WO2007099921A1 - PEPTIDE BINGING TO PILRα, POLYNUCLEOTIDE ENCODING THE SAME AND APPLICATION THEREOF - Google Patents

PEPTIDE BINGING TO PILRα, POLYNUCLEOTIDE ENCODING THE SAME AND APPLICATION THEREOF Download PDF

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Publication number
WO2007099921A1
WO2007099921A1 PCT/JP2007/053558 JP2007053558W WO2007099921A1 WO 2007099921 A1 WO2007099921 A1 WO 2007099921A1 JP 2007053558 W JP2007053558 W JP 2007053558W WO 2007099921 A1 WO2007099921 A1 WO 2007099921A1
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Prior art keywords
polypeptide
polynucleotide
seq
nos
amino acid
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PCT/JP2007/053558
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French (fr)
Japanese (ja)
Inventor
Hisashi Arase
Ikuo Shiratori
Katsumi Maenaka
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Osaka University
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Publication of WO2007099921A1 publication Critical patent/WO2007099921A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to control of an immune response.
  • the present invention particularly relates to polypeptides capable of binding to PILRa.
  • the property of the polypeptide of the present invention that binds to PILR o can be used to control the activation of antigen-presenting cells, such as rod-like cells and macrophages. It can be suitably used for pharmaceuticals that control immune responses, such as pharmaceuticals for medical use.
  • active receptors and inhibitory receptor paired receptors are expressed, and these paired receptors play an important role in controlling immune responses. It has become clear that it is responsible. Although these active receptor and inhibitory receptor have very high homology in the extracellular region, they have contradictory functions (activation'inhibition receptor).
  • immune cells are involved in immune responses such as T cells, B cells, NK cells, rod cells, macrophages, mast cells, basophils, eosinophils, or neutrophils. Including cells.
  • An active receptor expresses its ligand, recognizes a cell, and activates an immune cell.
  • To activate immune cells means to induce site-in production and cytotoxicity of immune cells.
  • the active receptor has a positively charged amino acid in the transmembrane region.
  • ITAM Immunorecept or Tyrosine—based Activation Motief
  • ⁇ ⁇ ⁇ ⁇ ti column such as DAP12, CD3z or FcR y
  • Y— x— x— L— x (6-8) — Y— x— x— x— L and x are arbitrary
  • the active receptor transmits an active signal.
  • the inhibitory receptor has an ITIM (Immunoreceptor Tyrosine-bas ed Inhibitory Motief) sequence [(I / V / L / S)-x- Y- x- x- (L / V) ( x is an arbitrary amino acid)], and by mobilizing a phosphatase such as SHP-1, the signal from the activated receptor is blocked.
  • ITIM Immunoreceptor Tyrosine-bas ed Inhibitory Motief
  • paired receptors that are recognized in immune cells include KIR (Killer cell Ig-like receptor) having an immunoglobulin-like structure in humans and Ly 49 having a C-type lectin-like structure in mice. Many of the inhibitory receptors recognize MHC class I and are thought to suppress the self-responsiveness of immune cells. On the other hand, the active type Ly49, KIR receptor hardly recognizes its own MHC, and the ligand recognition mechanism including the physiological meaning of its existence is clear.
  • PILR Porous Ig-like Type 2 Receptor
  • PILR is a pair-type receptor that also acts as an inhibitory receptor, PILR ⁇ (sometimes referred to as FDF03) and an activated receptor, mouse PILR ⁇ .
  • PILR is also widely expressed in immune cells such as rod cells, macrophages, mast cells, etc.
  • PILR o is clawed in humans and mice (Non-patent Documents 1 and 2).
  • the present inventors have cloned mouse PILR-L (PILR Ligand) which is a ligand of mouse PILR ⁇ and PILR
  • the inventors of the present invention have revealed that NK cells and dendritic cells are activated through the association between PILR-L and PILR
  • the present invention has been made in view of the above-described conventional problems, and an object thereof is to provide a polypeptide that binds to PILR a, a polynucleotide encoding the polypeptide, and use thereof. is there.
  • a polypeptide according to any one of the following (a) or (b), a polynucleotide encoding the polypeptide, or a cell expressing the polypeptide, and an immune cell A composition that activates or suppresses activation of immune cells.
  • a composition comprising the polynucleotide (e) or (f) below, which activates immune cells or suppresses activation of immune cells.
  • (f) consisting of a base sequence encoding a polypeptide that binds to PILR ⁇ under the stringent conditions and a polynucleotide that is misaligned under the following (iii) or (iv) Polynucleotide.
  • a composition containing a polypeptide that binds to PILRa which activates immune cells or suppresses the activity of immune cells.
  • a polypeptide according to any one of the following (g) or (h), a polynucleotide encoding the polypeptide, or a cell expressing the polypeptide, and an immune cell A composition that activates or suppresses activation of immune cells.
  • a composition comprising the polynucleotide (i) or (j) below, which activates immune cells or suppresses the activity of immune cells.
  • (V) A polynucleotide comprising the base sequence represented by any one of SEQ ID NOS: 16 to 18.
  • a polypeptide that binds to PILR a which is any of the following (a) or (b). [0031] (a) A polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 6.
  • a polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 2 to 4.
  • (C) A polynucleotide comprising the base sequence represented by any one of SEQ ID NOs: 8 to 10.
  • a polynucleotide comprising a base sequence encoding a polypeptide that hybridizes under stringent conditions with a non-regular polynucleotide of (i) or (ii) below and binds to PILRo; .
  • a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 8 to 10.
  • (j) A polynucleotide comprising a base sequence encoding a polypeptide that hybridizes under stringent conditions and binds to PILRo; with a polynucleotide of (V) or (vi) below: .
  • (v) A polynucleotide comprising the nucleotide sequence represented by any one of SEQ ID NOs: 16 to 18.
  • the polypeptide of the present invention can bind to PILR o ;. Therefore, the above-mentioned polypeptide can be suitably used for pharmaceuticals that modulate immune responses by, for example, activating immune cells or suppressing activity.
  • the present invention also includes a polynucleotide encoding the above polypeptide.
  • the polynucleotide can be used not only for the production of the polypeptide, but also for a pharmaceutical or the like that suppresses the immune response, as with the polypeptide.
  • FIG. 1 is a histogram showing the results of analysis of Ba / F3-L2 according to Example 1 of the present invention by flow cytometry.
  • FIG. 2 is a histogram showing the results of analyzing BaZF3-CD23 according to Example 2 of the present invention by flow cytometry.
  • FIG. 3 is a histogram showing the results of analyzing BaZF3-Mock according to Comparative Example 1 by flow cytometry.
  • FIG. 4 is a histogram showing the results of analyzing 293T-CD6 according to Example 3 of the present invention by flow cytometry.
  • FIG. 5 293T-CD45 according to Example 4 of the present invention was analyzed by flow cytometry. It is a histogram which shows the result.
  • FIG. 6 ULBP-Ba / F3-L2 according to Example 5 of the present invention and LBP-Ba / F3-Mock according to Comparative Example 2! /, Using NKL-Mock and Cr51 3 is a graph showing the results of a free cell injury assay.
  • FIG. 7 ULBP-Ba / F3-L2 according to Example 5 of the present invention and LBP-Ba / F3-Mock according to Comparative Example 2! /, Using NKL-PILRa and Cr51 3 is a graph showing the results of a free cell injury assay.
  • FIG. 8 shows the results of subjecting a coprecipitate with PILRa in a solubilized cell sample (human, mouse) to Western Blot using an anti-mouse CD45 antibody (anti-CD45Ab).
  • FIG. 9 shows the results of flow cytometry analysis of the binding of mouse CD45—Ig, mouse PILR—LI (CD 99) —Ig, and mouse PILR—L2—Ig chimeric proteins to cells expressing mouse PILR. .
  • the present invention relates to a polypeptide that is a ligand of PILR a, a polynucleotide encoding the polypeptide, and use thereof. Therefore, in the following, the polypeptide according to the present invention, then the polynucleotide encoding the polypeptide, and finally the method of using it will be described.
  • the present invention includes a polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4.
  • the present invention includes any of the following (a) and (b), and a polypeptide that binds to PILRa.
  • the present inventors newly identified a polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4.
  • it binds to the polypeptide power PILR a which is the amino acid sequence power shown by any one of SEQ ID NOs: 1 to 6. I found it.
  • SEQ ID NO: 1 shows the amino acid sequence of a polypeptide called hPILR-L2
  • SEQ ID NO: 2 shows the amino acid sequence of a polypeptide called human CD23
  • SEQ ID NO: 3 shows the amino acid sequence of a polypeptide called human CD6.
  • the amino acid sequence is shown.
  • SEQ ID NO: 4 shows the amino acid sequence of a polypeptide called mouse CD6.
  • SEQ ID NO: 5 shows the amino acid sequence of a polypeptide called human CD45
  • SEQ ID NO: 6 shows the amino acid sequence of a polypeptide called mouse CD45.
  • the term polypeptide may be used synonymously with protein.
  • the present inventors have found that the polypeptide functions as a ligand of the polypeptide force PILR o; consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6.
  • the PILR a is preferably derived from a human or a mouse, but may be a molecule derived from another organism such as a mammal other than a human and having a function control of immune cells.
  • the present invention includes a polypeptide that binds to PILR o; which is a variant of the polypeptide having the amino acid sequence ability shown in any one of SEQ ID NOs: 1 to 6. .
  • the polypeptide according to the present invention is preferably derived from a human or a mouse, but is a ligand of a molecule derived from another organism such as a mammal other than human and having a function control of immune cells, It can be different.
  • the polypeptide according to the present invention is a product purified from a natural cell, a product of chemical synthesis, and a prokaryotic or eukaryotic host (eg, bacterial cell, yeast cell, higher plant cell, insect cell, And products produced by recombinant techniques from mammalian cells).
  • a prokaryotic or eukaryotic host eg, bacterial cell, yeast cell, higher plant cell, insect cell, And products produced by recombinant techniques from mammalian cells.
  • the polypeptides according to the invention may be glycosylated or non-glycosylated.
  • the polypeptides according to the present invention may also contain an initiating modified methionine residue as a result of a host-mediated process.
  • polypeptide variant refers to "a substitution or deletion of one or several amino acids in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 6," It refers to a polypeptide that also has an amino acid sequence ability that is inserted or added.
  • mutant polypeptide production methods such as site-specific mutagenesis (preferably 10 or less, more preferably 7 or less, most preferably 5)
  • site-specific mutagenesis preferably 10 or less, more preferably 7 or less, most preferably 5
  • amino acids are substituted, deleted, inserted or added.
  • Such a mutant polypeptide is not limited to a polypeptide having a mutation artificially introduced by the above-mentioned known mutant polypeptide production method, but is a product obtained by isolating and purifying a naturally occurring polypeptide. There may be.
  • Such variants include deletions, insertions, inversions, repeats, and type substitutions (eg, one hydrophilic residue, substitution with another residue, but usually with high hydrophilicity).
  • the residue is not substituted with a highly hydrophobic residue having the opposite property.).
  • “neutral” amino acid substitutions in a polypeptide generally have little effect on the activity of the polypeptide, and therefore may be substituted with other amino acid residues.
  • One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a polypeptide using these well-known techniques. For example, according to a known point mutation introduction method, an arbitrary base of a polynucleotide encoding a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide. Furthermore, if the method described in this specification is used, it can be easily determined whether the produced mutant has a desired activity.
  • Preferred mutants include those having conservative or non-conservative amino acid substitutions, deletions, or added calories. More preferred are silent substitutions, additions and deletions, and even more preferred are conservative substitutions.
  • the polypeptide according to the present invention introduces a polynucleotide according to the present invention (a gene encoding the polypeptide according to the present invention) described later into a host cell, and allows the polypeptide to be expressed in the cell. Or isolated and purified from cells, tissues, etc. It may be.
  • the polypeptide according to the present invention may be chemically synthesized.
  • the polypeptides of the present invention may be recombinantly expressed in a modified form such as a fusion protein. That is, a polypeptide in which an amino acid sequence such as a tag is added to the end of the polypeptide according to the present invention, particularly the N-terminal side, is also included in the present invention.
  • glycosylation plays an important role in the functional expression of proteins, particularly proteins expressed on the cell surface.
  • the above-mentioned polypeptides are glycosylated. Included.
  • polynucleotide according to the present invention examples include a polynucleotide comprising a base sequence encoding a polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4.
  • a person skilled in the art can easily prepare the polynucleotide by referring to the description of the present specification, for example, the descriptions of SEQ ID NOs: 1 to 4.
  • the polynucleotide may be present in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA).
  • DNA can be double-stranded or single-stranded.
  • Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand).
  • the present invention also includes a polynucleotide which is a deviation from the following (c) or (d).
  • a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 7 to 10.
  • SEQ ID NO: 7 includes a polynucleotide encoding a polypeptide called hPILR-L2.
  • SEQ ID NO: 8 shows a polynucleotide encoding a polypeptide called human CD23
  • SEQ ID NO: 9 shows a polynucleotide encoding a polypeptide called human CD6
  • SEQ ID NO: 10 shows mouse CD6.
  • SEQ ID NO: 11 shows a polynucleotide encoding a polypeptide called human CD45
  • SEQ ID NO: 12 shows a polynucleotide encoding a polypeptide called mouse CD45.
  • the above hybridization can be performed by a known method such as the method described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989). it can. Generally, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained.
  • the conditions for the hybridization conventionally known conditions can be suitably used, and are not particularly limited.
  • the polynucleotide according to (d) above is not particularly limited in length or the like, and may be, for example, a fragment of the base sequence shown in SEQ ID NO: 7 to L0.
  • the fragment refers to a fragment of at least 12 nt (nucleotide), preferably about 15 nt, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably at least about 40 nt in length.
  • nucleotide sequences shown in SEQ ID NOs: 7 to 10 are provided, so that those skilled in the art can easily prepare DNA fragments based on SEQ ID NOs: 7 to 10.
  • restriction endonuclease cutting or ultrasonic shearing can be easily used to make fragments of various sizes.
  • such fragments can also be synthesized ihologically.
  • examples of the polynucleotide according to (d) above include variants in which one or several bases are deleted, substituted, or added in the polynucleotide of (c) above.
  • Variants can be mutated in coding or non-coding regions, or both. Mutations in the coding region can generate conservative or non-conservative amino acid deletions, substitutions or additions.
  • all the polynucleotides according to the present invention described above can be fused to the polynucleotide encoding the tag tag (tag sequence or marker sequence) described above on the 5 'side or 3' side.
  • the present invention includes a vector comprising the polynucleotide according to the present invention described in the section ⁇ 2> above. That is, the vector according to the present invention includes a polynucleotide comprising the base sequence encoding the polypeptide represented by any one of SEQ ID NOs: 1 to 4, or the above-mentioned (c) or (d). And a recombinant expression vector into which a polynucleotide is inserted.
  • Examples of a method for producing a recombinant expression vector include, but are not limited to, methods using a plasmid, phage, cosmid, or the like.
  • the specific type of vector is not particularly limited, and a vector that can be expressed in a host cell may be appropriately selected. That is, according to the type of host cell, an appropriate promoter sequence is selected to ensure expression of the polynucleotide according to the present invention, and a vector in which this and the polynucleotide according to the present invention are incorporated into various plasmids is expressed. It can be used as a vector.
  • the expression vector may comprise at least one selectable marker.
  • selectable markers include drug resistance genes such as tetracycline resistance gene or ampicillin resistance gene for eukaryotic cell culture, for dihydrofolate reductase or neomycin resistance, and for culture in E. coli and other bacteria. Can be mentioned. Of course, it is not limited to these selection markers, and it goes without saying that conventionally known selection markers that can be used by those skilled in the art in the state of the art at the time of the present application can be used. [0090] By using the above selectable marker, it is confirmed whether or not the polynucleotide according to the present invention has been introduced into the host cell, and whether or not it is reliably expressed in the host cell. Can do.
  • the polypeptide according to the present invention may be expressed as a fusion polypeptide.
  • a green fluorescent polypeptide GFP Green Fluorescent Protein
  • the polypeptide according to the present invention is used as a marker. It may be expressed as a GFP fusion polypeptide.
  • the host cell is not particularly limited, and various conventionally known cells can be preferably used. Specifically, for example, bacteria such as Escherichia coli, yeasts (development yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, mammals Examples of the cells can be mentioned, but are not particularly limited. Suitable culture media and conditions for the above host cells are well known in the art.
  • Methods for introducing the above expression vectors into host cells are not particularly limited. Conventionally known methods such as electroporation, calcium phosphate method, ribosome method, DEAE dextran method, retrovirus method, etc. Can be suitably used.
  • the vector according to the present invention should contain at least a polynucleotide encoding the polypeptide according to the present invention. That is, it should be noted that a vector other than the expression vector is also included in the technical scope of the present invention.
  • an object of the present invention is to provide a vector containing a polynucleotide encoding the polypeptide of the present invention, and the individual vector species specifically described in the present specification. And cell types, and vector production methods and cell introduction methods. Therefore, it should be noted that vectors obtained using vector species and vector production methods other than those described above also belong to the technical scope of the present invention.
  • the present invention also includes cells into which the polynucleotide described in the above ⁇ 2> column has been introduced so that it can be expressed.
  • This cell has the same expression vector power as described above in ⁇ 3>.
  • the cells are introduced into the host cells described above.
  • the vectors and host cells are as described above.
  • the present invention also includes a method for producing a protein that binds to PILR o; using the polynucleotide described in the section ⁇ 2>.
  • a method for producing this polypeptide a recombinant expression system using the polynucleotide described in the section ⁇ 2> above can be employed.
  • the method for producing a polypeptide is a method for purifying the polypeptide from the cells described in the section ⁇ 4>. That is, the polypeptide production method may include a step of introducing the vector described in the above ⁇ 3> column into a host cell. Details of the vector and the host cell are as described in the section ⁇ 3> above.
  • the method for producing a polypeptide according to the present invention further includes the step of purifying the polypeptide with the extractive force of cells or tissues containing the polypeptide.
  • the step of purifying the polypeptide is to prepare a cell extract from the cells or tissues by a well-known method (for example, to centrifuge the cells or tissues and then collect the soluble fraction by centrifugation). Hydrodynamics Well-known methods (e.g.
  • H PLC High performance liquid chromatography
  • the production method according to the present invention aims to provide a polypeptide having a binding activity to PILRa, and the production method including steps other than the various steps described above is also applicable to this production method. It goes without saying that it is included in the invention.
  • the polypeptide in column ⁇ 1> above is not limited to such a production method using a recombinant expression system, and is not limited to such a polypeptide. It may be produced by refining. As the purification method, the various techniques described above can be used.
  • the polypeptide according to the present invention may be produced by chemical synthesis. Those skilled in the art can easily understand that the polypeptide according to the present invention can be chemically synthesized by applying a well-known chemical synthesis technique based on the amino acid sequence of the polypeptide according to the present invention described in the present specification. to understand. That is, the polypeptide according to the present invention may be a naturally occurring mutant polypeptide or an artificially prepared mutant polypeptide.
  • the present invention includes any of the above-mentioned (a) and (b), a polypeptide that binds to PILRo; a polynucleotide that encodes the polypeptide; or a cell that expresses the polypeptide. Also included are compositions that activate or suppress activation of immune cells.
  • the polypeptide has the property of being able to bind to PILRa, which is a suppressor receptor of immune cells. Therefore, by utilizing this property, the composition according to the present invention can suppress the activity or activity of immune cells.
  • the phrase "activate immune cells” does not limit the mechanism or the like, and as long as immune cells are activated.
  • the above-mentioned polypeptide binds to PILRa or other receptor that controls the activity of immune cells, thereby directly activating immune cells via this receptor. It may be what you do.
  • the above-mentioned polypeptide inhibits immune cell inactivation by other ligands that suppress or inactivate immune cells via receptors. Alternatively, it may indirectly activate immune cells.
  • “suppressing the activity of immune cells” is not limited to the mechanism and the like, and as long as the activity of immune cells is suppressed.
  • a mechanism for suppressing activation it may be that by binding to a receptor of an immune cell, the activity of the immune cell is directly suppressed by a signal via the receptor. Further, the activity of the immune cell may be indirectly suppressed by inhibiting the ligand-receptor interaction that activates the immune cell.
  • the polypeptide preferably activates immune cells or suppresses activity by functioning as an agonist or an antagonist.
  • activation of immune cells means that, as described above, production of cytodynamic force from immune cells and cytotoxicity are induced.
  • suppression of immune cell activity means that immune cells are inactivated, or that activity does not occur even under conditions where immune cells are activated. It can also be said that it is.
  • the immune cells that are the target of the activity or suppression of the activity of the composition are activated by the polypeptide that is not particularly limited, such as the species of the organism. As long as it is an immune cell whose ability and activity are suppressed, it may be derived from humans or other mammals. In addition, the organs, tissues, and the like targeted by the composition are not particularly limited.
  • the composition can enhance or suppress the immune response by regulating the activity of immune cells. This is because the activity of individual immune cells and the ability to suppress activity can act in either direction of enhancing or suppressing the immune response, respectively. For example, when immune cells having a function of suppressing an immune response (such as cells that produce a suppressive cyto force-in) are inhibited by the above composition, the entire immune response is enhanced. Conversely, when immune cells having a function of activating an immune response are inhibited by the above composition, the entire immune response is suppressed.
  • the constitution other than those described above, that is, the content, shape, production method, and the like of the polypeptide, the polynucleotide, and the cell are not particularly limited.
  • the cells contained in the composition may be recombinant or non-recombinant cells, that is, cells into which no foreign gene has been introduced.
  • the polynucleotide contained in the composition may be contained in a state of being inserted into a vector.
  • a suitable conventionally known vector can be used as described in the section ⁇ 3> above.
  • these polypeptides, polynucleotides, and cells may be used alone or in appropriate combination.
  • the administration method, other composition, target organism, and the like are not particularly limited as long as the composition can exert the ability to activate the immune cells or the effect of suppressing the activity.
  • parenteral administration such as injection is preferred.
  • the polypeptide in the composition is associated with PILRa of immune cells at that part. Can control the function of immune cells.
  • a composition containing the polynucleotide according to the present invention is administered in vivo, the polynucleotide in the composition is expressed in vivo to produce the polypeptide according to the present invention.
  • the polynucleotide in the composition is preferably capable of being expressed in cells.
  • the same effect can be obtained by administering cells that express the polypeptide.
  • composition according to the present invention is used as a pharmaceutical agent for controlling the immune response by controlling the function of immune cells, and particularly the activity of rod cells, macrophages and mast cells. It is suitably used as a pharmaceutical product that suppresses
  • polynucleotide contained in the yarn composition of the present invention may be either (e) or (f) below.
  • composition containing the above-mentioned polynucleotide refers not only to administration of the polynucleotide in vivo but also to administration of the polynucleotide in vivo. It is also intended to express a polypeptide encoded by the nucleotide and to activate immune cells or suppress activity depending on the function of the polypeptide.
  • a fusion protein (chimeric protein) of the above polypeptide and another protein is also included in the present invention and can be used as an active ingredient of the above composition.
  • Examples of the chimeric protein include chimeric molecules of the polypeptide and IgG-Fc region. More specifically, for example, mouse CD45-Ig, mouse PILR-LI (CD99) -Ig, mouse PILR-L2-Ig, human CD45-Ig, human PILR-L2-Ig and the like can be mentioned.
  • Ig-chimeric molecules should be used for IJ to inhibit PILR function and to cross-link PILR and transmit inhibitory signals, as shown in Example 6 below. It is out.
  • the Ig-Fc region may be derived from a human or a mouse, but may be derived from other mammals and is not particularly limited. Desirably, an Ig-Fc region derived from the same organism as the application target of the composition is more preferably used.
  • the chimeric molecule having the Ig-Fc region is considered to form a dimer (dimer) from its structure. For this reason, it is considered that PILR is cross-linked to transmit an inhibitory signal or an activity signal. This function is unique to a chimeric molecule having an Ig-Fc region, and can be said to be an excellent effect not seen with a single ligand.
  • the present invention expresses a chimeric molecule of the polypeptide (a) or (b) above and an IgG-Fc region, a polynucleotide encoding the chimeric molecule, or the chimeric molecule. And a composition that contains a polypeptide that binds PILR ⁇ and activates immune cells or suppresses activation of immune cells.
  • Examples of the chimeric molecule include the polypeptides described in any one of SEQ ID NOs: 13 to 15. Specifically, it comprises a polypeptide according to any of the following (g) or (h), a polynucleotide encoding the polypeptide, or a cell that expresses the polypeptide, and activates immune cells. A composition that suppresses or suppresses the activity of immune cells.
  • polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOS: 13 to 15
  • the present invention also includes a composition comprising the polynucleotide (i) or (j) below, which activates immune cells or suppresses activation of immune cells.
  • (j) A polynucleotide comprising a nucleotide sequence that encodes a polypeptide that hybridizes under stringent conditions with a polynucleotide that is not shown in (V) or (vi) and binds to PILRo; .
  • (V) A polynucleotide comprising the nucleotide sequence represented by any one of SEQ ID NOS: 16 to 18.
  • a fusion protein that binds to PILR-a is produced using the above-mentioned polypeptide (a) or (b), or a partial polypeptide thereof, and an IgG-Fc region. The method of doing is also included. According to this method, a fusion protein that forms a dimer (dimer) and crosslinks PILR to transmit an inhibitory signal or an activity signal can be produced.
  • the cells contained in the composition of the present invention the cells described in the above item 4> can be used.
  • a cell into which the polynucleotide of any one of the above (e), (f), (i) and (j) has been introduced so as to be expressed can be used.
  • Molecules recognized by PILR-Ig were cloned from B16 melanoma (human-derived cells). Details are as follows.
  • human PILR a-Ig soluble human PILR
  • mouse PILR a extracellular region (amino acids 29 to 200) and human IgG-Fc region was prepared in the same manner, and soluble mouse PILR o; (hereinafter referred to as mouse PILR). o; —referred to as “I g”).
  • a cDNA library of B16 melanoma was prepared by the method described in the literature (Arase et al. J. Immunol. 167: 1141 200 1, Shiratori et al. J. Exp. Med 199: 525 2004). Specifically, mRNA was collected from B 16 melanoma cells, and cDNA was prepared using a cDNA synthesis kit (Invitrogen). By introducing the Sail-Notl fragment into the pMx-Sail retroviral library, a cDNA library with 1 X 10 6 clones was prepared. Then cD The cDNA library was introduced into Ba / F3 by introducing the NA library into packaging cells and mixing the culture supernatant with BaZF3 cells. PE / positive cells were collected by flow cytometry using Ba / F3 introduced with the cDNA library using PILR-Ig and PE-labeled anti-human IgG (Jackson laboratory).
  • the human cDNA showing high homology to this mouse cDNA clone contained an 831 bp ORF (SEQ ID NO: 7) encoding the 276 amino acid residues shown in SEQ ID NO: 1.
  • the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 is hereinafter referred to as hPILR-L2.
  • a retroviral vector pMx-PILR-L2 containing a polynucleotide (SEQ ID NO: 7) encoding PILR—L2 was prepared. That is, the ORF was inserted into the EcoRI-Notl site of pMX, a retroviral vector.
  • pMx—PILR—L2 was introduced into the packaging cells using Lipofectamine plus (Invitrogen). Three days after the introduction, the culture supernatant was collected. To this supernatant, 1Z100 amount of DOTAP (N- [l- (2,3-Dioleoyloxy)])- ⁇ , ⁇ , ⁇ -trimethylammonium plant methylsulfate) was mixed. By adding this mixed solution to the culture medium of target cells (BaZF3 cells), gene introduction into the target cells was performed. The obtained cells are referred to as BaZF3-L2.
  • DOTAP N- [l- (2,3-Dioleoyloxy)]
  • PILR a-Ig 50 ⁇ L of human PILR a-Ig was added to 2 X 10 5 transformed cells and left standing. After 30 minutes, the cells were washed twice with a HANKS 0.1% BSA solution, and a PE-labeled anti-human IgG Fc antibody (Jackson laboratory) was used as a secondary antibody and allowed to stand. After 30 minutes, the cells were washed twice with HANKS 0.1% BSA solution and analyzed by flow cytometry. The result is shown in Fig. 1 by a solid line.
  • CD200-Ig was used instead of human PILR a-Ig.
  • CD200—Ig is a chimeric molecule consisting of the extracellular region of the CD200 molecule and the human IgG—Fc region.
  • Figures 1-5 in the following description are histograms showing the results of flow cytometry.
  • the horizontal axis represents the fluorescence intensity
  • the vertical axis represents the number of cells at each fluorescence intensity
  • cell BaZF3-Mock was obtained in the same manner as in Example 1 except that an empty pMx betater was used instead of pMx-PILR-L2 and an empty pMx betater was used.
  • the BaZF3-Mock was analyzed by flow cytometry in the same manner as in Example 1. The results are shown in Figure 3.
  • the cells to be searched for the ligand were replaced with mouse B16 melanoma also with human 721.221 cells, and human PILR o; —Ig was used, and the same procedure as in (A-2) above was performed. ILR ligand was obtained.
  • This PILR ligand is designated CD23.
  • the amino acid sequence of CD23 is shown in SEQ ID NO: 2, and the nucleotide sequence is shown in SEQ ID NO: 8.
  • Example 1 BaZF3 cells were strongly recognized by PILR a-Ig by introducing the PILR-L2 gene.
  • FIG. 2 shows that BaZF3 cells were strongly recognized by human PILR a-Ig in Example 2 by introducing the CD23 gene.
  • PILR—L2 and CD23 can function as ligands for PILR. I understood.
  • human peripheral blood T cells were solubilized, and molecules associated with human PILR a-Ig were purified by immunoprecipitation. Specifically, human peripheral blood T cells were collected and made soluble by adding a solubilizer containing 1% Briji97. By adding Protein A Sepharose beads to which human PILR ⁇ -Ig was bound to this solubilized sample, the molecules associated with human PILR a-Ig were purified to obtain purified products.
  • nucleotide sequences were predicted.
  • the nucleotide sequences are shown in SEQ ID NOs: 9 and 11, respectively.
  • FIGS. 4 and 5 The analysis results of cells 293T-CD6 (Example 3) and 293T-CD45 (Example 4) are shown in FIGS. 4 and 5 respectively.
  • Human CD6 gene or mouse CD45 gene as shown in Figure 4 With the introduction of pups, 293T cells became strongly recognized by human and mouse PILR o;-Ig. In this case, the 293T cells were not recognized by PILR o; -Ig (data not shown), except that the vector introduced into the 293T cells was changed to the empty vector pMx.
  • PILRa recognizes human and mouse CD45.
  • human peripheral blood T cells and mouse spleen T cells that strongly express CD45 were solubilized.
  • immunoprecipitation with human PILR a-Ig or mouse PILR a-Ig was performed on the soluble yeast cell samples (human and mouse), respectively.
  • the precipitate obtained by this immunoprecipitation was electrophoresed on SDS-PAGE, followed by Western Blot with anti-mouse CD45 antibody and anti-human CD45 antibody (anti-CD45Ab).
  • anti-CD45Ab anti-human CD45 antibody
  • the precipitate was specifically recognized by the anti-CD45 antibody (FIG. 8).
  • the solubilized cell sample used is referred to as “human” or “mouse”
  • the control Ig used for immunoprecipitation is referred to as “C”
  • PILR a-Ig is referred to as “P”.
  • NKL cells which are human NK cell lines, using the same method as in (A-4) above.
  • the cell thus introduced with the gene is designated as NKL-PILR ⁇ .
  • PMx—PILR a is pMx Bam
  • PILR ⁇ 1 to 303 are inserted into the HI-Notl site.
  • NKL-Mock an empty pMx vector inserted with an insert was introduced into NKL cells in the same manner. This cell is called NKL-Mock.
  • pMx-PILR-L2 was introduced into ULBP-positive BaZF3 cells. This cell is designated as ULBP-BaZF3-L2.
  • ULBP is recognized by NKG2D, which is one of NK cell activation receptors, and activates NK cells. In other words, ULBP positive cells promote the activity of NK cells.
  • NKL-PIL Ra or NKL-Mock The cytotoxicity of NKL cells (NKL-PIL Ra or NKL-Mock) against ULBP-Ba / F3-L2 obtained in E-1> was analyzed by Cr51 free cytotoxicity assay.
  • Cr51 free cell injury assay a generally used method was used. Specifically, after labeling the target cells with Cr51, 1 x 10 4 target cells and various number of effector cells (NKL cells) shown in the figure were cultured for 4 hours, and 51 The amount of Cr was measured with a microbeta single scintillation counter.
  • Fig. 6 shows the results of the assembly using NKL-Mock
  • Fig. 7 shows the results of the assembly using NKL-PILR ⁇ .
  • Example 5 An empty pMx vector, which had been inserted, was used in the same manner as in Example 5 except that an empty pMx vector was used, and introduced into ULBP-positive BaZF3 cells. This cell is called ULBP-BaZF3-Mock.
  • the ULBP-BaZF3-Mock was subjected to 51 Cr free cell damage assay in the same manner as in Example 5. The results are shown in FIGS.
  • Fig. 6 ⁇ 7 the vertical axis indicates cytotoxicity, and the horizontal axis indicates the number of cells of Effector (NKL cells: NKL— ⁇ ILR a or NKL—Mock) ZTarget (BaZF3 cells: ULBP—BaZF3—L2 or ULBP—BaZF3) — Shows the ratio of the number of Mock cells. Cytotoxicity means the ratio of dead cells to all target cells.
  • cytotoxicity was remarkably suppressed by the combination of NKL—PILR a and ULBP—BaZF3—L2 (Example 5) (solid line in FIG. 7).
  • PILR-L2 has a function of suppressing the activity of NK cells by acting specifically on PILR.
  • the PILR ligand of the present invention can be used to control the activation of all immune cells that express PILR! [0175] ⁇ G.
  • mouse PILR gene was introduced into 293T cells using the pMx—IRES—GFP vector, and two days later, mouse CD45-Ig, mouse PILR-LI (CD99) —Ig, mouse PILR—L2 for the transfected cells (GFP positive cells) — Binding of Ig chimeric protein was analyzed by flow cytometry.
  • Mouse CD45 Ig
  • mouse PILR—LI (CD99) Ig
  • mouse PILR—L2 Ig chimeric proteins are mouse CD45 protein, mouse PILR—L1 (CD99) protein, mouse PILR—L2 protein A chimeric molecule (chimeric protein) with mouse IgG-Fc region.
  • mouse CD45-Ig The amino acid sequences of mouse CD45-Ig, mouse PILR-LI (CD99) -Ig, and mouse PILR-L2-Ig chimeric proteins are shown in SEQ ID NOs: 13 to 15, respectively.
  • the genes encoding mouse CD45-Ig, mouse PILR-L1 (CD99) -Ig, and mouse PILR-L2-Ig chimeric proteins are shown in SEQ ID NOs: 16-18, respectively.
  • Mouse CD45-Ig is a chimeric molecule consisting of a fragment from the 24th amino acid residue to the 377th amino acid residue of mouse CD45 protein and the mouse Ig-Fc region.
  • mouse PILR-LI (CD99) -Ig is composed of a fragment from the 26th amino acid residue to the 137th amino acid residue of mouse PILR-LI (CD99) protein and the mouse Ig-Fc region. It is a chimeric molecule.
  • PILR-L2-Ig is a chimeric molecule consisting of a fragment from the 28th amino acid residue to the 178th amino acid residue of mouse PILR-L2 protein and the mouse Ig-Fc region.
  • Advanced RPMI 1640 (Invitrogen) supplemented with 1% FCS was used as the culture solution. Introduce CD45—Ig, PILR-L1 (CD99) —Ig, PILR—L 2—Ig on the transfected cells for 30 minutes on ice, then mix with PE-labeled anti-human IgGAb and again on ice for 30 minutes. After standing, the fluorescence intensity was analyzed by flow cytometry.
  • CD45—Ig, PILR—LI—Ig, and PILR—L2—Ig specifically bind to PI LR gene-introduced cells. Therefore, it is considered that these Ig-chimeric molecules have inhibition of PILR function as ligands of suppressed PILR. Furthermore, since these Ig-chimeric molecules are thought to form dimers, PILR is cross-linked. It can be used to transmit an inhibitory signal. Regarding CD45 and PILR-L2, since human homologous molecules also bind to PILR, human CD45-Ig and human PILR-L2-Ig may be used as ligands for PILR as well.
  • the polynucleotide according to the present invention can be suitably used for a pharmaceutical agent for controlling an immune response, particularly a pharmaceutical agent for suppressing an immune response, such as a therapeutic agent for an autoimmune disease. Therefore, the structure including the polypeptide according to the present invention and the sugar chain added thereto has very promising industrial applicability.

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Abstract

The present inventors found that a polypeptide comprising an amino acid sequence shown in any one of SEQ ID NOS: 1 to 6 binds to PILRα and that the polypeptide activates an immune cell or suppresses the activation of the immune cell. The polypeptide can be preferably applied to a pharmaceutical or the like.

Description

PILR aと結合するポリペプチド、およびそれをコードするポリヌクレオチド 、並びにその利用  Polypeptide binding to PILR a, polynucleotide encoding the same, and use thereof
技術分野  Technical field
[0001] 本発明は、免疫応答の制御に関するものである。本発明は、特に、 PILR aに結合 することができるポリペプチドに関するものである。 PILR o;に結合するという本発明 のポリペプチドの特性は、抗原提示細胞である榭状細胞やマクロファージ等の活性 化を制御することに利用可能であるため、免疫応答抑制用医薬品および免疫応答 増強用医薬品等、免疫応答を制御する医薬品に好適に利用可能である。  [0001] The present invention relates to control of an immune response. The present invention particularly relates to polypeptides capable of binding to PILRa. The property of the polypeptide of the present invention that binds to PILR o; can be used to control the activation of antigen-presenting cells, such as rod-like cells and macrophages. It can be suitably used for pharmaceuticals that control immune responses, such as pharmaceuticals for medical use.
背景技術  Background art
[0002] 免疫細胞は、感染や腫瘍等に対する生体防御にお!、て重要な役割を担って 、る。  [0002] Immune cells play an important role in the defense of living bodies against infections and tumors.
その一方で、免疫細胞が異常に活性化されることで、自己免疫疾患やアレルギー疾 患が発症することが知られている。このため、免疫応答を適度に調節することが、生 体防御応答を制御する上で重要である。  On the other hand, it is known that autoimmune diseases and allergic diseases develop due to abnormal activation of immune cells. For this reason, moderate regulation of the immune response is important in controlling the biological defense response.
[0003] 免疫細胞にはその活性ィ匕を制御する分子として、活性ィ匕レセプターおよび抑制化 レセプター力 なるペア型レセプターが発現しており、これらペア型レセプターが免 疫応答の制御に重要な役割を担っていることが明らかになつてきた。これら活性ィ匕レ セプターおよび抑制化レセプターは、細胞外領域の相同性が非常に高いにもかかわ らず、相反する機能 (活性化'抑制ィ匕)を持っている。なお、本明細書中、免疫細胞と は、 T細胞、 B細胞、 NK細胞、榭状細胞、マクロファージ、マスト細胞、好塩基球、抗 酸球、または好中球等の、免疫応答に関与する細胞を含むものとする。  [0003] As a molecule that controls the activity of immune cells, active receptors and inhibitory receptor paired receptors are expressed, and these paired receptors play an important role in controlling immune responses. It has become clear that it is responsible. Although these active receptor and inhibitory receptor have very high homology in the extracellular region, they have contradictory functions (activation'inhibition receptor). In this specification, immune cells are involved in immune responses such as T cells, B cells, NK cells, rod cells, macrophages, mast cells, basophils, eosinophils, or neutrophils. Including cells.
[0004] 活性ィ匕レセプターは、そのリガンドを発現して 、る細胞を認識し、免疫細胞を活性 化する。免疫細胞を活性ィ匕するとは、免疫細胞力 のサイト力イン産生や細胞障害性 を誘起することを意味する。  [0004] An active receptor expresses its ligand, recognizes a cell, and activates an immune cell. To activate immune cells means to induce site-in production and cytotoxicity of immune cells.
[0005] 活性ィ匕レセプターは、細胞膜貫通領域に陽性荷電アミノ酸を有している。この陽性 荷電アミノ酸を介して、 DAP12、 CD3zまたは FcR yといった ITAM (Immunorecept or Tyrosine— based Activation Motief)酉 ti列 [Y— x— x— L— x(6〜8)— Y— x— x— x— L、xは任意の アミノ酸)]を有するアダプター分子と会合することで、活性ィ匕レセプターは活性ィ匕シグ ナルを伝達する。 [0005] The active receptor has a positively charged amino acid in the transmembrane region. Through this positively charged amino acid, ITAM (Immunorecept or Tyrosine—based Activation Motief) と い っ た ti column such as DAP12, CD3z or FcR y [Y— x— x— L— x (6-8) — Y— x— x— x— L and x are arbitrary In association with an adapter molecule having an amino acid)], the active receptor transmits an active signal.
[0006] 一方、抑制化レセプターは、細胞質内領域に ITIM (Immunoreceptor Tyrosine-bas ed Inhibitory Motief)配列 [(I/V/L/S)- x- Y- x- x- (L/V)(xは任意のアミノ酸)]を有し、 S HP— 1などのホスファターゼを動員することで、活性化レセプターからのシグナルを 遮断する。こうして、抑制化レセプターは免疫細胞の活性ィ匕を抑制する。  [0006] On the other hand, the inhibitory receptor has an ITIM (Immunoreceptor Tyrosine-bas ed Inhibitory Motief) sequence [(I / V / L / S)-x- Y- x- x- (L / V) ( x is an arbitrary amino acid)], and by mobilizing a phosphatase such as SHP-1, the signal from the activated receptor is blocked. Thus, the inhibitory receptor suppresses the activity of immune cells.
[0007] 免疫細胞に発現が認められるペア型レセプターとして、ヒトでは免疫グロブリン様構 造を持つ KIR (Killer cell Ig- like receptor)、マウスでは C型レクチン様構造を持つ Ly 49が挙げられる。抑制化レセプターの多くは MHCクラス Iを認識し、免疫細胞の自 己応答性を抑えると考えられている。一方、活性ィ匕型 Ly49、 KIRレセプターは自己 の MHCをほとんど認識せず、その存在の生理学的意味を含めリガンド認識機構は明 らかになつて ヽな 、ものが多 、。  [0007] Examples of paired receptors that are recognized in immune cells include KIR (Killer cell Ig-like receptor) having an immunoglobulin-like structure in humans and Ly 49 having a C-type lectin-like structure in mice. Many of the inhibitory receptors recognize MHC class I and are thought to suppress the self-responsiveness of immune cells. On the other hand, the active type Ly49, KIR receptor hardly recognizes its own MHC, and the ligand recognition mechanism including the physiological meaning of its existence is clear.
[0008] 上述したペア型レセプターの一つとして、 PILR (Paired Ig- like Type2 Receptor)が 知られている。 PILRは抑制型レセプターである PILR α (FDF03と称される場合もあ る）と、活性化型レセプターであるマウス PILR βと力もなるペア型レセプターである。 PILRも ΝΚ細胞に限らず、榭状細胞、マクロファージ、マスト細胞等の免疫細胞に幅 広く発現している  [0008] PILR (Paired Ig-like Type 2 Receptor) is known as one of the paired receptors described above. PILR is a pair-type receptor that also acts as an inhibitory receptor, PILR α (sometimes referred to as FDF03) and an activated receptor, mouse PILR β. PILR is also widely expressed in immune cells such as rod cells, macrophages, mast cells, etc.
PILR o;は、ヒトおよびマウスにおいてクローユングされている（非特許文献 1 · 2)。ま た、本発明者らは、非特許文献 3において、マウス PILR αと PILR |8のリガンドである マウス PILR— L (PILR Ligand)のクロー-ングを行っている。本発明者らは同文献に おいて、 PILR— Lと PILR |8との会合を介したシグナル力 NK細胞ゃ榭状細胞を活 性ィ匕することを明らかにしている。  PILR o; is clawed in humans and mice (Non-patent Documents 1 and 2). In addition, in the Non-patent Document 3, the present inventors have cloned mouse PILR-L (PILR Ligand) which is a ligand of mouse PILR α and PILR | 8. In the same document, the inventors of the present invention have revealed that NK cells and dendritic cells are activated through the association between PILR-L and PILR | 8.
〔非特許文献 1〕  [Non-Patent Document 1]
Mousseau D. D., et al, J. Biol. Chem. 275 (2000) p.4467- 4474  Mousseau D. D., et al, J. Biol. Chem. 275 (2000) p.4467-4474
〔非特許文献 2〕  [Non-Patent Document 2]
Fournier, N" et al., J. Immunol. 165 (2000) p.1197- 209.  Fournier, N "et al., J. Immunol. 165 (2000) p.1197-209.
〔非特許文献 3〕  [Non-Patent Document 3]
Shiratori, I., et al" J. Exp. Med. 199 (2004) p.525— 533 しかし、上記論文で同定したマウス PILR aおよび 13のリガンドと相同性の高いヒト 分子は存在せず、ヒト PILRのリガンドは不明であった。また、マウスにおいても上記 論文で同定したリガンドの非発現細胞の中に PILR— Igに認識される細胞が存在す ることから、本発明者らは、未知の PILRリガンドが存在すると考えた。 Shiratori, I., et al "J. Exp. Med. 199 (2004) p.525—533 However, there was no human molecule highly homologous to the mouse PILRa and 13 ligands identified in the above paper, and the ligand for human PILR was unknown. In addition, since there are cells recognized by PILR-Ig among the non-expressing cells of the ligand identified in the above paper in the mouse, the present inventors thought that an unknown PILR ligand was present.
発明の開示  Disclosure of the invention
[0009] 本発明は、上記従来の問題に鑑みてなされたものであり、その目的は、 PILR aと 結合するポリペプチド、およびそのポリペプチドをコードするポリヌクレオチド、並びに その利用を提供することにある。  [0009] The present invention has been made in view of the above-described conventional problems, and an object thereof is to provide a polypeptide that binds to PILR a, a polynucleotide encoding the polypeptide, and use thereof. is there.
[0010] 本発明者らは、上記課題を解決するために鋭意検討した結果、 PILR aと結合する ポリペプチドを独自に見出し、本発明を完成するに至った。本発明は上記新規な知 見に基づいて完成されたものであり、以下の発明を包含する。 [0010] As a result of intensive studies to solve the above-mentioned problems, the present inventors have found a polypeptide that binds to PILR a uniquely and completed the present invention. The present invention has been completed based on the above-mentioned novel knowledge and includes the following inventions.
[0011] ( 1)下記 (a)または (b)の 、ずれかに記載のポリペプチド、当該ポリペプチドをコー ドするポリヌクレオチド、または当該ポリペプチドを発現する細胞を含み、かつ免疫細 胞を活性化する、または免疫細胞の活性化を抑制する組成物。 [0011] (1) A polypeptide according to any one of the following (a) or (b), a polynucleotide encoding the polypeptide, or a cell expressing the polypeptide, and an immune cell: A composition that activates or suppresses activation of immune cells.
[0012] (a)配列番号 1〜6のいずれ力 1つに示されるアミノ酸配列からなるポリペプチド。 [0012] (a) A polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 1 to 6.
[0013] (b)配列番号 1〜6のいずれか 1つに示されるアミノ酸配列において、 1個または数 個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリべプチ ド、。 [0013] (b) a polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6 ,.
[0014] (2)下記 (e)または (f)の 、ずれかであるポリヌクレオチドを含み、かつ免疫細胞を 活性化する、または免疫細胞の活性化を抑制する組成物。  [0014] (2) A composition comprising the polynucleotide (e) or (f) below, which activates immune cells or suppresses activation of immune cells.
[0015] (e)配列番号 7〜12のいずれか 1つに示される塩基配列力もなるポリヌクレオチド。 [0015] (e) A polynucleotide having the nucleotide sequence ability represented by any one of SEQ ID NOs: 7 to 12.
[0016] (f)下記 (iii)または (iv)の 、ずれかであるポリヌクレオチドとストリンジェントな条件 下でノ、イブリダィズし、かつ PILR αと結合するポリペプチドをコードする塩基配列か らなるポリヌクレオチド。 [0016] (f) consisting of a base sequence encoding a polypeptide that binds to PILR α under the stringent conditions and a polynucleotide that is misaligned under the following (iii) or (iv) Polynucleotide.
[0017] (iii)配列番号 7〜12のいずれか 1つに示される塩基配列力もなるポリヌクレオチ ド、。  (Iii) A polynucleotide having a nucleotide sequence ability represented by any one of SEQ ID NOs: 7 to 12.
[0018] (iv)配列番号 7〜12の 、ずれか 1つに示される塩基配列と相補的な塩基配列 力もなるポリヌクレオチド。 [0019] (3)下記 (a)または (b)の 、ずれかのポリペプチドと IgG—Fc領域とのキメラ分子、 当該キメラ分子をコードするポリヌクレオチド、または当該キメラ分子を発現する細胞 を含むと共に、 PILR aと結合するポリペプチドを含有し、免疫細胞を活性化する、ま たは免疫細胞の活性ィ匕を抑制する組成物。 [0018] (iv) A polynucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 7 to 12. [0019] (3) including a chimeric molecule of any one of the following (a) or (b) and an IgG-Fc region, a polynucleotide encoding the chimeric molecule, or a cell expressing the chimeric molecule In addition, a composition containing a polypeptide that binds to PILRa, which activates immune cells or suppresses the activity of immune cells.
[0020] (a)配列番号 1〜6のいずれ力 1つに示されるアミノ酸配列からなるポリペプチド。 [0020] (a) A polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 1 to 6.
[0021] (b)配列番号 1〜6のいずれか 1つに示されるアミノ酸配列において、 1個または数 個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリべプチ ド、。 [0021] (b) A polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6 ,.
[0022] (4)下記 (g)または (h)の 、ずれかに記載のポリペプチド、当該ポリペプチドをコー ドするポリヌクレオチド、または当該ポリペプチドを発現する細胞を含み、かつ免疫細 胞を活性化する、または免疫細胞の活性化を抑制する組成物。  [0022] (4) A polypeptide according to any one of the following (g) or (h), a polynucleotide encoding the polypeptide, or a cell expressing the polypeptide, and an immune cell: A composition that activates or suppresses activation of immune cells.
[0023] (g)配列番号 13〜 15の!、ずれか 1つに示されるアミノ酸配列からなるポリペプチド  [0023] (g) a polypeptide consisting of the amino acid sequence of SEQ ID NOS: 13 to 15 and any one of them
[0024] (h)配列番号 13〜15のいずれ力 1つに示されるアミノ酸配列において、 1個または 数個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリぺプ チド。 [0024] (h) A polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence shown in any one of SEQ ID NOS: 13 to 15 .
[0025] (5)下記 (i)または (j)の 、ずれかであるポリヌクレオチドを含み、免疫細胞を活性化 する、または免疫細胞の活性ィ匕を抑制する組成物。  [0025] (5) A composition comprising the polynucleotide (i) or (j) below, which activates immune cells or suppresses the activity of immune cells.
[0026] (i)配列番号 16〜18のいずれ力 1つに示される塩基配列からなるポリヌクレオチド [0026] (i) a polynucleotide comprising the nucleotide sequence represented by any one of SEQ ID NOs: 16 to 18
[0027] (j)下記 (V)または (vi)の 、ずれかであるポリヌクレオチドとストリンジェントな条件下 でハイブリダィズし、かつ PILR o;と結合するポリペプチドをコードする塩基配列から なるポリヌクレオチド。 [0027] (j) A polynucleotide comprising a base sequence encoding a polypeptide that hybridizes under stringent conditions and binds to PILR o; with a polynucleotide of (V) or (vi) below: .
[0028] (V)配列番号 16〜18のいずれ力 1つに示される塩基配列からなるポリヌクレオ チド。  [0028] (V) A polynucleotide comprising the base sequence represented by any one of SEQ ID NOS: 16 to 18.
[0029] (vi)配列番号 16〜18のいずれか 1つに示される塩基配列と相補的な塩基配列 力もなるポリヌクレオチド。  [0029] (vi) A polynucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 16 to 18.
[0030] (6)下記（a)または (b)の 、ずれかであり、 PILR aと結合するポリペプチド。 [0031] (a)配列番号 1〜6のいずれ力 1つに示されるアミノ酸配列からなるポリペプチド。 [0030] (6) A polypeptide that binds to PILR a, which is any of the following (a) or (b). [0031] (a) A polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 6.
[0032] (b)配列番号 1〜6のいずれか 1つに示されるアミノ酸配列において、 1個または数 個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリべプチ ド、。 [0032] (b) A polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6 ,.
[0033] (7)配列番号 2〜4のいずれ力 1つに示されるアミノ酸配列からなるポリペプチド。  [0033] (7) A polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 2 to 4.
[0034] (8)上記（7)に記載のポリペプチドをコードする塩基配列力 なるポリヌクレオチド。 [0034] (8) A polynucleotide having a nucleotide sequence encoding the polypeptide of (7) above.
[0035] (9)下記（c)または（d)の!、ずれかであるポリヌクレオチド。 [0035] (9) A polynucleotide that is either! Or (d) of (c) or (d) below:
[0036] (c)配列番号 8〜10のいずれか 1つに示される塩基配列からなるポリヌクレオチド。  (C) A polynucleotide comprising the base sequence represented by any one of SEQ ID NOs: 8 to 10.
[0037] (d)下記 (i)または (ii)の 、ずれかであるポリヌクレオチドとストリンジェントな条件下 でハイブリダィズし、かつ PILR o;と結合するポリペプチドをコードする塩基配列から なるポリヌクレオチド。 [0037] (d) A polynucleotide comprising a base sequence encoding a polypeptide that hybridizes under stringent conditions with a non-regular polynucleotide of (i) or (ii) below and binds to PILRo; .
[0038] (i)配列番号 8〜 10のいずれ力 1つに示される塩基配列力 なるポリヌクレオチ ド、。  [0038] (i) A polynucleotide having the nucleotide sequence shown by any one of SEQ ID NOS: 8 to 10.
[0039] (ii)配列番号 8〜10の 、ずれか 1つに示される塩基配列と相補的な塩基配列か らなるポリヌクレオチド。  [0039] (ii) A polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 8 to 10.
[0040] (10)下記（g)または（h)の!、ずれかに記載のポリペプチドであって、 PILR aと結 合するポリペプチド。  [0040] (10) A polypeptide according to (1) or (2) below, which binds to PILR a.
[0041] (g)配列番号 13〜 15の!、ずれか 1つに示されるアミノ酸配列からなるポリペプチド  [0041] (g) a polypeptide comprising an amino acid sequence represented by any one of!
[0042] (h)配列番号 13〜15のいずれ力 1つに示されるアミノ酸配列において、 1個または 数個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリぺプ チド。 [0042] (h) A polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence shown in any one of SEQ ID NOS: 13 to 15 .
[0043] (11)下記 (i)または (j)の 、ずれかであるポリヌクレオチド。  [0043] (11) A polynucleotide which is any of the following (i) or (j):
[0044] (i)配列番号 16〜18のいずれ力 1つに示される塩基配列からなるポリヌクレオチド  [0044] (i) a polynucleotide comprising the nucleotide sequence represented by any one of SEQ ID NOs: 16 to 18
[0045] (j)下記 (V)または (vi)の 、ずれかであるポリヌクレオチドとストリンジェントな条件下 でハイブリダィズし、かつ PILR o;と結合するポリペプチドをコードする塩基配列から なるポリヌクレオチド。 [0046] (v)配列番号 16〜18のいずれ力 1つに示される塩基配列からなるポリヌクレオ チド。 [0045] (j) A polynucleotide comprising a base sequence encoding a polypeptide that hybridizes under stringent conditions and binds to PILRo; with a polynucleotide of (V) or (vi) below: . [0046] (v) A polynucleotide comprising the nucleotide sequence represented by any one of SEQ ID NOs: 16 to 18.
[0047] (vi)配列番号 16〜18の 、ずれか 1つに示される塩基配列と相補的な塩基配列 力もなるポリヌクレオチド。  [0047] (vi) A polynucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 16 to 18.
[0048] (12)上記（8) , (9)または（11)に記載のポリヌクレオチドを含むベクター。 [0048] (12) A vector comprising the polynucleotide according to (8), (9) or (11) above.
[0049] (13)上記（8) , (9)または（11)に記載のポリヌクレオチドが発現可能に導入されて なる細胞。 [0049] (13) A cell into which the polynucleotide according to (8), (9) or (11) is introduced so that it can be expressed.
[0050] (14)上記（8) , (9)または（11)に記載のポリヌクレオチドを用いて PILR o;に結合 するタンパク質を生産する方法。  [0050] (14) A method for producing a protein that binds to PILR o; using the polynucleotide described in (8), (9) or (11) above.
[0051] 上述のように、本願発明に係るポリペプチドは、 PILR o;に結合することができる。そ れゆえ、上記ポリペプチドは、例えば免疫細胞を活性化する、または活性ィ匕を抑制 することによって、免疫応答を調整する医薬品等に好適に利用することができる。 [0051] As described above, the polypeptide of the present invention can bind to PILR o ;. Therefore, the above-mentioned polypeptide can be suitably used for pharmaceuticals that modulate immune responses by, for example, activating immune cells or suppressing activity.
[0052] また、本発明には上記ポリペプチドをコードするポリヌクレオチドも含まれる。当該ポ リヌクレオチドは、上記ポリペプチドの生産に利用可能であるだけでなぐ上記ポリべ プチドと同様に免疫応答を抑制する医薬品等に利用可能である。 [0052] The present invention also includes a polynucleotide encoding the above polypeptide. The polynucleotide can be used not only for the production of the polypeptide, but also for a pharmaceutical or the like that suppresses the immune response, as with the polypeptide.
[0053] 本発明のさらに他の目的、特徴、および優れた点は、以下に示す記載によって十 分わ力るであろう。また、本発明の利益は、添付図面を参照した次の説明で明白にな るであろう。 [0053] Other objects, features, and advantages of the present invention will be sufficiently enhanced by the following description. The benefits of the present invention will become apparent from the following description with reference to the accompanying drawings.
図面の簡単な説明  Brief Description of Drawings
[0054] [図 1]本発明の実施例 1に係る Ba/F3— L2を、フローサイトメトリーによって解析した 結果を示すヒストグラムである。  [0054] FIG. 1 is a histogram showing the results of analysis of Ba / F3-L2 according to Example 1 of the present invention by flow cytometry.
[図 2]本発明の実施例 2に係る BaZF3— CD23を、フローサイトメトリーによって解析 した結果を示すヒストグラムである。  FIG. 2 is a histogram showing the results of analyzing BaZF3-CD23 according to Example 2 of the present invention by flow cytometry.
[図 3]比較例 1に係る BaZF3 - Mockを、フローサイトメトリ一によって解析した結果 を示すヒストグラムである。  FIG. 3 is a histogram showing the results of analyzing BaZF3-Mock according to Comparative Example 1 by flow cytometry.
[図 4]本発明の実施例 3に係る 293T— CD6を、フローサイトメトリーによって解析した 結果を示すヒストグラムである。  FIG. 4 is a histogram showing the results of analyzing 293T-CD6 according to Example 3 of the present invention by flow cytometry.
[図 5]本発明の実施例 4に係る 293T— CD45を、フローサイトメトリーによって解析し た結果を示すヒストグラムである。 [Fig. 5] 293T-CD45 according to Example 4 of the present invention was analyzed by flow cytometry. It is a histogram which shows the result.
[図 6]本発明の実施例 5に係る ULBP— Ba/F3— L2、および、比較例 2に係る LBP - Ba/F3 - Mockにつ!/、ての、 NKL - Mockを用 、た Cr51遊離細胞障害アツセ ィの結果を示すグラフである。  [Fig. 6] ULBP-Ba / F3-L2 according to Example 5 of the present invention and LBP-Ba / F3-Mock according to Comparative Example 2! /, Using NKL-Mock and Cr51 3 is a graph showing the results of a free cell injury assay.
[図 7]本発明の実施例 5に係る ULBP— Ba/F3— L2、および、比較例 2に係る LBP - Ba/F3 - Mockにつ!/、ての、 NKL - PILRaを用 、た Cr51遊離細胞障害アツセ ィの結果を示すグラフである。  [Fig. 7] ULBP-Ba / F3-L2 according to Example 5 of the present invention and LBP-Ba / F3-Mock according to Comparative Example 2! /, Using NKL-PILRa and Cr51 3 is a graph showing the results of a free cell injury assay.
[図 8]可溶化細胞試料（ヒト、マウス）中の、 PILR aとの共沈物を抗マウス CD45抗体 (anti-CD45Ab)による Western Blotに供した結果を示す図面である。  FIG. 8 shows the results of subjecting a coprecipitate with PILRa in a solubilized cell sample (human, mouse) to Western Blot using an anti-mouse CD45 antibody (anti-CD45Ab).
[図 9]マウス PILRを発現する細胞に対するマウス CD45— Ig、マウス PILR— LI (CD 99)— Ig、マウス PILR— L2— Igキメラタンパク質の結合をフローサイトメトリーで解析 した結果を示す図である。  FIG. 9 shows the results of flow cytometry analysis of the binding of mouse CD45—Ig, mouse PILR—LI (CD 99) —Ig, and mouse PILR—L2—Ig chimeric proteins to cells expressing mouse PILR. .
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0055] 本発明は、 PILR aのリガンドであるポリペプチド、そのポリペプチドをコードするポリ ヌクレオチド、およびその利用に関するものである。そこで、以下ではまず、本発明に 係るポリペプチド、次いでそのポリペプチドをコードするポリヌクレオチド、最後にその 利用方法等について説明する。 [0055] The present invention relates to a polypeptide that is a ligand of PILR a, a polynucleotide encoding the polypeptide, and use thereof. Therefore, in the following, the polypeptide according to the present invention, then the polynucleotide encoding the polypeptide, and finally the method of using it will be described.
[0056] < 1.ポリペプチド > [0056] <1. Polypeptide>
本発明は、配列番号 1〜4のいずれ力 1つに示されるアミノ酸配列からなるポリぺプ チドを含む。また、本発明は、下記 (a)または (b)のいずれかであり、 PILR aと結合 するポリペプチドを含む。  The present invention includes a polypeptide consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4. In addition, the present invention includes any of the following (a) and (b), and a polypeptide that binds to PILRa.
[0057] (a)配列番号 1〜6のいずれ力 1つに示されるアミノ酸配列からなるポリペプチド。 [0057] (a) A polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 6.
[0058] (b)配列番号 1〜6のいずれか 1つに示されるアミノ酸配列において、 1個または 数個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリぺプ チド。 [0058] (b) In the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6, a polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added .
[0059] 下記実施例で述べるように、本発明者らは、配列番号 1〜4のいずれ力 1つに示さ れるアミノ酸配列からなるポリペプチドを新たに同定した。また、配列番号 1〜6のい ずれ力 1つに示されるアミノ酸配列力 なるポリペプチド力 PILR aと結合することを 見出した。 [0059] As described in the following Examples, the present inventors newly identified a polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4. In addition, it binds to the polypeptide power PILR a which is the amino acid sequence power shown by any one of SEQ ID NOs: 1 to 6. I found it.
[0060] 配列番号 1には hPILR— L2と称するポリペプチドのアミノ酸配列を示し、配列番号 2にはヒト CD23と称するポリペプチドのアミノ酸配列を示し、配列番号 3にはヒト CD6 と称するポリペプチドのアミノ酸配列を示し、配列番号 4にはマウス CD6と称するポリ ペプチドのアミノ酸配列を示す。また、配列番号 5にはヒト CD45と称するポリペプチド のアミノ酸配列を示し、配列番号 6にはマウス CD45と称するポリペプチドのアミノ酸 配列を示す。なお、本明細書中、用語ポリペプチドはタンパク質と同義で用いる場合 もめる。  [0060] SEQ ID NO: 1 shows the amino acid sequence of a polypeptide called hPILR-L2, SEQ ID NO: 2 shows the amino acid sequence of a polypeptide called human CD23, and SEQ ID NO: 3 shows the amino acid sequence of a polypeptide called human CD6. The amino acid sequence is shown. SEQ ID NO: 4 shows the amino acid sequence of a polypeptide called mouse CD6. SEQ ID NO: 5 shows the amino acid sequence of a polypeptide called human CD45, and SEQ ID NO: 6 shows the amino acid sequence of a polypeptide called mouse CD45. In the present specification, the term polypeptide may be used synonymously with protein.
[0061] すなわち、本発明者らは、配列番号 1〜6のいずれか 1つに示されるアミノ酸配列か らなるポリペプチド力 PILR o;のリガンドとして機能することを見出した。上記 PILR aは、ヒト由来またはマウス由来であることが好ましいが、ヒト以外の哺乳類等、他の 生物に由来し、免疫細胞の機能制御を有する分子であってもよい。勿論、本発明に は、配列番号 1〜6のいずれか 1つに示されるアミノ酸配列力 なるポリペプチドの変 異体であって、 PILR o;と結合するポリペプチドも含まれることを付言しておく。  [0061] That is, the present inventors have found that the polypeptide functions as a ligand of the polypeptide force PILR o; consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6. The PILR a is preferably derived from a human or a mouse, but may be a molecule derived from another organism such as a mammal other than a human and having a function control of immune cells. Of course, it should be noted that the present invention includes a polypeptide that binds to PILR o; which is a variant of the polypeptide having the amino acid sequence ability shown in any one of SEQ ID NOs: 1 to 6. .
[0062] また、本発明に係るポリペプチドは、ヒトまたはマウス由来であることが好ましいが、ヒ ト以外の哺乳類等、他の生物に由来し、免疫細胞の機能制御を有する分子のリガン ド、とちなり得る。  [0062] The polypeptide according to the present invention is preferably derived from a human or a mouse, but is a ligand of a molecule derived from another organism such as a mammal other than human and having a function control of immune cells, It can be different.
[0063] 本発明に係るポリペプチドは、天然の細胞より精製された産物、化学合成の産物、 および原核生物宿主または真核生物宿主 (例えば、細菌細胞、酵母細胞、高等植物 細胞、昆虫細胞、および哺乳動物細胞を含む)から組換え技術によって産生された 産物を含む。組換え産生手順において用いられる宿主に依存して、本発明に係るポ リペプチドは、グリコシル化、または非グリコシルイ匕され得る。さら〖こ、本発明に係るポ リペプチドはまた、宿主媒介プロセスの結果として、開始の改変メチォニン残基を含 み得る。  [0063] The polypeptide according to the present invention is a product purified from a natural cell, a product of chemical synthesis, and a prokaryotic or eukaryotic host (eg, bacterial cell, yeast cell, higher plant cell, insect cell, And products produced by recombinant techniques from mammalian cells). Depending on the host used in the recombinant production procedure, the polypeptides according to the invention may be glycosylated or non-glycosylated. Furthermore, the polypeptides according to the present invention may also contain an initiating modified methionine residue as a result of a host-mediated process.
[0064] 本明細書でいう「ポリペプチドの変異体」とは、「配列番号 1〜6のいずれ力 1つに示 されるアミノ酸配列において、 1個または数個のアミノ酸が置換、欠失、挿入、または 付加されたアミノ酸配列力もなるポリペプチド」を指す。  [0064] The term "polypeptide variant" as used herein refers to "a substitution or deletion of one or several amino acids in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 6," It refers to a polypeptide that also has an amino acid sequence ability that is inserted or added.
[0065] 上記「1個もしくは数個のアミノ酸が置換、欠失、挿入、もしくは付加された」とは、部 位特異的突然変異誘発法等の公知の変異ポリペプチド作製法により置換、欠失、挿 入、もしくは付加できる程度の数 (好ましくは 10個以下、より好ましくは 7個以下、最も 好ましくは 5個以下）のアミノ酸が置換、欠失、挿入もしくは付加されていることを意味 する。このような変異ポリペプチドは、上述した公知の変異ポリペプチド作製法により 人為的に導入された変異を有するポリペプチドに限定されるものではなぐ天然に存 在するポリペプチドを単離精製したものであってもよい。 [0065] The above "one or several amino acids are substituted, deleted, inserted or added" means part A number that can be substituted, deleted, inserted, or added by known mutant polypeptide production methods such as site-specific mutagenesis (preferably 10 or less, more preferably 7 or less, most preferably 5) The following amino acids are substituted, deleted, inserted or added. Such a mutant polypeptide is not limited to a polypeptide having a mutation artificially introduced by the above-mentioned known mutant polypeptide production method, but is a product obtained by isolating and purifying a naturally occurring polypeptide. There may be.
[0066] 上記変異体としては、欠失、挿入、逆転、反復、およびタイプ置換 (例えば、親水性 残基につ 1、て他の残基への置換を含む。ただし通常は高親水性の残基を逆の性質 を有する高疎水性の残基には置換しない。）を含む変異体が挙げられる。特に、ポリ ペプチドにおける「中性」アミノ酸置換は、一般的にそのポリペプチドの活性にほとん ど影響しな 、ため、他のアミノ酸残基への置換されて 、てもよ 、。  [0066] Such variants include deletions, insertions, inversions, repeats, and type substitutions (eg, one hydrophilic residue, substitution with another residue, but usually with high hydrophilicity). The residue is not substituted with a highly hydrophobic residue having the opposite property.). In particular, “neutral” amino acid substitutions in a polypeptide generally have little effect on the activity of the polypeptide, and therefore may be substituted with other amino acid residues.
[0067] ポリペプチド中のいくつかのアミノ酸力 このポリペプチドの構造または機能に有意 に影響することなく容易に改変され得ることは、当該分野において周知である。さらに 、人為的に改変させるだけではぐ天然のタンパク質において、当該タンパク質の構 造または機能を有意に変化させない変異体が存在することもまた周知である。  [0067] Some amino acid forces in a polypeptide are well known in the art that can be readily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is also well known that there are mutants in natural proteins that do not significantly change the structure or function of natural proteins that need to be artificially modified.
[0068] 当業者は、これら周知技術を使用してポリペプチドのアミノ酸配列において 1または 数個のアミノ酸を容易に変異させることができる。例えば、公知の点変異導入法に従 えば、ポリペプチドをコードするポリヌクレオチドの任意の塩基を変異させることができ る。また、ポリペプチドをコードするポリヌクレオチドの任意の部位に対応するプライマ 一を設計して欠失変異体または付加変異体を作製することができる。さらに、本明細 書中に記載される方法を用いれば、作製した変異体が所望の活性を有するか否かを 容易に決定し得る。  [0068] One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a polypeptide using these well-known techniques. For example, according to a known point mutation introduction method, an arbitrary base of a polynucleotide encoding a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide. Furthermore, if the method described in this specification is used, it can be easily determined whether the produced mutant has a desired activity.
[0069] 好ましい変異体としては、保存性もしくは非保存性アミノ酸置換、欠失、または付カロ を有するものを挙げることができる。より好ましくは、サイレント置換、付加、および欠 失であり、さらに好ましくは、保存性置換である。  [0069] Preferred mutants include those having conservative or non-conservative amino acid substitutions, deletions, or added calories. More preferred are silent substitutions, additions and deletions, and even more preferred are conservative substitutions.
[0070] また、本発明に係るポリペプチドは、後述する本発明に係るポリヌクレオチド (本発 明に係るポリペプチドをコードする遺伝子）を宿主細胞に導入して、そのポリペプチド を細胞内発現させた状態であってもよいし、細胞、組織などから単離精製されたもの であってもよい。また、本発明に係るポリペプチドは、化学合成されたものであっても よい。 [0070] Further, the polypeptide according to the present invention introduces a polynucleotide according to the present invention (a gene encoding the polypeptide according to the present invention) described later into a host cell, and allows the polypeptide to be expressed in the cell. Or isolated and purified from cells, tissues, etc. It may be. The polypeptide according to the present invention may be chemically synthesized.
[0071] 他の実施形態において、本発明に係るポリペプチドは、融合タンパク質のような改 変された形態で組換え発現され得る。すなわち、上述した本発明に係るポリペプチド の末端、特に N末端側にタグ等のアミノ酸配列が付加されてなるポリペプチドも、本 発明に含まれる。  [0071] In other embodiments, the polypeptides of the present invention may be recombinantly expressed in a modified form such as a fusion protein. That is, a polypeptide in which an amino acid sequence such as a tag is added to the end of the polypeptide according to the present invention, particularly the N-terminal side, is also included in the present invention.
[0072] また、糖鎖修飾がタンパク質、特に細胞表面に発現するタンパク質の機能発現に 重要な役割を果たすことが知られており、本発明のポリペプチドとしては、上述したポ リペプチドが糖鎖修飾されたものも含まれる。  [0072] It is also known that glycosylation plays an important role in the functional expression of proteins, particularly proteins expressed on the cell surface. As the polypeptide of the present invention, the above-mentioned polypeptides are glycosylated. Included.
[0073] < 2.ポリヌクレオチド >  [0073] <2. Polynucleotide>
本発明に係るポリヌクレオチドとしては、まず、配列番号 1〜4のいずれか 1つに示さ れるアミノ酸配列からなるポリペプチドをコードする塩基配列からなるポリヌクレオチド が挙げられる。当業者は、本明細書の記載、例えば配列番号 1〜4の記載を参酌す ることにより、上記ポリヌクレオチドを容易に作製することができる。  Examples of the polynucleotide according to the present invention include a polynucleotide comprising a base sequence encoding a polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4. A person skilled in the art can easily prepare the polynucleotide by referring to the description of the present specification, for example, the descriptions of SEQ ID NOs: 1 to 4.
[0074] 上記ポリヌクレオチドは、 RNA (例えば、 mRNA)の形態、または DNAの形態（例 えば、 cDNAまたはゲノム DNA)で存在し得る。 DNAは、二本鎖または一本鎖であ り得る。一本鎖 DNAまたは RNAは、コード鎖（センス鎖としても知られる）であり得る 力 またはそれは、非コード鎖 (アンチセンス鎖としても知られる）であり得る。  [0074] The polynucleotide may be present in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand).
[0075] また、本発明には、下記の（c)または (d)の 、ずれかであるポリヌクレオチドも含まれ る。  [0075] Further, the present invention also includes a polynucleotide which is a deviation from the following (c) or (d).
[0076] (c)配列番号 7〜10のいずれか 1つに示される塩基配列からなるポリヌクレオチド。  [0076] (c) A polynucleotide comprising the base sequence represented by any one of SEQ ID NOs: 7 to 10.
[0077] (d)以下の（i)または（ii)とストリンジェントな条件下でハイブリダィズし、かつ PILR aと結合するポリペプチドをコードする塩基配列力 なるポリヌクレオチド： [0077] (d) A polynucleotide having a nucleotide sequence that hybridizes with the following (i) or (ii) under stringent conditions and encodes a polypeptide that binds to PILR a:
(i)配列番号 7〜 10の 、ずれか 1つに示される塩基配列力もなるポリヌクレオチ ド、。  (i) a polynucleotide having the nucleotide sequence ability shown in any one of SEQ ID NOs: 7 to 10;
[0078] (ii)配列番号 7〜10の 、ずれか 1つに示される塩基配列と相補的な塩基配列か らなるポリヌクレオチド。  [0078] (ii) A polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 7 to 10.
[0079] なお、配列番号 7には hPILR— L2と称するポリペプチドをコードするポリヌクレオチ ドを示し、配列番号 8にはヒト CD23と称するポリペプチドをコードするポリヌクレオチド を示し、配列番号 9にはヒト CD6と称するポリペプチドをコードするポリヌクレオチドを 示し、配列番号 10にはマウス CD6と称するポリペプチドをコードするポリヌクレオチド を示す。また、配列番号 11にはヒト CD45と称するポリペプチドをコードするポリヌクレ ォチドを示し、配列番号 12にはマウス CD45と称するポリペプチドをコードするポリヌ クレオチドを示す。 [0079] SEQ ID NO: 7 includes a polynucleotide encoding a polypeptide called hPILR-L2. SEQ ID NO: 8 shows a polynucleotide encoding a polypeptide called human CD23, SEQ ID NO: 9 shows a polynucleotide encoding a polypeptide called human CD6, and SEQ ID NO: 10 shows mouse CD6. The polynucleotide encoding the polypeptide referred to. SEQ ID NO: 11 shows a polynucleotide encoding a polypeptide called human CD45, and SEQ ID NO: 12 shows a polynucleotide encoding a polypeptide called mouse CD45.
[0080] 上記「ストリンジェントな条件」とは、少なくとも 90%以上の同一性、好ましくは少なく とも 95%以上の同一性、最も好ましくは 97%の同一性が配列間に存在するときにの みハイブリダィゼーシヨンが起こることを意味する。  [0080] The above "stringent conditions" are only when there is at least 90% identity, preferably at least 95% identity, most preferably 97% identity between sequences. This means that hybridization occurs.
[0081] 上記ハイブリダィゼーシヨンは、 Sambrookら、 Molecular Cloning, A Laborat ory Manual, 2d Ed. , Cold Spring Harbor Laboratory (1989)に記載さ れている方法のような周知の方法で行うことができる。通常、温度が高いほど、塩濃 度が低いほどストリンジエンシーは高くなり（ハイブリダィズし難くなる）、より相同なポリ ヌクレオチドを取得することができる。ノ、イブリダィゼーシヨンの条件としては、従来公 知の条件を好適に用いることができ、特に限定しないが、例えば、 42°C、 6 X SSPE 、 50%ホルムアミド、 1%SDS、 100 /z gZml サケ精子 DNA、 5 Xデンハルト液（た だし、 1 X SSPE ;0. 18M 塩ィ匕ナトリウム、 10mMリン酸ナトリウム、 pH7. 7、 ImM EDTA。 5 Xデンハルト液； 0. 1% 牛血清アルブミン、 0. 1% フイコール、 0. 1% ポリビュルピロリドン）が挙げられる。  [0081] The above hybridization can be performed by a known method such as the method described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989). it can. Generally, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained. As the conditions for the hybridization, conventionally known conditions can be suitably used, and are not particularly limited. For example, 42 ° C, 6 X SSPE, 50% formamide, 1% SDS, 100 / z gZml Salmon Sperm DNA, 5 X Denhardt's solution (However, 1 X SSPE; 0.18 M sodium chloride, 10 mM sodium phosphate, pH 7.7, ImM EDTA. 5 X Denhardt's solution; 0.1% bovine serum albumin 0.1% Ficoll, 0.1% polybulurpyrrolidone).
[0082] また、上記 (d)に係るポリヌクレオチドとしては、その長さ等も特に限定されるもので はなぐ例えば配列番号 7〜： L0に示される塩基配列のフラグメントであってもよい。  [0082] The polynucleotide according to (d) above is not particularly limited in length or the like, and may be, for example, a fragment of the base sequence shown in SEQ ID NO: 7 to L0.
[0083] 上記フラグメントは、少なくとも 12nt (ヌクレオチド）、好ましくは約 15nt、そしてより 好ましくは少なくとも約 20nt、なおより好ましくは少なくとも約 30nt、そしてさらにより 好ましくは少なくとも約 40ntの長さのフラグメントを指す。本明細書を参照すれば配 列番号 7〜10に示される塩基配列が提供されるので、当業者は，配列番号 7〜10に 基づく DNAフラグメントを容易に作製することができる。例えば、制限エンドヌクレア一 ゼ切断または超音波による剪断は、種々のサイズのフラグメントを作製するために容 易に使用され得る。あるいは、このようなフラグメントをィ匕学的に合成することもできる [0084] また、上記 (d)に係るポリヌクレオチドとしては、上記 (c)のポリヌクレオチドにおいて 、 1または数個の塩基が欠失、置換、または付加した変異体が挙げられる。変異体は 、コードもしくは非コード領域、またはその両方において変異され得る。コード領域に おける変異は、保存的もしくは非保存的なアミノ酸欠失、置換、または付加を生成し 得る。 [0083] The fragment refers to a fragment of at least 12 nt (nucleotide), preferably about 15 nt, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably at least about 40 nt in length. By referring to the present specification, the nucleotide sequences shown in SEQ ID NOs: 7 to 10 are provided, so that those skilled in the art can easily prepare DNA fragments based on SEQ ID NOs: 7 to 10. For example, restriction endonuclease cutting or ultrasonic shearing can be easily used to make fragments of various sizes. Alternatively, such fragments can also be synthesized ihologically. [0084] In addition, examples of the polynucleotide according to (d) above include variants in which one or several bases are deleted, substituted, or added in the polynucleotide of (c) above. Variants can be mutated in coding or non-coding regions, or both. Mutations in the coding region can generate conservative or non-conservative amino acid deletions, substitutions or additions.
[0085] また、上述した本発明に係るポリヌクレオチドは全て、その 5 '側または 3 '側で上述 のタグ標識 (タグ配列またはマーカー配列）をコードするポリヌクレオチドに融合され 得る。  [0085] In addition, all the polynucleotides according to the present invention described above can be fused to the polynucleotide encoding the tag tag (tag sequence or marker sequence) described above on the 5 'side or 3' side.
[0086] < 3.ベクター >  [0086] <3. Vector>
本発明には、上記 < 2 >欄で述べた本発明に係るポリヌクレオチドを含むベクター が含まれる。すなわち、本発明に係るベクターとしては、配列番号 1〜4のいずれか 1 つに示されるポリペプチドをコードする塩基配列からなるポリヌクレオチド、または、上 記 (c)または (d)の 、ずれかであるポリヌクレオチドが挿入された組換え発現ベクター などが挙げられる。  The present invention includes a vector comprising the polynucleotide according to the present invention described in the section <2> above. That is, the vector according to the present invention includes a polynucleotide comprising the base sequence encoding the polypeptide represented by any one of SEQ ID NOs: 1 to 4, or the above-mentioned (c) or (d). And a recombinant expression vector into which a polynucleotide is inserted.
[0087] 組換え発現ベクターの作製方法としては、プラスミド、ファージ、またはコスミドなどを 用いる方法が挙げられるが特に限定されな 、。  [0087] Examples of a method for producing a recombinant expression vector include, but are not limited to, methods using a plasmid, phage, cosmid, or the like.
[0088] ベクターの具体的な種類は特に限定されず、宿主細胞中で発現可能なベクターを 適宜選択すればよい。すなわち、宿主細胞の種類に応じて、確実に本発明に係るポ リヌクレオチドを発現させるために適宜プロモーター配列を選択し、これと本発明に係 るポリヌクレオチドを各種プラスミド等に組み込んだベクターを発現ベクターとして用 いればよい。  [0088] The specific type of vector is not particularly limited, and a vector that can be expressed in a host cell may be appropriately selected. That is, according to the type of host cell, an appropriate promoter sequence is selected to ensure expression of the polynucleotide according to the present invention, and a vector in which this and the polynucleotide according to the present invention are incorporated into various plasmids is expressed. It can be used as a vector.
[0089] 発現ベクターは、少なくとも 1つの選択マーカーを含んでもよい。このようなマーカー としては、真核生物細胞培養につ 、てはジヒドロ葉酸レダクターゼまたはネオマイシ ン耐性、および E. coliおよび他の細菌における培養についてはテトラサイクリン耐性 遺伝子またはアンピシリン耐性遺伝子等の薬剤耐性遺伝子が挙げられる。勿論、こ れらの選択マーカーに限られず、本出願当時の技術水準において当業者が使用可 能な従来公知の選択マーカーを用いることができることは 、うまでもな 、。 [0090] 上記選択マーカーを用いることにより、本発明に係るポリヌクレオチドが宿主細胞に 導入されたカゝ否カゝ、さらには宿主細胞中で確実に発現しているカゝ否かを確認すること ができる。 [0089] The expression vector may comprise at least one selectable marker. Such markers include drug resistance genes such as tetracycline resistance gene or ampicillin resistance gene for eukaryotic cell culture, for dihydrofolate reductase or neomycin resistance, and for culture in E. coli and other bacteria. Can be mentioned. Of course, it is not limited to these selection markers, and it goes without saying that conventionally known selection markers that can be used by those skilled in the art in the state of the art at the time of the present application can be used. [0090] By using the above selectable marker, it is confirmed whether or not the polynucleotide according to the present invention has been introduced into the host cell, and whether or not it is reliably expressed in the host cell. Can do.
[0091] また、本発明に係るポリペプチドを融合ポリペプチドとして発現させてもよぐ例えば 、ォワンクラゲ由来の緑色蛍光ポリペプチド GFP (Green Fluorescent Protein)をマー カーとして用い、本発明に係るポリペプチドを GFP融合ポリペプチドとして発現させ てもよい。  [0091] The polypeptide according to the present invention may be expressed as a fusion polypeptide. For example, a green fluorescent polypeptide GFP (Green Fluorescent Protein) derived from jellyfish is used as a marker, and the polypeptide according to the present invention is used as a marker. It may be expressed as a GFP fusion polypeptide.
[0092] 上記の宿主細胞は、特に限定されるものではなぐ従来公知の各種細胞を好適に 用いることができる。具体的には、例えば、大腸菌（Escherichia coli)等の細菌、酵母 (出 酵母 Saccharomyces cerevisiae、分裂酵母 Schizosaccharomyces pombe)、線 虫（Caenorhabditis elegans)、アフリカッメガエル（Xenopus laevis)の卵母細胞、哺乳 類由来の細胞等を挙げることができるが、特に限定されるものではない。上記の宿主 細胞のための適切な培養培地および条件は当分野で周知である。  [0092] The host cell is not particularly limited, and various conventionally known cells can be preferably used. Specifically, for example, bacteria such as Escherichia coli, yeasts (development yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, mammals Examples of the cells can be mentioned, but are not particularly limited. Suitable culture media and conditions for the above host cells are well known in the art.
[0093] 上記発現ベクターを宿主細胞に導入する方法、すなわち形質転換法も特に限定さ れるものではなぐ電気穿孔法、リン酸カルシウム法、リボソーム法、 DEAEデキストラ ン法、レトロウイルス法等の従来公知の方法を好適に用いることができる。  [0093] Methods for introducing the above expression vectors into host cells, that is, transformation methods are not particularly limited. Conventionally known methods such as electroporation, calcium phosphate method, ribosome method, DEAE dextran method, retrovirus method, etc. Can be suitably used.
[0094] このように、本発明に係るベクターは、少なくとも、本発明に係るポリペプチドをコー ドするポリヌクレオチドを含めばよいといえる。すなわち、発現ベクター以外のベクタ 一も、本発明の技術的範囲に含まれる点に留意すべきである。  [0094] Thus, it can be said that the vector according to the present invention should contain at least a polynucleotide encoding the polypeptide according to the present invention. That is, it should be noted that a vector other than the expression vector is also included in the technical scope of the present invention.
[0095] つまり、本発明の目的は、本発明に係るポリペプチドをコードするポリヌクレオチドを 含有するベクターを提供することにあるのであって、本明細書中に具体的に記載した 個々のベクター種および細胞種、ならびにベクター作製方法および細胞導入方法に 存するのではない。したがって、上記以外のベクター種およびベクター作製方法を用 いて取得したベクターも本発明の技術的範囲に属することに留意しなければならな い。  [0095] That is, an object of the present invention is to provide a vector containing a polynucleotide encoding the polypeptide of the present invention, and the individual vector species specifically described in the present specification. And cell types, and vector production methods and cell introduction methods. Therefore, it should be noted that vectors obtained using vector species and vector production methods other than those described above also belong to the technical scope of the present invention.
[0096] < 4.細胞 >  [0096] <4.Cell>
本発明には、上記 < 2 >欄に記載のポリヌクレオチドが発現可能に導入されてなる 細胞も含まれる。この細胞は、上記 < 3 >欄にて上述した発現ベクター力 同じく上 述した宿主細胞に導入されてなる細胞であると換言することもできる。ベクターおよび 宿主細胞としては既に述べた通りである。 The present invention also includes cells into which the polynucleotide described in the above <2> column has been introduced so that it can be expressed. This cell has the same expression vector power as described above in <3>. In other words, the cells are introduced into the host cells described above. The vectors and host cells are as described above.
[0097] < 5.ポリペプチドの生産方法 >  [0097] <5. Polypeptide production method>
本発明には、上記 < 2 >欄に記載のポリヌクレオチドを用いて、 PILR o;に結合する タンパク質を生産する方法も含まれる。このポリペプチドの生産方法として、上記 < 2 >欄に記載のポリヌクレオチドを用 、る組換え発現系を採用することができる。  The present invention also includes a method for producing a protein that binds to PILR o; using the polynucleotide described in the section <2>. As a method for producing this polypeptide, a recombinant expression system using the polynucleotide described in the section <2> above can be employed.
[0098] 組換え発現系を用いる場合、ポリペプチドの生産方法は、上記 < 4 >欄に記載の 細胞から、上記ポリペプチドを精製する方法であると換言することもできる。つまり、上 記ポリペプチドの生産方法は、上記 < 3 >欄に記載のベクターを宿主細胞に導入す る工程を含んでもよい。ベクターおよび宿主細胞についての詳細は、上記 < 3 >欄で 述べた通りである。  [0098] When a recombinant expression system is used, it can be said that the method for producing a polypeptide is a method for purifying the polypeptide from the cells described in the section <4>. That is, the polypeptide production method may include a step of introducing the vector described in the above <3> column into a host cell. Details of the vector and the host cell are as described in the section <3> above.
[0099] 本発明に係るポリペプチドの生産方法は、上記ポリペプチドを含む細胞または組織 の抽出液力も上記ポリペプチドを精製する工程をさらに包含することが好ましい。ポリ ペプチドを精製する工程は、周知の方法 (例えば、細胞または組織を破壊した後に 遠心分離して可溶性画分を回収する方法)で細胞や組織から細胞抽出液を調製し た後、この細胞抽出液力 周知の方法 (例えば、硫安沈殿またはエタノール沈殿、酸 抽出、免疫沈降、陰イオンまたは陽イオン交換クロマトグラフィー、ホスホセルロースク 口マトグラフィー、疎水性相互作用クロマトグラフィー、ァフィユティークロマトグラフィ 一、ヒドロキシアパタイトクロマトグラフィー、およびレクチンクロマトグラフィー）によって 精製する工程が好ましいが、これらに限定されない。高速液体クロマトグラフィー（「H PLC」）が精製のために好適に用いられる。宿主細胞内で産生されたポリペプチドを 精製する方法は、用いた宿主、ポリペプチドの性質によって異なる力 タグの利用等 によって比較的容易に目的のポリペプチドを精製することも可能である。  [0099] Preferably, the method for producing a polypeptide according to the present invention further includes the step of purifying the polypeptide with the extractive force of cells or tissues containing the polypeptide. The step of purifying the polypeptide is to prepare a cell extract from the cells or tissues by a well-known method (for example, to centrifuge the cells or tissues and then collect the soluble fraction by centrifugation). Hydrodynamics Well-known methods (e.g. ammonium sulfate precipitation or ethanol precipitation, acid extraction, immunoprecipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxy A step of purification by apatite chromatography and lectin chromatography) is preferred, but is not limited thereto. High performance liquid chromatography (“H PLC”) is preferably used for purification. As a method of purifying a polypeptide produced in a host cell, it is possible to purify the target polypeptide relatively easily by using a force tag that varies depending on the host used and the nature of the polypeptide.
[0100] 本発明に係る生産方法は、 PILR aへの結合活性を有するポリペプチドを提供する ことを目的にしているのであって、上述した種々の工程以外の工程を包含する生産 方法も、本発明に含まれることは言うまでもない。  [0100] The production method according to the present invention aims to provide a polypeptide having a binding activity to PILRa, and the production method including steps other than the various steps described above is also applicable to this production method. It goes without saying that it is included in the invention.
[0101] なお、上記く 1 >欄のポリペプチドは、組換え発現系を利用したこのような生産方 法に限定されるものではなぐ天然に発現する細胞または糸且織から当該ポリペプチド を精製することによって生産されるものであってもよい。精製方法としては、上述した 種々の技術を利用することができる。また、本発明に係るポリペプチドは、化学合成 によって生産されるものであってもよい。当業者は、本明細書中に記載される本発明 に係るポリペプチドのアミノ酸配列に基づいて周知の化学合成技術を適用すれば、 本発明に係るポリペプチドをィ匕学合成できることを、容易に理解する。すなわち、本 発明に係るポリペプチドは、天然に存在する変異ポリペプチドであっても、人為的に 作製された変異ポリペプチドであってもよ 、。 [0101] It should be noted that the polypeptide in column <1> above is not limited to such a production method using a recombinant expression system, and is not limited to such a polypeptide. It may be produced by refining. As the purification method, the various techniques described above can be used. The polypeptide according to the present invention may be produced by chemical synthesis. Those skilled in the art can easily understand that the polypeptide according to the present invention can be chemically synthesized by applying a well-known chemical synthesis technique based on the amino acid sequence of the polypeptide according to the present invention described in the present specification. to understand. That is, the polypeptide according to the present invention may be a naturally occurring mutant polypeptide or an artificially prepared mutant polypeptide.
[0102] く 6.本発明に係る利用 >  [0102] 6. Use according to the present invention>
本発明には、上記 (a)または (b)のいずれかであり、 PILR o;と結合するポリべプチ ド、当該ポリペプチドをコードするポリヌクレオチド、または当該ポリペプチドを発現す る細胞を含むと共に、免疫細胞を活性化する、または活性化を抑制する組成物も含 まれる。  The present invention includes any of the above-mentioned (a) and (b), a polypeptide that binds to PILRo; a polynucleotide that encodes the polypeptide; or a cell that expresses the polypeptide. Also included are compositions that activate or suppress activation of immune cells.
[0103] 上記ポリペプチドは、免疫細胞の抑制化レセプターである PILR aに結合すること ができるという特性を有する。それゆえ、この特性を利用することによって、本発明に 係る組成物は、免疫細胞を活性ィ匕または活性ィ匕を抑制することができる。  [0103] The polypeptide has the property of being able to bind to PILRa, which is a suppressor receptor of immune cells. Therefore, by utilizing this property, the composition according to the present invention can suppress the activity or activity of immune cells.
[0104] なお、ここで「免疫細胞を活性化する」とは、その機構等を限定されるものではなぐ 結果として免疫細胞が活性ィ匕されればよい。活性ィ匕する機構としては、上記ポリぺプ チドが、 PILR aまたは免疫細胞の活性ィ匕を制御する他のレセプターに結合すること によって、このレセプターを介して直接的に免疫細胞を活性ィ匕するものであってもよ い。また、活性ィ匕の機構としては他に、上記ポリペプチドが、レセプターを介して免疫 細胞の活性ィ匕を抑制したり不活性ィ匕したりする他のリガンドによる免疫細胞の不活性 化を阻害し、間接的に免疫細胞を活性化させるものであってもよい。  [0104] Here, the phrase "activate immune cells" does not limit the mechanism or the like, and as long as immune cells are activated. As a mechanism for activation, the above-mentioned polypeptide binds to PILRa or other receptor that controls the activity of immune cells, thereby directly activating immune cells via this receptor. It may be what you do. In addition, as a mechanism of activity, the above-mentioned polypeptide inhibits immune cell inactivation by other ligands that suppress or inactivate immune cells via receptors. Alternatively, it may indirectly activate immune cells.
[0105] また、「免疫細胞の活性ィ匕を抑制する」とは、同じくその機構等を限定されるもので はなぐ結果として免疫細胞の活性ィ匕が抑制されればよい。活性化抑制の機構として は、免疫細胞のレセプターに結合することで、レセプターを介したシグナルによって 直接免疫細胞の活性ィ匕を抑制するものであってもよい。また、免疫細胞を活性化さ せるリガンドーレセプターの相互作用を阻害することによって、間接的に免疫細胞の 活性ィ匕を抑制するものであってもよ 、。 [0106] このように、上記ポリペプチドは、ァゴ-ストまたはアンタゴ-ストとして機能すること によって、免疫細胞を活性化するか、または活性ィ匕を抑制するものであることが好ま しい。 Further, “suppressing the activity of immune cells” is not limited to the mechanism and the like, and as long as the activity of immune cells is suppressed. As a mechanism for suppressing activation, it may be that by binding to a receptor of an immune cell, the activity of the immune cell is directly suppressed by a signal via the receptor. Further, the activity of the immune cell may be indirectly suppressed by inhibiting the ligand-receptor interaction that activates the immune cell. [0106] Thus, the polypeptide preferably activates immune cells or suppresses activity by functioning as an agonist or an antagonist.
[0107] なお、「免疫細胞の活性化」とは、上述したように、免疫細胞からのサイト力イン産生 や細胞障害性が誘起されることを意味する。また、「免疫細胞の活性ィ匕の抑制」とは、 免疫細胞を不活性ィ匕することであるとも、免疫細胞が活性化される条件下にお ヽても 活性ィ匕が起こらな 、ようにすることであるとも言 、換えることができる。  It should be noted that “activation of immune cells” means that, as described above, production of cytodynamic force from immune cells and cytotoxicity are induced. In addition, “suppression of immune cell activity” means that immune cells are inactivated, or that activity does not occur even under conditions where immune cells are activated. It can also be said that it is.
[0108] なお、上記組成物の活性ィ匕または活性ィ匕の抑制の対象となる免疫細胞は、その由 来となる生物種等、特に限定されるものではなぐ上記ポリペプチドによって、活性化 される力、活性ィ匕が抑制される免疫細胞であれば、ヒト、またはその他の哺乳類由来 であってもよい。また、当該組成物の対象となる器官、組織等も特に限定されるもの ではない。  [0108] It should be noted that the immune cells that are the target of the activity or suppression of the activity of the composition are activated by the polypeptide that is not particularly limited, such as the species of the organism. As long as it is an immune cell whose ability and activity are suppressed, it may be derived from humans or other mammals. In addition, the organs, tissues, and the like targeted by the composition are not particularly limited.
[0109] また、上記組成物は、免疫細胞の活性を調節することによって、免疫応答を増強ま たは抑制することができる。これは、個々の免疫細胞の活性ィ匕および活性ィ匕の抑制 力 それぞれ、免疫応答の増強および抑制のどちらの方向にも作用し得るためであ る。例えば、免疫応答を抑制する機能を有する免疫細胞 (抑制化サイト力インを産生 する細胞等）について、該免疫細胞の活性化を上記組成物によって抑制すると、免 疫応答全体は増強される。逆に、免疫応答を活性化する機能を有する免疫細胞に ついて、該免疫細胞の活性化を上記組成物によって抑制すると、免疫応答全体は抑 制される。  [0109] Furthermore, the composition can enhance or suppress the immune response by regulating the activity of immune cells. This is because the activity of individual immune cells and the ability to suppress activity can act in either direction of enhancing or suppressing the immune response, respectively. For example, when immune cells having a function of suppressing an immune response (such as cells that produce a suppressive cyto force-in) are inhibited by the above composition, the entire immune response is enhanced. Conversely, when immune cells having a function of activating an immune response are inhibited by the above composition, the entire immune response is suppressed.
[0110] また、上記組成物において、上述した以外の構成、すなわちポリペプチド、ポリヌク レオチド、および細胞の含有量、形状、製造方法等は、特に限定されない。  [0110] In the above composition, the constitution other than those described above, that is, the content, shape, production method, and the like of the polypeptide, the polynucleotide, and the cell are not particularly limited.
[0111] なお、上記組成物中に含まれる細胞は、組換え体であってもよく、組換え体でな ヽ 細胞、すなわち外来遺伝子が導入されていない細胞であってもよい。また、組成物中 に含まれるポリヌクレオチドは、ベクターに挿入された状態で含まれていてもよい。ベ クタ一としては、上記 < 3 >欄で述べた通り、好適な従来公知のベクターを利用する ことができる。また、本発明の組成物において、これらポリペプチド、ポリヌクレオチド、 および細胞は、単独でも、適宜組み合わせて用いてもよい。 [0112] 上記糸且成物は、その精製物を生体に投与することで、生体内の免疫細胞の活性ィ匕 が抑制され、免疫反応を抑制することができる。すなわち、上記組成物は医薬品とし て利用可能である。上記組成物は、その免疫細胞を活性ィ匕する力 または活性ィ匕を 抑制する効果を発揮することができれば、投与方法、他の組成、対象となる生物等は 特に限定されな 、。投与方法としては注射等の非経口投与が好まし 、。 [0111] The cells contained in the composition may be recombinant or non-recombinant cells, that is, cells into which no foreign gene has been introduced. Moreover, the polynucleotide contained in the composition may be contained in a state of being inserted into a vector. As the vector, a suitable conventionally known vector can be used as described in the section <3> above. In the composition of the present invention, these polypeptides, polynucleotides, and cells may be used alone or in appropriate combination. [0112] By administering the purified product to the living body, the activity of immune cells in the living body can be suppressed and the immune reaction can be suppressed. That is, the composition can be used as a pharmaceutical product. The administration method, other composition, target organism, and the like are not particularly limited as long as the composition can exert the ability to activate the immune cells or the effect of suppressing the activity. As an administration method, parenteral administration such as injection is preferred.
[0113] 上記ポリペプチドを含む組成物を、例えば、生体の或る箇所に注射等によって投与 すると、組成物中のポリペプチドが、当該箇所で免疫細胞の PILR aに会合するので 、 目的の箇所で免疫細胞の機能を制御することができる。また、本発明に係るポリヌク レオチドを含む組成物を生体内に投与すると、組成物中のポリヌクレオチドが生体内 で発現し、本発明に係るポリペプチドを生成するので、上記ポリペプチドを含む組成 物と同様の効果を奏する。このとき、組成物中のポリヌクレオチドは、細胞内で発現可 能であることが好ましい。また、上記ポリペプチドを発現する細胞を投与しても、同様 の効果を得られる。  [0113] When a composition containing the above-mentioned polypeptide is administered to a part of a living body by injection or the like, for example, the polypeptide in the composition is associated with PILRa of immune cells at that part. Can control the function of immune cells. In addition, when a composition containing the polynucleotide according to the present invention is administered in vivo, the polynucleotide in the composition is expressed in vivo to produce the polypeptide according to the present invention. Has the same effect as. At this time, the polynucleotide in the composition is preferably capable of being expressed in cells. In addition, the same effect can be obtained by administering cells that express the polypeptide.
[0114] 以上のように、本発明に係る組成物は、免疫細胞の機能を制御することによって、 免疫応答を制御する医薬品として利用され、特に、榭状細胞、マクロファージ、マスト 細胞の活性ィ匕を抑制する医薬品として好適に利用される。  [0114] As described above, the composition according to the present invention is used as a pharmaceutical agent for controlling the immune response by controlling the function of immune cells, and particularly the activity of rod cells, macrophages and mast cells. It is suitably used as a pharmaceutical product that suppresses
[0115] また、本発明の糸且成物に含まれるポリヌクレオチドは、下記 (e)または (f)のいずれ かであってもよい。  [0115] Further, the polynucleotide contained in the yarn composition of the present invention may be either (e) or (f) below.
[0116] (e)配列番号 7〜12のいずれか 1つに示される塩基配列力もなるポリヌクレオチド  [0116] (e) a polynucleotide having a nucleotide sequence ability represented by any one of SEQ ID NOs: 7 to 12
[0117] (f)下記（iii)または（iv)とストリンジェントな条件下でハイブリダィズし、かつ PILR αと結合するポリペプチドをコードする塩基配列力 なるポリヌクレオチド。 [0117] (f) A polynucleotide having a nucleotide sequence that hybridizes with the following (iii) or (iv) under stringent conditions and encodes a polypeptide that binds to PILRα.
[0118] (iii)配列番号 7〜12のいずれか 1つに示される塩基配列力もなるポリヌクレオチ ド、。  [0118] (iii) a polynucleotide having the nucleotide sequence ability shown in any one of SEQ ID NOS: 7 to 12,
[0119] (iv)配列番号 7〜12のいずれか 1つに示される塩基配列と相補的な塩基配列 力もなるポリヌクレオチド。  [0119] (iv) A polynucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 7 to 12.
[0120] 上記ポリヌクレオチドを含む組成物とは、当該ポリヌクレオチドを単に生体内に投与 することのみならず、当該ポリヌクレオチドを生体内に投与することにより、当該ポリヌ クレオチドがコードするポリペプチドを発現させ、当該ポリペプチドの機能によって、 免疫細胞を活性化する、または活性ィ匕を抑制することをも意図して ヽる。 [0120] The composition containing the above-mentioned polynucleotide refers not only to administration of the polynucleotide in vivo but also to administration of the polynucleotide in vivo. It is also intended to express a polypeptide encoded by the nucleotide and to activate immune cells or suppress activity depending on the function of the polypeptide.
[0121] また、上記ポリペプチドと他のタンパク質との融合タンパク質 (キメラタンパク質)も、 本発明に含まれ、さらに上記組成物の有効成分として用いることができる。  [0121] A fusion protein (chimeric protein) of the above polypeptide and another protein is also included in the present invention and can be used as an active ingredient of the above composition.
[0122] 上記キメラタンパク質としては、例えば、上記ポリペプチドと IgG—Fc領域とのキメラ 分子を挙げることができる。より具体的には、例えば、マウス CD45— Ig、マウス PILR -LI (CD99)— Ig、マウス PILR— L2— Ig、ヒト CD45— Ig、ヒト PILR— L2— Ig等を 挙げることができる。  [0122] Examples of the chimeric protein include chimeric molecules of the polypeptide and IgG-Fc region. More specifically, for example, mouse CD45-Ig, mouse PILR-LI (CD99) -Ig, mouse PILR-L2-Ig, human CD45-Ig, human PILR-L2-Ig and the like can be mentioned.
[0123] これらの Ig—キメラ分子は、後述する実施例 6に示すように、抑制化 PILRのリガンド として PILRの機能阻害および PILRをクロスリンクし抑制化シグナルを伝達するのに 禾 IJ用することがでさる。  [0123] These Ig-chimeric molecules should be used for IJ to inhibit PILR function and to cross-link PILR and transmit inhibitory signals, as shown in Example 6 below. It is out.
[0124] なお、上記 Ig—Fc領域は、ヒト由来であってもよいし、マウス由来であってもよいが 、その他の哺乳動物由来であってもよぐ特に限定されるものではない。望ましくは、 組成物の適用対象と同一生物由来の Ig— Fc領域を用いることがより好ましい。  [0124] The Ig-Fc region may be derived from a human or a mouse, but may be derived from other mammals and is not particularly limited. Desirably, an Ig-Fc region derived from the same organism as the application target of the composition is more preferably used.
[0125] 上記 Ig-Fc領域を有するキメラ分子は、その構造からダイマー（二量体)を形成して いると考えられる。このため、 PILRをクロスリンクして抑制化シグナルあるいは活性ィ匕 シグナルを伝達すると考えられる。この機能は、 Ig-Fc領域を有するキメラ分子に特 有のものであり、リガンド単体では見られない、優れた効果といえる。  [0125] The chimeric molecule having the Ig-Fc region is considered to form a dimer (dimer) from its structure. For this reason, it is considered that PILR is cross-linked to transmit an inhibitory signal or an activity signal. This function is unique to a chimeric molecule having an Ig-Fc region, and can be said to be an excellent effect not seen with a single ligand.
[0126] それゆえ、本発明には、上記（a)または（b)のいずれかのポリペプチドと IgG— Fc 領域とのキメラ分子、当該キメラ分子をコードするポリヌクレオチド、または当該キメラ 分子を発現する細胞を含むと共に、 PILR αと結合するポリペプチドを含有し、免疫 細胞を活性化する、または免疫細胞の活性化を抑制する組成物も含まれる。  [0126] Therefore, the present invention expresses a chimeric molecule of the polypeptide (a) or (b) above and an IgG-Fc region, a polynucleotide encoding the chimeric molecule, or the chimeric molecule. And a composition that contains a polypeptide that binds PILRα and activates immune cells or suppresses activation of immune cells.
[0127] 上記キメラ分子として、例えば、配列番号 13〜15のいずれかに記載のポリペプチド を挙げることができる。具体的には、下記 (g)または (h)のいずれかに記載のポリぺプ チド、当該ポリペプチドをコードするポリヌクレオチド、または当該ポリペプチドを発現 する細胞を含み、かつ免疫細胞を活性ィ匕する、または免疫細胞の活性ィ匕を抑制する 組成物。  [0127] Examples of the chimeric molecule include the polypeptides described in any one of SEQ ID NOs: 13 to 15. Specifically, it comprises a polypeptide according to any of the following (g) or (h), a polynucleotide encoding the polypeptide, or a cell that expresses the polypeptide, and activates immune cells. A composition that suppresses or suppresses the activity of immune cells.
[0128] (g)配列番号 13〜15のいずれ力 1つに示されるアミノ酸配列からなるポリペプチド [0129] (h)配列番号 13〜15のいずれか 1つに示されるアミノ酸配列において、 1個または 数個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリぺプ チド。 [0128] (g) a polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOS: 13 to 15 [0129] (h) A polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence shown in any one of SEQ ID NOS: 13 to 15 .
[0130] また、下記 (i)または (j)の 、ずれかであるポリヌクレオチドを含み、免疫細胞を活性 化する、または免疫細胞の活性化を抑制する組成物も本発明に含まれる。  [0130] Further, the present invention also includes a composition comprising the polynucleotide (i) or (j) below, which activates immune cells or suppresses activation of immune cells.
[0131] (i)配列番号 16〜18のいずれ力 1つに示される塩基配列からなるポリヌクレオチド [0131] (i) a polynucleotide comprising the nucleotide sequence represented by any one of SEQ ID NOs: 16 to 18
[0132] (j)下記 (V)または (vi)の 、ずれかであるポリヌクレオチドとストリンジェントな条件下 でハイブリダィズし、かつ PILR o;と結合するポリペプチドをコードする塩基配列から なるポリヌクレオチド。 [0132] (j) A polynucleotide comprising a nucleotide sequence that encodes a polypeptide that hybridizes under stringent conditions with a polynucleotide that is not shown in (V) or (vi) and binds to PILRo; .
[0133] (V)配列番号 16〜18のいずれ力 1つに示される塩基配列からなるポリヌクレオ チド。  [0133] (V) A polynucleotide comprising the nucleotide sequence represented by any one of SEQ ID NOS: 16 to 18.
[0134] (vi)配列番号 16〜18のいずれか 1つに示される塩基配列と相補的な塩基配列 力もなるポリヌクレオチド。  [0134] (vi) a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOS: 16 to 18.
[0135] また、本発明には、上記 (a)または (b)のポリペプチドと、あるいはこれらの部分ポリ ペプチドと、 IgG— Fc領域とを用いて、 PILR- aと結合する融合タンパク質を作製 する方法も含まれる。本方法によれば、ダイマー（二量体)を形成し、 PILRをクロスリ ンクして抑制化シグナルあるいは活性ィ匕シグナルを伝達する融合タンパク質を生産 することができる。  [0135] Further, in the present invention, a fusion protein that binds to PILR-a is produced using the above-mentioned polypeptide (a) or (b), or a partial polypeptide thereof, and an IgG-Fc region. The method of doing is also included. According to this method, a fusion protein that forms a dimer (dimer) and crosslinks PILR to transmit an inhibitory signal or an activity signal can be produced.
[0136] また、本発明の組成物中に含まれる細胞としては、上記く 4 >欄に記載の細胞を用 いることができる。細胞としては他に、上記 (e)、（f)、（i)および (j)のいずれかである ポリヌクレオチドが発現可能に導入されてなる細胞を用いることができる。  [0136] Further, as the cells contained in the composition of the present invention, the cells described in the above item 4> can be used. In addition, as the cell, a cell into which the polynucleotide of any one of the above (e), (f), (i) and (j) has been introduced so as to be expressed can be used.
[0137] 以下、実施例を示し、本発明の実施の形態についてさらに詳しく説明する。もちろ ん、本発明は以下の実施例に限定されるものではなぐ細部については様々な態様 が可能であることはいうまでもない。さらに、本発明は上述した実施形態に限定される ものではなぐ請求項に示した範囲で種々の変更が可能であり、それぞれ開示された 技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲 に含まれる。 [0137] Hereinafter, examples will be shown and the embodiments of the present invention will be described in more detail. Needless to say, the present invention is not limited to the following examples, and it is needless to say that various modes are possible for details. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims, and the present invention also relates to embodiments obtained by appropriately combining the respective technical means disclosed. Technical scope of include.
〔実施例〕  〔Example〕
以下、実施例により、本発明をさらに詳細に説明するが、本発明はこれに限定され るものではない。  Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.
[0138] なお、以下の説明において特に言及しない操作は、従来公知の技術を用いた。ま た、試薬および機器は、添付の取扱説明書に沿って使用した。また、以下の実施例 で用いた細胞は、特に言及しない限り、 10%の FCSを含む RPMI1640培地で培養 した。  [0138] It should be noted that conventionally known techniques were used for operations not specifically mentioned in the following description. Reagents and equipment were used in accordance with the attached instruction manual. The cells used in the following examples were cultured in RPMI1640 medium containing 10% FCS unless otherwise specified.
[0139] <A.実施例 1 : B16メラノーマにおける PILR aリガンド（PILR— L2)のクローニン グおよび解析 >  [0139] <A. Example 1: Cloning and analysis of PILR a ligand (PILR—L2) in B16 melanoma>
B16メラノーマ（ヒト由来の細胞）より、 PILR—Igによって認識される分子をクロー- ングした。詳細は以下の通りである。  Molecules recognized by PILR-Ig were cloned from B16 melanoma (human-derived cells). Details are as follows.
[0140] <A- 1.可溶型ヒト PILR a (ヒト PILR a—Ig)および可溶型マウス PILR α (マウス[0140] <A- 1. Soluble human PILR a (human PILR a—Ig) and soluble mouse PILR α (mouse
PILR a— Ig)の作製 > Preparation of PILR a—Ig)>
非特許文献 1に記載のヒト PILR o;の細胞外領域 (アミノ酸 20番〜 196番）と、ヒト I gG—Fc領域とのキメラ分子の発現プラスミドを作製し、 COS7細胞に導入した。 3日 後、培養上清を採取し、この上清を可溶型のヒト PILRひ（以下、ヒト PILR a— Igと称 する）として以下の操作に用いた。  An expression plasmid for a chimeric molecule of the human PILR o; extracellular region (amino acids 20 to 196) described in Non-Patent Document 1 and a human IgG-Fc region was prepared and introduced into COS7 cells. Three days later, the culture supernatant was collected and used as the soluble human PILR (hereinafter referred to as human PILR a-Ig) for the following operations.
[0141] また、マウス PILR aの細胞外領域（アミノ酸 29番〜 200番）とヒト IgG— Fc領域と のキメラ分子を同様にして作製し、可溶型のマウス PILR o; (以下、マウス PILR o;— I gと称する）として以下の操作に用いた。 [0141] A chimeric molecule of mouse PILR a extracellular region (amino acids 29 to 200) and human IgG-Fc region was prepared in the same manner, and soluble mouse PILR o; (hereinafter referred to as mouse PILR). o; —referred to as “I g”).
[0142] <A- 2. cDNAライブラリーの作製および PILR aによって認識されるクローンの 探索 > [0142] <A- 2. Creation of cDNA library and search for clones recognized by PILR a>
B16メラノーマの cDNAライブラリーを文献（Arase et al. J. Immunol. 167:1141 200 1, Shiratori et al. J. Exp. Med 199:525 2004)に記載した方法で作成した。具体的に は B 16メラノーマ細胞より mRNAを採取し、 cDNAを cDNA合成キット (Invitrogen)を 用いて作製した。その Sail— Notl断片を pMx— Sailレトロウイルスライブラリーに導 入することにより 1 X 106のクローン力 なる cDNAライブラリーを作製した。次に、 cD NAライブラリーをパッケージング細胞に遺伝子導入し、その培養上精を BaZF3細 胞と混合することにより cDNAライブラリーを Ba/F3に導入した。 cDNAライブラリー を導入した Ba/F3を PILR— Igと PE標識抗ヒト IgG (Jackson laboratory社）を用いて 、 PE陽性細胞をフローサイトメトリーで採取した。 A cDNA library of B16 melanoma was prepared by the method described in the literature (Arase et al. J. Immunol. 167: 1141 200 1, Shiratori et al. J. Exp. Med 199: 525 2004). Specifically, mRNA was collected from B 16 melanoma cells, and cDNA was prepared using a cDNA synthesis kit (Invitrogen). By introducing the Sail-Notl fragment into the pMx-Sail retroviral library, a cDNA library with 1 X 10 6 clones was prepared. Then cD The cDNA library was introduced into Ba / F3 by introducing the NA library into packaging cells and mixing the culture supernatant with BaZF3 cells. PE / positive cells were collected by flow cytometry using Ba / F3 introduced with the cDNA library using PILR-Ig and PE-labeled anti-human IgG (Jackson laboratory).
[0143] その結果、マウス PILR a—Igによって認識される一つのクローンが得られた。この マウス cDNAクローンに高い相同性を示すヒト cDNAは、配列番号 1に示される 276 アミノ酸残基をコードする、 831bpの ORF (配列番号 7)を含んでいた。配列番号 1に 示されるアミノ酸配列を有するポリペプチドを以下、 hPILR— L2と称する。  [0143] As a result, one clone recognized by mouse PILR a-Ig was obtained. The human cDNA showing high homology to this mouse cDNA clone contained an 831 bp ORF (SEQ ID NO: 7) encoding the 276 amino acid residues shown in SEQ ID NO: 1. The polypeptide having the amino acid sequence shown in SEQ ID NO: 1 is hereinafter referred to as hPILR-L2.
[0144] <A- 3.ベクターの構築 >  [0144] <A- 3. Vector construction>
PILR— L2をコードするポリヌクレオチド（配列番号 7)を含むレトロウイルスベクター pMx- PILR- L2を作製した。すなわち、上記 ORFを、レトロウイルスベクターである pM Xの EcoRI— Notl部位に挿入した。  A retroviral vector pMx-PILR-L2 containing a polynucleotide (SEQ ID NO: 7) encoding PILR—L2 was prepared. That is, the ORF was inserted into the EcoRI-Notl site of pMX, a retroviral vector.
[0145] <A-4.細胞へのベクターの導入 >  [0145] <A-4. Introduction of vector into cells>
pMx— PILR— L2を、パッケージング細胞に、 Lipofectamine plus (Invitrogen社製） を用いて遺伝子導入した。導入してから 3日後に、培養液の上清を採取した。この上 清に 1Z100量の DOTAP (N-[l- (2,3- Dioleoyloxy)]) -Ν,Ν,Ν- trimethylammonium p ropane methylsulfate)を混ぜた。この混合液を標的細胞（BaZF3細胞）の培養液に 加えることによって、標的細胞への遺伝子導入を行った。得られた細胞を BaZF3— L2とする。  pMx—PILR—L2 was introduced into the packaging cells using Lipofectamine plus (Invitrogen). Three days after the introduction, the culture supernatant was collected. To this supernatant, 1Z100 amount of DOTAP (N- [l- (2,3-Dioleoyloxy)])-Ν, Ν, Ν-trimethylammonium plant methylsulfate) was mixed. By adding this mixed solution to the culture medium of target cells (BaZF3 cells), gene introduction into the target cells was performed. The obtained cells are referred to as BaZF3-L2.
[0146] <A— 5. PILR αリガンド発現細胞の解析 >  [0146] <A— 5. Analysis of PILR α-ligand expressing cells>
Ba/F3— L2について、遺伝子導入から 2日後、以下の操作を行い、フローサイト メトリーにて解析した。  For Ba / F3-L2, two days after gene transfer, the following operations were performed and analyzed by flow cytometry.
[0147] 2 X 105の形質転換細胞に、 50 μ Lのヒト PILR a—Igを加えて放置した。 30分後、 細胞を HANKS 0. 1% BSA溶液で 2回洗い、 2次抗体として、 PE標識された anti- human IgG Fc抗体（Jackson laboratory社）をカ卩えて放置した。 30分後、 HANKS 0 . 1% BSA溶液で 2回洗い、フローサイトメトリーで解析した。結果を図 1に実線で示 す。 [0147] 50 µL of human PILR a-Ig was added to 2 X 10 5 transformed cells and left standing. After 30 minutes, the cells were washed twice with a HANKS 0.1% BSA solution, and a PE-labeled anti-human IgG Fc antibody (Jackson laboratory) was used as a secondary antibody and allowed to stand. After 30 minutes, the cells were washed twice with HANKS 0.1% BSA solution and analyzed by flow cytometry. The result is shown in Fig. 1 by a solid line.
[0148] なお、コントロールとして、ヒト PILR a—Igの代わりに CD200— Igを用いた以外は 同様の実験を行った。結果を図 1に点線で示す。 CD200— Igとは CD200分子の細 胞外領域と、ヒト IgG— Fc領域とのキメラ分子である。 [0148] As a control, CD200-Ig was used instead of human PILR a-Ig. A similar experiment was conducted. The results are shown in Fig. 1 by dotted lines. CD200—Ig is a chimeric molecule consisting of the extracellular region of the CD200 molecule and the human IgG—Fc region.
[0149] 以下の説明における図 1〜5は、フローサイトメトリーの結果を示すヒストグラムであり[0149] Figures 1-5 in the following description are histograms showing the results of flow cytometry.
、横軸が蛍光強度、縦軸がそれぞれの蛍光強度における細胞数を示す。 The horizontal axis represents the fluorescence intensity, and the vertical axis represents the number of cells at each fluorescence intensity.
[0150] < B.比較例 1 > [0150] <B. Comparative Example 1>
比較例 1として、 pMx— PILR— L2の代わりにインサートを持たな 、空の pMxベタ ターを用いた以外は実施例 1と同様の操作を行って、細胞 BaZF3— Mockを得た。 この BaZF3— Mockについて、実施例 1と同様の操作により、フローサイトメトリーに よる解析を行った。結果を図 3に示す。  As Comparative Example 1, cell BaZF3-Mock was obtained in the same manner as in Example 1 except that an empty pMx betater was used instead of pMx-PILR-L2 and an empty pMx betater was used. The BaZF3-Mock was analyzed by flow cytometry in the same manner as in Example 1. The results are shown in Figure 3.
[0151] < C.実施例 2 :ヒト 721細胞における PILRリガンド（CD23)のクローユングおよび 解析〉 [0151] <C. Example 2: Cloning and analysis of PILR ligand (CD23) in human 721 cells>
リガンドの探索の対象となる細胞を、マウス B16メラノーマ力もヒト 721. 221細胞に 替え、ヒト PILR o;— Igを用いた以外は、上記 (A— 2)と同様の操作を行い、新規の P ILRリガンドを得た。この PILRリガンドを CD23とする。 CD23のアミノ酸配列を配列 番号 2に、塩基配列を配列番号 8に示す。  The cells to be searched for the ligand were replaced with mouse B16 melanoma also with human 721.221 cells, and human PILR o; —Ig was used, and the same procedure as in (A-2) above was performed. ILR ligand was obtained. This PILR ligand is designated CD23. The amino acid sequence of CD23 is shown in SEQ ID NO: 2, and the nucleotide sequence is shown in SEQ ID NO: 8.
[0152] この CD23について、上記 (A— 3)と同様の操作を行い、配列番号 8に示される配 列が挿入されたベクター pMx— CD23を作製した。上記 (A— 4)と同様の操作によつ て、 pMx— CD23を BaZF3細胞に導入し、細胞 BaZF3— CD23を得た。この細胞 BaZF3— CD23を BaZF3— L2の代わりに用いた以外は、上記（A— 5)と同様の 操作を行い、フローサイトメトリーによる解析を行った。結果を図 2に示す。  [0152] With respect to this CD23, the same operation as in the above (A-3) was carried out to prepare a vector pMx-CD23 in which the sequence shown in SEQ ID NO: 8 was inserted. By the same operation as in the above (A-4), pMx-CD23 was introduced into BaZF3 cells to obtain cells BaZF3-CD23. The cells were analyzed by flow cytometry in the same manner as in (A-5) except that BaZF3-CD23 was used instead of BaZF3-L2. The result is shown in figure 2.
[0153] <A〜Cにおけるフローサイトメトリーの結果〉  [0153] <Results of flow cytometry from A to C>
図 1より、実施例 1では、 PILR— L2遺伝子を導入することによって、 BaZF3細胞 が PILR a—Igによって強く認識されるようになったことが分かる。また、図 2より、実施 例 2では、 CD23遺伝子を導入することによって、 BaZF3細胞がヒト PILR a—Igに よって強く認識されるようになったことが分かる。  From FIG. 1, it can be seen that in Example 1, BaZF3 cells were strongly recognized by PILR a-Ig by introducing the PILR-L2 gene. FIG. 2 shows that BaZF3 cells were strongly recognized by human PILR a-Ig in Example 2 by introducing the CD23 gene.
[0154] 一方、図 3より、比較例 1では、ヒト PILR a—Igは空ベクターを導入した BaZF3細 胞に対して、コントロールとほぼ同じ程度の結合量しか示さな力つた。  [0154] On the other hand, from FIG. 3, in Comparative Example 1, human PILR a-Ig exerted only the same binding amount as the control against BaZF3 cells into which the empty vector was introduced.
[0155] 以上のことから、 PILR— L2および CD23が、 PILRのリガンドとして機能することが 分かった。 [0155] From the above, PILR—L2 and CD23 can function as ligands for PILR. I understood.
[0156] < D.実施例 3 ·4 :ヒト末梢血 T細胞における PILRリガンドのクローニングおよび解 析>  [0156] <D. Example 3 · 4: Cloning and analysis of PILR ligand in human peripheral blood T cells>
< D- 1.免疫沈降による PILRリガンド（CD6、 CD45)の探索〉  <D- 1. Search for PILR ligands (CD6, CD45) by immunoprecipitation>
本発明者らは、ヒト末梢血 T細胞を解析した結果、この細胞が PILR— L2および CD As a result of analyzing human peripheral blood T cells, the present inventors have found that these cells are PILR-L2 and CD.
23を発現して!/、な!/、のにもかかわらず、 PILRによって認識されることを見出した（デ ータ不図示)。 It was found that it was recognized by PILR despite expressing 23! /,! / (Data not shown).
[0157] そこで、 PILRによって認識される分子を明らかにするために、ヒト末梢血 T細胞を可 溶化し、ヒト PILR a—Igに会合する分子を免疫沈降によって精製した。具体的には 、ヒト末梢血 T細胞を回収し、 1% Briji97を含有する可溶化剤を加えることにより可 溶ィ匕した。この可溶化試料にヒト PILR α— Igを結合させた Protein A Sepharoseビー ズを加えることによって、ヒト PILR a—Igに会合する分子を精製し、精製物とした。  [0157] In order to clarify the molecules recognized by PILR, human peripheral blood T cells were solubilized, and molecules associated with human PILR a-Ig were purified by immunoprecipitation. Specifically, human peripheral blood T cells were collected and made soluble by adding a solubilizer containing 1% Briji97. By adding Protein A Sepharose beads to which human PILR α-Ig was bound to this solubilized sample, the molecules associated with human PILR a-Ig were purified to obtain purified products.
[0158] この精製物を SDS— PAGEで電気泳動し、ヒト PILR a—Igに特異的に会合するタ ンパク質をゲル力も切り出した。このタンパク質を質量分析によって同定したところ、 それぞれ配列表 3および 5に示すアミノ酸配列を有して 、た。それぞれのタンパク質 を CD6 (実施例 3)、 CD45 (実施例 4)とする。  [0158] This purified product was electrophoresed on SDS-PAGE, and the protein that specifically associates with human PILR a-Ig was also excised. This protein was identified by mass spectrometry and had the amino acid sequences shown in Sequence Listings 3 and 5, respectively. The respective proteins are designated as CD6 (Example 3) and CD45 (Example 4).
[0159] こうして得られた CD6、 CD45について、そのアミノ酸配列より、それぞれをコードす る塩基配列を予想した。この塩基配列を配列番号 9および 11にそれぞれ示す。  [0159] From the amino acid sequences of CD6 and CD45 thus obtained, nucleotide sequences encoding them were predicted. The nucleotide sequences are shown in SEQ ID NOs: 9 and 11, respectively.
[0160] < D- 2. CD45及び CD6の解析 >  [0160] <D-2. Analysis of CD45 and CD6>
PILR— L2の代わりに、ヒト CD6遺伝子（配列番号 9)またはマウス CD45遺伝子（ 配列番号 12)を pMxベクターに挿入した以外は、上記 (A— 3)と同様の操作を行つ て、ベクター pMx— CD6及び pMx— CD45を得た。  In the same manner as (A-3) above except that the human CD6 gene (SEQ ID NO: 9) or mouse CD45 gene (SEQ ID NO: 12) was inserted into the pMx vector instead of PILR—L2, the vector pMx — CD6 and pMx— CD45 were obtained.
[0161] これらベクターを Lipofectamine plusを用いて 293T細胞に導入し、細胞 293T—C D6及び 293T— CD45を得た。遺伝子導入から 2日後、上記 (A— 5)と同様の操作 を行ってフローサイトメトリーによる解析を行った。  [0161] These vectors were introduced into 293T cells using Lipofectamine plus to obtain cells 293T-CD6 and 293T-CD45. Two days after gene transfer, the same operation as in (A-5) above was performed, and analysis by flow cytometry was performed.
[0162] くフローサイトメトリーの結果〉  [0162] Results of flow cytometry>
細胞 293T— CD6 (実施例 3)および 293T— CD45 (実施例 4)の解析結果を、そ れぞれ図 4 · 5に示す。図 4 · 5に示すように、ヒト CD6遺伝子またはマウス CD45遺伝 子を導入することによって、 293T細胞がヒトおよびマウス PILR o;— Igに強く認識さ れるようになった。なお、 293T細胞に導入したベクターを空ベクター pMxとした以外 は同様の操作を行った力 この場合、 293T細胞は PILR o;— Igに認識されな力つた (データ不図示)。 The analysis results of cells 293T-CD6 (Example 3) and 293T-CD45 (Example 4) are shown in FIGS. 4 and 5 respectively. Human CD6 gene or mouse CD45 gene as shown in Figure 4 With the introduction of pups, 293T cells became strongly recognized by human and mouse PILR o;-Ig. In this case, the 293T cells were not recognized by PILR o; -Ig (data not shown), except that the vector introduced into the 293T cells was changed to the empty vector pMx.
[0163] <免疫沈降 >  [0163] <Immunoprecipitation>
さらに、 PILR aがヒトおよびマウスの CD45を認識することを、免疫沈降によって確 認した。まず、 CD45を強発現しているヒト抹消血 T細胞およびマウス脾臓 T細胞を可 溶化した。そして、この可溶ィ匕細胞試料 (ヒト、マウス）に対して、それぞれ、ヒト PILR a—Igまたはマウス PILR a—Igによる免疫沈降を行った。この免疫沈降によって得 られた沈降物を SDS— PAGEで電気泳動し、その後、抗マウス CD45抗体および抗 ヒト CD45抗体（anti- CD45Ab)による Western Blotを行った。その結果、沈降物は 抗 CD45抗体で特異的に認識された（図 8)。なお、図 8中に、用いた可溶化細胞試 料をそれぞれ「ヒト」または「マウス」と記し、免疫沈降に用いたコントロール用 Igを「C」 、 PILR a— Igを「P」と記す。  Furthermore, it was confirmed by immunoprecipitation that PILRa recognizes human and mouse CD45. First, human peripheral blood T cells and mouse spleen T cells that strongly express CD45 were solubilized. Then, immunoprecipitation with human PILR a-Ig or mouse PILR a-Ig was performed on the soluble yeast cell samples (human and mouse), respectively. The precipitate obtained by this immunoprecipitation was electrophoresed on SDS-PAGE, followed by Western Blot with anti-mouse CD45 antibody and anti-human CD45 antibody (anti-CD45Ab). As a result, the precipitate was specifically recognized by the anti-CD45 antibody (FIG. 8). In FIG. 8, the solubilized cell sample used is referred to as “human” or “mouse”, the control Ig used for immunoprecipitation is referred to as “C”, and PILR a-Ig is referred to as “P”.
[0164] < E.実施例 5 : PILRリガンドを介した NK細胞の活性ィ匕抑制 >  [0164] <E. Example 5: Inhibition of NK cell activity via PILR ligand>
< E- 1.細胞への遺伝子導入 >  <E- 1. Gene transfer to cells>
上記 (A— 4)と同様の手法を用いて、ヒト NK細胞株である NKL細胞に、インサート として PILR aが挿入された pMxベクター（pMx— PILR a )導入した。こうして遺伝 子導入された細胞を NKL— PILR αとする。なお、 pMx— PILR aは、 pMxの Bam A pMx vector (pMx-PILRa) in which PILRa was inserted as an insert was introduced into NKL cells, which are human NK cell lines, using the same method as in (A-4) above. The cell thus introduced with the gene is designated as NKL-PILR α. PMx—PILR a is pMx Bam
HI— Notl部位に PILR αの 1— 303までが挿入されてなる。 PILR α 1 to 303 are inserted into the HI-Notl site.
[0165] 一方、コントロールとして、インサートが挿入されて!ヽな 、空の pMxベクターを同様 の操作によって NKL細胞に導入した。この細胞を NKL— Mockとする。 On the other hand, as a control, an empty pMx vector inserted with an insert was introduced into NKL cells in the same manner. This cell is called NKL-Mock.
[0166] また、 BaZF3細胞の代わりに ULBP陽性 BaZF3細胞を用いた以外は、上記 (A [0166] The above (A) except that ULBP-positive BaZF3 cells were used instead of BaZF3 cells.
4)と同様の操作を行って、 ULBP陽性 BaZF3細胞に pMx— PILR— L2を導入 した。この細胞を ULBP— BaZF3— L2とする。  In the same manner as in 4), pMx-PILR-L2 was introduced into ULBP-positive BaZF3 cells. This cell is designated as ULBP-BaZF3-L2.
[0167] なお、 ULBPは、 NK細胞の活性化レセプターの一つである NKG2Dによって認識 され、 NK細胞を活性ィ匕する。つまり、 ULBP陽性細胞は、 NK細胞の活性ィ匕を促進し[0167] It should be noted that ULBP is recognized by NKG2D, which is one of NK cell activation receptors, and activates NK cells. In other words, ULBP positive cells promote the activity of NK cells.
、 NK細胞の細胞障害性を誘起する。 [0168] < E- 2.細胞障害アツセィ > Induces cytotoxicity of NK cells. [0168] <E-2. Cytotoxic activity>
上記く E— 1 >で得られた ULBP - Ba/F3— L2に対する NKL細胞（NKL - PIL R aまたは NKL— Mock)の細胞障害性を、 Cr51遊離細胞障害アツセィにて解析し た。  The cytotoxicity of NKL cells (NKL-PIL Ra or NKL-Mock) against ULBP-Ba / F3-L2 obtained in E-1> was analyzed by Cr51 free cytotoxicity assay.
[0169] Cr51遊離細胞障害アツセィは、通常一般に行われる方法を用いた。具体的には、 標的細胞を Cr51で標識後、 1 X 104個の標的細胞と図に示す種々の細胞数のエフ エタター細胞 (NKL細胞）とを 4時間培養し、培養上精中の51 Crの量をマイクロベータ 一シンチレーシヨンカウンターで計測した。 [0169] For Cr51 free cell injury assay, a generally used method was used. Specifically, after labeling the target cells with Cr51, 1 x 10 4 target cells and various number of effector cells (NKL cells) shown in the figure were cultured for 4 hours, and 51 The amount of Cr was measured with a microbeta single scintillation counter.
[0170] NKL— Mockを用いたアツセィ結果を図 6に、 NKL— PILR αを用いたアツセィ結 果を図 7に示す。  [0170] Fig. 6 shows the results of the assembly using NKL-Mock, and Fig. 7 shows the results of the assembly using NKL-PILRα.
[0171] < F.比較例 2 >  [0171] <F. Comparative Example 2>
インサートが挿入されて ヽな 、空の pMxベクターを、用いた以外は実施例 5と同様 の操作を行い、 ULBP陽性 BaZF3細胞に導入した。この細胞を ULBP— BaZF3 — Mockとする。この ULBP— BaZF3— Mockについて、実施例 5と同様に51 Cr遊 離細胞障害アツセィを行った。結果を実施例 5と同じく図 6 · 7に示す。 An empty pMx vector, which had been inserted, was used in the same manner as in Example 5 except that an empty pMx vector was used, and introduced into ULBP-positive BaZF3 cells. This cell is called ULBP-BaZF3-Mock. The ULBP-BaZF3-Mock was subjected to 51 Cr free cell damage assay in the same manner as in Example 5. The results are shown in FIGS.
[0172] < Ε · Fの Cr51遊離細胞障害アツセィの結果 >  [0172] <Result of Cr51 free cell injury at Ε · F>
図 6 · 7において、縦軸は細胞障害性を示し、横軸は Effector (NKL細胞： NKL— Ρ ILR aまたは NKL— Mock)の細胞数 ZTarget (BaZF3細胞： ULBP— BaZF3— L2または ULBP— BaZF3— Mock)の細胞数の比率を示す。なお、細胞障害性と は、全標的細胞に対する死細胞の比率を意味する。  In Fig. 6 · 7, the vertical axis indicates cytotoxicity, and the horizontal axis indicates the number of cells of Effector (NKL cells: NKL—Ρ ILR a or NKL—Mock) ZTarget (BaZF3 cells: ULBP—BaZF3—L2 or ULBP—BaZF3) — Shows the ratio of the number of Mock cells. Cytotoxicity means the ratio of dead cells to all target cells.
[0173] 図 6 · 7に示すように、 NKL— PILR aと ULBP— BaZF3— L2 (実施例 5)との組み 合わせ（図 7の実線)で、細胞障害性が著しく抑制された。すなわち、 PILR— L2は、 PILRに特異的に作用することで、 NK細胞の活性ィヒを抑制する機能を有することが 明らかとなった。  As shown in FIGS. 6 and 7, cytotoxicity was remarkably suppressed by the combination of NKL—PILR a and ULBP—BaZF3—L2 (Example 5) (solid line in FIG. 7). In other words, it was revealed that PILR-L2 has a function of suppressing the activity of NK cells by acting specifically on PILR.
[0174] 上記実験は NK細胞の活性ィ匕を例にとり PILRの抑制効果を示した力 抑制効果は PILRを発現しているすべての免疫細胞に認められると考えられる。従って、本発明 の PILRリガンドは、 PILRを発現して!/、るすべての免疫細胞の活性化を制御するの に利用することができる。 [0175] < G.実施例 6 > [0174] In the above experiment, taking the activity of NK cells as an example, the force-suppressing effect that showed the inhibitory effect of PILR is considered to be observed in all immune cells expressing PILR. Therefore, the PILR ligand of the present invention can be used to control the activation of all immune cells that express PILR! [0175] <G. Example 6>
293T細胞に、マウス PILR遺伝子を pMx— IRES— GFPベクターで遺伝子導入し 、 2日後、遺伝子導入細胞 (GFP陽性細胞）に対するマウス CD45-Ig、マウス PILR -LI (CD99)— Ig、マウス PILR— L2— Igキメラタンパク質の結合をフローサイトメト リーで解析した。  The mouse PILR gene was introduced into 293T cells using the pMx—IRES—GFP vector, and two days later, mouse CD45-Ig, mouse PILR-LI (CD99) —Ig, mouse PILR—L2 for the transfected cells (GFP positive cells) — Binding of Ig chimeric protein was analyzed by flow cytometry.
[0176] マウス CD45— Ig、マウス PILR— LI (CD99)— Ig、マウス PILR— L2— Igキメラタ ンパク質とは、マウス CD45タンパク質、マウス PILR— L1 (CD99)タンパク質、マウ ス PILR— L2タンパク質と、マウス IgG— Fc領域とのキメラ分子（キメラタンパク質)で ある。  [0176] Mouse CD45—Ig, mouse PILR—LI (CD99) —Ig, mouse PILR—L2—Ig chimeric proteins are mouse CD45 protein, mouse PILR—L1 (CD99) protein, mouse PILR—L2 protein A chimeric molecule (chimeric protein) with mouse IgG-Fc region.
[0177] マウス CD45— Ig、マウス PILR— LI (CD99)— Ig、マウス PILR— L2— Igキメラタ ンパク質のアミノ酸配列は、それぞれ配列番号 13〜 15に示す。また、マウス CD45 — Ig、マウス PILR— L1 (CD99)— Ig、マウス PILR— L2— Igキメラタンパク質をコー ドする遺伝子を、それぞれ配列番号 16〜 18に示す。  [0177] The amino acid sequences of mouse CD45-Ig, mouse PILR-LI (CD99) -Ig, and mouse PILR-L2-Ig chimeric proteins are shown in SEQ ID NOs: 13 to 15, respectively. In addition, the genes encoding mouse CD45-Ig, mouse PILR-L1 (CD99) -Ig, and mouse PILR-L2-Ig chimeric proteins are shown in SEQ ID NOs: 16-18, respectively.
[0178] マウス CD45— Igは、マウス CD45タンパク質の第 24番目のアミノ酸残基から第 37 7番目のアミノ酸残基までの断片と、マウス Ig—Fc領域とのキメラ分子である。また、 マウス PILR— LI (CD99)— Igは、マウス PILR— LI (CD99)タンパク質の第 26番 目のアミノ酸残基から第 137番目のアミノ酸残基までの断片と、マウス Ig—Fc領域と のキメラ分子である。また、 PILR— L2— Igは、マウス PILR— L2タンパク質の第 28 番目のアミノ酸残基から第 178番目のアミノ酸残基までの断片と、マウス Ig— Fc領域 とのキメラ分子である。  [0178] Mouse CD45-Ig is a chimeric molecule consisting of a fragment from the 24th amino acid residue to the 377th amino acid residue of mouse CD45 protein and the mouse Ig-Fc region. In addition, mouse PILR-LI (CD99) -Ig is composed of a fragment from the 26th amino acid residue to the 137th amino acid residue of mouse PILR-LI (CD99) protein and the mouse Ig-Fc region. It is a chimeric molecule. PILR-L2-Ig is a chimeric molecule consisting of a fragment from the 28th amino acid residue to the 178th amino acid residue of mouse PILR-L2 protein and the mouse Ig-Fc region.
[0179] 培養液は 1%FCSをカ卩えた Advanced RPMI 1640 (Invitrogen)を用いた。遺伝子導 入細胞に CD45— Ig、 PILR-L1 (CD99)— Ig、 PILR— L 2— Igをカ卩え on iceで 30 分間放置後、 PE標識 anti-human IgGAbを混ぜ再度 on iceで 30分間放置後、蛍光強 度をフローサイトメトリーで解析した。  [0179] Advanced RPMI 1640 (Invitrogen) supplemented with 1% FCS was used as the culture solution. Introduce CD45—Ig, PILR-L1 (CD99) —Ig, PILR—L 2—Ig on the transfected cells for 30 minutes on ice, then mix with PE-labeled anti-human IgGAb and again on ice for 30 minutes. After standing, the fluorescence intensity was analyzed by flow cytometry.
[0180] その結果、図 9に示すように、 CD45— Ig、 PILR— LI— Ig、 PILR— L2— Igは、 PI LR遺伝子導入細胞に特異的に結合することが判明した。従って、抑制化 PILRのリ ガンドとしてこれら Ig—キメラ分子は PILRの機能阻害があると考えられる。さらに、こ れら Ig—キメラ分子は、二量体を形成していると考えられるため、 PILRをクロスリンク し抑制化シグナルを伝達するのに利用できると考えられる。また、 CD45および PILR —L2に関しては、ヒト相同分子も PILRに結合することから、ヒト CD45— Ig、ヒト PILR — L2— Igも同様に PILRのリガンドとして利用できると考えられる。 As a result, as shown in FIG. 9, it was found that CD45—Ig, PILR—LI—Ig, and PILR—L2—Ig specifically bind to PI LR gene-introduced cells. Therefore, it is considered that these Ig-chimeric molecules have inhibition of PILR function as ligands of suppressed PILR. Furthermore, since these Ig-chimeric molecules are thought to form dimers, PILR is cross-linked. It can be used to transmit an inhibitory signal. Regarding CD45 and PILR-L2, since human homologous molecules also bind to PILR, human CD45-Ig and human PILR-L2-Ig may be used as ligands for PILR as well.
[0181] なお、発明を実施するための最良の形態の項においてなした具体的な実施態様ま たは実施例は、あくまでも、本発明の技術内容を明らかにするものであって、そのよう な具体例にのみ限定して狭義に解釈されるべきものではなぐ本発明の精神と次に 記載する特許請求の範囲内で、いろいろと変更して実施することができるものである 産業上の利用の可能性 [0181] Note that the specific embodiments or examples made in the section of the best mode for carrying out the invention are merely to clarify the technical contents of the present invention. The present invention is not limited to specific examples and should not be construed in a narrow sense, and can be implemented with various modifications within the scope of the following claims. possibility
[0182] 本発明に係るポリヌクレオチドは、免疫応答を制御する医薬品、特に、自己免疫疾 患の治療薬等、免疫応答を抑制するための医薬品に好適に利用することができる。 そのため、本発明に係るポリペプチドおよびそれに付加する糖鎖を含めた構造は、 非常に有望な産業上の利用可能性を備えている。 [0182] The polynucleotide according to the present invention can be suitably used for a pharmaceutical agent for controlling an immune response, particularly a pharmaceutical agent for suppressing an immune response, such as a therapeutic agent for an autoimmune disease. Therefore, the structure including the polypeptide according to the present invention and the sugar chain added thereto has very promising industrial applicability.

Claims

請求の範囲 The scope of the claims
[1] 下記 (a)または (b)の 、ずれかに記載のポリペプチド、当該ポリペプチドをコードす るポリヌクレオチド、または当該ポリペプチドを発現する細胞を含み、かつ免疫細胞を 活性化する、または免疫細胞の活性化を抑制することを特徴とする組成物。  [1] The polypeptide according to any one of the following (a) or (b), a polynucleotide encoding the polypeptide, or a cell expressing the polypeptide, and activating immune cells, Or the composition characterized by suppressing activation of an immune cell.
(a)配列番号 1〜6のいずれか 1つに示されるアミノ酸配列力 なるポリペプチド。 (a) A polypeptide having an amino acid sequence ability represented by any one of SEQ ID NOs: 1 to 6.
(b)配列番号 1〜6のいずれか 1つに示されるアミノ酸配列において、 1個または数 個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリべプチ ド、。 (b) A polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6.
[2] 下記 (e)または (f)の 、ずれかであるポリヌクレオチドを含み、かつ免疫細胞を活性 化する、または免疫細胞の活性化を抑制することを特徴とする組成物。  [2] A composition comprising the polynucleotide (e) or (f) below, which activates immune cells or suppresses activation of immune cells.
(e)配列番号 7〜 12のいずれか 1つに示される塩基配列力もなるポリヌクレオチド。 (e) a polynucleotide having the nucleotide sequence ability represented by any one of SEQ ID NOs: 7 to 12.
(f)下記 (iii)または (iv)の 、ずれかであるポリヌクレオチドとストリンジェントな条件 下でノ、イブリダィズし、かつ PILR o;と結合するポリペプチドをコードする塩基配列か らなるポリヌクレオチド。 (f) a polynucleotide comprising a nucleotide sequence encoding a polypeptide that binds to PILR o; under stringent conditions with a polynucleotide of any one of (iii) or (iv) below: .
(iii)配列番号 7〜 12のいずれか 1つに示される塩基配列力もなるポリヌクレオチ ド、。  (iii) a polynucleotide having a nucleotide sequence ability represented by any one of SEQ ID NOs: 7 to 12.
(iv)配列番号 7〜 12のいずれか 1つに示される塩基配列と相補的な塩基配列 力もなるポリヌクレオチド。  (iv) A polynucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 7 to 12.
[3] 下記 (a)または (b)のいずれかのポリペプチドと IgG—Fc領域とのキメラ分子、当該 キメラ分子をコードするポリヌクレオチド、または当該キメラ分子を発現する細胞を含 むと共に、 PILR aと結合するポリペプチドを含有し、免疫細胞を活性化する、または 免疫細胞の活性化を抑制することを特徴とする組成物  [3] A chimeric molecule comprising the polypeptide of any one of (a) or (b) below and an IgG-Fc region, a polynucleotide encoding the chimeric molecule, or a cell expressing the chimeric molecule, and PILR A composition comprising a polypeptide that binds to a, which activates immune cells or suppresses activation of immune cells
(a)配列番号 1〜6のいずれか 1つに示されるアミノ酸配列力 なるポリペプチド。 (a) A polypeptide having an amino acid sequence ability represented by any one of SEQ ID NOs: 1 to 6.
(b)配列番号 1〜6のいずれか 1つに示されるアミノ酸配列において、 1個または数 個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリべプチ ド、。 (b) A polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6.
[4] 下記 (g)または (h)の 、ずれかに記載のポリペプチド、当該ポリペプチドをコードす るポリヌクレオチド、または当該ポリペプチドを発現する細胞を含み、かつ免疫細胞を 活性化する、または免疫細胞の活性化を抑制することを特徴とする組成物。 [4] The polypeptide according to any one of the following (g) or (h), a polynucleotide encoding the polypeptide, or a cell expressing the polypeptide, and an immune cell: A composition that activates or suppresses activation of immune cells.
(g)配列番号 13〜 15の 、ずれか 1つに示されるアミノ酸配列からなるポリペプチド  (g) a polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 13 to 15
(h)配列番号 13〜 15の 、ずれか 1つに示されるアミノ酸配列にお 、て、 1個または 数個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリぺプ チド。 (h) In the amino acid sequence shown in any one of SEQ ID NOs: 13 to 15, a polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted or added Chido.
[5] 下記 (i)または (j)の 、ずれかであるポリヌクレオチドを含み、免疫細胞を活性化す る、または免疫細胞の活性化を抑制することを特徴とする組成物。  [5] A composition comprising a polynucleotide as defined in (i) or (j) below, which activates immune cells or suppresses activation of immune cells.
(i)配列番号 16〜18のいずれか 1つに示される塩基配列力もなるポリヌクレオチド  (i) a polynucleotide having a nucleotide sequence ability represented by any one of SEQ ID NOs: 16 to 18
(j)下記 (V)または (vi)の 、ずれかであるポリヌクレオチドとストリンジェントな条件下 でハイブリダィズし、かつ PILR o;と結合するポリペプチドをコードする塩基配列から なるポリヌクレオチド。 (j) A polynucleotide comprising a base sequence encoding a polypeptide that hybridizes under stringent conditions and binds to PILRo; in the following (V) or (vi):
(V)配列番号 16〜18のいずれ力 1つに示される塩基配列からなるポリヌクレオ チド。  (V) A polynucleotide comprising the base sequence represented by any one of SEQ ID NOs: 16 to 18.
(vi)配列番号 16〜18のいずれか 1つに示される塩基配列と相補的な塩基配列 力もなるポリヌクレオチド。  (vi) a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 16 to 18.
[6] 下記 (a)または (b)の 、ずれかであり、 PILR aと結合することを特徴とするポリぺプ チド。 [6] A polypeptide characterized by binding to PILR a, which is any of the following (a) or (b).
(a)配列番号 1〜6のいずれか 1つに示されるアミノ酸配列力 なるポリペプチド。 (a) A polypeptide having an amino acid sequence ability represented by any one of SEQ ID NOs: 1 to 6.
(b)配列番号 1〜6のいずれか 1つに示されるアミノ酸配列において、 1個または数 個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリべプチ ド、。 (b) A polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 6.
[7] 配列番号 2〜4のいずれか 1つに示されるアミノ酸配列からなることを特徴とするポリ ペプチド。  [7] A polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 2 to 4.
[8] 請求項 7に記載のポリペプチドをコードする塩基配列からなることを特徴とするポリ ヌクレオチド。  [8] A polynucleotide comprising a base sequence encoding the polypeptide of claim 7.
[9] 下記 (c)または (d)の 、ずれかであることを特徴とするポリヌクレオチド。 (c)配列番号 8〜10のいずれ力 1つに示される塩基配列力もなるポリヌクレオチド。[9] A polynucleotide according to (c) or (d) below: (c) a polynucleotide having the nucleotide sequence represented by any one of SEQ ID NOs: 8 to 10.
(d)下記 (i)または (ii)の 、ずれかであるポリヌクレオチドとストリンジェントな条件下 でハイブリダィズし、かつ PILR o;と結合するポリペプチドをコードする塩基配列から なるポリヌクレオチド。 (d) A polynucleotide comprising a base sequence encoding a polypeptide that hybridizes under stringent conditions and binds to PILRo; in the following (i) or (ii):
(i)配列番号 8〜 10の 、ずれか 1つに示される塩基配列力もなるポリヌクレオチ ド、。  (i) a polynucleotide having the nucleotide sequence shown in any one of SEQ ID NOs: 8 to 10;
(ii)配列番号 8〜10の 、ずれか 1つに示される塩基配列と相補的な塩基配列か らなるポリヌクレオチド。  (ii) A polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 8 to 10.
[10] 下記 (g)または（h)の 、ずれかに記載のポリペプチドであって、 PILR aと結合する ことを特徴とするポリペプチド。  [10] The polypeptide according to any one of (g) and (h) below, wherein the polypeptide binds to PILRa.
(g)配列番号 13〜 15の 、ずれか 1つに示されるアミノ酸配列からなるポリペプチド  (g) a polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 13 to 15
(h)配列番号 13〜 15の 、ずれか 1つに示されるアミノ酸配列にお 、て、 1個または 数個のアミノ酸が置換、欠失、挿入、または付加されたアミノ酸配列力もなるポリぺプ チド。 (h) In the amino acid sequence shown in any one of SEQ ID NOs: 13 to 15, a polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added. Chido.
[11] 下記 (i)または (j)の 、ずれかであることを特徴とするポリヌクレオチド。  [11] A polynucleotide according to (i) or (j) below:
(i)配列番号 16〜18のいずれか 1つに示される塩基配列力もなるポリヌクレオチド  (i) a polynucleotide having a nucleotide sequence ability represented by any one of SEQ ID NOs: 16 to 18
(j)下記 (V)または (vi)の 、ずれかであるポリヌクレオチドとストリンジェントな条件下 でハイブリダィズし、かつ PILR o;と結合するポリペプチドをコードする塩基配列から なるポリヌクレオチド。 (j) A polynucleotide comprising a nucleotide sequence that encodes a polypeptide that hybridizes under stringent conditions with a polynucleotide of any one of (V) or (vi) below and binds to PILRo;
(V)配列番号 16〜18のいずれ力 1つに示される塩基配列からなるポリヌクレオ チド。  (V) A polynucleotide comprising the base sequence represented by any one of SEQ ID NOs: 16 to 18.
(vi)配列番号 16〜18のいずれか 1つに示される塩基配列と相補的な塩基配列 力もなるポリヌクレオチド。  (vi) a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 16 to 18.
[12] 請求項 8, 9または 11に記載のポリヌクレオチドを含むことを特徴とするベクター。 [12] A vector comprising the polynucleotide according to claim 8, 9 or 11.
[13] 請求項 8, 9または 11に記載のポリヌクレオチドが発現可能に導入されてなることを 特徴とする細胞。 請求項 8, 9または 11に記載のポリヌクレオチドを用いて PILR o;に結合するタンパ ク質を生産する方法。 [13] A cell, wherein the polynucleotide according to claim 8, 9 or 11 is introduced so that it can be expressed. A method for producing a protein that binds to PILR o; using the polynucleotide according to claim 8, 9 or 11.
PCT/JP2007/053558 2006-02-28 2007-02-27 PEPTIDE BINGING TO PILRα, POLYNUCLEOTIDE ENCODING THE SAME AND APPLICATION THEREOF WO2007099921A1 (en)

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