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WO1996011212A1 - Nouveau ligand de tyrosine kinase de type recepteur - Google Patents

Nouveau ligand de tyrosine kinase de type recepteur Download PDF

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Publication number
WO1996011212A1
WO1996011212A1 PCT/JP1995/002069 JP9502069W WO9611212A1 WO 1996011212 A1 WO1996011212 A1 WO 1996011212A1 JP 9502069 W JP9502069 W JP 9502069W WO 9611212 A1 WO9611212 A1 WO 9611212A1
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Prior art keywords
amino acid
seq
sequence
acid sequence
ligand
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PCT/JP1995/002069
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English (en)
Japanese (ja)
Inventor
Seiji Sakano
Mitsuharu Ohno
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Asahi Kasei Kogyo Kabushiki Kaisha
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Application filed by Asahi Kasei Kogyo Kabushiki Kaisha filed Critical Asahi Kasei Kogyo Kabushiki Kaisha
Priority to AU36198/95A priority Critical patent/AU3619895A/en
Publication of WO1996011212A1 publication Critical patent/WO1996011212A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention relates to a novel receptor pig tyrosine kinase type ligand. More specifically, the present invention binds to the extracellular portion of a specific receptor tyrosine kinase involved in the differentiation and proliferation of blood undifferentiated cells, and reduces the tyrosine kinase activity of the intracellular portion.
  • the present invention relates to a novel receptor tyrosine kinase ligand that is activated to cause phosphorylation of the tyrosine kinase.
  • the present invention also provides a complex comprising the novel receptor tyrosine kinase ligand described above, and at least one member selected from the group consisting of the ligand and a compound other than the ligand.
  • red blood cells carry oxygen in the body
  • platelets provide a haemostatic effect
  • white blood cells make up the immune system to protect against infection.
  • These diverse cells are derived from hematopoietic stem cells in the bone marrow.
  • hematopoietic stem cells have been clearly influenced by various hematopoietic factors and environmental factors in the body and differentiated into various blood cells, osteoclasts, mast cells, and the like.
  • EPO Erythropoietin
  • G-CSF granulocyte colony-stimulating factor
  • Mp 1 -L platelet growth factor
  • Tyrosine kinase an enzyme that specifically phosphorylates tyrosine, an amino acid present in proteins, transmits signals from the outside of the cell to the inside of the cell, and the gene in the cell nucleus. It is an important substance that controls transcription. Recent studies have revealed that tyrosine kinases and activators that activate tyrosine kinase enzyme activity are significantly involved in the development and differentiation of animals and insects. It is considered that tyrosine kinase and its activator are greatly involved in cell differentiation and proliferation.
  • the physiologically active site of tyrosine kinase is composed of about 250 amino acid residues.
  • amino acid sequences of the tyrosine kinase group there is a very conserved amino acid sequence (Hanks et a 1. Science 241: 42, 1988).
  • C-kit which is a type of receptor pig type 1 osteosynkinase and is expressed on the surface of blood undifferentiated cells, is a mast cell growth factor and one of the growth factors of blood undifferentiated cells. It has been found that stem cell factor (SCF) is activated by binding to its extracellular portion (Witte. Cell 63: 5, 1992). Cell differentiation is controlled through the tyrosine kinase kit.
  • SCF stem cell factor
  • ELK a receptor type 1 tyrosine kinase
  • LERK-2 a receptor type 1 tyrosine kinase
  • the present inventors have previously described a novel receptor tyrosine kinase gene different from the receptor tyrosine kinase c-kit described above, which is involved in the differentiation and proliferation of blood undifferentiated cells. Cloning was performed from the blast leukemia cell line UT-7, and the entire nucleotide sequence was determined. Then, a method for genetically producing the receptor type 1 tyrosine kinase was established [refer to International Patent Publication No. WO 95/15386 (this receptor type tyrosine kinase). Nucleotide sequence and amino acid sequence The sequence considered to be the same is also described in Bennett el a 1., J. ⁇ i ⁇ 1. Chem., 269, 14211-14218 ⁇ , 1994)]).
  • a ligand for a receptor tyrosine kinase specifically binds to an extracellular portion of a certain receptor tyrosine kinase, activates a tyrosine kinase enzyme activity in that intracellular portion, and It is defined as a substance having the property of inducing the phosphorylation of tyrosine kinases.
  • one kind of receptor-type tyrosine kinase has one kind of ligand in the living body.
  • organs expressing the receptor tyrosine kinase, specific cells expressing the same, and changes in expression due to some stimuli are detected by the antisense oligonucleotide of the receptor tyrosine kinase.
  • organs expressing the receptor tyrosine kinase, specific cells expressing the same, and changes in expression due to some stimuli are detected by the antisense oligonucleotide of the receptor tyrosine kinase.
  • In situ using a nucleotide Detecting by a staining method using hybridization, Northern blot, or an antibody, etc., in the body where the ligand acts Since the site and the cell can be specified, it is possible to estimate the physiological action of the ligand to some extent.
  • the extracellular domain of the receptor pig type 1 ostium syncinase polypeptide containing the amino acid sequence of SEQ ID NO: 1 in the attached sequence listing previously disclosed by the present inventors was specific.
  • Cells that express a novel compound (ligand) that bind specifically and promote the proliferation of blood undifferentiated cells were successfully identified, and the ligand was purified and isolated from the expressed cells.
  • the amino acid sequence of the amino terminal (N-terminal) of the ligand is determined, a primer is prepared based on the amino acid sequence, and c A DNA probe was prepared and cDNA encoding the full-length amino acid sequence was isolated. Using this cDNA, a cell expressing the ligand was prepared, and the ligand was further isolated.
  • the resettiva type 1 tyrosine kinase ligand of the present invention significantly promoted the proliferation of blood undifferentiated cells.
  • the extracellular domain of the receptor type 1 oral synthase polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 is added to other known receptor type tyrosine kinase ligands.
  • the receptor tyrosine kinase ligand of the present invention bound very tightly, causing phosphorylation of the tyrosine kinase, compared to almost no binding.
  • the ribose pig type 1 tyrosine kinase ligand of the present invention comprises the amino acid sequence of SEQ ID NO: 1.
  • the receptor tyrosine kinase ligand of the present invention is one of the factors that significantly promote the differentiation and proliferation of blood undifferentiated cells.
  • the present inventors further provide a complex of one or more molecules of the ligand of the present invention with a heterologous protein or a complex comprising two or more molecules of the ligand of the present invention (dimer or more) Was found to have an activity equal to or greater than the physiological activity of one molecule of the ligand.
  • an antibody was produced that specifically recognizes and binds to the novel receptor pig type 1 tyrosine kinase ligand described above. The present invention has been completed based on the above findings.
  • one object of the present invention is to provide a novel ligand that specifically binds to a specific receptor tyrosine kinase involved in the differentiation of blood undifferentiated cells and activates the tyrosine kinase activity, And a DNA encoding the same.
  • Another object of the present invention is to provide one or more molecules of the above-mentioned receptor type tyrosine kinase ligand having a bioactivity equivalent to or higher than that of the above-mentioned receptor type 1 tyrosine kinase ligand and a heterologous protein. And a complex comprising two or more molecules of the ligand.
  • Still another object of the present invention is to specifically bind to the ligand of the present invention, which can be advantageously used for purification of the ligand. To provide an antibody.
  • Still another object of the present invention is to provide a sense DNA fragment and an antisense which can be advantageously used for confirming the gene expression of the ligand of the present invention and for regulating gene expression in cells.
  • An object of the present invention is to provide a DNA fragment and a derivative thereof, and a sense RNA fragment and an antisense RNA fragment and a derivative thereof.
  • the amino acid sequence of SEQ ID NO: 1 is the sequence of the extracellular domain of the receptor pig type 1 tyrosine kinase to which the ligand of the present invention specifically binds, excluding the signal peptide.
  • the amino acid sequence of the receptor type 1 tyrosine kinase shown in SEQ ID NO: 3 corresponds to amino acid numbers 1 to 522 of the entire amino acid sequence;
  • the amino acid sequence of SEQ ID NO: 2 corresponds to the extracellular domain and membrane permeation of the ribosomal type 1 lipoprotein kinase to which the ligand of the present invention specifically binds, excluding signal peptide.
  • SEQ ID NO: 3 is the entire amino acid sequence of the receptor tyrosine kinase to which the ligand of the present invention specifically binds, and the entire cDNA sequence of the receptor tyrosine kinase. ;
  • SEQ ID NO: 4 is the sequence of the 8 amino acid residues at the N-terminal side of the ligand of the present invention, and the amino acid sequence of the entire amino acid sequence of the ligand of the present invention shown in SEQ ID NO: 7 is SEQ ID NO: 7.
  • SEQ ID NO: 5 corresponds to the amino acid Nos. 1 to 8;
  • SEQ ID NO: 5 is the sequence of the extracellular domain of the ligand of the present invention excluding the signal peptide, and is represented by SEQ ID NO: 7.
  • SEQ ID NO: 6 is the sequence of all the domains of the extracellular domain, transmembrane domain, and intracellular domain of the ligand of the present invention except for the signal peptide.
  • 7 corresponds to amino acid numbers 1 to 308 of the entire amino acid sequence of the ligand of the present invention shown in FIG. 7;
  • SEQ ID NO: 7 is the entire amino acid sequence and the entire cDNA sequence of the ligand of the present invention.
  • SEQ ID NO: 8 is a nucleotide sequence of sense primer used in Reference Example 2;
  • SEQ ID NO: 9 is the base sequence of antisense primer used in Reference Example 2;
  • SEQ ID NO: 10 is the nucleotide sequence of primer 11 used in Reference Example 8
  • SEQ ID NO: 11 is the nucleotide sequence of primer 2 used in Reference Example 8;
  • SEQ ID NO: 12 is the nucleotide sequence of primer 3 used in Reference Example 8;
  • SEQ ID NO: 13 is the nucleotide sequence used in Reference Example 8 and the amino acid sequence of the oligopeptide FLAG which it encodes;
  • SEQ ID NO: 14 is the primer sequence used in Reference Example 8. A sequence of 14 bases;
  • SEQ ID NO: 15 is the nucleotide sequence of primer 15 used in Reference Example 8;
  • SEQ ID NO: 16 is the base sequence of primer 6 used in Reference Example 8;
  • SEQ ID NO: 17 is the nucleotide sequence of primer 7 used in Reference Example 8;
  • SEQ ID NO: 18 is the nucleotide sequence of primer 8 used in Reference Example 8;
  • SEQ ID NO: 19 is the N-terminal sequence of the ligand of the present invention determined by the peptide sequencer in Example 9, and comprises the entirety of the ligand of the present invention shown in SEQ ID NO: 7.
  • SEQ ID NO: 20 is the nucleotide sequence of primer 19 used in Example 10;
  • SEQ ID NO: 21 is a fragment of the primer 10 used in Example 10 In base sequence
  • SEQ ID NO: 22 is the nucleotide sequence of the cDNA probe prepared in Example 10 and the amino acid sequence encoded by it.
  • SEQ ID NO: 23 is the LERK-2 gene of Example 19 in Example 19. A sense primer used for crossing; and
  • SEQ ID NO: 24 is an antisense primer used in Example 19 to close the LERK-2 gene.
  • each amino acid sequence represented by SEQ ID NOs: 1 to 4 and 7 are the N-terminal and C-terminal, respectively, and the base sequence represented by SEQ ID NOs: 4 to 7 Are the 5 'and 3' ends, respectively.
  • FIG. 1 is a graph showing the results of measuring the binding activity of the ligand in the cell supernatant of C-11 cells (unstimulated) to ostium synkinase in Example 2;
  • FIG. 2 is a graph showing the results of measurement of the fluorescence peak of the cells expressing the ligand of the present invention by the flow site meter in Example 3;
  • FIG. 3 is a photograph of a Western blot of cells expressing the ligand of the present invention by phosphorylation in Example 4;
  • FIG. 4 is a photograph showing the result of electrophoresis of the primary purified ligand of the present invention in Example 5;
  • FIG. 5 is a graph showing a chromatographic pattern obtained by gel filtration in Example 6.
  • FIG. 6 is a graph showing the measurement results of the ligand binding activity of each fraction of the separated solution by gel filtration in Example 6;
  • FIG. 7 is a photograph showing the result of electrophoresis of the ligand of the present invention purified by gel filtration in Example 6;
  • FIG. 8 is a photograph showing the result of electrophoresis of the finally purified ligand of the present invention in Example 7;
  • FIG. 9 is a graph showing the result of comparing the ligand binding activity of the ligand of the present invention and LERK-2 in Example 19;
  • FIG. 10 shows a photograph of a Western blot comparing the ligand of the present invention with LERK-2 ⁇ for phosphorylation activity.
  • an isolated amino acid sequence having a receptor tyrosine kinase activity and containing an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 and 2 in the sequence listing is provided.
  • Has the ability to bind to a polypeptide and a homologous mutant having a receptor tyrosine kinase activity of the polypeptide is analyzed by polyacrylamide gel electrophoresis.
  • an isolated polypeptide comprising at least a portion of the polypeptide containing the amino acid sequence of SEQ ID NO: 6 in the sequence listing is provided.
  • Compounds are provided.
  • a compound containing at least a part of a polypeptide containing an amino acid sequence of SEQ ID NO: 6 in a sequence listing a compound containing at least a part of a polypeptide containing an amino acid sequence of SEQ ID NO: 6 in a sequence listing
  • At least one kind of the compound containing at least a part of the polypeptide containing the amino acid sequence of SEQ ID NO: 6 in the sequence listing and the amino acid of SEQ ID NO: 6 in the above sequence listing At least one member selected from the group consisting of compounds other than compounds containing at least a part of the polypeptide containing an acid sequence;
  • a conjugate comprising:
  • an isolated DNA encoding at least a part of a polypeptide containing the amino acid sequence of SEQ ID NO: 6 in the sequence listing.
  • a method is provided.
  • the present invention comprises the above-described compound of the present invention or the amino acid sequence of SEQ ID NO: 5 in the sequence listing.
  • the present invention provides an antibody that specifically recognizes a compound containing at least a part of the polypeptides.
  • Item 1 having the property of phosphorylating at least one tyrosine residue of the polypeptide when reacted with a cell expressing a homologous mutant having a septa type 1 tyrosine kinase activity.
  • At least a part of the polypeptide containing the amino acid sequence of SEQ ID NO: 6 in the above sequence listing is at least partially containing the amino acid sequence of SEQ ID NO: 5 in the sequence listing. 7.
  • At least a part of the polypeptide containing the amino acid sequence of SEQ ID NO: 6 in the above sequence listing is at least partially derived from the polypeptide containing the amino acid sequence of SEQ ID NO: 5 in the sequence listing.
  • At least one kind of a compound containing at least a part of the polypeptide containing the amino acid sequence of SEQ ID NO: 6 in the sequence listing and the amino acid of SEQ ID NO: 6 in the above sequence listing At least one member selected from the group consisting of compounds other than compounds containing at least a part of a polypeptide having an acid sequence;
  • At least a portion of the polypeptide containing the amino acid sequence of SEQ ID NO: 6 in the above sequence listing is at least partially a protein of SEQ ID NO: 5 in the sequence listing. 21.
  • At least a part of the polypeptide containing the amino acid sequence of SEQ ID NO: 6 in the above sequence listing is a polypeptide containing the amino acid sequence of SEQ ID NO: 5 in the sequence listing.
  • a method for producing an isolated compound comprising at least a portion of a polypeptide containing the amino acid sequence of SEQ ID NO: 6 in the sequence listing:
  • DNA encoding at least a part of the polypeptide is linked to a replicable expression vector, and the DNA is operably incorporated into the replicable expression vector.
  • At least a part of the polypeptide containing the amino acid sequence of SEQ ID NO: 6 in the above sequence listing is a polypeptide containing the amino acid sequence of SEQ ID NO: 5 in the sequence listing.
  • the method of item 14 which is at least part of the code.
  • a single DNA selected from the group consisting of a sense DNA containing at least 12 consecutive nucleotide sequences of the nucleotide sequence of SEQ ID NO: 7 in the sequence listing, and an antisense DNA complementary to the sense DNA
  • the isolated DNA fragment, and the sense DNA and the antisense DNA are methylated, methylphosphorylated, thiophosphorylated, or deaminated, respectively.
  • An antisense RNA comprising at least 12 consecutive nucleotide sequences complementary to the nucleotide sequence of SEQ ID NO: 7 in the sequence listing, and a sense RNA complementary to the antisense RNA.
  • the selected isolated RNA fragment, and the antisense RNA and the sense RNA are methylated, methylphosphorated, thiophosphorylated, or deamino, respectively.
  • preparation of cDNA necessary for gene manipulation examination of expression by Northern blot, screening by hybridization, preparation of recombinant DNA, determination of DNA base sequence,
  • a series of molecular biology experiments, such as preparation of a cDNA library can be performed by the method described in an ordinary experiment manual.
  • the usual test book mentioned above is, for example,
  • the receptor tyrosine kinase ligand of the present invention has an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 and 2 in the sequence listing, having receptor type 1 tyrosine kinase activity. And has the ability to bind to the isolated polypeptide and a homologous mutant of the polypeptide having a receptor-type thymic synthase activity, and is capable of binding to a polyacrylamide gel.
  • the term “receptor tyrosine kinase activity” refers to an enzyme activity that tyrosine kinase originally possesses for phosphorylating tyrosine residues, and the extracellular domain of receptor tyrosine kinase.
  • the “reset pig type 1 tyrosine kinase ligand activity” refers to a receptor type 1 tyrosine kinase having at least the amino acid sequence of SEQ ID NO: 1 in the sequence listing.
  • An activity of binding to a polypeptide that is, an extracellular domain of the receptor pig type 1 tyrosine kinase
  • a receptor type containing the amino acid sequence of SEQ ID NO: 2 in the sequence listing Tyrosine kinase lipopeptide (ie, a polypeptide composed of an extracellular domain, a transmembrane domain, and an intracellular domain excluding the signal peptide of the receptor-type tyrosine kinase).
  • mutations such as intra-species mutations and allelic mutations that are known to occur in nature and can be artificially produced Homologous mutants having a receptor tyrosine kinase ligand activity, which are caused by mutations such as point mutations, are also included in the receptor tyrosine kinase ligand of the present invention.
  • the chromosomal DNA or cDNA isolated from the natural environment can cause the amino acid sequence encoded by the DNA to be reduced due to the degeneracy of the genetic code. There are often cases where the DNA base sequence is mutated without change.
  • the DNA sequences of those regions are easily mutated.
  • the nucleotide sequence obtained by such degeneracy or mutation of the gene, and the amino acid sequence of the above-described ligand homologous mutant having the receptor type 1 tyrosine kinase activity are described below.
  • the nucleotide sequence to be coded is also included in the DNA of the present invention.
  • the step of obtaining the cDNA of the receptor type 1 tyrosine kinase used in the present invention is performed as described in Reference Examples 1 to 6 described below. You.
  • PCR is performed using primers corresponding to the amino acid sequence characteristic of the tyrosine kinase gene.
  • Preparation of PCR primers and PCR can be performed by the method of Wi 1 ks (Proc. Nat 1. Acad. Sci. USA 86: 1603, 1989). Specifically, an oligonucleotide is synthesized and purified using a commercially available DNA synthesizer to obtain a primer for PCR.
  • the PCR primer was used to transform human megakaryoblastic leukemia cell line UT-7 (Japan, Kumamoto University School of Medicine, Division of Developmental Medicine, Division of Developmental Medicine, Toshio Suda (Available from Lecturer, Department of Hematology, Jichi Medical University, Japan, Lecturer Norio Komatsu) and perform PCR to amplify specific sites. As a result, about 200 bp, which is a part of a specific tyrosine kinase, can be expanded.
  • the PCR product DNA fragment is separated by agarose gel electrophoresis or the like, and purified and recovered.
  • nucleotides 2644 to 2812 of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing encodes the central portion of the active site of the chicokinase enzyme and encodes an amino acid sequence that plays an important role in intracellular signaling.
  • the cDNA fragment cloned by the method described above is labeled with an isotobe, and the cDNA of the UT-7 cell line is cloned. It can be obtained by screening a DNA library by a method such as hybridization. Is a labeling eye Seo bets Ichipu was example, if [32 P] y - how to label the end with ATP and T 4 port I J click Reochi Dokina one peptidase and other two click DOO Labeling methods such as the lanceration method or the primer extension method can be used.
  • the cDNA base sequence of the receptor tyrosine kinase used in the present invention is shown as SEQ ID NO: 3 in the sequence listing together with the amino acid sequence encoded by it.
  • the nucleotide sequence is longer than 409 bases. 5 'untranslated region, followed by a region coding for the receptor tyrosine kinase consisting of 2961 bases, and further followed by 3' consisting of 919 bases Consists of untranslated regions.
  • the amino acid sequence of this receptor tyrosine kinase is a signal peptide composed of 15 amino acids corresponding to amino acids 15 to 11 of SEQ ID NO: 3 in the sequence listing. And the extracellular portion composed of amino acids 5 to 22 of amino acid sequence No. 1 to SEQ ID No.
  • amino acid sequence of SEQ ID No. 3 in the sequence listing amino acid sequence of SEQ ID No. 3 in the sequence listing
  • the transmembrane portion composed of 26 amino acids corresponding to Nos. 52 3 to 54 8 of the amino acid sequence of SEQ ID NO: 3 in the sequence listing
  • the intracellular portion composed of 4 2 4 amino acids is composed of the same.
  • 260 amino acid corresponding to amino acids 600 to 859 of the amino acid sequence of SEQ ID NO: 3 in the sequence listing is a tyrosine kinase enzyme active portion.
  • the transformed cell obtained by transfecting Escherichia coli JM109 (manufactured by Toyobo, Japan) with the vector pBSRTKFULL containing the entire nucleotide sequence of this resettle pig type 1 tyrosine kinase cDNA was produced by the Ministry of International Trade and Industry of Japan. Deposited at the National Institute of Advanced Industrial Science and Technology, National Institute of Biotechnology, under the deposit number FE RM BP—4883, on January 11, 1994.
  • polypeptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is obtained by removing the amino acid in the portion excluding the signal peptide in the extracellular portion of the receptor type 1 lipoprotein kinase. Has an acid sequence Equivalent to a polypeptide.
  • polypeptide having the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing is a polypeptide having an amino acid sequence excluding the signal peptide of the receptor tyrosine kinase described above. Equivalent to a peptide.
  • the receptor tyrosine kinase used in the present invention is human EPH (Hirai eta, Science, 238, 1717—1720, 1987), a cloned receptor type 1 tyrosine kinase. ECK (Lindberg and Hunter, Mo 1. Cel l.
  • the methods for obtaining the receptor tyrosine kinase gene used in the present invention include the methods described in Reference Examples 1 to 6 and commercially available human placenta or fetal liver cDNA. Uses the cDNA library directly as a template, and A sense primer and an antisense primer of about 2 O mer of the DNA sequence described in No. 3 are prepared and subjected to PCR, or by using this PCR product as a probe.
  • the library can be obtained by screening using the method described in Reference Examples 1 to 6.
  • the cDNA of the receptor type 1 tyrosine kinase used in the present invention is not only UT-7 but also a human chronic myelogenous leukemia cell line, K5662, a blood cell line.
  • RCB 027 and human acute megakaryoblastic leukemia cell line CMK (Blood 74: 42, 1989), and liver for non-blood cell lines.
  • Cell carcinoma cell line Hep3B American 'Type' Culture 'Collection (hereinafter referred to as ATCC) available as HB8064] and human fetal lung fibroblast cell line MRC — 5 (available from the National Institute of Physical and Chemical Research, Cell Development Bank, No. RCB0211), etc., and can be obtained in substantially the same manner as Reference Examples 1 to 6 described below. You.
  • the degeneracy of the gene code causes The desired recombinant DNA can be prepared by changing the gene sequence without changing the acid sequence.
  • the properties of the receptor tyrosine kinase used in the present invention are as described in Reference Example 7. That is, when an undifferentiated blood-derived cell line is differentiated by a stimulator and loses its proliferative ability, Expression of mRNA of receptor tyrosine kinase gene is not observed. Therefore, it is presumed that this receptor-type tyrosine kinase is a receptor for a differentiation / growth factor of particularly unknown blood undifferentiated cells.
  • the receptor type 1 tyrosine kinase when the receptor type 1 tyrosine kinase is to be expressed under more stable conditions, it is preferable to use a nucleated cell, particularly an animal cultured cell, as a host.
  • the polypeptide outside the cell can be purified by affinity chromatography, and an antibody can be produced by using this as an antigen.
  • three mouse monoclonal antibody-producing hybridomas that specifically recognize this receptor type 1 tyrosine kinase, namely, clones 38-1E and 66-6-3 6 8 — 38 are deposit numbers FERBP—4884, BP—4885, and BP—488 at the Research Institute of Biotechnology, Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry of Japan. 6 deposited on January 11, 1974.
  • cells producing ligands that specifically bind to the receptor tyrosine kinase are identified and selected.
  • Examples of the method include a method using a biosensor BIACORE (manufactured by FANORE Masia Co., Sweden), as described in Example 1, Cara 4.
  • This instrument can detect protein binding based on the principle of plasmon resonance on the surface of a substance. That is, a ligand molecule present in the culture of various human cell lines or on the cell membrane and a receptor type 1 tyrosine kinase containing the amino acid sequence described in SEQ ID NO: 1 in the sequence listing.
  • a cell line producing a substance that binds to the receptor tyrosine kinase polypeptide can be selected.
  • a supernatant or cells of various cell lines and a cell line expressing the receptor type 1 lipoprotein kinase polypeptide described in SEQ ID NO: 2 in the sequence listing may be used. Were cultured together and the presence or absence of phosphorylation of tyrosine residues in the intracellular domain of the receptor type 1 tyrosine synthase polypeptide was determined. You can select a cell line that expresses the ligand that binds to the kinase.
  • an antibody recognition site for example, an antibody recognition site such as a receptor tyrosine kinase polypeptide described in SEQ ID NO: 1 in the sequence listing or a nucleotide sequence encoding this polypeptide is used.
  • Examples 1 to 4 of the present application as the ligand-expressing cell of the present invention, a human colon cancer-derived cell line C-1 (reproducible from the Japan Institute of Immunology and Biology, Inc. Te Sato medicine Ayumi, 9 6:. 8 7 6, 1 9 7 6 years) t identified the following cells:
  • the ligand of the present invention is purified from cells expressing the same. Purification of the ligand, measurement of molecular weight, evaluation of physical properties, and identification of the N-terminal amino acid sequence in the present invention are described in Example 5. This is the communication described in Nos. To 9. That is, a culture supernatant or a cell membrane fraction of a ligand-expressing cell line, a solution prepared by treating cells with a specific enzyme, or the like contains an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. And purified using a carrier such as affinity gel to which the polypeptide is bound, and further purified by using the amino acid sequence of SEQ ID NO: 1 in the sequence listing.
  • a carrier such as affinity gel to which the polypeptide is bound
  • the ligand of the present invention can be purified by a separation operation such as gel filtration or ion exchange using the activity of phosphorylating the cysteine synthyl residue of the polypeptide as an index. It can be.
  • the thus purified compound (ligand) of the present invention is an isolated polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2 in the sequence listing. And a binding ability of the polypeptide to a homologous mutant having a receptor tyrosine kinase activity, and having a molecular weight of at least 4 as measured by polyacrylamide gel electrophoresis.
  • the ligand of the present invention an isolated polypeptide having a receptor type 1 tyrosine kinase activity and containing the amino acid sequence of SEQ ID NO: 2 in the sequence listing, Alternatively, when the polypeptide is reacted with a cell expressing a homologous mutant having a receptor tyrosine kinase activity, at least one tyrosine residue of the polypeptide is phosphorylated. Those having characteristics are preferred.
  • the ligand of the present invention also includes a polypeptide containing the amino acid sequence described in SEQ ID NO: 4 in the sequence listing, and the amino acid sequence is preferably More preferably, it is located at the amino terminal of the above-mentioned polypeptide.
  • the ligand of the present invention is preferably capable of undergoing both a Coomassie brilliant staining reaction and a PAS staining reaction (detecting the presence or absence of sugar chain binding).
  • the amino acid sequence described in SEQ ID NO: 4 in the sequence listing was determined by the Protein Sequencer (manufactured by Applied Biosystems, USA), and the amino terminal (N The amino acid sequence of amino acid Nos. 1 to 8 of the sequence at the end) is shown.
  • the amino acid sequence of SEQ ID NO: 4 in this sequence listing was converted into a protein database, NBRF-PDB (National Institute of Biomedical Research, Research Foundation, Release 4.0, 1). March 1994) and SWISS — PROT (Eurobian 'Molecular' Biology 'Laboratories, Release 29.0, June 1994). At that time, no known protein having the same sequence was found, and it was a novel amino acid sequence.
  • the ligand of the present invention that is, an isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2 in the sequence listing, It has the ability to bind the peptide to a homologous mutant having receptor type 1 tyrosine kinase activity, and has a molecular weight of at least 41 measured by polyacrylamide gel electrophoresis.
  • SEQ ID NO: 19 in the sequence listing contains the purified ligase of the present invention.
  • the amino acid sequence identified from the N-terminus of the protein by the protein sequencer is shown.
  • a sense primer and an antisense primer were synthesized based on the amino acid sequence of SEQ ID NO: 19 in the sequence listing, and the cDNA of the entire cDNA sequence of the ligand of the present invention was synthesized. Can be used for cloning.
  • the ligands can be fragmented by treatment with a protease such as trypsin or a chemical treatment to determine their amino acid sequences, or to determine the amino acid sequence at the carboxyl terminus (C-terminus). It is possible to determine the acid sequence.
  • a protease such as trypsin or a chemical treatment to determine their amino acid sequences, or to determine the amino acid sequence at the carboxyl terminus (C-terminus). It is possible to determine the acid sequence.
  • the ligand of the present invention is a cell membrane-associated protein as evident from the analysis results of the amino acid sequence, but as shown in Examples 5 to 7,
  • the ligands of the invention can be purified from the cell culture supernatant of the expression cells. This is considered to be because extracellular domains were released into the cell supernatant due to cleavage of the amino acid sequence on the cell membrane by a protease such as metalob lipase.
  • the ligand-expressing cell of the present invention also contains the amino acid sequence of SEQ ID NO: 2 (which has the amino acid sequence of SEQ ID NO: 3) of the receptor type 1 tyrosine kinase. (Excluding extracellular domains, transmembrane domains, and intracellular domains, excluding domains), and binding to polypeptides; A receptor containing the amino acid sequence described in SEQ ID NO: 3 (the entire amino acid sequence of the receptor type 1 tyrosine kinase); When a cell expressing the tyrosine kinase polypeptide is reacted with a cell expressing the ligand, the tyrosine residue of the receptor tyrosine kinase zepolipide is phosphorylated; As shown, the results of analysis of the amino acid sequence deduced from the total cDNA sequence of the ligand indicate that the ligand of the present invention has a transmembrane portion.
  • the ligand of the present invention can be purified from cell membranes.
  • the ligand of the present invention contains not only extracellular domains but also transmembrane domains and intracellular domains. The size also increases.
  • the amino acid sequence of the ligand is modified by a genetic engineering technique.
  • the gene can be obtained by obtaining the gene to be cloned, introducing the gene into a nucleated cell such as an animal cell, or a prokaryotic cell such as Escherichia coli, producing and isolating the ligand. .
  • an oligo DNA probe was prepared according to the method described in Example 10.
  • One-shot method or PC The gene encoding the amino acid sequence of the full length of the ligand can be screened by the method or a method combining the both.
  • a cDNA library of a ligand-expressing cell is incorporated into an appropriate expression vector and expressed in a COS cell or the like.
  • An example of a method for isolating the cDNA of the ligand of the present invention is an expression cloning technique for screening genes. The expression cloning is performed by a cell sorter utilizing the binding of the receptor-type tyrosin kinase polypeptide containing the amino acid sequence described in SEQ ID NO: 1 in the sequence listing to the ligand.
  • a method such as a fractionation method, a detection method using a film emulsion using a radioisotope, or a cell line transfected with the cDNA library itself or its cell culture.
  • a cell line expressing a receptor tyrosine kinase polypeptide containing the amino acid sequence of SEQ ID NO: 2 in the sequence listing, the tyrosine residue of the polypeptide was reduced.
  • a method based on the fact that phosphorylation is used can be used.
  • the cDNA base sequence coding for the ligand thus obtained is shown in the Sequence Listing along with the non-coding region before and after it and the amino acid sequence coding for it.
  • Figure 7 shows the results.
  • the amino acid sequence predicted from the cDNA sequence was determined by the method of Kyte — Doo 1 itt 1 e U. Mol. Biol. 157: 105, 19 82), the hydrophobic part and the hydrophilic part were analyzed.
  • the ligand of the present invention has a permeable domain consisting of a hydrophobic amino acid sequence and an intracellular and extracellular domain consisting of a hydrophilic amino acid sequence. It was revealed that it was a peptide anchor type cell membrane protein.
  • the extracellular domain of the receptor type 1 tyrosine kinase is released from the intracellular domain of the receptor type 1 tyrosine kinase. May cause oxidation.
  • the amino acid sequence of the ligand of the present invention is obtained from the amino acid sequence of SEQ ID NO: 7 from the 25th amino acid residue to the 25th amino acid residue of the 1st amino acid sequence.
  • Signal peptide extracellular domain consisting of 1-95 amino acid residues from amino acids 1 to 195 in the amino acid sequence of SEQ ID NO: 7 in the sequence listing, SEQ ID NO in the sequence listing
  • the amino acid sequence of No. 7 is a transmembrane ifi domain consisting of amino acids Nos. 196 and 225 of the amino acid sequence No. 22 and No. 25 of the amino acid sequence No. 7 in the sequence listing. It is composed of an intracellular domain consisting of 83 amino acid residues from No.226 to No.308.
  • each part other than the signal peptide part has the domain configuration deduced from the amino acid sequence, and its actual form may be slightly different from the above configuration.
  • the constituent amino acids of each domain specified above may be around 5 to 10 amino acids.
  • amino acid sequence described in SEQ ID NO: 5 in the sequence listing is the amino acid sequence of amino acids 1 to 195 of the amino acid sequence in SEQ ID NO: 7 in the sequence listing.
  • Amino acid sequence, ie the amino acid sequence of the above-mentioned extracellular domain excluding the signal peptide
  • amino acid sequence of SEQ ID NO: 6 in the sequence listing is SEQ ID NO: 7 in the sequence listing.
  • 1 shows the amino acid sequence from No. 1 to No. 308 of the amino acid sequence of the present invention, that is, the amino acid sequence of the entire ligand of the present invention excluding the signal peptide.
  • amino acid sequence at the N-terminus determined by the above-described blot sequencer that is, an amino acid sequence consisting of 48 amino acid residues shown in SEQ ID NO: 19, and a sequence number deduced from the cDNA sequence
  • amino acid sequences Nos. 1 to 48 of the amino acid sequence No. 7 are the same as those of the amino acid sequence No. 19 in the sequence listing except for the three XXX portions that could not be determined.
  • the noic acid sequences were completely identical.
  • the transformed cell obtained by transfecting Escherichia coli DH5 ⁇ with vector pUCMEKL containing the entire nucleotide sequence of the receptor type 1 tyrosine kinase ligand of the present invention was produced by the Ministry of International Trade and Industry of Japan. Deposit number FER ⁇ at Biotechnology Research Institute
  • the polypeptide that is an extracellular domain of the receptor type 1 tyrosine kinase containing the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing contains At least a part of the extracellular domain of the ligand of the invention, that is, the polypeptide containing the amino acid sequence shown in SEQ ID NO: 5 binds to the receptor type 1 tyrosome. It is thought to activate receptor tyrosine kinase activity of synkinase.
  • the term “part” used herein refers to at least 12 consecutive amino acid residues that are the active centers of ligands having receptor-type synthase ligand kinase activity. Can be used as an indicator to identify polyreceptor tyrosine kinase activity.
  • N-acetyl-D-darcosamine is a part capable of N-glycosidic bond, and is defined as amino acids 11 and 114 of the amino acid sequence of SEQ ID NO: 7 in the sequence listing.
  • Asparagine residue did not affect the measurement at all in the amino acid sequence analysis by the protein sequencer performed in Example 9 and thus at least C-paragine used in the present invention was not used.
  • the asparagine No. 11 did not affect the measurement at all in the amino acid sequence analysis by the protein sequencer performed in Example 9 and thus at least C-paragine used in the present invention was not used.
  • the ligand produced by one cell it is considered that no sugar chain is added to the asparagine residue at position 11 in the amino acid sequence of SEQ ID NO: 7.
  • N-acetyl-D-galactosamine is an O-dalicoside-bondable moiety, and the amino acid of SEQ ID NO: 7 in the frequently occurring sequence list of serine or threonine residues is used.
  • the most probable parts are the threonine at position 155 and the serine from position 168 to position 172 of the amino acid sequence of SEQ ID NO: 7 in the sequence listing.
  • Serine residue or threonine And a serine residue from serine No. 18 to threonine No. 183 or a threonine residue are more stable to degradation in vivo and have stronger bioactivity than those without sugar chains. It is considered that
  • the ligand of the present invention is purified from the cell culture supernatant, and this ligand is produced by proteolytic enzymes such as metalloproteases.
  • proteolytic enzymes such as metalloproteases.
  • the extracellular domain is considered to be a molecule released into the cell supernatant.
  • the cleavage site is located between the extracellular domain and the transmembrane domain described above, that is, isoleucine 195 and 196 in the amino acid sequence of SEQ ID NO: 7 in the sequence listing. It is thought to be between the lysines, but it may have been digested and digested with proteolytic enzymes in other parts as well.
  • this LERK-2 is a binding type of receptor type 1 tyrosine kinase ELK having a relatively homologous receptor type 1 tyrosine kinase to which the ligand of the present invention specifically binds. It is a substance identified as a protein or ligand.
  • the common points between the amino acid sequence encoded by the entire cDNA sequence of the ligand of the present invention and the above amino acid sequence of LERK-2 are as follows. First, four cysteine residues expected to have a disulfide bond, that is, amino acids 37, 64, 76, and 12 of the amino acid sequence of SEQ ID NO: 7 in the sequence listing The position of cystine residue 8 on the amino acid sequence is common. Also, the amino acid at the portion where N-acetyl-D-dalcosamine can bind N-glycosidic, ie, the asparagine residue at position 114 of the amino acid sequence of SEQ ID NO: 7 in the sequence listing. The positions on the array are also common. In addition, the sequence consisting of 33 amino acid residues from amino acids 276 to 308 of the amino acid sequence of SEQ ID NO: 7 in the sequence listing, which is an intracellular domain, is completely absent. It is the same.
  • the ligand of the present invention and LERK-2 may have the same physiological action, as shown in Example 19, the ligand of the present invention and the above LERK-2
  • the gene of LERK-2 was obtained from human placenta cDNA by PCR and expressed, and the effect on the receptor tyrosine kinase bound by the ligand of the present invention was obtained. Analysis was performed. As a result, although the receptor was weakly bound to the receptor type 1 tyrosine kinase, the binding was much weaker than that of the ligand of the present invention, and the action of causing the phosphorylation of the receptor was extremely low. It was weak. Therefore, LERK-2 is a substance that has structural similarity to the ligand of the present invention, but has a completely different physiological action.
  • the ligand of the present invention is preferably at least a part of the polypeptide containing the amino acid sequence of SEQ ID NO: 6 in the sequence listing.
  • a genetic engineering technique may be used. It can be prepared as at least a part of a polypeptide containing the amino acid sequence of SEQ ID NO: 6 or 5.
  • the ligand of the present invention can be produced by genetic engineering techniques.
  • the obtained ligand can be purified by the above-mentioned known method.
  • the ligand of the present invention expresses cDNA encoding a polypeptide having an amino acid sequence described in SEQ ID NO: 6 in the sequence listing.
  • SEQ ID No. 5 in the sequence listing which is a domain containing a bioactive center, was used. It is preferable to produce only the amino acid sequence, ie, the extracellular domain of the ligand. That is, it is preferable to use DNA encoding at least a part of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing, and to produce the protein as a secretory protein.
  • the form in which the ligand of the present invention is expressed may be a ligand.
  • the compound may be a single compound, or may be in the form of a complex.
  • the “complex” refers to a compound containing at least a part of the polypeptide containing the amino acid sequence of SEQ ID NO: 6, preferably SEQ ID NO: 5 in the sequence listing.
  • the conjugate of the present invention can be preferably used because it has the same or higher physiological activity as compared with one molecule of the ligand described above.
  • the ligand is expressed as a chimera protein of the Fc portion of human IgG, Furthermore, a dimer that forms a disulfide bond with the Hinge region of the IgG Fc; a specific antigen (for example, Fc portion of human IgG, FLAG, etc.) is added to the ligand. It is expressed as a chimera protein expressed at the C-terminal or N-terminal side of the amino acid sequence, and reacted with an antibody that specifically recognizes the antigen to form an antigen-antibody complex.
  • SEQ ID NO: 6 in the sequence listing preferably May be a multimer obtained by linking two or more DNAs encoding at least a part of the amino acid sequence of SEQ ID NO: 5 so as to match the reading frame of translation.
  • the complex of the present invention is obtained by cross-linking the extracellular domain of the ligand, which is expressed and purified, using a cross-linking agent, in addition to the above.
  • a cross-linking agent such as Daltar aldehyde that cross-links amino and amino groups, it is possible to form a ligand complex by performing a cross-linking reaction of protein and carbohydrate.
  • Example 13 and Example 16 when expressed using an expression vector having a DNA encoding the amino acid sequence of SEQ ID NO: 6 in the sequence listing, it was expressed on a natural cell membrane.
  • a ligand having the same physiological activity as the existing ligand is obtained.
  • Polypeptide having sequence has micellar or liposomal morphology by mimicking biological membranes such as lipids, phospholipids, etc. When present on a substance, it can exert the same physiological activity as when expressed as a molecule on the cell membrane.
  • the ligand of the present invention and the complex containing the ligand have an action of promoting the differentiation and proliferation of undifferentiated blood cells, they are effective for leukemia, an early recovery agent at the time of bone marrow transplantation, and a bone marrow at the time of administration of an anticancer agent. Suppression It is useful as an active ingredient of pharmaceuticals such as a recovery agent and its preventive agent. Further, as described above, a substance in which the ligand is present on a substance having a morphology similar to a biological membrane (eg, micellar liposome) is also useful as an active ingredient of a similar drug. It is.
  • a lipid is covalently bonded to a part of the ligand, and this part is ligated to the lipid of the liposome micelle.
  • the ligand of the present invention can be used for a medical method in which blood cells extracted from a human body are proliferated and activated in an extracorporeal medium, and the cells are returned to the body again.
  • the ligand of the present invention can be added directly to the medium, but it is desirable that the ligand be immobilized in a cell culture vessel.
  • the ligand is shared with the culture vessel by using the amino group / carboxyl group of the ligand and using an appropriate spacer. It can be immobilized by bonding.
  • Examples of the spacer when an amino group of a ligand is used include an ⁇ -carboxyalkyl group derivative, a bromoacetyl derivative, a diazonium derivative and the like.
  • An example of a spacer when the carboxyl group of the ligand is used is an ⁇ -aminoalkyl derivative / hydrazide derivative.
  • As another immobilization method there is a method based on CNB r activation. For details of the ligand immobilization method, see, for example, “Experiment and Application Affinity Chromatography” Chiba
  • the present invention also includes a compound containing at least a part of the polypeptide.
  • the ligand of the present invention produced by a genetic engineering technique can utilize the polypeptide of the extracellular portion of the receptor type 1 tyrosinase used in the present invention as described above. It can be purified, but as shown in Example 15, a polyclonal mono- or monoclonal antibody prepared by immunizing a suitable animal with the ligand. Purification can also be performed by using an affinity-based matrix using antibodies.
  • the ligand of the present invention has an effect of promoting colony formation of human cord blood mononuclear cells, thereby promoting the proliferation of undifferentiated blood cells (the ligand of the present invention and the ligand). Since it has the effect of promoting the differentiation and proliferation of complex blood undifferentiated cells containing gand, it can be used as an early recovery agent for leukemia bone marrow transplantation, a myelosuppressive recovery agent for administration of anticancer drugs and its preventive agent. When the ligand of the present invention is used as a pharmaceutical, the ligand of the present invention is useful. It is desirable to use the lyophilized product of the solution dissolved or suspended in distilled water for injection.
  • the physiological activity of the inviting mouth of the ligand of the present invention can be confirmed by administering various disease model mice or animals such as rats and monkeys exhibiting symptoms similar to those of a similar disease.
  • various disease model mice or animals such as rats and monkeys exhibiting symptoms similar to those of a similar disease.
  • a bone marrow suppression model mouse was prepared by administering a 5-FU anticancer drug, and the compound of the present invention was administered to this mouse. This is clarified by examining the number of bone marrow cells and peripheral blood cells and the physiological function of each group.
  • mouse bone marrow cells were cultured using an incubator or the like, and the compound of the present invention was added at that time. After culture in the group and the group without Cells can be transplanted to a lethal irradiation mouse, and the degree of recovery can be examined by using the survival rate, blood cell count fluctuation, etc. as indicators. Since the results of these experiments can be extrapolated to humans, they can be used as effective data for evaluating the efficacy of the compound of the present invention.
  • Example 12 which showed in which organ the mRNA encoding the polypeptide of the compound of the present invention is expressed, and in which organ the mRNA of the receptor was expressed Comparing the results of Reference Example 7 showing whether or not the compound is expressed, lung and kidney are mentioned as organs in which the compound of the present invention may produce some physiological activity or a similar drug effect. Organ-related diseases are expected to be treated with the compounds of the present invention. Regarding the liver, it was found that the receptor type 1 tyrosine kinase gene of the present invention was strongly expressed in liver cancer. 3 B Northern blot results and a paper by Bennett et al. (Bennetteta 1., J. Bio 1.
  • the ligand of the present invention is related to liver cancer, the growth of normal liver tissue, etc., and is a drug effective for diseases related to these, or a screening system for an effective drug. Applicable to
  • drugs for diseases associated with hematopoietic cells such as leukemia, an early recovery agent at the time of bone marrow transplantation, a bone marrow suppression / recovery agent at the time of administration of an anticancer agent, and a prophylactic agent thereof, may be used.
  • the dosage at this time depends on the form of the drug and the activity of the ligand, but the dose may be about 0.1 mg / kg to 10 Omg / kg.
  • Example 12 an isolated antisense DNA fragment paired with a sequence from 12 mer to 16 mer or more, which is a part of the gene sequence of SEQ ID NO: 7 in the sequence listing. And the antisense RNA fragments and derivatives thereof that are methylated, methylphosphorylated, deaminated, or thiophosphorylated, and the ligands of the present invention.
  • Gene expression can be examined by Northern blot. In a manner similar to that described in Example 12, it can be used for detection of homologues of the gene of the present invention in other organisms such as mice and rats, and for gene cloning. .
  • the present invention can be similarly used for detection and cloning of genes on genomics including human genomics.
  • inventive ligand can also be revealed. For example, with the use of recent gene manipulation techniques, all methods such as transgenic mice, jet-getting mice, and double knockouts in which the gene related to the gene of the present invention is inactivated are used. You can do this. If there is an abnormality in a portion on the genome corresponding to the gene of the present invention, Applications to gene diagnosis and gene therapy are also possible.
  • an isolated sense DNA fragment and an antisense DNA fragment having a part of the base sequence of SEQ ID NO: 7 in the sequence listing or derivatives thereof, and an isolated sense RNA fragment and an antisense having a sequence corresponding thereto Gene expression in cells can also be regulated by RNA fragments or their derivatives.
  • the compounds of the present invention can be detected and measured, and the above-mentioned diseases and the like can be diagnosed.
  • Useful as medicine
  • the culture and subculture of UT-7 cells contain 10% fetal bovine serum (FCS; all FCSs used in this patent are manufactured by Finoletron, Australia) as a medium.
  • FCS fetal bovine serum
  • hGM-CSF Human granulocyte macrophage colony stimulating factor
  • IMDM Skov-modified Dulbecco's medium
  • RNA Lithium Chloride / U Tota 1 RNA was extracted by the rea method (Eur. J. Biochei 107: 303 (1980)). Next, Poly (A) + RNA was separated and purified using Oligotex-dT30 (Takara Shuzo, Japan).
  • Synthetic oligonucleotides were prepared using a fully automatic DNA synthesizer based on the solid-phase method. As a fully automatic DNA synthesizer, Applied Biosystems Inc. 39.1 PCR-MATE in the United States was used. Nucleotide, 3'-nucleotide-immobilized carriers, solutions, and reagents were used according to the company's instructions. Complete the specified coupling reaction, and remove the 5'-terminal protecting group with trichloroacetic acid to remove the oligonucleotide carrier in concentrated ammonia. The oligonucleotide was released from the carrier by allowing to stand at room temperature for 1 hour at room temperature.
  • the reaction solution containing the nucleic acid is left in a sealed vial at 55 ° C for 14 hours or more in a concentrated ammonia solution in a sealed vial.
  • Purification of each of the oligonucleotides from which the carrier and protecting groups were released was performed using an OPC force cartridge from Applied Biosystems in the United States and detrityled with 2% trifluoroacetic acid. It has become.
  • the purified primer was dissolved in deionized water to a final concentration of 1 ⁇ g / ⁇ and used for PCR.
  • CDNA was synthesized using the Po1y (A) + RNA obtained in Reference Example 1. That is, 2 g of Poly (A) + RNA was dissolved in 12.3 £ of deionized water, and 10X buffer solution (500 mM MKC 1, 100 mM MTris — HC)
  • Amplification by PCR was performed as follows. Using 20 ⁇ of the cDNA solution obtained in Reference Example 3, 10 ⁇ buffer (500 mM MKC 1, 100 mM M ris—HC 1 (pH 8.3), 1 ⁇ 5 mM g C 1 2, 0 . 0 1% gelatin) 8 ⁇ ⁇ , d NTPM ixture ( Japan, Takara Shuzo) 6. 4 ⁇ ⁇ , sense primer specific for the above-mentioned tyrosine kinases [rho T kappa
  • the entire amount of the PCR product was electrophoresed on a 2% agarose gel prepared using low melting point agarose (GIBCO-BRL, USA). After staining with Etudemub mouth, a band of about 210 bp was cut out under UV irradiation, and the same volume of distilled water as the gel was added. After heating for 1 minute to completely dissolve the gel, add an equal volume of TE-saturated phenol (manufactured by Nippon Gene, Japan), centrifuge at 1500 rpm for 5 minutes, and remove the supernatant. Separation was performed, and the same separation operation was performed using a TE-saturated phenol: chloroform (1: 1) solution, and furthermore, chlorophore.
  • TE-saturated phenol manufactured by Nippon Gene, Japan
  • cDNA was recovered by ethanol precipitation.
  • the recovered cDNA was digested with restriction enzymes EcoRI (Takara Shuzo, Japan) and Sa1I (Takara Shuzo, Japan), and then used for incorporation into vectors.
  • pBIuescript IIKS manufactured by Stratagene, USA; hereinafter, referred to as pBluescript
  • pBluescript digest with the restriction enzymes EcoRI and Sa1I before incorporating the cDNA above. Then, it was refined by the above method. A mixture of the vector subjected to these treatments and the preceding cDNA in a molar ratio of 1: 5 was mixed with T4 DNA ligase manufactured by New England BioLab in the United States. The cDNA was incorporated into the vector with the enzyme.
  • pBIuescript into which E. coli was incorporated was introduced into E.
  • the nucleotide sequence of the incorporated cDNA was determined for the confirmed clone using a fluorescence sequencer of Applied Biosystems, Inc. of the United States.
  • a tyrosine kinase gene fragment was obtained that was cloned from unstimulated UT-7 cells, but was not cloned when UT-7 was differentiated into megakaryocytes.
  • This gene fragment was No. 2664 of the SEQ ID No. 3 in the sequence listing, and No. 2812 of the sequence.
  • these cDNA libraries are Li Daizesho down of also rather plaque hive Li Daizeshi ® 5 X 1 0 5 corresponding to the search phrase loans with a total length c DNA in emissions co-Roni one also properly went from plaque.
  • the resulting colonies or plaques were transferred to nylon finole letters (Hybond N +: manufactured by Amersham, UK), and the transcribed nylon finole letters were treated with alkali (1.5 MNaCl, 0.1 M NaCl).
  • a cDNA probe labeled with 32 P radioisotope was prepared as follows. That is, pB1uescript in which the partial cDNA of tyrosine kinase was incorporated was cut out from vector with Sa1I and EcoRI, and the DNA fragment was obtained from the low melting point agarose gel. Was purified and recovered. The obtained cDNA fragment was used as a DNA labeling kit (Megaprime DNA). labeling system: Amersham, UK). That is, to 5 ng of DNA, add 5 ⁇ l of the primer solution and deionized water to make a total volume of 33 ⁇ , perform a boiling water bath for 5 minutes, and then add 10 mL of reaction buffer containing dNT 1.
  • the filter prepared by the above method is used for the SSP E solution with the final concentration of each component of 5 times and the Denhardt solution with the 5 times concentration.
  • pre-hybridase a denatured salmon sperm DNA denatured in a lOmg Z ml boiling water bath. After immersion in shaking solution and shaking at 65 ° C for 2 hours, a prehybridization solution containing the 32 P-labeled probe by the above-mentioned method was used. The sample was immersed in the rehydration solution, shaken at 65 ° C for 16 hours, and subjected to hybridization.
  • the final letter was immersed in an SSPE solution containing 0.1% SDS, shaken at 65 ° C, washed twice, and further diluted 10-fold containing 0.1% SDS. It was immersed in the SSPE solution and washed four times at 65 ° C. ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ Autoradiography was performed using As a result, the strongly exposed part of the clone was picked up, the colony and the black were sown again, and the screening was performed in the manner described above, thereby completely separating the single clone. .
  • cDNA was purified by low melting point agarose electrophoresis and incorporated into pB1uescript. The size of the incorporated cDNA was approximately 3.0 kbp and 1.6 kbp.
  • phage DNA was purified from two clones isolated from the human placenta library, which had a large insert size, according to the method described in the experimental manual by Maniatis et al. It was digested with coRI and similarly incorporated into pB1uescript. The size of the incorporated cDNA was approximately 3.8 kbp (clone 2) and 3.5 kbp (clone 9).
  • the gene sequences at both ends of the cDNAs of these clones were used for labeling kits for ALFDNA Sequencer (Swedish, Pharmacia) and ALF Sequencer (Swedish, Pharmacia) using the attached kit. The decision was made according to the book. In order to determine the full-length nucleotide sequence, there is also a delugetion module for kilosequence manufactured by Takara Shuzo Co., Ltd. in Japan. Using a kit, according to the attached instruction manual, a dilution medium was prepared, and the nucleotide sequence in both directions of the cDNA was determined.
  • the nucleotide sequence of the gene prepared as described above is shown in SEQ ID NO: 3 in the sequence listing.
  • MOLT-4 available from ATCC, CCL1582
  • this gene was expressed in an undifferentiated state of blood cells, and its expression disappeared as blood cells differentiated. Therefore, it was considered that this receptor type 1 tyrosine kinase is linked to fV: maintenance of blood stem cells and megakaryocyte differentiation.
  • Step 1 A cell line expressing a receptor tyrosine kinase polypeptide having an amino acid sequence of SEQ ID NO: 2 in the sequence listing, Ba /
  • DNA encoding the polypeptide was transformed into an expression vector containing the SRct promoter and the neomycin resistance gene pMK IT neo (Maruyama et al., Proceedings of the 91st Molecular Biology Society of Japan, (Available from Tokyo Medical and Dental University Maruyama), respectively, to produce expression vectors.
  • the PCR product is purified by the method described in Reference Example 5, and treated with the restriction enzymes XhoI and EcoRI.
  • the purified gene was ligated to the treated pBluescript using T4 DNA ligase manufactured by New England BioLab, USA, and subcloned. Then four clones
  • the plasmid DNA is purified from the colony and sequenced to confirm the gene sequence.
  • the gene sequence is correct and the target gene sequence, that is, the DNA sequence of SEQ ID NO: 3 in the sequence listing It was confirmed that the fragment had the sequence from No. 3230 to No. 3337.
  • the vector having this fragment is referred to as p BSF1.
  • r Lys Asp Asp Asp Asp Asp An expression vector having a gene sequence encoding a polypeptide to which a polypeptide (FLAG, described in SEQ ID NO: 13 in the sequence listing) is added.
  • primers were used in the same manner as described above, and the primers were primers of the primers 1 and 5'-GGGAATTCATTTATCATCATCATCTTTATAATCGTACTG CGGGGCCGGTCCTCCTGT-3 '(primer 13 and SEQ ID NO: 12 in the sequence listing).
  • PCR subcloning into pB1uescript, sequencing the 4 clones, confirming the gene sequence, and confirming the gene sequence of interest, that is, the DNA sequence of SEQ ID NO: 3 in the Sequence Listing. 3 2 3 0 3 to 3 3 0 It was confirmed that the fragment was a fragment having a sequence in which 5′—GATTATA AAGATGATGATGATAAATGA-3 ′ (described in Iris No.
  • PBSF2 the vector having this fragment
  • p BSRTKFULL was digested with the restriction enzyme BamHI (Takara Shuzo Co., Ltd., Japan) and purified by the same method as described above.
  • the PUCRTKFULL thus prepared is digested with the restriction enzymes Sa1I and Bg1II, and the fragment of about 3 kbp is subjected to agarose gel electrophoresis. Fragment in which the portion from Sa1I to BamHI of the restriction enzyme site for PUC19 is connected to the end of the 5th end of the sequence from No. 384 to No. 3230 of the DNA sequence No. 3
  • the sample was cut out and purified by the method described above. This gene fragment is designated as F 3.
  • restriction enzymes Sa1I and Bg1II were digested with pBSF1 and PBSF2, and the fragment Bg1II in the vector from the Sa1I siteka of the multi-cloning site.
  • the gene fragment except for the 30 bP gene fragment was purified by the above method. These gene fragments are referred to as F 1 and F 2, respectively. Then, F1 and F3, and F2 and F3 are connected by the above-described method, respectively, and the polypeptide of SEQ ID NO: 3 in the sequence listing is coded by restriction enzymes Sa1I and NotI.
  • the vector from which the gene fragment is cut out is p FLAG amino acid sequence at the C-terminus of the polypeptide of SEQ ID NO: 2 with BSFULLl and restriction enzymes Sa1I and N0tI.
  • a vector PBSFULL2 from which a gene fragment corresponding to a portion coding for a polypeptide having the above was cut out was prepared.
  • the thus prepared vectors-PBSFULLl and pBSFULL2- are digested with restriction enzymes Sa1I, NotI and Pvul, and a gene fragment of approximately 3 kbp is obtained by electrophoresis. That is, a gene fragment containing the gene fragment encoding the amino acid sequence of receptor type 1 tyrosine kinase was separated and purified. These two gene fragments were treated with the above-mentioned pMKIT neo using the restriction enzymes Sa1I and NotI to remove a portion of the staff, and then subjected to T4 DNA ligation. As a result, an expression vector for the receptor tyrosine kinase was constructed.
  • the vector prepared without the FLAG sequence as described above is referred to as p MK FULL, and the vector including the FLAG sequence is referred to as p MK FULLFLAG.
  • the cell lines to be transfected are mouse pro-Bce11 1 ineBano F3 (available from RIKEN, Cell Development Bank, Japan, No. RCB0805) and mouse. Fibroblast B alb // 3 T 3 clone A 31 (available from RIKEN, Cell Development Bank, Japan, No. RCB 005).
  • Ba / F3 was RPM 1 1640 (GIBCO—BRL, USA), 10% FCS, 100 OOig / ml mouse IL—3 (US, Intergen Ba1bZ3T3 was cultured in D-MEM (Dalbecco's modified MEM medium, GIBCO-BRL, USA) 10% FCS.
  • the expression vector was digested with a restriction enzyme NruI (Takara Shuzo Co., Ltd., Japan) to make it into a linear form and then transfected.
  • NruI restriction enzyme
  • the cells were cultivated in a 0 cm cell culture dish with 10 ml of the above medium. The next day, centrifuge the above medium into G418 (US, S),
  • a cell line which transfects the thus-produced Ba / F3 and stably expresses the polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing is obtained from Ba / F3 / FULL, stable expression of a polypeptide having a FLAG amino acid sequence at the C-terminus of a polypeptide containing the amino acid sequence described in SEQ ID NO: 2 in the sequence listing
  • the cell line was named BanoF3 / FULLFLAG.
  • a control transformed cell line into which 1 ⁇ 1: 1 1116 itself was transfected was prepared and named Ba / F3ZCON.
  • Preparation of a transgenic cell line using Ba1b / 3T3 was carried out as follows. That is, the medium of the cells was changed the day before, the number of cells was changed to 5 ⁇ 10 6 cells for a cell culture dish with a diameter of 10 cm, 10 ml of the above medium was added, and the cells were cultured overnight. . The next day, to precipitate the cells by centrifugation fraction away, PBS (-) 2 times after centrifugation washed with, 1 m MM g C 1 2 , PBS - a to 1 X 1 0 7 eel 1 s Z ml () Thus, cells were prepared.
  • Gene transfer was performed by an electroporation method using a gene transfer device manufactured by Bio-Rad, USA. Transfer the above cell suspension (500 ⁇ fi) to a dedicated electroporation cell (0.4 mm), add 20 / g of the expression vector, and place on ice for 5 minutes. put. Thereafter, the soda was allowed to stand at room temperature for 1 minute, and two 3 F, 450 V voltages were applied. Then, after leaving it on ice for 5 minutes, it is cultured for 3 days in the above culture medium (10 ml) in a 10 cm diameter cell culture dish. I got it.
  • the medium was replaced with a medium containing G418 (Sigma, USA) at a concentration of 400 ⁇ g / m ⁇ , and the medium was replaced every other day with 1Z3
  • the culture was continued for about 10 days after the exchange. After 10 days, the colonies formed on the dish were peeled off using a Tribsin solution (GIBCO — BRL, USA) to obtain a cell line stably expressing the cloned gene. .
  • the cell line that stably expresses the polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing of Ba1b / 3T3 prepared in this manner was transformed into Ba1FULL.
  • a cell line that stably expresses a polypeptide having a FLAG amino acid sequence at the C-terminus of the polypeptide containing the amino acid sequence described in SEQ ID NO: 2 in the sequence listing. was named Balb ZFULLFLAG.
  • a control-transformed cell line into which PMKIT neo itself was transfected was prepared, and named Ba1bZCON.
  • Step 2 Preparation of a cell line expressing a receptor tyrosine kinase polypeptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing Also, the extracellular portion of receptor type 1 tyrosine kinase, namely, An expression vector was constructed which allows animal cells to produce a polypeptide containing the polypeptide described in SEQ ID NO: 1 of the Sequence Listing.
  • oligo DNA (primer 15, described in SEQ ID NO: 15 in the sequence listing) as the primer.
  • PCR was performed using pB1uescript containing the gene of SEQ ID NO: 3 in the column list as a template. After confirming that a DNA of about 150 bp is amplified by agarose gel electrophoresis, the PCR product is purified by the method described in Reference Example 5 and digested with the restriction enzyme EcoRI. restriction enzyme treatment was, by connecting gene p B 1 uescript that was de-phosphate processing terminal in United States N ew E ngland B io L ab Co.
  • a clone was selected so that the S a1 I site of the 1 pt multi-cloning site and the N0tI site were located downstream of the gene sequence encoding the 3 'FLAG sequence.
  • this vector is referred to as pBSF4.
  • the above vector pUCRTKFULL was digested with restriction enzymes Sa1I and AatII (Toyobo, Japan), and a gene fragment of about 1.6 kbp was purified by the above method.
  • This gene fragment contains the sequence from No. 384 to No. 182 of the DNA sequence of SEQ ID NO: 3 in the Sequence Listing.
  • PBSF3 was digested with the restriction enzymes Sa1I and AatII, and approximately 3.
  • the 2 kbp gene fragment was purified.
  • the vector prepared by connecting these two gene fragments was further digested with restriction enzymes Sa1I and NotI and connected to the above-mentioned expression vector PMKIT neo.
  • a vector producing a polypeptide having a FLAG sequence at the C-terminus of the amino acid sequence was prepared. This expression vector is designated as PMKEXFLAG.
  • the Fc portion of the polypeptide below the hinge portion of the immunoglobulin IgG1 is located at the C-terminus of the polypeptide having the amino acid sequence described in SEQ ID NO: 1 in the sequence listing.
  • a vector capable of producing a polypeptide having an amino acid sequence was prepared as follows. 73
  • the fusion protein was synthesized with the method of Zett 1 meiss 1 et al. (Zet t lmeissl eta 1.
  • a gene using intron-containing genomic DNA was used in accordance with DNA Cell Bio, 9, 347-354, 1990), and the gene was prepared by PCR. That is, using the human genome DNA as a template, the gene sequence encoding the human IgGI Fc portion was replaced with the 5'-A ACCATCCCCGAGGGTGTCTGCTGGAAGCCAGGCTCA with restriction enzyme BamHI site.
  • oligo DNA (primer 7, described in SEQ ID NO: 17 in the sequence listing), restriction enzyme XbaI site, 5'-CCTCTAGAGTCGCGGCCGT CGCACTCATTTACC-3, PCR was performed using oligo DNA (primer—8, described in SEQ ID NO: 18 in the sequence listing) having a systematic primer as a primer, and a band of about 1.4 kbp was purified.
  • oligo DNA primer—8, described in SEQ ID NO: 18 in the sequence listing
  • Treated with HI and XbaI (Takara Shuzo Co., Ltd., Japan) and sent to the same restriction enzyme-treated PB1uescript using T4 DNA1 igase from New England BioLab, USA. The child was connected and sub-cropped.
  • the brassmid DNA was purified and sequenced to confirm the gene sequence, confirming that the gene sequence was indeed genomic DNA corresponding to the hinge portion of the heavy chain of human IgGI. (See Kabateta I., Sequence of Immunological Interest, NIH pub cat ion No 91-3242, 1991 for the sequence.)
  • this vector is referred to as p BS hlg F c.
  • the above vector p BSF5 is replaced with the restriction enzymes BamHI and Ec o Digestion with RI, purify a gene fragment of approximately 150 bp by the above method, and then convert the above vector pBShIgFc to restriction enzymes BamHI and EcoRI. Then, a gene fragment of about 4.4 kbp is purified by the above method, and the both are ligated to each other with T4D ⁇ ligase manufactured by New England BioLab in the United States.
  • the vector thus prepared was digested with restriction enzymes Sa1I and AatII, and a gene fragment of about 4.4 kbp was purified by the above method.
  • the above vector PUCRTKFULL was digested with restriction enzymes Sa1I and AatII, and a gene fragment of about 1.6 kb P was purified by the above method.
  • the vector prepared by connecting these two gene fragments was further digested with restriction enzymes Sa1I and N0tI, and connected to the above-described expression vector PMKIT neo.
  • a polypeptide production vector having the amino acid sequence of human IgGI Fc at the C-terminus of the amino acid sequence was prepared. This expression vector is referred to as pMKEXIg.
  • the expression vectors pMKEXFLAG and pMKEXIg were transfected with COS-7 (RCB0539, available from RIKEN, Cell Development Bank, Japan). The method of culturing COS-7 and introducing the gene were performed in the same manner as in BaIb / 3T3, and cells into which the gene had been introduced were obtained.
  • cell culture supernatant serum-free D-MEM (GIBCO-BRL, USA) was replaced every 4 days, and the culture supernatant was collected for 2 weeks. Each of the collected culture supernatants was exchanged with PBS (-) from the medium and centrifuged 10 times with Centricon 30 (Amicon, USA).
  • the concentrated culture supernatant was purified using an SDS-PAGE electrophoresis tank and SDS-PAGE polyacrylamide gel (gradient gel 5 to 10%) manufactured by ACI Japan of Japan. SDS-PAGE was performed in accordance with the instructions.
  • the sample was prepared by adding 2-mercaptoethanol (2-ME) and heat-treating the sample under non-reducing conditions, and the non-reducing sample under non-reducing conditions.
  • the sample was prepared using a Rainbow Marker 1 (for high molecular weight) manufactured by Amersham, Inc., according to the instruction manual attached to the sample, the electrophoresis buffer, and the end plate. After completion of SDS—PAGE, acrylamide gel is applied to the PVDF membrane label.
  • a Rainbow Marker 1 for high molecular weight manufactured by Amersham, Inc.
  • the filter thus prepared was subjected to 5% BSA (manufactured by Sigma, USA), TBS-T (20 mM MTris, 1337 mNaC1 (pH 7.6)) , 0.1% T weem 20) Blocked while rubbing at 4 ° C.
  • BSA manufactured by Sigma, USA
  • TBS-T 20 mM MTris, 1337 mNaC1 (pH 7.6)
  • T weem 20 0.1% T weem 20
  • polypeptide-producing cell containing the amino acid sequence described in SEQ ID NO: 1 of the target sequence listing was obtained.
  • the extracellular portion was approximately 20 kilodaltons larger than the molecular weight expected from the amino acid composition, and was expected to be glycosylated.
  • the vectors PMKEXFLAG obtained in Step 2 of Reference Example 8 and Five liters of each of the culture supernatants of the cells transformed with pKEXIg were prepared. These culture supernatants were applied to an Anti-FLAGM 2 Affinity Gel column manufactured by Kodak, USA and a pr0tein G Sepharose column or a Protein A Sepharose column manufactured by Pharmacia, Sweden. Each protein was absorbed into the column. The size of each column was 1 cm ⁇ 3 cm, the volume was about 2 ml, and the flow rate was all 1 ml Zmin. After the adsorption, the column was washed with PBS (-) 2 O ml, and then eluted with 0.5 Tris-glycine (PH 3.0).
  • the FLAG chimera and IgGFc chimera protein purified by the above method were purified by gel filtration.
  • the eluate from the affinity column is exchanged for buffer with PBS (-) at Centricon 30 manufactured by Amicon, USA.
  • the gel flow was carried out using a FPLC system manufactured by Pharmacia in Sweden and at the company's Superose 12 column.
  • Reference Example 8-2 The main peak eluted at the same molecular weight position as the result of the Western blot under the condition without ME was collected.
  • EXFLAG PTN
  • EXI g Shown as PTN.
  • Reference Example 10 Establishment of a monoclonal antibody that recognizes receptor type 1 thyrokinase and confirmation of expression of receptor type thycosine kinase
  • a mouse monoclonal antibody was prepared according to the method described in the written document. Immunization was performed three times, and one animal was immunized with 30 / ig each time. Blood was collected before the third immunization, blood samples were collected, and the antibody titer using the immunized EXFLAG-PTN was used. The cell culture supernatant was used for screening, and the cell culture supernatant was used to recognize both EXFLAG-PTN and EXIg-PTN. As a result, three hybridomas producing mouse monoclonal antibodies, namely clones 38-1E, 66-3A and 68-3A, were established.
  • the following experiments were performed using the monoclonal antibody 38 produced by the clone 38-1E.
  • XJT_7, MRC-5, and the above-mentioned transformed cell lines BaZF3 / FULL, Ba / F3 / FULLFLAG, Balb FULL and Ba1bZFULLFLAG were subjected to cell surface antigen analysis using a flow cytometer EPICS Elite manufactured by Coulter, USA.
  • Antibody staining was performed according to the method described in the written literature. The antibody was purified from the hybridoma culture supernatant as a primary antibody using Matratra GII (Pharmacia, Sweden) according to the attached instructions, and the secondary antibody was used as a secondary antibody.
  • the assay was performed with a goat anti-mouse Ig FITC label manufactured by Incorporated. As a result, it was confirmed that cells transfected with UT-7, MRC-5, and Receptor type 1 osteosynkinase gene were stained with the above-mentioned monoclonal antibody.
  • the preparation of the cell lysate was performed as follows.
  • the 2 X 1 0 6 cells cells re Shisuba' file one (5 0 mM H epes (p H 7. 5), 1% T riton X 1 0 0, 1 0% glycerol, 1 0 mM N a 4 P 407, 100 mM NaF, 4 mM EDTA, 2 mM Na3VO4, 50 ⁇ g / m ⁇ A protinin, 100 ⁇ ML eupe ptin, 25 ⁇ Pepstatin A, 1 m MPMSF) Suspend in 200 / fi, leave on ice for 20 minutes, centrifuge at 140 rpm for 20 minutes, remove supernatant, and disrupt cells Thing was obtained.
  • a sample to be measured with the BIA core was prepared as follows. .
  • Table 1 summarizes cell names, cell types, culture media for cell culture passages, and cell sources.
  • EXI g — PTN and EXFLAG — PTN prepared and purified by the method described in Reference Example 9 were combined with 100 mM sodium acetate buffer (pH 4) to give a concentration of 100 / g Zml. 0) and activated BIA core sensor chip CC-certified (Fanoremasia, Sweden) according to the instructions in the attached instruction manual. And bonded. Injection was performed twice with 5O ⁇ , combined, blocked with ethanolamine, and washed with HSB buffer until the baseline was stable. The flow rate was 5 ⁇ min, including the measurement of the following samples. The difference between the response unit (RU) before and after binding of SenShip to EXIg-PTN and EXFLAG-PTN was about 100 RU.
  • BIA c 0 re The conditions for sample introduction and measurement of BIA c 0 re were as follows. A flow of 30 ⁇ (6 min) was applied to the sensor chip to which the above peptide was bound. As a representative profile, a graph showing the measurement results of the binding activity of ligand contained in the culture supernatant of C-11 cells (unstimulated) to tyrosine kinase is shown in Fig. 1.
  • the RU value is the part of the baseline before the sample is introduced, the mass transit due to the entry of the sample (the top of the baseline is re-aligned) and the ligand is " t: Changes occur in the part that binds to the tyrosine kinase, which is a puter, and in the part that is replaced by the normal HSB buffer, as indicated by the two points indicated by the arrows in Fig. 1.
  • the value of the baseline (30 seconds before sample introduction; indicated by arrow A in Figure 1) and the value immediately after the final replacement with HSB buffer (ie, 6 minutes after sample introduction)
  • the difference between the two values of the value after 10 seconds was defined as the ligand binding activity.Every time the sample was measured once, 5 OmM sodium acetate was added. 0.5 M Flow N a C 1 (pH 4.0) by 15 ⁇
  • C-1 is a typical cell line of candidate ligand-expressing cells that showed high ligand binding activity to both EXIg—PTN and EXFLAG—PTN.
  • BT-20 cells were revealed as a cell line that did not express the cells or ligand.
  • Example 2 the ligand-expressing cells were identified by examining various cell culture conditions.If the ligand was also present on the cell membrane, cells were prepared in Reference Example 9. Ligand-expressing cells should be stained with polypeptides containing the extracellular portion of the receptor tyrosine kinase. Therefore, identification of a ligand-expressing cell was carried out using a polypeptide EXIg—PTN containing the amino acid sequence of SEQ ID NO: 1 in the sequence listing prepared as shown in Reference Example 9. It was performed at a low-site meter.
  • Figure 2 shows the results.
  • the vertical axis in FIG. 2 indicates the number of cells.
  • the horizontal axis shows the relative fluorescence intensity, which becomes stronger as going to the right. From this result, a shift in the fluorescence peak was observed for C-1 as compared to the control to which EXIg-PTN was not added, and it was found that ligand was also present on the cell surface. With respect to BT-20, such a shift of the peak was not observed, indicating that the ligand was not expressed on the cell membrane.
  • C-1 cells which are one of the cell lines that are considered to express the ligand, are not only bound, but also transformed cells B described in Step 1 of Reference Example 8
  • the following experiment was performed to determine whether the phosphorylation of receptor type 1 tyrosin kinase expressed on the cell membrane surface of a / F3ZFULLFLAG was caused.
  • the C-1 cell prepared by the method described in Example 2 Cell culture supernatant concentrate, C-11 cells themselves, and mouse anti-blood sample collected at the time of antibody titer measurement during monoclonal antibody production described in Reference Example 10 react with receptor tyrosine kinase. And examined whether it could be phosphorylated.
  • Reaction solution 1 Medium (RPMI 1640, 10% FCS, 100 / g Zml mouse IL-13) only;
  • Reaction mixture 2 suspension of 5 ⁇ 10 6 C-11 cells in the medium of reaction mixture 1;
  • Reaction solution 3 A solution obtained by diluting the mouse anti-blood serum described in Reference Example 10 100-fold with the medium of reaction solution 1;
  • Reaction Solution 4 A solution obtained by diluting the mouse antiserum described in Reference Example 10 by 500 times with the medium of Reaction Solution 1;
  • Reaction solution 5 The cell culture supernatant of C-11 cells prepared by the method described in Example 2 was concentrated 50-fold with Centriprep 10 (Amicon, USA). And the solution was exchanged for the medium of Reaction Solution 1.
  • the cells are immediately placed on ice, and the cells containing 2 mM sodium vanadate (Na 3 VOford), which has been cooled in advance, are added.
  • a cell disruption solution was obtained in the manner described.
  • This gel precipitate was subjected to Western blotting according to the method described in Reference Example 8.
  • Gel precipitate 30 ⁇ SDS-PAGE sample buffer was added, and the mixture was subjected to a boiling water bath for 5 minutes in the presence of 2—ME, and 15 ⁇ was passed through one lane for SDS-PAGE.
  • Two sets of PVDF membranes to which the same sample was transcribed were prepared, and one set was a polypeptide having the amino acid sequence of SEQ ID NO: 2 in the sequence listing having the target FLAG sequence.
  • the presence of phosphorylation of tyrosine residues It was used for the judgment of nothing.
  • Figure 3 shows a photograph of this Western blot.
  • a and B are the results of the polypeptide B a ZF3 ZFULLFLAG containing the amino acid sequence of SEQ ID NO: 2 in the target sequence listing, and C and D are the controls thereof, Ba / This is the result of F 3 CON.
  • Antibody staining was performed with A and C using an anti-phosphorylated tyrosine antibody (UBI, USA), and B and D with Anti-FLAGM2 (Kodak, USA).
  • the number of each lane corresponds to the number of the reaction solution described above.
  • the arrow indicates the position of the polypeptide described in SEQ ID NO: 2 in the sequence listing having the FLAG sequence.
  • the concentration condition of mouse antiserum was changed, it was not phosphorylated as strongly as co-culture with C-1 cells.
  • the effect of the ligand of the present invention is apparently stronger than that of the ligand of the present invention, but it clearly increases the enzymatic activity of receptor tyrosine kinase and strongly reduces the phosphorylation of tyrosine residues. It is thought to cause a bioactive effect on cells through receptor tyrosine kinase.
  • the ligand was narrow. It can be seen that it is present in the cell culture supernatant, but the 50-fold concentrate from the culture of C-11 cells caused only very weak phosphorylation of tyrosine residues. This is powerful because the amount of ligand in the cell culture supernatant is extremely small and a concentration of about 50-fold concentration does not sufficiently increase the tyrosine kinase activity of the intracellular portion of the receptor type 1 tyrosine kinase. It is unknown whether the ligand has a high activity, which is partially high on the cell surface, but it is presumed that the result was very weak phosphorylation. Was.
  • Example 4 From the results up to Example 4, it is considered that the colon cancer-derived cell line C-11 surely expresses the receptor tyrosine kinase ligand. — PT An attempt was made to purify using an N-linked affinity gel column.
  • the supernatant of C-11 was prepared by the method described in Example 1 and finally collected in an amount of 54 O ml.
  • the concentrated solution obtained in Step 1 was used.
  • the value of BIAc0 ree of this concentrated liquid measured by the method described in Example 2 was 134 RU.
  • the affinity column was prepared using CNBr-activated Sepharose 4B manufactured by Pharmacia, Sweden, according to the attached instruction manual. Finally, an affinity gel in which 4.1 mg of EXIg-PTN was bound to a 3.5 ml gel was produced. The coupling efficiency was 99.6%. A column with a size of 2 cm 2 ⁇ 1 cm was prepared from 2 ml of this gel.
  • the above cell culture supernatant concentrate is passed through the column at a flow rate of 20 m 1 / hr, and then washed with the same flow of PBS (-) at a flow rate of 15 m 1. Eluted with sodium, 0.5 MNaCl (PH 4.0). The eluate was fractionated at a rate of 1 ml, and each fraction was neutralized by adding 200 ⁇ of lMTris-HCl (pH9.5) to each fraction.
  • each column of the column before washing, the washing solution, and the fractionated eluate (in this case, one-fifth dilution) was transferred to Ultraflow by Millipore, USA.
  • the solution was exchanged into the HBS buffer using C 3 LGC (10 K cuts), and the measurement was carried out again with the BIA c 0 re by the method described in Example 2.
  • the column for gel filtration used was Superdex 75 HR10Z30, manufactured by Pharmacia, Sweden, and the separation buffer used was PBS (-) containing 0.02% Tween20. The flow rate was 40 ⁇ ⁇ ⁇ ⁇ .
  • the molecular weight at the elution position was measured using a molecular weight marker measurement kit LMW manufactured by Pharmacia, Sweden, and gel filtration of the sample was performed. fractionated into ⁇ .
  • the sample thus fractionated in molecular weight was diluted 10-fold with an HBS buffer, and BIAc0 ree was measured.
  • FIG. 5 shows the change in the absorbance of the gel fraction at 214 nm
  • FIG. 6 shows the measurement results of the BIA core of each fraction. From this result, the peak of the ligand binding to the polypeptide having the amino acid sequence of SEQ ID NO: 2 in the sequence listing having peaks at the fraction numbers 13 and 14 was found. was detected. In addition, small peaks were similarly observed in the fraction numbers 21 and 22.
  • the fraction between fraction numbers 11 and 12 is 63 000 daltons and the force between 13 and 14 ⁇ 540 000 danoletone , 15 and 16 S 4 3 0 0 Daltons, fraction numbers 13 and 14
  • the molecular weight of the receptor type 1 tyrosine kinase ligand of the present invention having a peak of was determined to be 540,000 soil, 900,000 daltons from the results of gel filtration.
  • the ligand of the present invention was a smear band (shown by the right arrow in FIG. 7) at approximately 42 K daltons. Therefore, the molecular weight of the receptor type 1 tyrosin kinase ligand of the present invention was estimated to be 4150 ⁇ 7500 daltons.
  • the result of gel filtration does not match the molecular weight value of SDS-PAGE, but the estimated molecular weight of gel filtration may differ from the actual substance due to interaction with the gel. It was inferred that 410,000 ⁇ 750 daltons was the molecular weight of the receptor tyrosine kinase ligand of the present invention.
  • active fractions fraction numbers 12 to 15
  • Purification was carried out with an affinity column using a polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 in the table.
  • the column was eluted by gravity flow using a small-scale, 150- ⁇ bet volume.
  • the above active fraction fraction numbers 12 to 15 were collected and loaded on the small-scale column described above. After that, washing was performed by flowing 8 times of PBS (-) containing 150% of 0.02% Tween 20 and then washing with 150M of 0.1 M sodium acetate, 0.5%. MNaCl (PH4.0) was flowed four times to fractionate and elute each fraction.
  • the eluate was neutralized by adding 30 ⁇ of lMTris-HCl (pH9.5). In order to confirm the purity of the final purified ligand in this way, each fraction was subjected to Ultrafree C3 LGC (10 K cut from Millipore, USA).
  • the sample buffer used here did not contain 2-ME, was used under non-reducing conditions without heating, and the gel was used using a 15-20% gradient gel.
  • the molecular weight marker LMW KitE manufactured by Pharmacia, Sweden was used.
  • silver staining was performed using a silver staining kit II manufactured by Wako Pure Chemical Industries, Japan according to the attached staining method.
  • Figure 8 shows the results.
  • one of the three mixtures was one of four hundred and fifty-five ⁇ seven hundred and fifty-five danoleston banding force; fraction number It was confirmed in 1 and 2 that it was purified as a single substance.
  • the above results indicate that the receptor tyrosine kinase ligand of the present invention can be purified by these methods, and a pure product can be obtained.
  • Example 7 The pure product of the ligand purified by the method described in Example 7 was subjected to SDS-PAGE in the same manner as described in Example 5.
  • the polyacrylamide gel, on which the new ligand has been migrated in this way, is used for the protein staining method, Kumasi-Pri Lant Blue, a Wako Pure Chemical Industries, Japan
  • staining was carried out using a Quick CBB kit manufactured in accordance with the attached experimental method, the band of about 420,000 daltons, which is the ligand of the present invention, was stained. It was found that the ligand of the invention contained protein.
  • the polyacrylamide gel prepared in the same manner was detected by PAS staining, which is a specific staining method for sugar chains. That is, the polyacrylamide amide gel on which the new receptor type 1 tyrosine kinase ligand was run was immersed in a 12.5% trichloroacetic acid solution with shaking for 30 minutes. Then, the sample was immersed in distilled water while shaking for 30 seconds, and then immersed in a 3% acetic acid solution containing 1% periodic acid for 50 minutes. Thereafter, washing was performed by shaking well with distilled water. Wash once for 10 minutes I went eight times.
  • PAS staining is a specific staining method for sugar chains. That is, the polyacrylamide amide gel on which the new receptor type 1 tyrosine kinase ligand was run was immersed in a 12.5% trichloroacetic acid solution with shaking for 30 minutes. Then, the sample was immersed in distilled water while shaking for 30 seconds, and then immersed in a ace
  • fuchsin solution (1 g of basic fuchsin (Wako Pure Chemical Industries, Japan) was dissolved in 200 ml of distilled water heated to 60 ° C in a dark place.
  • it was prepared by adding lg of sodium bisulfite and 20 ml of 1N HCl 1.) for about 1 hour to stain.
  • the plate was washed by shaking with a 0.5% sodium sulfite solution for 10 minutes, and this was repeated twice.
  • the band was placed in distilled water and the band was observed.
  • a red band stained with PAS was confirmed to have a molecular weight of about 42,000 daltons. From these results, it was found that the ligand of the present invention contains sugar.
  • the ligand of the present invention was a compound containing a glycoprotein.
  • the solution containing the ligand purified according to the method described in Example 7 was placed on a prospin (manufactured by Applied Biosystems, USA), centrifuged, and the PVDF in the prospin was removed using a special punch. Removed the final note. The final letter was thoroughly washed with a 50% aqueous methanol solution and purified water, and then the N-terminal sequence was determined using a peptide sequencer analyzer. The peptide sensor was analyzed according to the attached instruction manual using Protein 'theta enhancer mode 1492 of ABB Biosystems, Inc. of the United States.
  • amino acids that have undergone some modification such as disulfide-bonded cystine residues or amino acid residues that have been modified with sugar chains, etc., have undergone some modification. It is probable that the sequence could not be determined.
  • a cDNA probe was prepared by a PCR method using an oligonucleotide mixed primer. That is, the mRNA of the C-11 cells cultured under the conditions containing the PMA described in Example 1 NA was prepared, and a mixed primer of sense and antisense 2 O mer was synthesized from the amino acid sequence found in Example 9, RT-PCR was performed, and subcloning was performed to pB1uescript The gene sequence was determined, the amino acid sequence encoded by the gene was compared with the amino acid sequence determined in Example 9, and the amino acid sequence of the ligand was certainly determined. It was confirmed that the cDNA probe partially encoded.
  • RNA separation and purification of mRNA, ie, Po y (A) + RNA, from C-1 cells were performed as follows.
  • the C-11 cells cultured under the conditions of Example 1 were detached with a cell scraper (manufactured by Corning, USA) and washed twice by centrifugation with PBS (-).
  • Cell Number 3 X 1 0 8 pieces of 4 M guanidine-I O Shiane preparative This cell precipitate, 2 0 mM acetate Na Application Benefits um (p H 5. 2), 0. 1 MDTT, 0. 5% N — Suspend in 20 ml of total RNA extraction solution composed of Raurirsarco silka, and completely dissolve and disperse by passing through a 20 G needle manufactured by Terumo of Japan.
  • Centrifugation was performed at 500 g for 20 minutes, and the supernatant was recovered. Next, after adjusting the total volume to 25 ml with the above total RNA extraction solution, centrifugation was performed with 12 ml of 5.7 M cesium chloride and 0.1 MEDTA (pH 80) solution. The mixture was layered on a tube and centrifuged at 250 ° C at 180 ° C. As a result of centrifugation, a precipitate containing total RNA was recovered. Apply 720 ⁇ of TES solution (10 mM Tris (pH 7.4), 5 mM MEDTA, 1% SDS), dissolve by leaving at room temperature for 10 minutes, and dissolve this solution.
  • TES solution 10 mM Tris (pH 7.4), 5 mM MEDTA, 1% SDS
  • the total RNA thus obtained was washed with 70% ethanol, air-dried, and then dissolved in 10 ⁇ l of sterile distilled water, and the absorbance was measured at 260: 1111. 1 ⁇ 1 ⁇ octamer was determined. As a result, it was confirmed that 24 mg of the total RNA was recovered.
  • mRNA that is, Poy (A) + RNA
  • mRNA was isolated from total RNA of C-1 cells obtained by the above method. Separation was performed using an oligo dT column according to the attached instruction using an mRNA purification Kit manufactured by Phanolemasia, Sweden. As a result, 60 ⁇ g of the total RNA 15 / ig of Poly (A) + RNA was recovered.
  • the purified Po1y (A) + RNA was purified using Life Sciences, Inc.'s First—strandc DNAS synthesis Kit (purchased from Takara Shuzo Co., Ltd., Japan). CDNA was synthesized according to the attached instruction manual. That is, 2 ⁇ g of the above Po1y (A) * RNA was diluted to a final body contamination of 16 ⁇ , whereas the oligo d attached to this kit was Apply T (12-18) solution (0.5 yg / fi) or random hexamer (100 ng / ⁇ ) for 10 minutes at 70 ° C. The mixture was allowed to stand, and then left on ice for 10 minutes.
  • the 5X buffer solution supplied with the kit was added for 5 £
  • the RNase inhibitor RN asin solution was 1 ⁇
  • 0.25 ⁇ DTT was 1 ⁇
  • Abiamero Avianmyeloblastosis reverse transcriptase (25 / ⁇ ) solution was added to each of 1 ⁇ , and left at 41 ° C for 60 minutes to synthesize cDNA.Then, at 72 ° C The enzyme was inactivated by leaving it for 10 minutes, and this was used for the next PCR.
  • PCR for preparing a cDNA probe encoding a part of the amino acid sequence of the ligand was performed as follows.
  • amino acid sequence from the N-terminus of the ligand described in SEQ ID NO: 19 to the 5th leucine to 11th asparagine in the amino acid sequence ie, Leu-Gl U- Pro-11 Encodes the amino acid sequence of e-Tyr-Trp-Asn.
  • a 20-mer oligonucleotide mixed-sense primer corresponding to all possibilities of cDNA sequence i.e.
  • Asp-11 e-1 I 20-mer oligonucleotide corresponding to all possibilities of cDNA sequence encoding amino acid sequence of e-Cys-Pro-Lys-VaI Mixed antisense primer that is, the sequence shown in SEQ ID NO: 21 in the sequence listing (5'-AC (TC) TT (ACGT) GG (AG) CA (AGT) AT (AGT) AT (AG) TC-3 ', The above two types of synthetic DNAs having primers 10) were prepared according to the method described in Reference Example 2. Probe production by PCR was performed as follows. Mix two kinds of C-11 cDNA solutions prepared by the above method and use ⁇ ⁇ ⁇ of this mixture to prepare a 10-fold concentration buffer solution (500 mM
  • KC 1 100 mM MT ris — HCl (pH 8.3), 15 mM MgC 12, 0.01% gelatin) 10 ⁇ , d NTPM ixture (Takara Shuzo, Japan) 8 / ⁇ , and Taq DNA polymerase (Ampli Taq: Perkin-Elmer, USA, 5 ⁇ / ⁇ ) 0.5 ⁇ , and the above primer 9 And 10 to a final concentration of 10 and finally add deionized water to bring the total volume to 100 ⁇ , at 95 ° C for 0.5 min at 42 ° C.
  • Taq DNA polymerase Ampli Taq: Perkin-Elmer, USA, 5 ⁇ / ⁇
  • a cycle consisting of 0.5 minute and 72 ° C for 1 minute was taken as one cycle, and this cycle was performed for 5 cycles, followed by 0.5 minute at 48 ° C at 95 ° C.
  • the process consisting of 0.5 min at 72 ° C for 1 minute is defined as 1 cycle, this process is performed for 35 cycles, and finally the PCR is left at 72 ° C for 7 minutes.
  • a portion of this PCR product was subjected to 3% agarose gel electrophoresis, stained with Etch Dumbu Mide (manufactured by Nippon Gene, Japan), and observed under ultraviolet light. It was confirmed that the DNA was amplified.
  • this PCR product was electrophoresed on a 3% agarose gel prepared with low-melting point agarose, stained with ethidium bromide, and then subjected to a band of about 11 Obp under ultraviolet irradiation. And add 3 times the volume of the TE solution to the gel. After heating for 1 minute to completely dissolve the gel, add an equal volume of TE-saturated phenol (Nippon Gene, Japan), and centrifuge at 1500 rpm for 5 minutes to separate the upper layer. Further, the same separation work was performed using a TE-saturated phenol: chlorophonolem (1: 1) solution, and furthermore, a cologloform. The DNA was recovered from the final solution by ethanol precipitation.
  • the recovered DNA is blunt-ended using the DNAB1 unting Kit from Takara Shuzo, Japan, according to the attached instruction manual, and then T4P01 ynuc 1 eotide from Takara Shuzo, Japan Using Kinase, terminal phosphorylation was performed according to the method described in the company's Guide to Genetic Engineering Products (1991-1995 edition).
  • the blunt-ended and phosphorylated PCR product is digested beforehand with the restriction enzyme Eco RV (Takara Shuzo, Japan), and the end is Alka 1ine Phosphatasase from Takara Shuzo, Japan.
  • Eco RV Transcription Reduction enzyme
  • the terminally dephosphorylated PB 1 uescript according to the method described in the Genetic Engineering Product Guide (1994--1995 version) issued by Takara Shuzo Co., Ltd. Ligation was performed using igation Kit Ver 2 according to the attached instruction manual.
  • a cDNA library of C-11 cells was prepared using the PoIyA + RNA purified in Example 10.
  • c DNA preparation is performed by GIBCO in the United States.
  • Scrit IIRNase H was used. Dilute 2 ⁇ g of the PoIyA + RNA purified in Example 10 with 5 mM MTris (pH 7.5) to a final volume force of 6 ⁇ , After standing at 65 ° C for 5 minutes and then on ice for 5 minutes, the following solution was added.
  • cDNA was blunt-ended using a DNAB1 unting Kit manufactured by Takara Shuzo Co., Ltd. in Japan according to the attached instruction manual, and finally a 20 ⁇ TE buffer was used. Dissolved.
  • a cDNA cloning system manufactured by Amersham in the UK; Igt10, C-11 cells; Lgtl Oc DNA library according to the attached experimental method. was prepared.
  • Thai data one finally fabricated c DNA is (attached to the kit) NM 5 1 4 as a host and were determined using rollers, approximately 1 X 1 0 8 pfu / ⁇ gpoly A + RNA.
  • the cDNA library prepared by the above method was screened by the black hybridization method in the same manner as described in Reference Example 6. Plaques number of the found line bra rie is 4 X 1 0 to five, making the DNA pro one blanking used in disk cleanings is having a DNA sequence set forth in SEQ ID NO: 2: 2 in the sequence listing by PCR and were 32 P labeled with the label-ring method label-ring method described in the same manner as in reference example 6.
  • the clone names were T2, T3, T5, and ⁇ 6.
  • the phage DNA of each clone was purified according to the method described in the above-mentioned compiling, edited by Maniatis et al. These four types of DNAs are digested with the restriction enzyme Ec0RI, and the insert part of the cDNA of the ligand is cut out from ⁇ gt10 by agarol gel electrophoresis, and then agarol gel electrophoresis is performed. When the size was examined at, three bands were confirmed for all clones. Approximately their size of the DDNN AA is TT22 respectively; 3300 000 bbpp, 50 Obp, 180 bp, T3 force; 3 0 bp, 800 bP,
  • the gene sequence was determined using a ring kit.
  • each restriction enzyme site is mapped, and the resulting gene fragment is subjected to subcloning by the above method.
  • Kilo Kiense made by Takara Shuzo in Japan Using a Drilling Mutant kit, a combination of manufacturing a Drilling Mutant in accordance with the attached instruction manual, and transmitting the wire from both directions The sequence was determined.
  • the directions of each gene fragment of the clone are connected to the direction of each gene fragment using the PCR method and the above-described mapping of the phage DNA restriction enzyme sites, and furthermore, those genes.
  • the cDNA obtained by coding for the ligand is a gene fragment of about 500 to 800 bp of each clone in the 5 'direction. , Then a gene fragment of about 300 bp is located, and the remaining gene fragment of about 1500 to 400 bp is located in the 3 ′ direction. Or it becomes.
  • the cDNA having the full length of the present gene sequence is the above-described clone T3
  • the cDNAs should be connected in the same orientation at each restriction enzyme Ec0RI site (about 500 bP).
  • the plasmid prepared by connecting it to the pB1uescript multi-cloning site is called PBS-LIG-FULL, and another vector, PUC 18 (Pharmacia, Sweden)
  • PUCMEKL the plasmid created by subcloning was called PUCMEKL.
  • Example 12 2 mR of ligand by Northern blotting
  • human ulti- ple tissue is a final letter to which mRNA is transcribed in advance.
  • Human Mult iple Tissue Northern Blot II and Human Fetal Mult iple Tissue Northern Blot the DNA sequence of SEQ ID NO: 7 in the sequence listing separated in Example 11 was used.
  • a gene fragment of 46 bP that is, a gene fragment of approximately 300 bP which is cut out with the restriction enzyme EcoRI at the portion encoding the amino acid sequence of the ligand, as described above.
  • the size of the mRNA and the expression organ were examined after 32 P labeling by the method described above.
  • the size of the mRNA is approximately 520b, Only one band was identified.
  • expression organs expression is observed in the heart, brain, placenta, lung, skeletal muscle, kidney, spleen, prostate, testis, ovary, and small intestine of human adult tissues. However, expression was slightly stronger in the kidney. However, no extremely weak or no expression was observed in liver, pancreas, thymus, large intestine, and peripheral blood lymphocytes. In human fetal tissues, expression was observed in the heart, brain, lung, and kidney, but not in the liver.
  • the size of the mRNA was approximately 50%.
  • the expression was 00b, and expression was observed in the heart, brain, spleen, lung, skeletal muscle, and testis, and particularly strong expression was observed in the lung. However, expression was not observed in liver and kidney.
  • a ligand expression vector and a chimera protein expression vector with the ligand were prepared, and the ligand gene was prepared.
  • the morphology and method of expression were raised in 1) to 5).
  • 1) The DNA sequence described in SEQ ID NO: 7 in the sequence listing, that is, the full-length cDNA was transfected into animal cells using an expression vector to form a membrane-bound type containing a signal peptide. And finally expressed on the cell surface as a ligand that does not contain a signal peptide, that is, a form that contains the amino acid sequence of SEQ ID NO: 6 in the sequence listing, and is finally cultured A method for expressing in the supernatant.
  • Expression vectors capable of taking the forms 1) to 5) were prepared using the expression vector pMKITNeo shown in Reference Example 8. The details of the fabrication were in accordance with the same method as that shown in Reference Example 8, and fabricated by using the PCR method or the like.
  • the expression vectors of 1) carapara 5) were prepared, 1) pama p MK Lig FULL FLAG, 2) pMK Lig FULLFLAG, 3) pMK Lig EX, 4) p MKLig EXFLAG, 5) is pMKLig EXIg.
  • the C-11 cells prepared by the method described in 1) were cultured for 1 day in a condition containing 10% FCS, and then replaced with a serum-free medium, and the culture medium was replaced every 4 days. Three times were taken. The same procedure was used to perform gene transfer using an expression vector that did not contain the ligand gene, and used as a control.
  • the two cultures prepared in this way were concentrated 10-fold by the method described in Example 1 and their binding A using a BIA core according to the method described in Example 2.
  • ctivity was measured on EX lg — PTN conjugated chips. As a result, the binding activity was 98 RU in the control, and the binding activity was 310 RU in the ligand-transfected sample.
  • the ligand was purified.
  • the ligand was purified.
  • the amount of purified ligand was visually confirmed based on the results of silver staining on a polyacrylamide gel electrophoresis gel. About three times that of Was.
  • the binding affinity of the purified ligand was measured again using a BIA core, the binding activity was approximately three times higher than that of the control. It was clear that I was working.
  • the ligand can be produced by introducing the gene of SEQ ID NO: 7 into a cell line that does not express or weakly express the ligand. What I can do is clearly better.
  • COS-7 cells transfected with the gene using the expression vector described in 2) were cultured for 4 days in FCS-containing medium, followed by cell disruption by the method described in Reference Example 9. And described in Reference Example 8.
  • Prepare a sample under reducing conditions according to the method perform Western blotting by SDS-PAGE and antibody staining using an anti-FLAG antibody, and obtain a sequence It was examined whether a ligand containing the amino acid sequence of SEQ ID NO: 6 was expressed on cells.
  • C-1 cells prepared by the method described in 3) were cultured under the conditions of 10% FCS for 1 day after gene transfer, and replaced with a serum-free medium. Three samples were taken every day.
  • the culture supernatant thus prepared is concentrated 10-fold by the method described in Example 1, and the ligand is purified by the same method as described in Examples 5 to 7.
  • a ligand of 410,000 ⁇ 750,000 daltons was purified.
  • the N-terminal amino acid sequence of this purified ligand was determined by the method described in Example 9, it had the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing. Was.
  • the ligand was expressed by introducing at least the gene described in 3) into a cell line that does not express or weakly express the ligand. It was clear that it could be produced.
  • the C _ 1 cells prepared by the method described in 4) were cultured under the conditions of 10% FCS for 1 day after the gene transfer, and replaced with a serum-free medium, and the culture supernatant was replaced for 4 days. Three samples were taken at intervals. The culture supernatant thus prepared was concentrated 10-fold by the method described in Example 1. A sample was prepared from this concentrated solution under non-reducing conditions according to the method described in Reference Example 8, and subjected to Western blotting by SDS-PAGE and an antibody staining method using an anti-FLAG antibody.
  • the ligand containing the polypeptide containing the amino acid sequence of SEQ ID NO: 5 in the sequence listing having the FLAG sequence at the C-terminus shown in 4) above was contained in the culture supernatant.
  • an extremely wide range of the molecular weight from about 20000 to 500,000 daltons was dyed, and especially strong dyed areas were centered at 420,000 daltons. Part and a part centered at 240,000 daltons.
  • the strain having a FLAG sequence at the C-terminal shown in 4) above was used.
  • the C-1 cells produced by the method described in 5) were After the introduction of the cells, the cells were cultured under the conditions of 10% FCS for 1 day, then replaced with a serum-free medium, and the culture was collected three times every 4 days.
  • the culture supernatant thus prepared was concentrated 10-fold by the method described in Example 1.
  • the concentrated solution was subjected to Western blotting by SDS-PAGE and an antibody staining method using a peroxidase-labeled anti-human Ig sheep antibody described in Reference Example 8.
  • two sample preparation conditions were used for SDS-PAGE: a sample under reducing conditions and a sample under non-reducing conditions.
  • the port containing the amino acid sequence of SEQ ID NO: 5 in the sequence listing having the amino acid sequence of the Fc portion of human IgG at the C-terminus shown in 5) above was obtained. It was examined whether the ligand containing the receptor was expressed in the culture supernatant. As a result, under a reducing condition, a band of an extremely wide band having a center of a molecular weight of about 800,000 daltons was stained, and under a non-reducing condition, a band of about 2,000 daltons was obtained. A very wide band was stained. However, since no such band was observed in the culture supernatant of control COS-7 cells, human Ig was added to the C-terminus shown in 5) above.
  • a compound containing a part or all of a polypeptide having the amino acid sequence of SEQ ID NO: 5 or 6 in the sequence listing can be prepared, and a dimer of the compound can be prepared. It has been shown that a complex having a multimeric structure higher than that can be produced.
  • 51 of the culture supernatant was passed through a Protein A Sepharose scalar manufactured by Pharmacia, Sweden, to obtain the same strength as above. The size and flow rate were used. In addition, washing and elution were performed in the same manner.
  • a ligand containing a polypeptide containing the amino acid sequence of SEQ ID NO: 5 in the sequence listing having a FLAG sequence at the C-terminus purified by the above method, and a human at the C-terminus The ligand containing the polypeptide containing the amino acid sequence of SEQ ID NO: 5 in the sequence listing having the amino acid sequence of the Fc portion of IgG was purified by gel filtration. .
  • the eluate from the affinity column is concentrated and centrifuged to PBS (-) with Centricon 30 manufactured by Amicon of the United States, and gel filtration is performed by Pharmacia, Sweden. Using FPLC system manufactured by the company, the Superose 12 column was used.
  • Lig FLAG-PTN a ligand containing a polypeptide containing the amino acid sequence of SEQ ID NO: 5 in the sequence listing having a FLAG sequence at the C-terminus
  • the amino acid sequence C Ligand containing a polypeptide containing the amino acid sequence of SEQ ID NO: 5 in the sequence listing having the amino acid sequence of the Fc portion of human IgG at the end
  • Purified material Lig Ig-PTN was obtained.
  • Oligopeptide consisting of amino acid sequence Nos. 11 to 37 of the amino acid sequence described in SEQ ID NO: 7 in the Sequence Listing can be obtained from Applied Biosystems, U.S.A. in a conventional manner. It is prepared by a mosquito synthesizer and conjugated to the horseshoe crab hemosyanin (KLH) (Sigma, USA) with terminal cystine residues, and immunized to rabbits as an immunogen. After measuring the antibody titer, the whole blood is collected, the blood is collected, and the attached handling is performed using an Econopack serum IgG purification kit manufactured by BioRad Inc. of the United States. According to the instructions, an anti-ligand rabbit polyclonal antibody was purified and produced.
  • KLH horseshoe crab hemosyanin
  • Lig FLAG-PTN purified by the method described in Example 14 was used as an immunogen, and in the same manner as described above, rabbits were immunized with an anti-ligand rabbit polyclonal antibody. Produced.
  • mouse monoclonal antibodies were prepared in accordance with the method described in a written document using LigFLAG-PTN purified by the method described in Example 14 as an immunogen. That is, 1 g of Lig FLAG- PTN purified as described above is applied subcutaneously or intradermally to a Ba1b / c mouse (manufactured by Yomoto SLC, Japan). Immunized. After the second immunization, blood was collected from the fundus and an increase in the antibody titer in the serum was observed. After the third immunization, the spleen cells of the mouse were removed, and the mouse myeloma cell line P 3 X Cell fusion was performed with 63 Ag8 (ATCCTIB 9) by the polyethylene glycol method.
  • LigFLAG-PTN purified by the method described in Example 14 as an immunogen. That is, 1 g of Lig FLAG- PTN purified as described above is applied subcutaneously or intradermally to a Ba1b / c mouse (manu
  • Hybridomas are selected in HAT medium (Japan Institute for Immunity and Biological Sciences, Japan), and hybridoma strains producing antibodies that recognize the extracellular part of the ligand in the medium by enzyme-linked immunosorbent assay And a hybridoma-producing strain that produces a mouse monoclonal antibody that specifically recognizes the ligand was established.
  • Example 7 the ligand purified in Example 7 and the cell culture of the transfected COS-7 cells prepared by the methods of 1), 2) and 3) of Example 13 were used.
  • the concentrates of the respective ligands purified by the methods described in Examples 5 to 7 were subjected to Western blotting under the conditions described in Reference Example 8.
  • an anti-Persian Ig sheep antibody manufactured by Amersham, UK was used as the secondary antibody. As a result, it was possible to stain all bands at a broad portion near 420 000 daltons, and it was clarified that all of these antibodies could specifically recognize ligands. .
  • Lig FLAG-PTN and Lig Ig-PTN prepared by the method described in 4) and 5) of Example 13 and prepared by the method described in Example 14 A dot blot was performed using a PVDF membrane manufactured by ioRad according to the attached instruction manual. That is, 5 / x ⁇ of the solution of Lig FLAG-PTN and LigIg-PTN was dropped on PVDF manufactured by BioRad in the United States, air-dried, and then subjected to the Western blotting method described in Example 8. Antibody staining was performed in the same manner as in the above conditions. As a result, it was evident that both were not stained with the control antibody, but were stained with all the purified antibodies.
  • the gene was transfected with the C-1 cells and the method of Example 13-1), particularly for the above monoclonal antibody, and 4 days later.
  • COS-7 cells the expression of ligand on the cell surface was analyzed using a flow site meter EPICS Elite manufactured by Coulter, USA in the same manner as described in Reference Example 10. Staining with the antibody was performed according to the method described in the written literature. The purified monoclonal antibody described above was used as a primary antibody, and a goat anti-mouse Ig FITC label manufactured by Vector Dockton Sons, Inc. of the United States was used as a secondary antibody. As a result, it was confirmed that all of the C-11 cells and about 20% of the transfected COS-7 cells were stained with the above-mentioned monoclonal antibody.
  • the antibody shown in this example was expressed under the conditions of the ligand purified in Example 7, the ligand present on C-1 cells, and the conditions of Example 13 respectively. It was confirmed that the recognized ligand was recognized specifically.
  • Ligand purified in Example 7 and LigFLAG-PTN and LigIg-PTN obtained in Example 14 are reference examples.
  • Reaction solution 1 Medium (RPMI 1640, 10% FCS, 100 / ig / ml mouse IL-13) only;
  • Reaction mixture 2 suspension of 5 ⁇ 10 6 C-1 cells suspended in the medium of reaction mixture 1;
  • Reaction solution 3 Medium of reaction solution 1 containing the ligand purified in Example 7 at a concentration of 500 ⁇ g / m 1;
  • Reaction solution 4 Medium of reaction solution 1 containing the ligand purified in Example 7 at a concentration of 100 g / m 1;
  • Reaction solution 5 Medium of reaction solution 1 containing LigFLAG-PTN at a concentration of gZml;
  • Reaction Solution 6 Medium of Reaction Solution 1 containing Lig FLAG— ⁇ at a concentration of 100 g / ml;
  • Reaction solution 7 Medium of reaction solution 1 containing Lig Ig-PTN at a concentration of 500 g / m];
  • Reaction solution 8 Liglg — Medium of reaction solution 1 containing PTN at lOOg / ml
  • the gel precipitate was subjected to a Westcut by the method described in Reference Example 8.
  • the SDS-PAGE was carried out by adding a SDS-PAGE sample buffer of 30 ⁇ of the gel precipitate, running a boiling water bath for 5 minutes in the presence of 2—ME, and flowing 15 ⁇ into one lane.
  • Two sets of PVDF membranes to which the same sample was transcribed were prepared, and one set was obtained from a polypeptide having the amino acid sequence of SEQ ID NO: 3 in the sequence listing having the target FLAG sequence. And the other was used to determine if the tyrosine residue was phosphorylated.
  • Example 8 As a result, phosphorylation was confirmed in reaction solutions 2, 3, 5, 7, 7 and 8, and the extent of phosphorylation was in reaction solutions 2, 7, and 8 was strong, and reaction solutions 3 and 5 were weaker, and were visually less than about half. Therefore, the ligand purified in Example 8 as well as the co-culture with C-1 cells, the ig FLAG-PTN and LigIg-PTN obtained in Example 14 were It has been shown that it activates the synthase activity of the synkinase, causing phosphorylation of its own tyrosine residue. However, no phosphorylation of such tyrosine residues was observed in the medium alone.
  • Example 8 did not cause phosphorylation under the diluted conditions, that is, the conditions of Reaction Solutions 4 and 6. .
  • LigIg-PTN had sufficient phosphorylation activity even under the condition diluted to the same concentration, and had stronger activity than the former two.
  • reaction solution 9 (compared to the reaction solution 5 with Monodox manufactured by Kodak Corporation in the United States) was used for the Lig FLAG-PTN obtained in Example 14.
  • Reaction solution 10 (reaction solution with the addition of the lonal antibody Anti-FLAGM 2 so as to obtain 100 g Zml).
  • Antibody Anti-FLAGM2 was added to the reaction solution to give a concentration of 100 g / ml), and the reaction was performed using reaction solution 11 (reaction solution 1 in the United States).
  • reaction solution 11 reaction solution 1 in the United States
  • reaction solutions 9 and 10 were sufficiently phosphorylated and had the same level of phosphorylation activity as the reaction solutions 2, 7, and 8. No phosphorus oxidation was observed in the reaction solution 11 of the control.
  • the ligand purified by the method of Example 8 had an activity of phosphorylating a polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing.
  • Mosquito The same was true for LigFLAG-PTN and LigIg-PTN obtained in Example 14.
  • the form having the highest activity is the form of LigIg-PTN, and the LigFLAG-PTN is also administered with an anti-FLAG antibody that recognizes the C-terminal part. Since these compounds exhibit similar strong activities, the form having a dimer or multimer structure is the form of a ligand having a stronger activity. What I wanted was clear.
  • these compounds are compounds that can act on cells expressing the type I tyrosine kinase of the receptor to promote the phosphorylation of tyrosine residues and transmit a signal for proliferation.
  • Example 14 Using the ligand purified in Example 7 and Lig FLAG-PTN and Lig Ig-PTN obtained in Example 14 The physiological activity was confirmed.
  • PBS (-) An equal amount of PBS (-) is added to the cord blood to which heparin has been added, and the mononuclear cells in the cord blood are centrifuged using Fikonorenock (Fanore Macia, Sweden). Well separated.
  • the cells separated in this manner are further washed twice by centrifugation in PBS (-) twice, and finally the cell density is 5 ⁇ 10 6 ce 11 s / m 1 in IMDM medium. This was suspended to prepare a cord blood mononuclear cell suspension.
  • the reagents required for Koguchi Nee Assy were previously prepared as follows.
  • a 3% methylcellulose solution was prepared by dissolving methylcellulose (Wako Pure Chemical Industries, Japan) with stirring in boiling water, cooling to about body temperature, and preparing a double-concentration I prepared in advance.
  • An equal amount of MDM medium was added and left at 4 ° C for about 5 days to prepare.
  • the 10% BSA solution was prepared by adding Sigma BSA in distilled water to distilled water and leaving it at 4 ° C for 2 days with gentle stirring. It was prepared by framing with a ⁇ 's Membrane Final Letter (Millipore, USA).
  • a 7.5% sodium bicarbonate solution (manufactured by GIBCO-BRL, USA) was added to this solution at a concentration of 20 ⁇ m ⁇ 2 in 5 ml of a 10% BSA solution.
  • the condition medium (5663C C) is 10% from the human cell line 56637 manufactured by Coning, Inc. of the United States. ? 5 containing 1 ⁇ ⁇ 1 1 16 40 medium in 50 ml for 5 days. It was prepared by filtration through a 22 ⁇ membrane finoletter (Millipore, USA).
  • reaction solution 2 (600 ⁇ H of 5637 CM), reaction solution 3 (900 ⁇ g of the ligand purified in Example 7), reaction solution 4 (Example 14 And the reaction solution 5 (Liglg—PTN obtained in Example 14 is 300 / ig), respectively, of the Lig FLAG-PTN obtained in the above, and the final volume.
  • IMDM medium was added so that the volume became 1.8 ml.
  • reaction solution 1 was 0 and 0, reaction solution 3 was 7 and 10, reaction solution 4 was 15 and 12, reaction solution 5 was 55 and 68, and reaction solution was 5 and 68.
  • the number 6 was 63 and 75, and the number of reaction solutions 7 was 80 and 89.
  • the colony of the reaction solution 7 was about 20 to 50% larger than that of the other reaction solutions, as judged visually.
  • the following experiment was performed to examine the effect of the ligand on the growth of blood cells in a liquid medium.
  • cord blood mononuclear cells expressing the receptor type 1 tyrosine kinase used in the present application were used.
  • the cells were stained with the 38 antibody described in Reference Example 10, and a FACSV antage (Vectin Deckinson, USA) was used.
  • the cells were analyzed and separated according to the instruction manual attached.
  • receptor type 1 tyrosine kinase was expressed in about a few percent of bone marrow mononuclear cells, and these cells were weakly positive for CD34, a marker for hematopoietic stem cells. , C — Kit positive cells.
  • cord blood mononuclear cells were also expressed.
  • bone marrow and cord blood mononuclear cells were treated with 100 ng / m 1 stem self-actor (International, Inc., USA) to obtain 10% fetal bovine serum (ICN Japan, Japan).
  • Lee scan co full modification da behenate Tsu co medium containing (US, GIBCO-BRL Inc.) at 1 X 1 0 7 ce 1 1 s / 1 5 ml in the culture is performed when re ceptor first die tyrosinate Nkinaze expressing cells And the proportion of the tyrosin kinase-expressing cells is increased by about 10 times or more in the culture for about one week as compared to before the culture. After one week of culture under the above conditions, to separate cells expressing the tyrosine kinase at FACSV antae e Was. Similarly, the cells expressing the tyrosine kinase before culturing were isolated in the same manner.
  • the separated cells were cultured in the presence or absence of the ligand, and the increase in the number of cells was examined.
  • the culture conditions were as follows: 1 ng / m 1 of Interlokin 3 (Intagen, USA) and 2 Unit / ml of erythropoietin (Japan) And Chugai Pharmaceutical Co., Ltd.).
  • 500 ng / m 1 of Lig Ig — PTN and 5 g of anti-human Ig GF c goat antibody prepared by the above method were added.
  • the number of cells increased to about 1.76 ⁇ 10 5 ce 11 s / 6 m 1 approximately 5-fold in the ligand presence section, and increased to 7.0 in the comparison section.
  • the new receptor pig type 1 tyrosine kinase ligand has a 54% homologue with LERK-2, a known molecule, and is supposed to have the same effect as the ligand. The effect on the receptor was compared.
  • the LERK-2 gene was obtained as follows. That is, based on the gene sequence described in the paper of Beckmann et al. (EMBO J, 13, 3757-3762, 1994) or in the specification of W094Z113384, the sequence in the sequence listing is used. No. 23 (corresponding to the sequence of the sense strand 20 bases from the gene sequence 29 bases upstream from the first methionine encoding amino acid of LERK-2) and SEQ ID Nos.
  • LERK corresponds to the sequence of the 23-base antisense strand from the gene sequence upstream of the 23 base upstream of the termination codon to the termination codon of the amino acid sequence coding portion of LERK-2)
  • oligonucleotide having the gene sequence of SEQ ID NO: 1 as a primer, and using human placenta cDNA (Ciontech, USA) as a template the PCR method described above was used to generate the gene. Lone link.
  • the LERK-2 gene isolated in this manner was purified using a method completely similar to that described in Examples 13 and 8.
  • a LERK-2 recombinant protein expression vector was constructed in the same manner as the ligand, produced by gene transfer, and further purified from the cell culture supernatant by the method described in Reference Example 9. And purified.
  • These expression vectors are the extracellular portion of LERK-2, that is, the portion up to the amino acid at position 237 of the amino acid sequence of LERK-2 described in Beekmann et al., Supra.
  • the molecules are LERK-2Ig-PTN and LERK-2FLAG-PTN.
  • the calculation method was calculated in accordance with the computer software BIA eva 1 ation 2.1 attached to BIA c0 re and the harmful explanation of its handling.
  • K d values of ⁇ Ri cancer de is 1.
  • LERK- 2 is 2 3 X 1 0- 9 M 1 . 3 5 X 1 0- 7 M der Li, the Li cancer de The LERK- 2 The ability to bind approximately 100 times as much as possible was clearly demonstrated.
  • Example 16 and Reference Example 8 The results are shown in FIG.
  • 1 was a control, 2 lig Ig — PTN, 100 ng / m 1 and 100 ⁇ g / ml of anti-human Ig GF c goat antibody manufactured by Organontechniki, USA 1 ⁇ g Zml, 3 ⁇ g ig_PTN, 1 ⁇ g / m1 with anti-human Ig GFc goat antibody manufactured by Organon Techni Riki, USA 1 O jug / m 1, 4 LERK — 2 Ig — PTN, 1 OO ng / ml, 1 ⁇ g / m 1, 5 of anti-human Ig gc goat antibody manufactured by Organontech Niki, USA is LERK-2 I g — PTN, 1 ⁇ g / m ⁇ against anti-human Ig GF c 1 4 1
  • the antibody was added to each of I 0 jug / ml and reacted with the reaction solution described in Example 16 to carry out the reaction.
  • a in Fig. 10 is stained with antiphosphorylated tyrosine monoclonal antibody (UBI, USA), and B is stained with anti-FLA GM2 monoclonal antibody (Kodak, USA).
  • the film was printed on the X-ray film under the conditions of Example 8.
  • the tyrosin kinase that has been blotted from the molecular weight is indicated by an arrow.
  • LERK-2 does not have the physicochemical properties possessed by the resebuta-type tyrosine kinase ligand of the present application, and thus does not have the physiological action as shown in the present application.
  • Specific receptor involved in differentiation and proliferation of undifferentiated blood cells Specific receptor type 1 binds to the extracellular portion of tyrosine kinase, activates the tyrosine kinase enzyme activity of the intracellular portion, and activates the tyrosine kinase. Since the ligand of the present invention that causes oxidation and the complex containing the ligand have an action of promoting the differentiation and proliferation of undifferentiated blood cells, they can enhance blood-producing stem cells such as leukemia and bone marrow transplantation. It can be used for research and treatment of problems related to undifferentiated blood cells.
  • Organism name human
  • Organism name human
  • Lys lie Leu Ala Se r Va 1 Gin His Met Lys Se r Gin Ala Lys Pi Gly
  • Sequence type c DNA to mRNA, and amino acid
  • Organism name human
  • TTC TAC AAA AAG TGC GCC CAG CTG ACT CTG AAC CTG ACT CGA TTC CCG 1033 Phe Tyr Lys Lys Cys Ala Gin Leu Tin Va 1 Asn Leu Thr Arg Phe Pro
  • Ser r Se Leu H is Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg SKI OVO ⁇ :) ⁇ 33;) 030 003 V10 9V3 010 91D :) VI 33V DDO VOO OOD OVV 013 o S 0
  • GGC CAC TGG CTT CGG GCC ATC AAA ATC GGA AGA TAC GAA GAA ACT TTC 3193 Gl Glu Trp Leu Arg Ala lie Lys Met Gly Arg Tyr Glu Glu Se Phe
  • Organism name human Sequence: Lys Ser lie Va 1 Leu Glu Pro lie
  • Organism name human
  • Lys Ser lie Va 1 Leu Glu Pro lie Tyt Trp Asn Ser Ser Asn Ser Lys
  • Gin Thr ⁇ ⁇ l Ala Met Lys lie Leu Met Lys Va 1 Gly Gin Asp Ala Ser 130 135 140 1
  • Organism name human
  • Lys Ser lie Val Leu C 1 u Pro lie Ty r Tr p Asn Ser Ser Asn Ser Lys
  • Th r lye Lys Lys G I u Asn Th r Pro Leu Leu Asn Cys A I a Lys Pro Asp 65 70 75 80
  • Sequence type c DNA to m RNA, and amino acid
  • Organism name human

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Abstract

La présente invention concerne un ligand de tyrosine kinase de type récepteur se liant à la partie extracellulaire d'une tyrosine kinase spécifiée de type récepteur et permettant d'accroître l'activité enzymatique de la tyrosine kinase dans ladite partie intracellulaire afin d'induire la phosphorylation de la tyrosine kinase. Cette kinase spécifiée en est une qui, d'une part, s'exprime dans des cellules sanguines indifférenciées, mais subit une baisse de niveau d'expression accompagnant la différenciation progressive des cellules sanguines, et d'autre part participe à la différenciation et à la croissance des cellules indifférenciées. L'invention concerne également un complexe constitué du ligand de la présente invention et de l'un au moins des éléments appartenant à un groupe constitué du ligand et d'autres composés. L'invention concerne un ADN codant pour ce ligand. L'invention concerne ensuite un procédé de production du ligand par génie génétique. L'invention concerne enfin un anticorps reconnaissant spécifiquement ce ligand. L'accélération de la croissance des cellules sanguines indifférenciées sous l'effet de ce ligand est imputable à l'accélération de la formation de colonies de monocytes du sang cordonal humain. Etant donné que le ligand, ainsi que le complexe à base de ce ligand, ont pour effet d'accélérer la différenciation et la croissance des cellules sanguines indifférenciées, ils conviennent particulièrement, d'une part, à l'étude des problèmes associés aux cellules sanguines indifférenciées incluant les cellules souches hématopoïétiques, par exemple dans le cas de la leucémie et de la greffe de la moelle osseuse, et d'autre part à l'application thérapeutique appropriée.
PCT/JP1995/002069 1994-10-07 1995-10-09 Nouveau ligand de tyrosine kinase de type recepteur WO1996011212A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU36198/95A AU3619895A (en) 1994-10-07 1995-10-09 Novel receptor-type tyrosine kinase ligand

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP6/244433 1994-10-07
JP24443394 1994-10-07
JP26288294 1994-10-26
JP6/262882 1994-10-26
JP6/320712 1994-12-22
JP32071294 1994-12-22

Publications (1)

Publication Number Publication Date
WO1996011212A1 true WO1996011212A1 (fr) 1996-04-18

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PCT/JP1995/002069 WO1996011212A1 (fr) 1994-10-07 1995-10-09 Nouveau ligand de tyrosine kinase de type recepteur

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AU (1) AU3619895A (fr)
WO (1) WO1996011212A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0770088A4 (fr) * 1994-07-08 1999-10-13 Immunex Corp Nouvelle cytokine designee lerk-5
US6291210B1 (en) 1996-07-16 2001-09-18 Asahi Kasei Kabushiki Kaisha Differentiation inhibitor
US6337387B1 (en) 1995-11-17 2002-01-08 Asahi Kasei Kabushiki Kaisha Differentiation-suppressive polypeptide

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM., (13 May 1994), Vol. 269, No. 19, BENNETT B.D. et al., "Cloning and Characterization of HTK, a Novel Transmembrane Tyrosine Kinase of the EPH Subfamily", p. 14211-18. *
MOL. CELL. BIOL., (Sep. 1995), Vol. 15, No. 9, BERGEMANN A.D., "ELF-2, a New Member of the Eph Ligand Family, is Segmentally Expressed in Mouse Embryos in the Region of the Hindbrain and Newly Forming Somites", p. 4921-9. *
PROC. NATL. ACAD. SCI. U.S.A., (14 Mar. 1995), Vol. 92, No. 6, BENNETT B.D. et al., "Molecular Cloning of a Ligand for the EPH-Related Receptor Protein Tyrosine Kinase HtK", p. 1866-70. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0770088A4 (fr) * 1994-07-08 1999-10-13 Immunex Corp Nouvelle cytokine designee lerk-5
US6479459B1 (en) 1994-07-08 2002-11-12 Immunex Corporation Cytokine designated lerk-5
US6492140B2 (en) 1994-07-08 2002-12-10 Immunex Corporation Polynucleotides encoding cytokine designated LERK-5
US6596852B2 (en) 1994-07-08 2003-07-22 Immunex Corporation Antibodies that bind the cytokine designated LERK-5
US6337387B1 (en) 1995-11-17 2002-01-08 Asahi Kasei Kabushiki Kaisha Differentiation-suppressive polypeptide
US7141379B2 (en) 1995-11-17 2006-11-28 Asahi Kasei Kabushiki Kaisha Method of suppressing differentiation by administering a human serrate-1 polypeptide
US7179622B2 (en) 1995-11-17 2007-02-20 Asahi Kasei Kabushiki Kaisha Method of suppressing differentiation by administering a human serrate-1 polypeptide
US7198918B2 (en) 1995-11-17 2007-04-03 Asahi Kasei Kabushiki Kaisha Nucleic acid encoding a human serrate-1 polypeptide
US7253265B2 (en) 1995-11-17 2007-08-07 Asahi Kasei Kabushiki Kaisha Antibody and a method for producing an antibody that specifically binds to a full length amino acid sequence of human Delta-1
US6291210B1 (en) 1996-07-16 2001-09-18 Asahi Kasei Kabushiki Kaisha Differentiation inhibitor
US6638741B2 (en) 1996-07-16 2003-10-28 Asahi Kasei Kabushiki Kaisha Differentiation-suppressive polypeptide serrate-2
US7138276B2 (en) 1996-07-16 2006-11-21 Asahi Kasei Kabushiki Kaisha Differentiation-suppressive polypeptide serrate-2 and methods of use

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Publication number Publication date
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