically identifying proteins that prevent apoptosis following superantibody induction, as well as proteins that regulate lymphocyte homeostasis, are described by Darzynkiewicz et al., Cytometry 13: 795-808, 1992; Gorczyca et al.
, Leukemia 7: 659-670, 1993; Gorczyca et al., Cancer Research 53: 1945-1
951, 1993; Itoh et al., Cell 66: 233-243, 1991; Zacharchuk, Journal of I
mmunology 145: 4037-4045, 1990; Zamai et al., Cytometry 14: 891-897, 199
3; including but not limited to the assays described in Gorczyca et al., International Journal of Oncology 1: 639-648, 1992. Early stage T cell commitment and development assays
Antica et al., Blood 84: 111-117, 1994; Fine et al., Cellular Immunology 15
5: 111-122, 1994; Galy et al., Blood 85: 2770-2778, 1995; Toki et al., Pr
USA 88: 7548-7551, 1991, including but not limited to oc. Natl. Acad. Sci.

Hematopoietic modulatory activity The proteins of the present invention are useful in regulating hematopoiesis and, therefore, in treating bone marrow and lymphocyte deficiencies. Even minimal biological activity supporting colony forming cells or factor-dependent cell lines has been implicated in regulating hematopoiesis.
For example, supporting the proliferation of erythroid progenitor cells alone, or in combination with other cytokines, for example, showing usefulness in the treatment of various anemias, or in combination with radiation therapy / chemotherapy, erythroid progenitor cells And / or stimulating the production of erythroblasts; supporting the proliferation of bone marrow cells such as granulocytes and monocytes / macrophages (ie, traditional CSF activity), eg, in combination with chemotherapy, Useful in preventing myelosuppression that occurs; supporting proliferation of megakaryocytes and then platelets, and thereby preventing or treating various platelet disorders such as thrombocytopenia; It is also commonly used instead of or in complement to platelet infusion; and / or hematopoietic stem cells (mature Becomes all of hematopoietic cells, therefore, a variety of stem cell disease (
It is usually a disease that is treated by transplantation and is useful in supporting the growth of, for example, aplastic anemia and paroxysmal nocturnal hemoglobinuria), which is useful in vivo or ex vivo. It is also useful in repopulating the stem cell compartment as normal cells after radiation / chemotherapy with (ie, combined with bone marrow transplantation), or after being genetically engineered for gene therapy. In particular, the activity of the protein of the present invention may be measured by the following method. Assays suitable for the proliferation and differentiation of various hematopoietic cell lines have already been cited. Assays for embryonic stem cell differentiation, particularly those that identify proteins that affect embryonic hematopoiesis, are described in Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al.
, Molecular and Cellular Biology 13: 473-486, 1993; McClanahan et al., B
lood 81: 2903-2915, 1993, including, but not limited to. Assays for stem cell survival and differentiation (particularly identifying proteins that regulate lympho-hematopoiesis) are described in Methylcellulose colony forming assays, Freshney, MG In Cultu
re of Hematopoietic Cells.RI Freshney, et al. eds.Vol pp. 265-268,
Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad.
Sci. USA 89: 5907-5911, 1992; Primitive hematopoietic colony forming ce
lls with high proliferative potential, McNiece, IK and Briddell, RA
In Culture of Hematopoietic Cells.RI Freshney, et al. Eds.Vol pp. 2
3-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental H
ematology 22: 353-359, 1994; Cobblestone area forming cell assay, Ploema
cher, RE In Culture of Hematopoietic Cells.RI Freshney, et al. eds.
Vol pp. 1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone ma
rrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M.
and Allen, T. In Culture of Hematopoietic Cells.RI Freshney, et al.
eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term c
ulture initiating cell assay, Sutherland, HJ In Culture of Hematopoiet
ic Cells.RI Freshney, et al. eds.Vol pp. 139-162, Wiley-Liss, Inc.,
Includes, but is not limited to, the assays described in New York, NY. 1994.

Research Uses and Utility The polynucleotides provided by the present invention can be used for various purposes by a research population. For analysis, characterization or therapeutic use, as a marker of tissue in which the corresponding protein is preferentially expressed (constitutively or at a particular stage of tissue differentiation or development or in a disease state), and To identify potential genetic diseases as compared to the patient's endogenous DNA, as chromosomal markers or tags (if labeled) for identification of chromosomes or mapping of relevant gene locations, as molecular weight markers for Southern gels The use of known sequences in the process of discovering other novel polynucleotides as a source for obtaining PCR primers for genetic fingerprinting to find new related DNA sequences to use as hybridization probes "Gene chips" or other probes as probes for subtraction To select and generate oligomers for binding to carriers, to test expression patterns, to generate anti-protein antibodies using DNA immunization techniques, and to generate anti-DNA antibodies or to generate additional immunizations. Polynucleotides can be used as antigens to induce a response. If encoding a protein that binds or potentially binds to another protein (e.g., as in a receptor-ligand interaction), identify the other protein that binds, and / or an inhibitor of the binding interaction Interaction trap assays (eg, Gyuris et al., Cell 75: 791-803 (1993) and Rossi et al., 1)
997, Proc. Natl. Acad. Sci. USA 94: 8405-8410). The proteins provided by the present invention may be used in assays to determine biological activity, including a group of multiple proteins for high-throughput screening; to generate antibodies or induce other immune responses; As a reagent (including a labeling reagent) in an assay designed to quantitatively determine the level of a protein (or its receptor) in a biological fluid; the corresponding protein is preferentially expressed (constitutively Or expressed at a particular stage of tissue differentiation or development, or in a disease state) as a marker for tissue; and, of course, may be used to isolate relevant receptors or ligands. Where a protein binds or potentially binds to another protein, the protein can be used to identify other proteins that bind, or to identify inhibitors of the binding interaction. Using proteins involved in these binding interactions, screening for peptides or small molecular inhibitors or agonists for the binding interaction can also be performed. Any or all of these research applications can be evolved into reagent grade or kit formats for commercialization as research products. Methods for performing the above uses are well known to those skilled in the art. A literature disclosing such a method can be found in "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Sp.
ring Harbor Laboratory Press, Sambrook, J., EF Fritsch and T. Maniatis
eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Tec
hniques ", Academic Press, Berger, SL and AR Kimmel eds., 1987.

Use as Nutrition The polynucleotides and proteins of the present invention can be used as nutrient sources or additives. Such uses include, but are not limited to, use as a protein or amino acid additive, use as a carbon source, use as a nitrogen source, and use as a carbohydrate source. In such cases, the proteins or polynucleotides of the present invention may be added as food for a particular organism, or may be administered as a separate solid liquid formulation such as in the form of a powder, pill, solution, suspension or capsule. . In the case of a microorganism, the protein or polynucleotide of the present invention can be added to a medium in which the microorganism is cultured.

An animal may be immunized with the U4 protein of the present invention to obtain polyclonal and monoclonal antibodies that can specifically react with the U4 protein and inhibit ligand binding to the receptor. Such antibodies may be obtained using whole U4 as an immunogen or by using fragments of U4. Animals may be immunized with smaller U4 fragments. The peptide immunogen may further include a cysteine residue at the carboxyl terminus and is conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Additional peptide immunogens may be obtained by replacing thiine residues with sulfated tyrosine residues. Methods for synthesizing such peptides are known in the art, for example, RP Merrifiel, J. Amer. Chem. Soc. 8
5, 2149-2154 (1963); JL Krstenansky, et al., FEBS Lett. 211, 10 (1987
)It is described in. Neutralizing or non-neutralizing antibodies (preferably monoclonal antibodies) that bind to U4 protein are also useful as therapeutics for certain tumors and may be useful in treating the above conditions. These neutralizing monoclonal antibodies may have the ability to block ligand binding of the U4 receptor chain.

[0028] The DNA coding for expression full length murine U4 protein of Example U4 protein, Gly-Ser-G by PCR
ly, and then fused to the COS-1 expression vector pED. It was ligated in frame to the sequence encoding the CH2 CH3 region of human IgG1 in Fc. The DNA was transfected into COS cells, and the expression of the fusion protein was detected by ELISA using an antibody that detects the IgG1 portion of the protein. This indicated that the protein could be expressed and secreted in COS cells.

All patents and publications cited herein are hereby incorporated by reference.

[0030]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 A61K 37/02 C12P 21/02 C12N 5/00 B (81) Designated countries EP (AT, BE) , CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA , GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU , LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Mary Collins, No. 54, Rusburn Road, Natick, Massachusetts, USA 01760 (72) Inventor Matthew Whiters, No. 14, Brenton Wood Road, Hudson, Mass. 72) Inventor Thamrin Niven Danforth Lane 1115 F-term (reference) 4B024, Walnut Creek, CA 94598, USA AA01 AA11 CA04 CA07 DA02 GA11 GA27 4B064 AG20 AG27 CA10 CA19 CA20 CC01 CC24 DA01 DA13 4B065 AA01X AA58X AA72X AA87X AA90X AA91Y AB01 AC14 BA01 BA24 CA24 CA44 CA46 4C084 AA02 AA06 AA07A19 A22 MA22 BA10 BA41 CA40 DA50 DA75 EA20 EA50 FA71 FA74

Claims (19)

[Claims] 1. (a) the nucleotide sequence from nucleotide 242 to nucleotide 1396 of SEQ ID NO: 4; (b) the nucleotide sequence from nucleotide 71 to nucleotide 1225 of SEQ ID NO: 6; (c) the nucleotide sequence of SEQ ID NO: 9 A nucleotide sequence from 136 to nucleotide 1401; (d) a nucleotide sequence altered from the sequence of the nucleotide sequence shown in (a), (b) or (c) as a result of the degeneracy of the genetic code; A nucleotide sequence capable of hybridizing under mild conditions to a nucleotide shown in (a) or (b); (f) a nucleotide sequence encoding a species homolog of the sequence shown in (a), (b) or (c); And (g) an allele of the nucleotide sequence shown in (a), (b) or (c) An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of a variant. 2. The polynucleotide of claim 1 wherein said nucleotide sequence encodes a protein having the biological activity of the U4 hematopoietin receptor superfamily chain. 3. The polynucleotide of claim 1, wherein said nucleotide sequence is operably linked to an expression control sequence. 4. The nucleotide sequence from nucleotide 122 to nucleotide 139 of SEQ ID NO: 4.
The polynucleotide of claim 1 comprising up to 6 nucleotide sequences. 5. Nucleotide 11 to nucleotide 1225 of SEQ ID NO: 6
2. The polynucleotide of claim 1, comprising the nucleotide sequence of 6. A host cell transformed with the polynucleotide of claim 3. 7. The host cell according to claim 6, which is a mammalian cell. 8. A method for producing a U4 protein, comprising: (a) growing a culture of the host cell of claim 6 in a suitable medium; and (b) purifying the U4 protein from the culture. 9. An amino acid sequence of SEQ ID NO: 5; (b) an amino acid sequence of amino acids 41 to 425 of SEQ ID NO: 5; (c) an amino acid sequence of SEQ ID NO: 7; (E) amino acid sequence of SEQ ID NO: 10; (f) amino acid sequence of amino acids 38 to 421 of SEQ ID NO: 10; and (g) U4 hematopoietin receptor superfamily chain An isolated U4 protein comprising an amino acid sequence selected from the group consisting of: a fragment having the biological activity of (a) to (f). 10. The protein of claim 9, which comprises the amino acid sequence of SEQ ID NO: 5. 11. The protein of claim 9 comprising the sequence of amino acids 41 to 425 of SEQ ID NO: 5. 12. The protein of claim 9, comprising the amino acid sequence of SEQ ID NO: 7. 13. The protein of claim 9, comprising the sequence of amino acids 24 to 408 of SEQ ID NO: 7. 14. A pharmaceutical composition comprising the protein of claim 9 and a pharmaceutically acceptable carrier. 15. A protein produced by the method of claim 8. 16. A composition comprising an antibody that specifically reacts with the protein of claim 9. 17. (a) the amino acid sequence of SEQ ID NO: 5; (b) the amino acid sequence of amino acids 41 to 425 of SEQ ID NO: 5; (c) the amino acid sequence of SEQ ID NO: 7; A peptide comprising an amino acid sequence selected from the group consisting of: (a) a fragment of (a) to (d) having the biological activity of the U4 hematopoietin receptor superfamily chain; Alternatively, an isolated polynucleotide comprising a nucleotide sequence encoding a protein. 18. The protein of claim 9, wherein said amino acid sequence is part of a fusion protein. 19. The protein of claim 18, comprising an Fc fragment.