JP2001508309A - 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            Member of the hematopoietin receptor superfamilyField of the invention   The present invention relates to a new type of mammalian hematopoietin superfamily among proteins. Members (including but not limited to human and murine receptor proteins) , Fragments thereof and recombinant polynucleotides useful for the expression of such proteins And cells.Background of the Invention   Various regulatory molecules known as hematopoietin have been identified, It is involved in the development and proliferation of hematopoietic cells or various populations of blood cells. Most Hematopoietin is a specific organism that interacts with receptors on the surface of target cells. It shows biological activity. Usually, cytokine receptors consist of one, two or three chains. ing. Many cytokine receptors and some, such as IL-12p40 Cytokines are among the proteins in the hematopoietin receptor superfamily Be a member. New members of the hematopoietin receptor superfamily Identification of hematopoietic preparations, regulation of the immune response, and encapsulation of cytokines and receptors Useful in identifying other members of the hematopoietin superfamily containing It can be.   A previously unknown member of the hematopoietin receptor superfamily It may be desirable to identify and determine the DNA and protein sequence of the DNA.Summary of the Invention   According to the present invention, the U4 hematopoietin receptor superfamily chain is encoded. Disclosed are polynucleotides that include those of murine and human origin But not limited to these. In certain embodiments, the present invention provides   (A) nucleotides from nucleotide 242 to nucleotide 1396 of SEQ ID NO: 4 K Leotide sequence;   (B) the nucleotide sequence from nucleotide 71 to nucleotide 1225 of SEQ ID NO: 6 Leotide sequence;   (C) the nucleoside shown in (a) or (b) as a result of the degeneracy of the genetic code A nucleotide sequence that differs from the nucleotide sequence;   (D) hybrids under stringent conditions to the nucleotides shown in (a) or (b) A nucleotide sequence that can be redistributed;   (E) a nucleotide sequence encoding a species homolog of the sequence shown in (a) or (b) Columns; and   (F) an allelic variant of the nucleotide sequence shown in (a) or (b) Providing an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: I do.   Preferably, the nucleotide sequence is a U4 hematopoietin receptor superfamily. Encodes a protein having the biological activity of the Millie chain. Expression of nucleotide sequence It may be operatively connected to an array. In a preferred embodiment, the polynucleotide Is the nucleotide from nucleotide 242 to nucleotide 1396 of SEQ ID NO: 4. A nucleotide from nucleotides 122 to 1396 of SEQ ID NO: 4 Nucleotide sequence from nucleotide 71 to nucleotide 1225 of SEQ ID NO: 6 A nucleotide sequence; or nucleotides 11 to 12 of SEQ ID NO: 6 Includes up to 25 nucleotide sequences.   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;   (D) an amino acid sequence of amino acids 24 to 408 of SEQ ID NO: 7;   (E) possesses the biological activity of the U4 hematopoietin receptor superfamily chain Fragments of (a) to (d) Encodes a peptide or protein containing an amino acid sequence selected from the group consisting of Nu An isolated polynucleotide comprising a nucleotide sequence is provided. Other preferred embodiments are The amino acid sequence of SEQ ID NO: 5; the amino acid sequence of amino acids 41 to 425 of SEQ ID NO: 5 Amino acid sequence; amino acid sequence of SEQ ID NO: 7; and amino acid 2 of SEQ ID NO: 7 Encodes an amino acid sequence from 4 to 408.   A host cell, preferably a mammalian cell, transformed with the polynucleotide is also provided. Provided.   In another embodiment, the present invention provides a method for producing a U4 protein. The method comprises:   (A) growing a culture of the host cell of the present invention in a suitable medium;   (B) Purifying human U4 protein from the culture Including.   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;   (D) an amino acid sequence of amino acids 24 to 408 of SEQ ID NO: 7;   (E) possesses the biological activity of the U4 hematopoietin receptor superfamily chain Fragments of (a) to (d) An isolated U4 protein comprising an amino acid sequence selected from the group consisting of:   Preferably, the protein is an amino acid sequence of SEQ ID NO: 5; an amino acid sequence of SEQ ID NO: 5 Amino acid sequence from acids 41 to 425; amino acid sequence of SEQ ID NO: 7; Contains the amino acid sequence of amino acids 24 to 408 of SEQ ID NO: 7. Other preferred In a specific example, the amino acid sequence is a fusion protein (not derived from U4, Sequence fusion protein). Preferred fusion proteins are such as Fc fragments. Sometimes includes antibody fragments.   A pharmaceutical composition comprising the protein of the present invention and a pharmaceutically acceptable carrier is also provided.   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 embodiment   The present inventors have determined that the U4 hematopoietin receptor superfamily chain (hereinafter referred to as "" U4 ”or a polynucleotide encoding“ U4 protein ”(rat and rat). Including, but not limited to, polynucleotides encoding U4 and human U4) And provided it for the first time.   Human IL-5 receptor (LMTNAFISIIDDLSKYDVQVRAAVSSMCREAGLWSEWSQPIYVGNDEHKP Using a region of 79 amino acids of LREWFVIVIMATICFILLIL, SEQ ID NO: 1), TB Search the GenBank EST database using the LASTN algorithm Was. EST W66776 was identified as having homology to this region, It was suggested that it might encode a novel hematopoietin receptor. GCG map Translation of the reverse complement of this EST using a preprogram provides a hematopoietin receptor. A protein in the second reading frame containing a conserved WSXWS motif found in the body A white arrangement was revealed. However, in this reading frame, nucleotide 22 There is also a stop codon at 7 and this EST is a novel hematopoietin receptor Either it was not a body or the DNA sequence in the EST was incorrect.   Whether this EST sequence may be related to the hematopoietin receptor To determine if we have the sequence CTTGGCTTGG AAGAGGAAAT CCTTGAGAGC (SEQ ID NO: : 2) The mouse embryo library was screened using the oligonucleotide probe I was leaning. A full-length cDNA clone U6-3 (1A) was identified and its complete sequence was gotten. The DNA sequence and the deduced amino acid sequence of the murine protein were No .: 4 and SEQ ID NO: 5. The murine protein is a hematopoietin receptor family Codes a new member of Lee. It has a leader sequence and this family Conserved cysteine pair PP and WSXWS motif Have. This clone has no transmembrane or cytoplasmic domain. this When the clone was juxtaposed and compared with EST in GenBank, No shift mutation was found.   SEQ ID NO: 4 shows the nucleotide sequence of cDNA encoding murine U4 . SEQ ID NO: 5 shows the predicted amino acid sequence of the receptor chain, from amino acids 1 to 40 Guess Includes constant signal sequence. Mature mouse U4 is derived from amino acids 41-38 of SEQ ID NO: 5. It is thought to have three sequences. Identify additional related sequences in GenBank Genome using the BLASTN algorithm with the W66776 sequence Bank was searched. Closely related ESTs from human genomic DNA, H14 009 was identified. Then, the EST-derived oligonucleotide CTGAGCGTGC Using GCTGGGTGTCGCCAC (SEQ ID NO: 3), c A DNA clone was isolated. CD encoding full-length mature protein homolog (homolog) The NA clone (HU4-3B) was completely sequenced. This clone is complete It has no signal sequence, but encodes the entire putative full-length mature protein. The Human clones have 85% homology at the DNA level to mouse clones . The deduced amino acid sequence has 95% identity between human and mouse. Human U SEQ ID NO: 6 and SEQ ID NO: 4, respectively. FIG.   SEQ ID NO: 6 shows the nucleotide sequence of cDNA encoding human U4. SEQ ID NO: 7 shows the predicted amino acid sequence of the receptor chain, from amino acids 1 to 23 Contains a predicted signal sequence. Mature human U4 has amino acids 24-3 of SEQ ID NO: 7. It is believed to have 80 sequences.   Murine and human clones were transferred to American Thai on January 1997. Deposit with the PUC Culture Collection, with accession numbers ATCC and And ATCC.   By replacing the human leader sequence with a murine leader sequence, or Amino acid 1-14 of the murine sequence (MPAGRPGPVA QSAR, SEQ ID NO: : 8) to express human U4 protein . In addition, using the sequences disclosed herein as probes, Encoding longer cDNA or genomic clones can also be isolated You.   Any form of U4 protein of less than full length is encompassed by the present invention, And together with the mature form are referred to as "U4" or "U4 protein". full length Of the polynucleotide (SEQ ID NO: 4 or SEQ ID NO: 6) encoding the U4 protein Less than full-length U4 protein may be produced by expressing the corresponding fragment. No. this These corresponding polynucleotide fragments are also encompassed by the present invention. Suitable desirable Standard molecular biology techniques, including deletion mutant construction and site-directed mutagenesis Or polymerase chain using appropriate oligonucleotide primers The modified polynucleotides described above may be produced by a strand reaction.   For the purposes of the present invention, one or more biological activities of the corresponding mature U4 protein. If the protein has the property, the protein is referred to as “U4 hematopoietin receptor superfamily. It has "biological activity of the chain".   U4 or its active fragment (U4 protein) is It may be fused to a rear molecule. For example, the soluble form of U4 is replaced by a "linker" sequence. May be fused to the Fc portion of an immunoglobulin. GST, Lex-A Alternatively, another fusion protein such as a fusion protein with MBP may be used.   The present invention also relates to a pair of nucleotide sequences shown in SEQ ID NO: 4 or SEQ ID NO: 6. A progenitor variant, ie, the isolated polynucleotide of SEQ ID NO: 4 or SEQ ID NO: 6 A naturally occurring variant of Ud (also encoding a U4 protein, preferably U4 Which encodes a protein having the biological activity of Also very SEQ ID NO: 4 under stringent conditions (eg, 0.1 × SSC at 65 ° C.) An isolated polynucleotide hybridizing to the nucleotide sequence shown in SEQ ID NO: 6. Nucleotides are also included in the present invention. It encodes the U4 protein but has a genetic code Differs from the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6 due to degeneracy of The isolated polynucleotide is also encompassed by the present invention. Point mutation or induction SEQ ID NO: 4 or SEQ ID NO: 6 resulting from the modifications made Variations in the sequence are also encompassed by the present invention.   The invention also relates to rodents or humans from other animal species, in particular from other mammalian species. And a polynucleotide encoding a homologue to U4 of U.S.A. Disclosure in this specification Probes or primers derived from murine or human sequences A suitable library such as a library constructed from PBMC, thymus or testis of a specific species Species homologues can be identified by screening libraries .   The isolated polynucleotide of the present invention can be prepared as described in Kaufman et al., Nucleic Acids Res. 19, Expression control, such as the pMT2 or pED vector disclosed in 4485-4490 (1991) The U4 protein may be recombinantly produced by operably linking the sequence. Many decent Expression control sequences are known in the art. General for recombinant protein expression Is also known, R. Kaufman, Methods in Enzymology 185, 537-566 (199 0). As used herein, "operably linked" refers to (Transfection) with the polynucleotide / expression control sequence The enzymatically or chemically linked U4 protein is expressed such that the U4 protein is expressed by the host cell. That a covalent bond is formed between the isolated polynucleotide of the invention and the expression control sequence. Means   Many cells can serve as suitable host cells for expression of the protein. Mammalian host Cells include, for example, monkey COS cells, Chinese hamster ovary (CHO) cells , Human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T 3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, 1 Cell lines, primary explants, HeLa cells, mice derived from in vitro culture of the following tissues L cell, BHK, HL-60, U937, HaK, Rat2, BaF3, 32D , FDCP-1, PC123, M1x or C2C12 cells.   Actuation of U4 protein on appropriate regulatory sequences in one or more insect expression vectors The protein may be obtained by ligating as possible and using an insect expression system. Baculoi Materials and methods for the Rus / insect cell expression system are described, for example, in Invitrogen, San Dieg. o, kit form from California, USA (MaxBac^RKit) Such methods are well known in the art and are described by Summers and Smith, Texas Ag. ricultural Experiment Station Bulletin No. 1555 (1987) (hereby incorporated by reference) It is regarded as described in the book). The appropriate unit above Soluble forms of U4 protein were produced in insect cells using isolated polynucleotides. You may.   Alternatively, in lower eukaryotic cells such as yeast or prokaryotic cells such as bacteria. It is also possible to produce proteins. A potentially suitable yeast strain is Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strain, Candida, or Yeast strains capable of expressing heterologous proteins are included. A potentially suitable bacterial strain is Escher ichiacoli, Bacillus subtilis, Salmonella typhimurium, or heterologous protein Ability Bacterial strains that have   Expression in bacteria may result in inclusion bodies containing the recombinant protein. Therefore, to obtain an active substance or a more active substance, it is necessary to regenerate the recombinant protein. May be needed. Obtaining correctly folded heterologous proteins from bacterial inclusions Methods are known in the art. In general, these methods Solubilization of the protein obtained from the input, followed by complete denaturation of the protein using chaotropic agents Is included. If a cysteine residue is present in the primary amino acid sequence of the protein, Often requires regeneration in an environment (redox system) where sulfide bonds can be formed correctly It is. General methods for regeneration are described in Kohno, Meth. Enzym., 185: 187-195 (1990) Is disclosed. EP 0433225 and co-pending application USSN 08/1 63877 describes another suitable method.   By somatic or germ cells containing a polynucleotide sequence encoding the U4 protein Transgenic animal products that can be characterized Expression of the U4 protein of the present invention as a component in milk of cows, goats, pigs or sheep May be.   Growing the transformed host cell culture in culture conditions necessary for the expression of the desired protein Thus, the U4 protein of the present invention may be produced. Then, the obtained expressed protein is cultured. It may be purified from ground or cell extracts. Conditioning soluble U4 protein of the present invention It can be purified from the medium. The whole membrane fraction was obtained from the expressing cells and then Extraction of the membrane with a non-ionic surfactant such as Triton X-100, The U4 protein of the present invention in a conformed form can be purified. Anyone known in the field The U4 protein can be purified using the method. For example, a commercially available protein concentration filter The invention U4 using, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Protein can be concentrated. After the concentration step, a purification matrix such as gel filtration media The concentrate can be applied to the mix. Alternatively, an anion exchange resin, for example, Pendant diethylaminoethyl (DEAE) or polyethyleneimine (PEI) A matrix or substrate with groups can be used. The matrix is Commonly used for claramide, agarose, dextran, cellulose or protein purification Other types used May be. Alternatively, a cation exchange step may be used. Suitable cation exchanger Are various insoluble matrices containing sulfopropyl or carboxymethyl Embrace. Sulfopropyl group (for example, S-Sepharose^RColumn) is preferred New Purification of U4 protein from the culture supernatant was performed using Concanavalin A-agarose, Lin-Toyo Pearl^ROr Cibachrome Blue 3GA Sepharose^RAffi One or more column steps with a phenolic resin or phenyl ether Interaction with resins such as butyl, butyl ether, or propyl ether Chromatography or immunoaffinity chromatography May be. Finally, a hydrophobic reversed-phase high quality liquid chromatography medium, such as a suspension One or more reversals using silica gel with pendant methyl or other aliphatic groups U4 protein was analyzed using a two-phase high quality liquid chromatography (RP-HPLC) process. It can be further purified. According to known methods, it contains an antibody against the U4 protein. An affinity column may be used for purification. Some of the above purification steps or Are all used in various combinations or in combination with other known methods, A substantially purified isolated recombinant protein can also be obtained. Preferably, the other The isolated U4 protein is purified to be substantially free of milk protein.   An agent capable of binding to U4 may be screened using the U4 protein of the present invention. No. Binding assays using the desired binding protein, either immobilized or not, The U4 protein of the present invention is well known in the art and used for this purpose. it can. Screening assays with purified cells or with purified proteins (cell-free ) Such agents may be identified using screening assays. For example, U 4 protein is immobilized on a carrier in a purified form, and is bound to purified U4 protein. The ligand may be measured.   Purified U4 protein or combination derived from cells when mixed with a pharmaceutically acceptable carrier The purified and purified U4 protein may be used as a pharmaceutical composition. Such composition Are diluents, fillers, salts, buffers (in addition to U4 or inhibitors and carriers) Containing additives, stabilizers, solubilizers, and other materials well known in the art. May be No. The term "pharmaceutically acceptable" does not interfere with the effectiveness of the biological activity of the active ingredient. Means non-toxic substance. The characteristics of the carrier will depend on the route of administration.   The pharmaceutical composition of the present invention comprises M-CSF, GM-CSF) IL-1, IL-2, IL -3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12, IL-13, IL-14, IL-15, GC SF, stem cell factor, and cytokines such as erythropoietin, lymphokines Or other hematopoietic factors. Pharmaceutical compositions may further include anti-sites It may contain a caine antibody. The pharmaceutical composition comprises plasminogen activator Contains thrombolytic or antithrombotic factors such as You may. Further, the pharmaceutical composition may contain other anti-inflammatory agents. Like that The following factors and / or agents are included in the pharmaceutical composition to provide a phase with the isolated U4 protein: Exerts a synergistic effect or minimizes side effects caused by isolated U4 protein May be. Conversely, certain cytokines, lymphokines, other hematopoietic factors, thrombolytics Including the isolated U4 protein in the formulation of an anti-thrombotic factor or an anti-thrombotic agent, Cytokines, lymphokines, other hematopoietic factors, thrombolytic or antithrombotic factors, Alternatively, the side effects of the anti-inflammatory agent may be minimized.   The pharmaceutical composition of the present invention may be in the form of liposome, in which the protein of the present invention is contained. And other pharmaceutically acceptable carriers, amphipathic agents such as lipids (mice in aqueous solution). Agglomerate form, such as insoluble monolayer, liquid crystal or lamellar layer) Have been combined. Suitable lipids for liposome formulations are monoglycerides, diglycerides , Sulfatizide, lysolecithin, phospholipids, saponins, bile acids, etc. However, it is not limited to these. The preparation of such liposome formulations is within the level of ordinary skill in the art. And, for example, U.S. Pat. Nos. 4,235,871, 4501728, 4837028, And 4737323, all of which are described herein by reference. Is considered).   As used herein, the term "therapeutically effective amount" refers to a significant patient benefit, e.g., Sufficient pharmaceutical composition or method to show improvement in symptoms of the condition, healing, increased rate of healing It means the total amount of each active ingredient. Used for individual active ingredients administered alone If The term refers only to the component. When used in a combination of active ingredients, Or, it refers to the total amount of active ingredients that produce a therapeutic effect regardless of simultaneous administration.   In practicing the therapeutic methods or uses of the present invention, a therapeutically effective amount of the isolated U4 protein is obtained. Administered to mammals. According to the method of the present invention, alone or with a cytokine, lymphokine Administering the isolated U4 protein along with other therapeutic agents such as in or other hematopoietic factors Is also good. One or more cytokines, lymphokines or other hematopoietic factors When co-administered with U4, the U4 protein can be used for cytokines, lymphokines, other hematopoietic factors, It may be administered simultaneously or sequentially with the thrombolytic or antithrombotic factor. Administer sequentially If you are a physician, you may be asked for cytokines, lymphokines, other hematopoietic factors, Alternatively, determine an appropriate administration order of U4 protein in combination with an antithrombotic factor. Will be.   Administration of the U4 protein in the pharmaceutical composition of the present invention or used in the practice of the present invention can be accomplished by various conventional techniques. Method, for example, oral feeding, inhalation, or intradermal, subcutaneous, or intravenous injection be able to. Intravenous injection into a patient is preferred.   When a therapeutically effective amount of U4 protein is administered orally, the U4 protein may be in tablets, capsules, powders, Powder, solution or elixir form. When administered in tablet form, the physician of the present invention The pharmaceutical composition may further comprise a solid carrier such as gelatin or an adjuvant. Is also good. Tablets, capsules, and powders contain about 5 to 95% U4 protein, preferably Or about 25-90% U4 protein. When administered in liquid form, water, Petroleum, oils of animal or vegetable origin, such as peanut oil, mineral oil, soybean oil Or sesame oil or synthetic oils and fats may be added. Pharmaceutical compositions in liquid form include: Saline, dextrose or other sugar solutions, or glycols (eg, Ethylene glycol, propylene glycol or polyethylene glycol ) May be further contained. When administered in liquid form, the pharmaceutical composition comprises about 0 . 5 to 90% by weight U4 protein, preferably about 1 to 50% U4 protein. Have.   When a therapeutically effective amount of U4 protein is administered by intravenous, intradermal or subcutaneous injection, The four proteins will be pyrogen-free and in the form of a parenterally acceptable aqueous solution. Suitable Preparation of such a parenterally acceptable protein solution having a good pH, isotonicity and stability Is It is within the purview of those skilled in the art. Preferred pharmaceutical compositions for intravenous, intradermal, or subcutaneous injection are , U4 protein, sodium chloride for injection, Ringer's solution, dextro for injection Dextrose and sodium chloride solution for injection, lactate containing lactate for injection Solution, or other carriers known in the art. The present inventor The drug composition may be a stabilizer, preservative, buffer, antioxidant, or other And other additives.   The amount of U4 protein in the pharmaceutical composition of the invention will depend on the nature and severity of the condition being treated. It depends on the nature of the treatment the patient has already received. Ultimately, the doctor in charge The amount of U4 protein at which the patient should be treated will be determined. First, your doctor should Administer U4 protein and observe patient response. Until the optimal therapeutic effect is obtained, High doses of U4 protein may be administered, and the dose may be further increased once optimal therapeutic effects are achieved. Do not increase. Various pharmaceutical compositions for carrying out the method of the invention have a body weight of 1 kg. It should contain from about 0.1 μg to about 100 mg of U4 protein per.   The duration of treatment by intravenous administration using the composition of the present invention depends on the severity of the disease to be treated. It will vary depending on the condition and unique response of each patient. U4 protein Is considered to range from 12 to 24 hours for continuous intravenous administration. It is. Eventually, the attending physician will perform treatment by intravenous administration using the pharmaceutical composition of the present invention. Will determine the duration of the   The polynucleotides and proteins of the present invention may have one or more of the following uses or Exhibiting biological activity (including those associated with the assays cited herein) Conceivable. The uses or activities described for the proteins of the invention may be attributed to the administration of such proteins. Supply or use, or a polynucleotide encoding such a protein. Administration or use (e.g., a vector suitable for gene therapy or for DNA transfer) ).Cytokines and cell proliferation / differentiation activity   The protein of the present invention has cytokine activity, cell growth activity (induction or inhibition) or cell activity. Can show blast differentiation activity (induction or inhibition) or in certain cell populations Other Can induce the production of cytokines. Many protein factors discovered to date Offspring (including all known cytokines) are one or more factor-dependent cells Activity in the proliferation assay, and thus the assay It is useful as a convenient confirmation method. The activity of the protein of the present invention is determined in cell lines (32D, DA2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (preB M +), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, (Including but not limited to TF-1, Mo7e and CMK) It is confirmed by any of the 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: Current Protocols in Immunology, Ed by J.E.Coligan, A.M.Kruisbeek, D.H.Marg ulies, E.M.Shcvach, W Strobcr, Pub.Grccnc Publishing Associates and Wiley-I nterscience (Chapter3, In Vitroassays for Mouse Lymphocyte Function3.1-3.1 9; Chapter 7, Immunologic studics in Humans); Takai et al., J. Immunol. 137: 349 4-3500,1986; Bertagnolli et al., J.Immunol. 145: 1706-1712,1990; Bertagnolli et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli, et al., J. Immunol .149: 3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994. But not limited thereto.   Cytokine production and / or expansion of spleen cells, lymph node cells or thymocytes Assays for breeding Polyclonal T cell stimulation, Kruisbeek, A.M.and Shevach, E.M.In Current Protocols in Immunology.J.E.e.a.Coligan eds.Vol 1 pp.3.12.1-3.12.14, John  Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interfer on γ, Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a.Coligan eds.Vol 1 pp.6.8.1-6.8.8, John Wiley and Sons, Toronto.1994 But not limited thereto.   Assays for proliferation and differentiation of hematopoietic and lymphogenic cells Measurcmcnt of Human and Muine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S.and Lipsky, P.E.In Current Protocols in Immunology.J.E.e.a.C oligan eds.Vol 1 pp.6.3.1-6.3.12, John Wiley and Sons, Toronto.1991; deVrie s et al., J. Exp.Med. 173: 1205-1211, 1991; Moreau et al., Nature 336: 690-692, 1988; Green berger et al., Proc. Natl. Acad. Sci. U.S.A. 80: 2931-2938,1983; Measurement of mouse and human interleukin 6-Nordan, R. In Current Protocols in Immunology.J.E.e.a.Coligan eds.Vol 1 pp.6.6.1-6.6.5 , John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83: 1857-1861,1986; Measurement of human Interleukin 11-Bennett, F., Giannot ti, J., Clark, S.C.and Tumer, K.J.In Current Protocols in Immunology.J.E.e.a .Coligan eds.Vol 1 pp.6.15.1 John Wiley and Sons, Toronto.1991; Measurem ent of mouse and humman Interleukin9-Ciarletta, A., Giannotti, J., Clark, S.C .and Turner, K.J. In Current Protocols In Immunology.J.E.c.a.Colligateds. Vol 1pp.6.13.1, John Wiley and Sons, Toronto.1991 But not limited thereto.   Assays for the response of T cell clones to antigens (particularly proliferation and APC-T cell interaction as well as direct Identifying the effects of T cells) Current Protocols in Immnunology, Ed by J.E.Coligan, A.M.Kruisbeek, D.H.Mar gulies, E.M.Shevach, W Strobcr, Pub.Greene Publishing Associatcs and Wiley- Interscience (Chapter3, In Vitroassays for Mouse Lymphocyte Function; Chapt er 6, Cytokines and their cellular receptors; Chapter 7, Immunlogic studies  in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77: 6091-6095,1980; Weinberger et al., Eur. J. Immunol. 11: 405-411, 1981; Takai et al., J. Immunol. 137 : 3494-3500,1986; Takai et al., J.Immunol.140: 508-512,1988 But not limited thereto.   Immunostimulatory or suppressive activity   Further, the protein of the present invention has an activity in the assay described in the present specification (not limited thereto). ), And may exhibit immunostimulatory or immunosuppressive activity. Tongue In various deficiencies and disorders, including severe immunodeficiency complications (SCID) May be useful, for example, to increase the proliferation of T and / or B lymphocytes In regulating or down-regulating, and N Useful in enhancing the cytolytic activity of K cells and other cell populations It is possible. These immunodeficiencies can be genetic and can include viral ( For example, HIV) And may be caused by a bacterial or fungal infection, or May be caused by an autoimmune disease. More specifically, Infectious diseases caused by ruth, bacteria, fungi or other infectious agents, such as , HIV, hepatitis virus, herpes virus, mycobacteria, Leishmania, Various fungal infections such as malaria and Candida species can be treated using the protein of the present invention. Can be treated. Of course, in this regard, a boost to the immune system is indicated In other words, the protein of the present invention may be used 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, Guilla in-Barre syndrome, autoimmune thyroiditis, insulin-dependent diabetes, myasthenia gravis , Graft-versus-host disease and autoimmune inflammatory eye diseases. Of the present invention Such proteins can be used to treat allergic reactions such as asthma or other respiratory disorders and It may be used to treat symptoms. Other conditions for which immunosuppression is desired (eg, asthma (especially Allergic asthma) and related respiratory problems) using the protein of the present invention. You may be treated. Other conditions in which immunosuppression is desired (including, for example, organ transplantation) The treatment may be performed using the inventive protein.   The proteins of the present invention can also be used to enable an immune response in a number of ways. Da Unregulation can block or block an immune response already in progress Or includes interfering with the induction of an immune response. There may be. By suppressing the T cell response, or Inhibits activated T cell function by inducing resistance or both May be. Generally, immunosuppression of T cell responses is an active, non-antigen specific Process, which requires continuous exposure of T cells to the inhibitor. Resistance and Means non-responsive or anergic in T cells, generally antigen-specific In that it persists after exposure to the tolerizing agent has ceased. Operationally, the lack of a T cell response when exposed to a specific antigen in the absence of a resistance agent More resistance may be shown.   One or more antigen functions (eg, B lymphocyte antigen function (eg, B7) Including, but not limited to, down-regulating Ruko For example, disruption of high levels of lymphokine synthesis by activated T cells can be caused by tissue, Useful in skin and organ transplantation and graft versus host disease (GVHD) U. For example, blocking T cell function reduces tissue destruction in tissue transplantation. It is. Typically, in tissue transplants, the rejection of the graft is a Begun by being recognized as foreign by the vesicle, and then destroying the graft An immune response occurs. B7 lymphocyte antigen and its natural ligand on immune cells Molecules that inhibit or block interaction with another B lymphocyte antigen (eg, B 7-1, B7-3) mixed with a peptide in the monomer form having the activity. Or alone, a soluble monomeric peptide with B7-2 activity) Administration does not transmit a responsive costimulatory signal, but does not May induce binding of the molecule to gand. In this regard, B lymphocyte antigen Blocking function involves cytokine synthesis by immune cells such as T cells. Interferes, and thus acts as an immunosuppressant. Moreover, the lack of co-stimulation is T It may be sufficient to cause the cells to lose their response. For B lymphocyte antigen blocking reagent Induces long-term tolerance, thus requiring repeated administration of these blocking reagents Sex can be avoided. To achieve sufficient immunosuppression or tolerance in a subject It may also be necessary to block the function of the combination of B lymphocyte antigens.   Individual blocking in preventing organ transplant rejection or GVHD Evaluate the effectiveness of the reagents using animal models that can predict their effectiveness in humans be able to. Examples of suitable systems that can be used include allografts in rats and allografts. Xenogeneic pancreatic islet cell grafts in mice and Was used to test the immunosuppressive effect of the CTLA4Ig fusion protein (Lenscho w et al., Science 257: 789-792 (1992) and Turka et al., Proc Natl. Acad. Sci. USA, 89: 11102-11105 (1992)). In addition, GVHD mice Mimodel (Pauled., Fundamental Immunology, Ravan Press, New York, 1989, p. p. 846-847) using the B lymphocyte antigen for the progression of the disease in vivo. The blocking effect of the function can be determined.   In addition, blocking antigen function is therapeutically useful for treating autoimmune diseases so It is possible. Many autoimmune diseases are reactive against self-tissues and cause disease pathology. Activity of T cells that promotes the production of cytokines and autoantibodies involved in cytokines It is the result of conversion. Blocking activation of self-reactive T cells reduces signs of disease Or it can be removed. Disrupting B lymphocyte antigen receptor: ligand interactions Inhibits T cell activation using administration of reagents that block costimulation of T cells And production of autoantibodies or T cell-derived cytokines that may be involved in the disease process Can be disturbed. In addition, blocking reagents can lead to long-term remission of the disease. May induce antigen-specific resistance of potentially self-reactive T cells. Autoimmune disease The effectiveness of blocking reagents in preventing or ameliorating disease in human autoimmune diseases Can be determined using a number of well-characterized animal models for . Examples are murine experimental autoimmune encephalitis, MRL1pr / 1pr mice or NZ Systemic deep erythematosus and murine autoimmunity in B hybrid mice Collagen arthritis, diabetes in NOD mice and BB rats, and mice Experimental Myasthenia Gravis in M. (Paul ed., Fundamental Immunology, Raven Press, N. ew York, 1989, pp. 840-856).   Antigen function as a means of up-regulating the immune response (preferred In particular, up-regulation of B lymphocyte antigen function) is also therapeutically useful. Up-regulation of the immune response may be May be in a form that eliminates the initial immune response. For example, B lymphocyte antigen function Enhancing the immune response by stimulating may be useful in the case of a viral infection. In addition, systemic viral such as influenza, common cold, and encephalitis Disease may be ameliorated by systemic administration of a stimulatory form of B lymphocyte antigen .   Alternatively, T cells are taken from a patient and tagged with ACPs that express a peptide of the invention. Co-stimulate T cells in vitro with added viral antigens, or Is combined with a stimulating form of the soluble peptide of the invention and then activated in vitro Re-introduced T cells into the patient, the anti-virus in infected patients It may enhance the immune response. Another method of promoting an antiviral immune response is Infected cells are taken from the patient and the nucleic acid encoding the protein of the invention described herein is Transfer to So that the cell expresses all or part of the protein on its surface, Would reintroduce the transfected cells into the patient. Then, feeling Stained cells can transmit costimulatory signals to T cells in vivo, This could activate T cells.   In another application, antigen function (preferably B lymphocyte antigen function) Pre-regulation or promotion may be useful in inducing tumor immunity. Transfected with a nucleic acid encoding at least one peptide of the invention Tumor cells (eg, sarcomas, melanomas, lymphomas, leukemias, neuroblastomas, carcinomas) ) Can be administered to a subject to overcome tumor-specific resistance in the subject. If desired Transfection of tumor cells to express the peptide combination. Can be. For example, a tumor cell obtained from a patient is transformed into a peptide having B7-2-like activity. Having peptide-only or B7-1-like activity and / or B7-3-like activity An expression vector that directs expression in combination with Can be transfected. Transfected tumor cells Return to the patient to express the peptide on the surface of the transfected cells. You. Alternatively, transfection in vivo using gene therapy techniques Tumor cells can be targeted.   Existence of the peptide of the present invention having B lymphocyte antigen activity on the surface of tumor cells Provides the necessary co-stimulatory signals for T cells to provide T cell-mediated trafficking. Induces an immune response against the transfected tumor cells. In addition, MHC Tumor cells lacking class I or MHC class II molecules, or sufficient MHC Tumor cells that cannot reexpress class I or MHC class II molecules are I α chain protein and β_TwoMicroglobulin protein or MHC class II α chain or Or all or part of the MHC class II β chain protein (eg, Transfection with the nucleic acid encoding the Class I or class II MHC proteins can be expressed on the cell surface . A marker having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-3) Expression of the appropriate class I or class II HMC in combination with the peptide is Vesicle-mediated trans Induces an immune response against the transfected tumor cells. Invariant chain, if desired Antisense constructs that block the expression of MHC class II binding proteins Encodes a peptide having B lymphocyte antigen activity Co-transfection with existing DNA to enhance presentation of tumor-associated antigens However, it can also induce tumor-specific immunity. Therefore, T cells in human subjects Induction of a more mediated immune response is sufficient to overcome tumor-specific resistance in the subject. Minutes.   The activity of the protein of the invention may be measured, inter alia, by the following method:   Suitable assays for thymocyte or spleen cell cytotoxicity include: Current Protocols in Immunology, Ed by J.E.Coligan, A.M.Kruisbeek, D.H.Marg ulics, E.M.Shevach, W Strober, Pub.Greene Publishing Associates and Wiley-I nterscience (Chapter 3, In Vitroassays for Mouse Lymphocyte Function3.1-3. 19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Ac ad. Sci. USA 78: 2488-2492, 1981; Hermmann et al., J. Am. Immunol. 128: 1968-1974,19 82; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 13 7: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512, 1988; Herrnmnann et a USA, 78: 2488-2492, 1981, Hemnarm et al., J. L., Proc. Immunol.12 8: 1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 137: 3494-3500, 1986: Bowmanet al., J. Virology 61: 1992-1998; Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., Cellular Immunolo gy 133: 327-341, 1991; Brown et al., J. Immunol. 153: 3079-3092, 1994. Including, but not limited to, customized assays.   Updates for T cell-dependent immunoglobulin response and isotype switching Say (especially to modulate the T cell dependent antibody response to a Th1 / Th2 profile Influencing proteins are identified in Maliazewski, J. Immunol. 144: 3028-3033, 1990. Includes, but is not limited to, the assays described, and further enhances B cell function. Say, In vitro antibody production, Mond, J.J. and Brunswick, M. In Current  Protocols in Immunology J.E.Coligan eds.Val 1 pp.3.8.1-3.8.16, John Wile y and Sons, Tronto 1994.   Mixed lymphocyte reaction (MLR) assays (especially primarily Th1 and CTL Identify the protein that produces the answer) Current Protocols in Immunology, Ed by J.E.Coligan, A.M.Kruisbeek, D.H.Margu lics, E.M.Shevach, W Strober, Pub.Greene Publishing Associates and Wiley-In terscience (Chapter 3, In Vitroassays for Mouse Lymphocyte Function 3.1-3. 19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137: 3 494-3500,1986; Takai et al., J.Immunol.140: 508-512,1988; Bertagnolli et al. ., J.Immunol. 149: 3778-3783,1992 Including but not limited to the assays described in   Dendritic cell-dependent assays (especially for dendritic cells that activate untreated T cells) To identify more expressed proteins) Guery et al., J. Immunol. 134: 536-544, 1995; Inaba et al., Journal of Experime. ntal Medicine 173: 549-559,1991; Macatonia et al., Joumal of Immunology 154 : 5071-5079,1995; Porgador et al., Joumal of Experimental Medicine 182: 255- 260, 1995; Nair et al., Jounlal of Virology 67: 4062-4069, 1993; Huang et al., Science 264: 961-965,1994; Macatonia et al., Joumal of Experimental Medici ne 169: 1255-1264,1989; Bhardwajctal., Journal of Clinical Invcstigation 94 : 797-807,1994; and Inaba et al., Journal of Experimental Medicine 172: 631 -640,1990 Including but not limited to the assays described in   Lymphocyte survival / apoptosis assays (especially apoptosis after superantibody induction) Identify Proteins That Prevent Lysis and that Regulate Lymphocyte Homeostasis ) Is Darzynkiewicz et al., Cytometry 13: 795-808, 1992; Gorczyca et al., Leu kemia 7: 659-670, 1993; Gorczyca et al., Cancer Research 53: 1945-1951, 1993; I toh et al., Cell 66: 233-243,1991; Zacharchuk, Joumal of Immunology 145: 4037 -4045,1990; Zamai et al., Cytometry 14: 891-897,1993; Gorczyca et al., Intern ational Joumal of Oncology 1: 639-648,1992 But not limited thereto.   Early stage T cell commitment and developmental assays are described in An tica et al., Blood 84: 111-117, 1994; Fine et al., Cellular Immunology 155: 11 1-122, 1994; Galy et al., Blood 85: 2770-2778, 1995; Toki et al., Proc. Natl. Aca d.Sci. USA 88: 7548-7551, 1991, including but not limited to No.Hematopoietic regulatory activity   The proteins of the present invention are useful in regulating hematopoiesis and are therefore Useful in the treatment of bulb deficiency. Colony forming cells or factor-dependent cell lines Even minimal biological activity in support of has been implicated in regulating hematopoiesis. For example, to support the growth of erythroid progenitor cells alone, or to In combination with, for example, treatment of various anemias, or release Production of erythroid progenitors and / or erythroblasts in combination with radiation therapy / chemotherapy Stimulating viability; proliferation of bone marrow cells such as granulocytes and monocytes / macrophages Support (ie, traditional CSF activity), eg, combined with chemotherapy To prevent subsequent myelosuppression; megakaryocyte proliferation And then to support platelet proliferation and thereby thrombocytopenia It is useful for preventing or treating various platelet diseases, and generally Used instead of or in addition to plate transfusion; and / or hematopoietic stem cells Vesicles (mature to all of the above hematopoietic cells, and therefore various stem cell It is always a disease treated by transplantation, for example, aplastic anemia and seizures It is useful for supporting the growth of thymic hemoglobinuria) And even after radiation / chemotherapy in vivo or ex vivo (ie, Combined with bone marrow transplantation), or after being genetically engineered for gene therapy It is also useful in the repopulation of the stem cell compartment as a normal cell.   In particular, the activity of the protein of the present invention may be measured by the following method.   Assays suitable for proliferation and differentiation of various hematopoietic cell lines have already been cited .   Assays for embryonic stem cell differentiation, specifically identifying proteins that affect embryonic hematopoiesis ) Is Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al., Mol ecular and Cellular Biology 13: 473-486,1993; McClanahan et al., Blood81: 29 Includes, but is not limited to, the assays described in 03-2915,1993.   Assays for stem cell survival and differentiation, specifically identifying proteins that regulate lymphoper hematopoiesis Do) Methylcellulose colony forming assays, Freshney, M.G.In Culture of Hematop oietic Cells.R.I. Freshtey, et al. Eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Himy ama et al., Proc. Natl. Acad. Sci. USA 89: 5907-5911, 1992; Primitive hematopoie tic colony forming cells with high proliferative potential, McNiece, I.K.a nd Briddell, R.A.In Culture of Hematopoietic Cells.R.I.Frecshney, et al ed s. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experiment al Hematology 22: 353-359,1994; Cobblestone area forming cell assay, Ploema cher, R.E.In Culture of Hematopoietic Cells.R.I.Freshney, et al.eds.Vol pp .1-21, Wiley-Liss, Inc .., New York, NY.1994: Long term bone marrow cultures i n the presence of stromal cells, Spooncer, E., Dexter, M.and Allen, T.In Cult ure of Hematopoietic Cells.R.I.Freshney, et al.eds.Vol pp.163-179, Wiley- Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay, Suthe rland, H.J.In Culture of Hematopoietic Cells.R.I.Freshney, et al.eds.Vol pp.139-162, Wiley-Liss, Inc., New York, NY.1994 Including but not limited to the assays described inResearch applications and usefulness   The polynucleotide provided by the present invention can be used for various purposes depending on the research population. Can be used. Preferential expression of the corresponding protein for analysis, characterization or therapeutic use (Constitutively, at a specific stage of tissue differentiation or development, or (Expressed in disease states) As a quantity marker, it can be used to identify chromosomes or map related gene locations. Compared to the patient's endogenous DNA as a chromosome marker or tag (if labeled) Probes to identify potential genetic diseases Genetic fingerprinting to discover new related DNA sequences As a source of information for obtaining PCR primers for A probe to "subtract" known sequences in the process of finding nucleotides Oligomers to bind to a “gene chip” or other support DNA immunization to test expression patterns to select and create To generate protein antibodies, as well as to generate anti-DNA antibodies, or The use of polynucleotides as antigens to induce immune responses it can. Binds to or binds to another protein Proteins that specifically bind (eg, as in receptor-ligand interactions) If so, identify other proteins that bind and / or Interaction trap assays to identify inhibitors (see, eg, Gyuris et al., C ell 75: 791-803 (1993)). Can be   The proteins provided by the present invention are a family of multiple proteins for high-throughput screening. For assays to determine biological activity, including proteins of interest; production of antibodies or other A protein (or its receptor) in a biological fluid Reagents (including labeling reagents) in assays designed to quantitatively determine The corresponding protein is preferentially expressed (constitutively or with tissue differentiation or Are expressed at specific stages of development or in disease states) As well as, of course, to isolate the relevant receptor or ligand You may. Bind when a protein binds or potentially binds to another protein Proteins to identify other proteins and / or inhibitors of binding interactions White can be used. Using proteins involved in these binding interactions, Of peptide or small molecular inhibitor or agonist for binding interaction Training can also be performed.   Any or all of these research uses may be commercialized as research products. Can be developed to reagent grade or kit format.   Methods for performing the above uses are well known to those skilled in the art. Open this method References shown are "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spr. ing Harbor Laboratory Press, Sambrook, J., E.F.Fritsch and T. Maniatis e ds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techni ques ", including Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987 However, it is not limited to these.Nutritional uses   Use of the polynucleotide and protein of the present invention as a nutrient source or additive But it can. Such uses include protein or amino acid additives, carbon sources , The use as a nitrogen source and the use as a carbohydrate source Not limited to them. In such a case, the protein or polynucleotide of the present invention May be added as food, or powder, pill, solution, suspension or capsule Can be administered as a separate individual or liquid formulation, such as Weakness Product, the protein or polynucleotide of the present invention is added to the medium in which the microorganism is cultured. can do.   An animal is immunized with the U4 protein of the present invention, and can specifically react with the U4 protein. Polyclonal and monoclonal capable of inhibiting ligand binding to the body Antibodies may be obtained. Either use the entire U4 as an immunogen or use the U4 fragment Such an antibody may be obtained by using an antibody. A small fragment of U4 The animal may be used to immunize the animal. In addition, peptide immunogens have a carboxyl terminus It may contain a cysteine residue, and may contain keyhole limpet hemocyanin (KLH ) And conjugated with a hapten. Replace tyrosine residues with sulfated tyrosine residues Alternatively, additional peptide immunogens may be obtained. The combination of such peptides Synthesis methods are known in the art, for example, R.P.Merrifield, J. Amer. Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987 )It is in.   Neutralizing or non-neutralizing antibodies that bind to U4 protein (preferably monoclonal antibodies) May also be useful therapeutics for certain types of tumors, and may also cure the above symptoms. It may be a useful therapeutic agent in medical treatment. These neutralizing monoclonal antibodies Can block ligand binding to the U4 receptor chain.                                  Example Expression of U4 protein   DNA encoding the full-length murine U4 protein was coded by Gly-Ser-Gly. Is fused to the spacer sequence to be cloned by PCR, and then the COS-1 expression vector -PED. Sequence coding for hinge IgG2 and CH3 regions of human IgG1 in Fc And the frame together. Transfection of DNA into COS cells And Expression of the fusion protein was determined by ELISA using an antibody to detect the white IgG1 portion. Detected. This allows expression and secretion of proteins in COS cells It was shown that.   The patents and publications cited herein are hereby incorporated by reference in their entirety. It is assumed that

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C07K 16/28 C12P 21/02 C C12N 5/10 21/08 C12P 21/02 C12N 5/00 A // C12P 21/08 A61K 37/02 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG) , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, H, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, 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, US, UZ, VN, YU, ZW (72) Inventor Collins, Mary United States 01760 Massachusetts Natick, Euclid Avenue 27th (72) Inventor Neben, Thamrin United States 01720 Daghan Road, Acton, MA No.13 (72) Inventor Whittiers, Matthew United States01749 Haddons, Mass.

Claims (1)

[Claims]   1. (A) from nucleotide 242 to nucleotide 1396 of SEQ ID NO: 4 A nucleotide sequence of   (B) the nucleotide sequence from nucleotide 71 to nucleotide 1225 of SEQ ID NO: 6 Leotide sequence;   (C) the nucleoside shown in (a) or (b) as a result of the degeneracy of the genetic code A nucleotide sequence that differs from the nucleotide sequence;   (D) hybrids under stringent conditions to the nucleotides shown in (a) or (b) A nucleotide sequence that can be redistributed;   (E) a nucleotide sequence encoding a species homolog of the sequence shown in (a) or (b) Columns; and   (F) an allelic variant of the nucleotide sequence shown in (a) or (b) An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:   2. The nucleotide sequence is a U4 hematopoietin receptor superfamily chain; 2. The polynucleotide of claim 1 which encodes a protein having the biological activity of De.   3. 2. The method of claim 1, wherein said nucleotide sequence is operably linked to an expression control sequence. Polynucleotide.   4. Nuku from nucleotide 122 to nucleotide 1396 of SEQ ID NO: 4 2. The polynucleotide of claim 1, comprising a reotide sequence.   5. Nucleotides from nucleotide 11 to nucleotide 1225 of SEQ ID NO: 6 2. The polynucleotide of claim 1, comprising an otide sequence.   6. A host cell transformed with the polynucleotide of claim 3.   7. 7. The host cell of claim 6, wherein said cell is a mammalian cell.   8. (A) growing a culture of the host cell of claim 6 in a suitable medium;   (B) Purify U4 protein from culture A method for producing a U4 protein.   9. (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;   (D) an amino acid sequence of amino acids 24 to 408 of SEQ ID NO: 7;   (E) Determine the biological activity of the U4 hematopoietin receptor superfamily chain Fragments of (a) to (d) An isolated U4 protein comprising an amino acid sequence selected from the group consisting of:   10. 10. The protein of claim 9, comprising the amino acid sequence of SEQ ID NO: 5.   11. 10. The protein of claim 9 comprising the sequence of amino acids 41 to 425 of SEQ ID NO: 5. White.   12. 10. The protein of claim 9, comprising the amino acid sequence of SEQ ID NO: 7.   13. 10. The protein of claim 9 comprising the sequence of amino acids 24 to 408 of SEQ ID NO: 7. White.   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;   (D) an amino acid sequence of amino acids 24 to 408 of SEQ ID NO: 7;   (E) Determine the biological activity of the U4 hematopoietin receptor superfamily chain Fragments of (a) to (d) Encodes a peptide or protein containing an amino acid sequence selected from the group consisting of An isolated polynucleotide comprising a nucleotide sequence.   18. 10. The protein of claim 9, wherein said amino acid sequence is part of a fusion protein.   19. 19. The protein of claim 18, comprising an Fc fragment.