WO2022080305A1 - Anti-ptdss2 antibody - Google Patents

Anti-ptdss2 antibody Download PDF

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Publication number
WO2022080305A1
WO2022080305A1 PCT/JP2021/037546 JP2021037546W WO2022080305A1 WO 2022080305 A1 WO2022080305 A1 WO 2022080305A1 JP 2021037546 W JP2021037546 W JP 2021037546W WO 2022080305 A1 WO2022080305 A1 WO 2022080305A1
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antibody
ptdss2
antigen
treatment
amino acid
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PCT/JP2021/037546
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French (fr)
Japanese (ja)
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秀雄 幸長
尚之 前田
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第一三共株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention comprises a novel anti-PTDSS2 antibody, a functional fragment of the antibody, a modified version of the antibody, a nucleotide containing a base sequence encoding the amino acid sequence of the antibody, a vector into which the nucleotide is inserted, the nucleotide or a vector.
  • the present invention relates to introduced cells, a method for producing the antibody including a step of culturing the cells, a pharmaceutical composition, a composition for diagnosis or a test, and the like.
  • Phosphatidylserine is one of the acidic phospholipids having a negative charge on its polar head under physiological conditions, and occupies about 5 to 15% of the cell membrane phospholipids. Phosphatidylserine is known to be produced in mammalian cells by two enzymes, phosphatidylserine synthase 1 (sometimes referred to as PTDSS1) and phosphatidylserine synthase 2 (sometimes referred to as PTDSS2). (Non-Patent Documents 1 and 2).
  • PTDSS1 is an enzyme that produces phosphatidylserine by exchanging the choline portion of phosphatidylcholine with L-serine
  • PTDSS2 is an enzyme that produces phosphatidylserine by a parallel base exchange reaction of phosphatidylethanolamine (Non-Patent Documents 1 and 2).
  • PTDSS1 and PTDSS2 have about 28% in common in amino acid sequences (Non-Patent Documents 3 and 4).
  • PTDSS1 and PTDSS2 have a synthetic lethal relationship (Patent Document 1). Therefore, treatment that inhibits PTDSS1 is a promising approach for the treatment of cancers in which PTDSS2 function is suppressed (hereinafter, may be referred to as "PTDSS2 function-suppressing cancer").
  • PTDSS2 function-suppressing cancer when the expression of PTDSS2 in the cancer tissue is lower than that of other cancers, or when it is not expressed at all, the function is further reduced due to gene mutation or the like, or the function is reduced. Can be mentioned if you have lost.
  • PTDSS1 and PTDSS2 have a synthetic lethal relationship, and it is necessary to find a PTDSS2 function-suppressing cancer as a target for treatment that inhibits PTDSS1.
  • An immunohistochemical staining method using an antibody against PTDSS2 can be mentioned as a method for evaluating the presence or absence of PTDSS2 expression in cancer tissue and the expression level. By using the immunohistochemical staining method, it becomes possible to detect cancers that do not express PTDSS2 or cancers that have a low expression level.
  • One object of the present invention is to provide an antibody that specifically binds to PTDSS2.
  • the subject of the present invention includes a nucleotide encoding the amino acid sequence of the antibody, a vector into which the nucleotide is inserted, a cell into which the nucleotide or the vector has been introduced, and a step of culturing the antibody. Methods etc. are included.
  • the present invention includes the following inventions.
  • (1-1) The antibody according to (1) or an antigen-binding fragment of the antibody, wherein the antibody is a monoclonal antibody.
  • (2) The antibody according to (1) or an antigen-binding fragment of the antibody, wherein the partial peptide of human PTDSS2 is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing.
  • the light chain sequence comprises a variable region having CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 13, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 15.
  • the antibody according to any one of (3) to (3) or an antigen-binding fragment of the antibody.
  • the antibody according to any one of (1) to (5) which comprises a light chain composed of a light chain, or an antigen-binding fragment of the antibody.
  • the antibody according to any one of (1) to (5) which is a chimeric antibody, or an antigen-binding fragment of the antibody.
  • composition according to (12), which is used in. (14) Used in a method for detecting or measuring PTDSS2 in a specimen, which comprises a step of contacting the test specimen with the antibody according to any one of (1) to (11) or an antigen-binding fragment of the antibody. , (12) or (13).
  • the above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  • the method for detecting or measuring PTDSS2 is such that PTDSS2 is detected or measured in the test specimen, or the expression level of PTDSS2 in the test specimen is equal to or higher than a predetermined criterion, the subject is said to be the subject.
  • test sample is judged to be positive and PTDSS2 is not detected or measured in the test sample, or if the expression level of PTDSS2 in the test sample is equal to or lower than a predetermined criterion, the test sample is concerned.
  • the above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  • the composition according to any one of (12) to (15) which is used for a test or diagnostic method for a PTDSS2-negative disease (cancer).
  • PTDSS2-negative disease using a fragment
  • a PTDSS2-negative disease due to a PTDSS1 inhibitor including determining that a subject determined to be PTDSS2 negative is responsive to treatment of a PTDSS2-negative disease (cancer) with a PTDSS1 inhibitor. How to predict the responsiveness to treatment.
  • the above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  • a subject whose PTDSS2 is determined to be negative using the test specimen for the determination, the antibody according to any one of (1) to (11) or an antigen-binding fragment of the antibody is used.
  • a method for predicting the responsiveness of a PTDSS1 inhibitor to the treatment of a PTDSS2-negative disease which comprises determining that the PTDSS1 inhibitor is responsive to the treatment of a PTDSS2-negative disease (cancer).
  • the above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment. (23) Using the test sample, the presence or absence of PTDSS2 in the test sample is detected (for the detection, the antibody according to any one of (1) to (11) or the antigen binding property of the antibody.
  • PTDSS2-negative disease cancer
  • PTDSS1 inhibitor Treatment of PTDSS2-negative disease (cancer) with PTDSS1 inhibitor, including selecting subjects who are determined to be negative for PTDSS2 (using fragments) for treatment of PTDSS2-negative disease (cancer) with PTDSS1 inhibitor. How to select the target of.
  • the above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment. (24) A subject whose PTDSS2 is determined to be negative using the test specimen (for the determination, the antibody according to any one of (1) to (11) or an antigen-binding fragment of the antibody is used.
  • a method for selecting a target for treatment of a PTDSS2-negative disease (cancer) with a PTDSS1 inhibitor which comprises selecting the target for treatment of a PTDSS2-negative disease (cancer) with a PTDSS1 inhibitor.
  • the above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment. (25) Using the test specimen, the presence or absence of PTDSS2 in the test specimen is detected (for the detection, the antibody according to any one of (1) to (11) or the antigen binding property of the antibody.
  • a method for treating a PTDSS2-negative disease which comprises administering a PTDSS1 inhibitor to a subject who is determined to be negative for PTDSS2 (using a fragment).
  • the above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  • the antibody according to any one of (1) to (11) or an antigen-binding fragment of the antibody was detected in a subject in which the suppression of PTDSS2 function was detected using the test specimen.
  • a method for treating a PTDSS2-negative disease which comprises administering a PTDSS1 inhibitor.
  • the above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  • FIG. 13-1 shows a stained image of a rabbit anti-PTDSS2 monoclonal antibody.
  • the immunostaining image of PTDSS2 in A375 cells wild type, WT; heterologus KO, Hetero; Homologus KO, Homo
  • FIG. 13-2 shows a stained image of the rabbit anti-PTDSS2 monoclonal antibody.
  • the immunostaining image of PTDSS2 in the human breast cancer tissue is shown.
  • FIG. 14-1 shows a stained image of a rabbit anti-PTDSS2 monoclonal antibody and a stained image of Normal Rabbit IgG as a negative control.
  • the immunostaining image of PTDSS2 in the human breast cancer tissue is shown.
  • FIG. 14-2 shows a stained image of a rabbit anti-PTDSS2 monoclonal antibody and a stained image of Normal Rabbit IgG as a negative control.
  • the "gene” means a nucleotide containing a base sequence encoding a protein's amino acid sequence or a complementary strand thereof, and for example, a nucleotide containing a base sequence encoding a protein's amino acid sequence or its complement. Strands such as polynucleotides, oligonucleotides, DNAs, mRNAs, cDNAs, and cRNAs are included in the meaning of "gene”.
  • Such genes are single-stranded, double-stranded or triple-stranded or higher nucleotides, which are aggregates of DNA strands and RNA strands, in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed on one nucleotide strand. Nucleotides and double-stranded or triple-stranded or higher nucleotides containing such nucleotide chains are also included in the meaning of "gene”.
  • Examples of the "PTDSS2 gene" of the present invention include DNA, mRNA, cDNA, cRNA and the like containing a base sequence encoding the amino acid sequence of the PTDSS2 protein.
  • nucleotide and “nucleic acid” are synonymous, and for example, DNA, RNA, probe, oligonucleotide, polynucleotide, primer and the like are also included in the meaning of “nucleotide”.
  • a nucleotide is a nucleotide consisting of a single strand, a double strand, or three or more strands, and an aggregate of a DNA strand and an RNA strand, a ribonucleotide (RNA) and a deoxyribonucleotide (DNA) on the single nucleotide strand. Consolidations and aggregates of double or three or more strands containing such nucleotide chains are also included in the meaning of "nucleotide”.
  • polypeptide In the present invention, “polypeptide”, “peptide” and “protein” are synonymous.
  • antigen may be used to mean “immunogen”.
  • the "cell” also includes various cells derived from individual animals, subcultured cells, primary cultured cells, cell lines, recombinant cells, microorganisms and the like.
  • anti-PTDSS2 antibody the antibody that recognizes PTDSS2 may be referred to as "anti-PTDSS2 antibody”.
  • Such antibodies include polyclonal antibodies, monoclonal antibodies, chimerized antibodies, rabbitized antibodies, humanized antibodies, human antibodies and the like.
  • the "functional fragment of an antibody” in the present invention means an antibody fragment that plays at least a part of the function of the original antibody.
  • Examples of the "functional fragment of the antibody” include, but are not limited to, Fab, F (ab') 2, scFv, Fab', single-chain immunoglobulin and the like.
  • the functional fragment of such an antibody is a recombinant protein produced in a suitable host cell using a recombinant gene in addition to the one obtained by treating the full-length molecule of the antibody protein with an enzyme such as papain or pepsin. May be.
  • the "site" to which the antibody binds that is, the "site” recognized by the antibody means a partial peptide or a partial higher-order structure on the antigen to which the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or an antibody binding site.
  • the site on the PTDSS2 protein to which the anti-PTDSS2 antibody of the present invention binds or recognizes include a partial peptide or a partial higher-order structure on the PTDSS2 protein.
  • CDRs complementarity determining regions
  • Complementarity determining regions also called hypervariable domains, are sites within the variable regions of the heavy and light chains of an antibody that are particularly highly variable in primary structure and of the heavy and light chains.
  • CDRH1, CDRH2, and CDRH3 from the amino-terminal side of the heavy chain amino acid sequence
  • the complementarity determining regions of the light chain are represented by light chain amino acids. From the amino terminal side of the sequence, they are referred to as CDRL1, CDRL2, and CDRL3. These sites are close to each other on the three-dimensional structure and determine the specificity for the antigen to which they bind.
  • the "antibody variant” has an amino acid sequence in which an amino acid is substituted, deleted, added and / or inserted (hereinafter collectively referred to as "mutation") in the amino acid sequence of the original antibody. And, it means a polypeptide that binds to the PTDSS2 protein of the present invention.
  • the number of mutant amino acids in such antibody variants is 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9. 1 to 10 pieces, 1 to 12 pieces, 1 to 15 pieces, 1 to 20 pieces, 1 to 25 pieces, 1 to 30 pieces, 1 to 40 pieces or 1 to 50 pieces.
  • Such antibody variants are also included in the "antibodies" of the present invention.
  • "several pieces" in “1 to several pieces” means 3 to 10 pieces.
  • Examples of the activity / property exhibited by the antibody of the present invention include biological activity, physicochemical property, etc. Specifically, various biological activities, binding activity to antigens and epitopes, stability during production and storage. Properties, thermal stability, etc. can be mentioned.
  • hybridizing under stringent conditions means that hybridization is performed at 65 ° C. in a solution containing 5 ⁇ SSC, and then in an aqueous solution containing 2 ⁇ SSC-0.1% SDS. 65 ° C. for 20 minutes at 65 ° C. in an aqueous solution containing 0.5 ⁇ SSC-0.1% SDS and 65 ° C. in an aqueous solution containing 0.2 ⁇ SSC-0.1% SDS. It means that the cells are hybridized under the conditions of washing or equivalent for 20 minutes.
  • SSC is an aqueous solution of 150 mM NaCl-15 mM sodium citrate, and n ⁇ SSC means n times the concentration of SSC.
  • cell injury refers to causing pathological changes in cells in some way, and is not limited to direct trauma, but also DNA cleavage, base dimer formation, chromosomal cleavage, etc. It means damage to the structure and function of all cells, such as damage to the cell division device and decreased activity of various enzymes.
  • cytotoxic activity means to cause the above-mentioned cytotoxicity.
  • antibody-dependent cellular cytotoxicity (ADCC) activity refers to “antibody dependent cellular cytotoxicity (ADCC) activity", and means an action activity in which NK cells damage target cells such as tumor cells via an antibody.
  • tumors malignant tumors, cancers, malignant neoplasms, cancers, sarcomas and the like may be collectively referred to as “tumors” or “cancers”.
  • immunohistochemistry means a histological (histochemical) method for detecting an antigen in a tissue sample, and is synonymous with “immunohistochemical method” and is synonymous with “immunostaining (immunostaining (iHC)”. "Immunostaining)” is also used interchangeably.
  • modified PTDSS2 means a PTDSS2 molecule in a specimen fixed with formalin.
  • the PTDSS2 molecules in the specimens that have been fixed with formalin and then paraffinized and deparaffinized are also referred to as "modified PTDSS2".
  • non-denatured PTDSS2 means PTDSS2 in a sample not fixed with formalin.
  • the PTDSS2 molecule in the specimen not fixed with formalin is also called “non-denatured PTDSS2”.
  • test specimen means a biological sample derived from a subject.
  • the "subject” means a human and a non-human mammal undergoing a test by a method for predicting responsiveness to a PTDSS1 inhibitor.
  • it means humans and non-human mammals suffering from a disease that is expected to have a therapeutic effect by a PTDSS1 inhibitor (preferably humans).
  • the disease in which the therapeutic effect of the PTDSS1 inhibitor is expected is not particularly limited as long as it is a disease in which the therapeutic effect of the PTDSS1 inhibitor is expected, but cancer can be preferably exemplified.
  • the non-human mammal may be any organism as long as it is classified as a mammal, and includes, for example, guinea pigs, rats, mice, rabbits, pigs, sheep, cows, monkeys, and the like.
  • guinea pigs rats, mice, rabbits, pigs, sheep, cows, monkeys, and the like.
  • humans and non-human mammals suspected of having cancer, and humans and non-human mammals diagnosed with cancer can be preferably exemplified.
  • a "biological sample” refers to a tissue, fluid, cell, or a mixture of two or more thereof isolated from an individual, for example, tumor biopsy, spinal fluid, intrathoracic fluid, intraperitoneal fluid, etc. Examples thereof include, but are not limited to, lymph, skin sections, blood, urine, feces, sputum, respiratory organs, intestinal tract, urogenital tract, saliva, milk, digestive organs, or cells collected from these.
  • a "biological sample” is, for example, a part of excised tissue obtained during surgery performed for the purpose of treating cancer, or tissue collected by biopsy from a subject suspected of having cancer. Cells derived in part from blood or intrathoracic or intraperitoneal fluid can be preferably exemplified.
  • the biological sample may be a protein extract or a nucleic acid extract prepared from a tissue, a liquid, a cell, or a mixture of two or more kinds thereof isolated from an individual.
  • the protein extract and the nucleic acid extract can be prepared by using a protein preparation method or a nucleic acid preparation method known per se.
  • the biological sample is preferably a biological sample collected before treatment with a PTDSS1 inhibitor.
  • the use of such biological samples makes it possible to predict susceptibility to PTDSS1 inhibitors prior to treatment with PTDSS1 inhibitors, and as a result, apply treatments containing PTDSS1 inhibitors to subjects. It is possible to determine whether or not the subject is to be treated, that is, to select a subject to which the treatment containing the PTDSS1 inhibitor is applied.
  • phosphatidylserine synthase 1 is a protein encoded by the PTDSS1 gene.
  • the human PTDSS1 gene is registered in NCBI as Gene ID: 9791, RefSEQ: accession NM_014754.2 (protein: RefSeq NP_055569.1).
  • phosphatidylserine synthase 2 is a protein encoded by the PTDSS2 gene.
  • the human PTDSS2 gene is registered in NCBI as Gene ID: 81490, RefSEQ: accession NM_030783.1_ (protein: RefSeq NP_110410.1).
  • PTDSS2 Whether PTDSS2 used in the present invention is directly purified from humans, non-human mammals (eg, guinea pigs, rats, mice, rabbits, pigs, sheep, cows, monkeys, etc.) or chicken T cells or mast cells.
  • the cell membrane fraction of the above cells can be prepared and used, or can be obtained by synthesizing PTDSS2 in vitro or by genetically manipulating it in a host cell.
  • the PTDSS2 cDNA is incorporated into an expressible vector and then synthesized in a solution containing the enzymes, substrates and energy substances required for transcription and translation, or other prokaryotes, or eukaryotes.
  • the protein can be obtained by expressing PTDSS2 by transforming the host cell of an organism.
  • the nucleotide sequence of the cDNA of human PTDSS2 is based on the above-mentioned human PTDSS2 gene.
  • the cDNA of PTDSS2 is, for example, a polymerase chain reaction using a cDNA library of an organ expressing the mRNA of PTDSS2 or a genomic DNA extracted from human cells as a template and a primer that specifically amplifies the cDNA of PTDSS2 (hereinafter referred to as "" It can be obtained by the so-called PCR method in which (referred to as PCR) (Saiki, R.K., et al., Science, (1988) 239, 487-491) is performed.
  • a polynucleotide encoding a protein having a biological activity equivalent to that of PTDSS2, which hybridizes with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence encoding human PTDSS2 under stringent conditions is also included in the cDNA of PTDSS2. included.
  • a splicing variant transcribed from the human PTDSS2 locus or a polynucleotide that hybridizes to the splicing variant under stringent conditions and encodes a protein having the same biological activity as PTDSS2 is also included in the cDNA of PTDSS2. included.
  • a protein consisting of an amino acid sequence in which one, two, three, or four or five amino acids are substituted, deleted, or added in the amino acid sequence of human PTDSS2 and having the same biological activity as PTDSS2 is also added to PTDSS2. included.
  • PTDSS2 also contains a protein consisting of PTDSS2 and having the same biological activity as PTDSS2.
  • the antibody against PTDSS2 of the present invention is obtained by immunizing an animal with an arbitrary polypeptide selected from the amino acid sequence of PTDSS2 or PTDSS2 using a conventional method, and collecting and purifying the antibody produced in vivo. Can be obtained by doing.
  • the species of PTDSS2 as an antigen is not limited to humans, and PTDSS2 derived from non-human animals such as monkeys, mice, and rats can be immunized against animals. In this case, by testing the crossing property between the obtained antibody that binds to the heterologous PTDSS2 and human PTDSS2, an antibody applicable to human diseases can be selected.
  • the PTDSS2 as an antigen can also be obtained by causing the host cell to produce the PTDSS2 gene by genetic engineering. Specifically, a vector capable of expressing the PTDSS2 gene may be prepared, introduced into a host cell to express the gene, and the expressed PTDSS2 may be purified.
  • the 401-P-17 antibody can be mentioned.
  • the heavy chain nucleotide sequence of the 401-P-17 antibody is described in SEQ ID NO: 8 of the sequence listing, nucleotide number 1-57 is the signal sequence, 58-384 is the heavy chain variable region, and 385-1356 is the heavy chain constant. Shows the area.
  • SEQ ID NO: 8: 130-153 indicates CDRH1, 205-225 indicates CDRH2, and 336-351 indicates CDRH3.
  • the heavy chain amino acid sequence of the 401-P-17 antibody is shown in SEQ ID NO: 2 and FIG.
  • amino acid numbers 1-19 are the signal sequence
  • 20-128 are the heavy chain variable region
  • 129-451 Indicates the heavy chain constant region.
  • the amino acid sequence of CDR determined by IMGT http://www.imgt.org/ indicates that amino acid numbers 44-51 of SEQ ID NO: 2 indicate CDRH1, 69-75 indicates CDRH2, and 112-116 indicate CDRH3.
  • the amino acid sequence of the heavy chain variable region of the 401-P-17 antibody is shown in SEQ ID NO: 3 and FIG. 2 in the sequence listing.
  • the amino acid sequence of the heavy chain constant region of the 401-P-17 antibody is shown in SEQ ID NO: 4 and FIG. 3 of the sequence listing.
  • the amino acid sequence of CDRH1 of the 401-P-17 antibody is shown in SEQ ID NO: 5 and FIG. 4 of the sequence listing.
  • the amino acid sequence of CDRH2 of the 401-P-17 antibody is shown in SEQ ID NO: 6 and FIG. 5 of the sequence listing.
  • the amino acid sequence of CDRH3 of the 401-P-17 antibody is shown in SEQ ID NO: 7 and FIG. 6 of the sequence listing.
  • the light chain nucleotide sequence of the 401-P-17 antibody is set forth in SEQ ID NO: 16 of the sequence listing, nucleotide number 1-66 is the signal sequence, 67-405 is the light chain variable region, and 406-720 is the light chain constant. Shows the area.
  • SEQ ID NO: 16: 145-168 indicates CDRL1, 220-228 indicates CDRL2, and 337-375 indicates CDRL3.
  • the light chain amino acid sequence of the 401-P-17 antibody is shown in SEQ ID NO: 10 and FIG. 7 of the sequence listing, where amino acid numbers 1-22 are the signal sequence, 23-135 is the light chain variable region, 136-239.
  • amino acid numbers 49-56 of SEQ ID NO: 10 indicate CDRL1, 74-76 indicates CDRL2, and 113-125 indicate CDRL3. ..
  • the amino acid sequence of the light chain variable region of the 401-P-17 antibody is shown in SEQ ID NO: 11 and FIG. 8 of the sequence listing.
  • the amino acid sequence of the light chain constant region of the 401-P-17 antibody is shown in SEQ ID NO: 12 and FIG. 9 of the sequence listing.
  • the amino acid sequence of CDRL1 of the 401-P-17 antibody is set forth in SEQ ID NO: 13 and FIG. 10 of the sequence listing.
  • the amino acid sequence of CDRL2 of the 401-P-17 antibody is set forth in SEQ ID NO: 14 and FIG. 11 of the sequence listing.
  • the amino acid sequence of CDRL3 of the 401-P-17 antibody is set forth in SEQ ID NO: 15 and FIG. 12 of the sequence listing.
  • the antibody of the present invention specifically recognizes the PTDSS2 protein.
  • the preferred antibody of the invention specifically binds to the PTDSS2 protein.
  • the suitable antibody specifically binds to both the non-denatured human PTDSS2 and the denatured human PTDSS2 in a formalin-fixed specimen.
  • more suitable antibodies include antibodies that specifically bind to both non-denatured human PTDSS2 and denatured human PTDSS2 in formalin-fixed specimens, but do not specifically bind to other PTDSS families. However, it is not limited to them.
  • the antibody of the present invention includes a recombinant antibody artificially modified for the purpose of reducing heterologous antigenicity, for example, a Chimeric antibody, a humanized antibody.
  • Antibodies, mouse antibodies, etc. are also included. These antibodies can be produced using known methods.
  • chimeric antibody examples include an antibody in which the variable region and the constant region of the antibody are different from each other, for example, a chimeric antibody in which the variable region of a mouse or rat-derived antibody is bonded to the constant region of human origin (Proc. Natl. Acad). .Sci. USA, 81, 6851-6855, (1984)).
  • the antibody of the present invention includes an antibody obtained by modifying the CDR of the antibody. These antibodies can be produced using known methods.
  • the present invention also includes the modified antibody, which is a deletion in which one or two amino acids are deleted at the heavy chain carboxyl terminus, and the amidated deletion (for example, at the carboxyl terminus site).
  • a heavy chain in which a proline residue is amidated) and the like can be mentioned.
  • the deletion of the carboxyl terminus of the heavy chain of the antibody according to the present invention is not limited to the above types.
  • the two heavy chains constituting the antibody according to the present invention may be any one of the heavy chains selected from the group consisting of the full length and the above-mentioned deletion product, or a combination of the two heavy chains. It may be a thing.
  • the amount ratio of each deletion substance may be affected by the type of cultured mammalian cells producing the antibody according to the present invention and the culture conditions, but the main component of the antibody according to the present invention is carboxyl in both of the two heavy chains.
  • the case where one amino acid residue at the terminal is deleted can be mentioned.
  • the antibody obtained by the above method can evaluate the binding property to the antigen and select a suitable antibody.
  • the stability of the antibody can be mentioned.
  • Differential scanning calorimetry (DSC) is a method that can quickly and accurately measure the heat denaturation midpoint (Tm), which is a good indicator of the relative structural stability of proteins. By measuring the Tm value using DSC and comparing the values, the difference in thermal stability can be compared. It is known that the storage stability of an antibody shows a certain degree of correlation with the thermal stability of the antibody (Lori Burton, et. Al., Pharmaceutical Development and Technology (2007) 12, p.265-273). Suitable antibodies can be selected using the stability as an index.
  • Other indicators for selecting antibodies include high yields in suitable host cells and low cohesiveness in aqueous solution.
  • the antibody with the highest yield does not always show the highest thermal stability, so it is necessary to make a comprehensive judgment based on the above-mentioned indicators and select the most suitable antibody.
  • an antibody gene When an antibody gene is once isolated and then introduced into an appropriate host to prepare an antibody, a combination of an appropriate host and an expression vector can be used.
  • Specific examples of the antibody gene include a combination of a gene encoding a heavy chain sequence of an antibody described herein and a gene encoding a light chain sequence.
  • the heavy chain sequence gene and the light chain sequence gene When transforming a host cell, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or can be inserted into different expression vectors. be.
  • animal cells, plant cells, and eukaryotic microorganisms can be used. Examples of animal cells include mammalian cells such as human infant kidney cells 293 (HEK293), monkey cells COS cells (Gluzman, Y.
  • mice Dihydrofolate reductase-deficient strains of fibroblastic NIH3T3 (ATCC No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chain, LA Proc. Natl. Acad. Sci. USA (1980) 77, p. 4126-4220) can be mentioned.
  • prokaryotic cells for example, Escherichia coli and Bacillus subtilis can be mentioned.
  • Antibodies can be obtained by introducing the target antibody gene into these cells by transformation and culturing the transformed cells in vitro. In the above culture method, the yield may differ depending on the sequence of the antibody, and it is possible to select an antibody having the same binding activity that is easy to produce as a pharmaceutical by using the yield as an index.
  • the isotype of the antibody of the present invention is not limited, and examples thereof include IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, IgE, etc., but IgG or IgM is preferable. More preferably, IgG1 or IgG2 can be mentioned.
  • the antibody of the present invention may be an antigen-binding fragment of an antibody having an antigen-binding portion of the antibody or a modified product thereof. Fragments of the antibody can be obtained by treating the antibody with a proteolytic enzyme such as papain or pepsin, or by modifying the antibody gene by a genetic engineering technique and expressing it in a suitable cultured cell. Among such antibody fragments, a fragment that retains all or part of the functions of the full-length antibody molecule can be referred to as an antigen-binding fragment of the antibody.
  • the functions of antibodies are generally antigen-binding activity, activity that neutralizes antigen activity, activity that enhances antigen activity, antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and complement-dependent cytotoxicity. Cell-mediated cytotoxic activity can be mentioned.
  • the function retained by the antigen-binding fragment of the antibody in the present invention is the binding activity to PTDSS2.
  • antibody fragments include Fab, F (ab') 2, Fv, or single chain Fv (scFv), diabody (diabodies), linear antibodies in which heavy and light chain Fv are linked with an appropriate linker. , And a multispecific antibody formed from an antibody fragment.
  • Fab' which is a monovalent fragment of the variable region of the antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the antibody fragment.
  • the obtained antibody can be purified uniformly.
  • the separation and purification methods used for ordinary proteins may be used. For example, by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, polyacrylamide gel electrophoresis for preparation, isoelectric point electrophoresis, etc., antibodies can be separated and purified (Strategies).
  • Protein Purification and Characterization A Laboratory Course Manual, Daniel R.Marshak et al.eds, Cold Spring Harbor Laboratory Press (1996); Antibodies:. A Laboratory Manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988))
  • chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography and the like. These chromatographies can be performed using liquid chromatography such as HPLC or FPLC.
  • column used for affinity chromatography examples include a protein A column and a protein G column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose F. F. (Pharmacia) and the like. It is also possible to purify the antibody by utilizing the binding property to the antigen by using the carrier on which the antigen is immobilized.
  • Diagnostic Composition The present invention also provides a test or diagnostic composition (hereinafter collectively referred to as "diagnostic composition") containing an anti-PTDSS2 antibody or a functional fragment thereof or a modified product thereof.
  • diagnostic composition containing an anti-PTDSS2 antibody or a functional fragment thereof or a modified product thereof.
  • the diagnostic composition of the present invention can detect the presence or absence of expression of PTDSS2 in a sample, and can further evaluate the expression level of PTDSS2 in a sample. Therefore, the diagnostic composition of the present invention is useful for detecting cancer or sarcoma in which PTDSS2, which is a target of synthetic lethal treatment with a PTDSS1 inhibitor, is not expressed or the expression level is low.
  • the test or diagnosis includes, for example, determination of the presence or absence of illness, measurement or determination of the effect of drug treatment with a pharmaceutical composition such as PTDSS1 inhibitor, measurement or determination of the effect of treatment other than drug treatment, and risk of recurrence. It includes, but is not limited to, measurement, determination of the presence or absence of recurrence, etc., as long as it is an examination or diagnosis.
  • the diagnostic composition may contain a pH buffer, an osmotic pressure regulator, salts, a stabilizer, a preservative, a color developer, a sensitizer, an anti-aggregation agent and the like.
  • the present invention relates to a method for testing or diagnosing a disease related to PTDSS2 such as cancer, the use of the antibody of the present invention for preparing a composition for diagnosing the disease, and the antibody of the present invention for testing or diagnosing the disease. Use, also provide.
  • Test or diagnostic kits comprising the antibodies of the invention are also included in the invention.
  • Sandwich ELISA is desirable as a method of examination or diagnosis using the diagnostic composition of the present invention, but ordinary ELISA method, RIA method, ELISA (Enzyme-Linked ImmunoSpot) method, dot blot method, octalony method, CIE (Counterimneuometry).
  • ELISA Enzyme-Linked ImmunoSpot
  • CIE Counterimneuometry
  • a detection method using an antibody such as CLIA (Chemiluminescence immunoassay) or FCM (Flow Cytometry) can be used.
  • CLIA Click immunoassay
  • FCM Flow Cytometry
  • the antibody labeling method in addition to biotin, a labeling method that can be carried out for biochemical analysis such as HRP, alkaline phosphatase, fluorescent groups such as FITC and ALEXA, and labels such as radioactive isotopes can be used.
  • TMB (3,3', 5,5'-tetramethylbenzidine)
  • BCIP (5-blomo-4-chlor-3-indolyl phosphate)
  • ⁇ -NPP ⁇ -nitrophenyl phosphate
  • OPD O-Phylenediamine
  • ABTS (3-Ethylbenzothiazoline-6-sulfonic acid)
  • SuperSignal ELISA PicoChemilluminescent Substrate Substrate (Thermo-Fischer Scientific Substrate)
  • a chemically luminescent substrate can be used.
  • test sample derived from an individual organism include, but are not limited to, blood, joint fluid, ascites, lymph fluid, cerebrospinal fluid, tissue homogenate supernatant, tissue section, and the like.
  • the test or diagnostic sandwich ELISA kit containing the antibody of the present invention contains a control (standard solution of PTDSS2-derived peptide), a coloring reagent, a buffer solution for dilution, an antibody for a solid phase, an antibody for detection, a washing solution, and the like. It's okay.
  • a control standard solution of PTDSS2-derived peptide
  • a coloring reagent e
  • the target test samples include solubilized proteins prepared from cells, circulating tumor cells in the blood, exosomes secreted by various cells including cancer cells, etc. contained in body fluids such as blood. It is not limited to them.
  • the present invention provides an antibody useful for analysis of immunohistochemistry (IHC), a functional fragment thereof and a modified product thereof, and a composition containing them. Such compositions are also included in the "diagnostic composition" of the present invention.
  • IHC immunohistochemistry
  • Immunohistochemistry is not particularly limited as long as it is a method of reacting a tissue section with an antibody (primary antibody) that binds to an antigen and detecting the primary antibody bound to the antigen.
  • Tissue sections are preferably paraffin-embedded after being fixed in formalin. After embedding in paraffin, the sliced tissue sections are deparaffinized, and then subjected to antigen activation treatment and non-specific reaction suppression treatment.
  • antigen activation treatment heat treatment, enzyme treatment with a protease or the like can be exemplified, and heat treatment is preferable.
  • a temperature range of 90 to 110 ° C., a pH of 8 to 10, and a treatment time of 20 to 60 minutes are usually suitable.
  • Tris-EDTA buffer for example, 10 mM Tris buffer containing 1 mM EDTA
  • a method of inactivating an endogenous enzyme having the same or similar catalytic activity as the enzyme used for color development is usually used.
  • the color is developed by the peroxidase reaction, it is preferable to inhibit the endogenous peroxidase existing in the tissue in advance with H2O2 or the like.
  • H 2 O 2 Water, methanol or the like can be used as the solvent for H 2 O 2 , and the concentration of H 2 O 2 is 0.1 to 3%, preferably 0.3 to 3%.
  • Sodium azide can be added to the H2O2 solution .
  • a method of blocking with serum or casein can also be used as a non-specific reaction suppression treatment. Serum and casein can be treated with tissue prior to the primary antibody reaction, but can also be contained in a solvent that dilutes the primary antibody.
  • the reaction conditions of the primary antibody are not particularly limited, but the temperature is 4 to 50 ° C, preferably 20 to 37 ° C, and more preferably 24 ° C.
  • the reaction time is 5 minutes to one day and night, preferably 10 minutes to 4 hours, and more preferably 30 minutes to 1 hour.
  • an antibody that can be visualized and binds to the primary antibody can be preferably used.
  • an antibody that binds to the secondary antibody itself (tertiary antibody) is used to perform the reaction three or more times.
  • an enzyme such as peroxidase or alkaline phosphatase is bound to those antibodies, or biotin or the like is added to those antibodies to bind to streptavidin or the like to which the enzyme is bound.
  • a method of reacting with a color-developing substrate corresponding to those enzymes can be preferably used.
  • a method for binding an enzyme to a secondary antibody or a tertiary antibody a method using a reagent in which a large number of the enzyme and the secondary antibody are bound to a dextrin polymer or an amino acid polymer can be exemplified (polymer method).
  • a biotinylated secondary antibody and peroxidase-labeled streptavidin (LSAB method) DAB or the like can be used as a color-developing substrate.
  • LSAB method biotinylated secondary antibody and peroxidase-labeled streptavidin
  • a secondary antibody labeled with a fluorescent dye or the like can also be used. When treated with a fluorescently labeled secondary antibody, positive cells are detected using a fluorescence microscope after the treatment.
  • the excised cells are coated on glass or separated by a centrifuge, separated into cell components and humoral components, and immunostaining is performed on the cell components. That is, after applying the cell component on a slide glass and fixing it with an ethanol solution or a 10% formalin solution, immunostaining similar to that of a tissue section can be performed.
  • the excised tissue is embedded in OCT compound or the like, then rapidly frozen in liquid nitrogen or the like, and sliced with a cryostat to prepare a slide specimen. After fixing this specimen with 10% formalin or ethanol solution, immunostaining similar to that of a tissue section can be performed.
  • Operations related to immunohistochemistry can be performed automatically by programming the reaction solution, reaction conditions, number of washings, etc., incorporating them into the immune device.
  • an antibody is labeled with a pharmaceutically acceptable radionuclide or luminescent material, the antibody is administered to the subject, an image is taken using diagnostic imaging techniques such as PET / CT, and the presence of PTDSS2 is present. Can be determined or inspected.
  • the antibody contained in the diagnostic composition of the present invention, a functional fragment thereof or a modified product thereof is preferably an antibody that binds to PTDSS2, that is, an antibody having PTDSS2 selectivity, a functional fragment thereof or a modified product thereof.
  • the present invention provides a method for detecting or measuring human PTDSS2 in a test sample.
  • the diagnostic composition of the present invention can be used for these detection or measurement methods. Such a measuring method and a diagnostic composition are also included in the present invention for diagnostic or testing for human PTDSS2-positive or negative cancer.
  • the present invention also includes a method for identifying a patient having cancer in which PTDSS2 is not expressed as a therapeutic target of a PTDSS1 inhibitor.
  • human PTDSS2 in the sample derived from the individual is measured and compared with the amount of human PTDSS2 detected in the sample, either negative of human PTDSS2 or detected in the sample derived from a healthy individual. If less or no negative is detected, the individual can be determined to be negative and identified as an individual to which the PTDSS1 inhibitor is administered.
  • the diagnostic composition of the present invention can be used in the method.
  • a pharmaceutical composition such as a PTDSS1 inhibitor can be administered to an individual determined to be negative by such an identification method in one embodiment thereof.
  • Reagent The antibody of the present invention, an antigen-binding fragment thereof, or a modified product thereof is also useful as a reagent. Such reagents are used for the above-mentioned tests or diagnostics, research and other uses.
  • PTDSS1 inhibitor of the present invention is not particularly limited, and is, for example, a synthetic small molecule compound library, an expression product of a gene library, a peptide library, siRNA, an antibody, a bacterial release substance, a cell (microorganism, plant cell). , Animal cell) extracts and culture supernatants, purified or partially purified polypeptides, marine organisms, plant or animal derived extracts, PTDSS1 inhibitors derived from random phage peptide display libraries. Examples of the PTDSS1 inhibitor include WO / 2020/179859, WO2016 / 148115, and Biochem. J. , 418, 421-429 (2009).
  • the PTDSS1 inhibitor can be made into a pharmaceutical product by mixing it with a pharmaceutically acceptable carrier and formulating it by a known pharmaceutical method.
  • Pharmaceutically acceptable carriers include, for example, sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, fragrances, excipients, vehicles, etc.
  • Preservatives, binders, diluents, tonics, soothing agents, bulking agents, disintegrants, buffers, coatings, lubricants, colorants, sweeteners, thickeners, flavoring agents, dissolution aids examples include, but are not limited to, agents or other additives.
  • Example 1 Preparation of rabbit anti-human PTDSS2 antibody 1
  • PTDSS2 partial peptide A peptide in which cysteine is added to the N-terminal side of the amino acid sequences 449 to 487 of human PTDSS2 (synthetic peptide sequence: [H] CQNKDDQGSTVGNGDQHPLGLDEDLLGPGVAEGEGAPTPN [OH]) (SEQ ID NO: 1) was synthesized according to a conventional method using a peptide synthesizer. This synthetic peptide was bound to the carrier protein KLH (keyhole limpet hemocyanin) or BSA (bovine serum albumin). In the following steps, the one bound to KLH was used as an immune antigen, and the one bound to BSA was used as a screening antigen.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the above-mentioned collected 2 ⁇ 10 5 lymphocytes were seeded, and then Goat anti-Labbit IgG (H + L) Highly Cross-Absorbed Secondary Antibody, Alexa Fluor 488 (Thermo10A10F).
  • 1 mL of a solution diluted 500-fold with RPMI-1640 containing% FBS was added, and the cells were allowed to stand for 45 minutes under the conditions of 37 ° C. and 5% CO 2 .
  • the solution was washed with RPMI-1640 containing 10% FBS, and lymphocytes from wells with positive signals were collected using AS ONE Cell Picking System (AS ONE Corporation).
  • pCEC3.1 vector a vector to which a constant region sequence of a CAG promoter and a rabbit antibody gene was preliminarily added.
  • a pCEC3.1 plasmid sample containing the antibody gene was prepared with E. coli. After transforming into coli JM109 Competent Cells (TAKARA, # 9052) and incubating on LB agar containing ampicillin at 37 ° C. overnight, it was confirmed that the gene was inserted into the plasmid sample by the colony PCR method.
  • a plasmid sample was purified using a GenElute (registered trademark) Prismid Miniprep Kit (SIGMA) and introduced into HEK293 cells using polyethyleneimine according to the transfection procedure.
  • GenElute registered trademark
  • SIGMA Prismid Miniprep Kit
  • the cells were statically cultured for 48 hours under the conditions of 37 ° C. and 5% CO 2 , and the culture supernatant secreted by the antibody was collected.
  • the ELISA test described below was performed using the culture supernatant, and a positive signal in the ELISA test was confirmed, and it was confirmed that the antibody gene of interest was inserted into the plasmid sample.
  • Peptide not homologous to the amino acid sequence from 449 to 487 of human PTDSS2 (SEQ ID NO: 18: [AC] RRGERRDAGGPRPESPVPAGRASLEEPPDGPSAGQATGPGEGRRC [OH]) was prepared as a negative control for the screening antigen, and the test was performed simultaneously under the same conditions as above. The negative control did not react on the plate immobilized on the solid phase, and was specific to the target screening antigen. The clone that reacted with was judged to be positive.
  • clone 401-P-17 was obtained as a positive clone.
  • 401-P-17 antibody which is an anti-PTDSS2 antibody derived from clone 401-P-17
  • sequence analysis of clone 401-P-17 was performed, and the signal sequences and variable regions of heavy and light chains were performed.
  • the nucleotide sequence of the cDNA was obtained.
  • the heavy chain nucleotide sequence of the 401-P-17 antibody is described in SEQ ID NO: 8 of the sequence listing, nucleotide number 1-57 is the signal sequence, 58-384 is the heavy chain variable region, and 385-1356 is the heavy chain constant. Shows the area.
  • SEQ ID NO: 8: 130-153 indicates CDRH1
  • 205-225 indicates CDRH2
  • 336-351 indicates CDRH3.
  • the heavy chain amino acid sequence of the 401-P-17 antibody is shown in SEQ ID NO: 2 and FIG. 1 of the sequence listing, where amino acid numbers 1-19 are the signal sequence, 20-128 are the heavy chain variable region, and 129-451. Indicates the heavy chain constant region.
  • the amino acid sequence of CDR determined by IMGT http://www.imgt.org/
  • indicates that amino acid numbers 44-51 of SEQ ID NO: 2 indicate CDRH1, 69-75 indicates CDRH2, and 112-116 indicate CDRH3. There is.
  • the amino acid sequence of the heavy chain variable region of the 401-P-17 antibody is shown in SEQ ID NO: 3 and FIG. 2 in the sequence listing.
  • the amino acid sequence of the heavy chain constant region of the 401-P-17 antibody is shown in SEQ ID NO: 4 and FIG. 3 of the sequence listing.
  • the amino acid sequence of CDRH1 of the 401-P-17 antibody is shown in SEQ ID NO: 5 and FIG. 4 of the sequence listing.
  • the amino acid sequence of CDRH2 of the 401-P-17 antibody is shown in SEQ ID NO: 6 and FIG. 5 of the sequence listing.
  • the amino acid sequence of CDRH3 of the 401-P-17 antibody is shown in SEQ ID NO: 7 and FIG. 6 of the sequence listing.
  • the light chain nucleotide sequence of the 401-P-17 antibody is set forth in SEQ ID NO: 16 of the sequence listing, nucleotide number 1-66 is the signal sequence, 67-405 is the light chain variable region, and 406-720 is the light chain constant. Shows the area.
  • SEQ ID NO: 16: 145-168 indicates CDRL1, 220-228 indicates CDRL2, and 337-375 indicates CDRL3.
  • the light chain amino acid sequence of the 401-P-17 antibody is shown in SEQ ID NO: 10 and FIG. 7 of the sequence listing, where amino acid numbers 1-22 are the signal sequence, 23-135 is the light chain variable region, 136-239.
  • amino acid numbers 49-56 of SEQ ID NO: 10 indicate CDRL1, 74-76 indicates CDRL2, and 113-125 indicate CDRL3. ..
  • the amino acid sequence of the light chain variable region of the 401-P-17 antibody is shown in SEQ ID NO: 11 and FIG. 8 of the sequence listing.
  • the amino acid sequence of the light chain constant region of the 401-P-17 antibody is shown in SEQ ID NO: 12 and FIG. 9 of the sequence listing.
  • the amino acid sequence of CDRL1 of the 401-P-17 antibody is set forth in SEQ ID NO: 13 and FIG. 10 of the sequence listing.
  • the amino acid sequence of CDRL2 of the 401-P-17 antibody is set forth in SEQ ID NO: 14 and FIG. 11 of the sequence listing.
  • the amino acid sequence of CDRL3 of the 401-P-17 antibody is set forth in SEQ ID NO: 15 and FIG. 12 of the sequence listing.
  • Example 2 IHC suitability evaluation of rabbit anti-human PTDSS2 antibody 2) -1 Stainability evaluation of PTDSS2 using wild-type A375 cells expressing PTDSS2 and A375 cells knocked out from the PTDSS2 gene 2) -1-1 Cell block Preparation of A375 cells were selected by knocking out the PTDSS2 gene and inactivating the gene in heterologus or homologus. Pellets of A375 cells of the wild type (wild type), heterologus or homologus knockout of the PTDSS2 gene were fixed with formalin and then used as a paraffin-embedded block.
  • the primary antibody 401-P-17 was diluted with Reaction Buffer with Antibody diluentz (Cat. No. 251-018, manufactured by Ventana) to the concentration shown in Table 2, and reacted at 37 ° C. for 60 minutes. After washing with Reaction Buffer, OptiView HQ Universal Linker or DISCOVERY anti-Rabbit HQ (manufactured by Ventana) was added and incubated for 8 minutes. After washing with Reaction Buffer, OptiView HRP Multimer (manufactured by Ventana) was added and incubated for 8 minutes. After washing with Reaction Buffer, OptiView H2O2 and OptiVier DAB (manufactured by Ventana) were added and incubated for 8 minutes.
  • FIGS. 13-1 and 13-2 The results of staining using the anti-PTDSS2 antibody (401-P-17 antibody) are shown in FIGS. 13-1 and 13-2.
  • Table 2 shows the results of dyeing evaluation in FIGS. 13-1 and 13-2.
  • the primary antibody concentration was 1, 2.5, 5 and 10 ⁇ g / mL, which were stained in four stages. WT and Hetero were positive, but WT showed stronger staining intensity compared to Hetero. In Homo, all were judged to be negative, but faint background staining was observed at 5 and 10 ⁇ g / mL. Therefore, it was confirmed that the stainability was sufficiently specific at 2.5 ⁇ g / mL or less. In WT, sufficient staining intensity was confirmed even at 1 ⁇ g / mL.
  • the stainability of the rabbit monoclonal anti-PTDSS2 antibody (clone 401-P-17) was examined.
  • an automatic immunostaining apparatus BenchMark Ultra (manufactured by Ventana) was used.
  • antigen activation was carried out at 100 ° C. for 56 minutes using an antigen activation solution ULTRA CC1 (manufactured by Ventana).
  • Reaction Buffer manufactured by Ventana
  • OptiView Peroxidase Inhibitor manufactured by Ventana
  • the primary antibody 401-P-17 was diluted with Reaction Buffer with Antibody diluentz (Cat. No. 251-018, manufactured by Ventana) to a concentration of 1 ⁇ g / mL, and reacted at 37 ° C. for 60 minutes.
  • Reaction Buffer with Antibody diluentz (Cat. No. 251-018, manufactured by Ventana) to a concentration of 1 ⁇ g / mL, and reacted at 37 ° C. for 60 minutes.
  • adjacent sections were similarly reacted with Normal Rabbit IgG (cat. # 2729, CST) instead of the primary antibody 401-P-17.
  • OptiView HQ Universal Linker or DISCOVERY anti-Labbit HQ manufactured by Ventana was added and incubated for 8 minutes.
  • FIGS. 14-1 and 14-2 The results of staining using the anti-PTDSS2 antibody (clone 401-P-17) are shown in FIGS. 14-1 and 14-2.
  • Table 3 shows the results of dyeing evaluation in FIGS. 14-1 and 14-2.
  • B4 and F9 showed strong positive tumors. At J4, the tumor was weakly positive. At E5, the tumor was negative.
  • the section in which Normal Rabbit IgG was added to the primary antibody as a negative control none showed slight staining property including non-specific staining. From the above, it was confirmed that positive or negative PTDSS2 expression can be discriminated even in human tumor tissue sections.
  • PTDSS2 Breast tissue tumor array, BR20811, US Biomax
  • the anti-PTDSS2 antibody provided by the present invention, it is possible to test or diagnose a disease related to the expression of PTDSS2.
  • a kit for testing or diagnosis of a disease related to the expression of PTDSS2 which comprises the anti-PTDSS2 antibody of the present invention.

Abstract

The present invention provides, inter alia, an antibody which specifically binds to PTDSS2, a PTDSS2 detection reagent containing an anti-PTDSS2 antibody, and a composition for examining or reagent for diagnosing diseases related to the expression of PTDSS2, said composition or reagent containing the anti-PTDSS2 antibody.　The present invention provides, inter alia, an antibody, antigen binding fragment thereof, or chimeric antibody thereof which specifically binds to human PTDSS2 or a partial peptide thereof; provides a composition containing the antibody; etc.

Description

抗ＰＴＤＳＳ２抗体Anti-PTDSS2 antibody
　本発明は、新規な抗ＰＴＤＳＳ２抗体、該抗体の機能断片、該抗体の修飾体、該抗体の有するアミノ酸配列をコードする塩基配列を含むヌクレオチド、該ヌクレオチドが挿入されたベクター、該ヌクレオチドまたはベクターが導入された細胞、該細胞を培養する工程を含む該抗体の製造方法、医薬組成物、診断用または検査用組成物等に関する。 The present invention comprises a novel anti-PTDSS2 antibody, a functional fragment of the antibody, a modified version of the antibody, a nucleotide containing a base sequence encoding the amino acid sequence of the antibody, a vector into which the nucleotide is inserted, the nucleotide or a vector. The present invention relates to introduced cells, a method for producing the antibody including a step of culturing the cells, a pharmaceutical composition, a composition for diagnosis or a test, and the like.
　ホスファチジルセリンは生理的条件下でその極性頭部に負電荷を持つ酸性リン脂質の一つで、細胞膜リン脂質の約５から１５％を占める。ホスファチジルセリンは哺乳動物細胞においてはホスファチジルセリンシンターゼ１（ＰＴＤＳＳ１と表記することがある。）およびホスファチジルセリンシンターゼ２（ＰＴＤＳＳ２と表記することがある）の２つの酵素により産生されることが知られている（非特許文献１，２）。 Phosphatidylserine is one of the acidic phospholipids having a negative charge on its polar head under physiological conditions, and occupies about 5 to 15% of the cell membrane phospholipids. Phosphatidylserine is known to be produced in mammalian cells by two enzymes, phosphatidylserine synthase 1 (sometimes referred to as PTDSS1) and phosphatidylserine synthase 2 (sometimes referred to as PTDSS2). (Non-Patent Documents 1 and 2).
　ＰＴＤＳＳ１はホスファチジルコリンのコリン部分をＬ－セリンに交換することでホスファチジルセリンを産生する酵素であり、ＰＴＤＳＳ２はホスファチジルエタノールアミンの並行塩基交換反応によりホスファチジルセリンを産生する酵素である（非特許文献１、２）。ＰＴＤＳＳ１とＰＴＤＳＳ２はアミノ酸配列において２８％程度共通している（非特許文献３、４）。 PTDSS1 is an enzyme that produces phosphatidylserine by exchanging the choline portion of phosphatidylcholine with L-serine, and PTDSS2 is an enzyme that produces phosphatidylserine by a parallel base exchange reaction of phosphatidylethanolamine (Non-Patent Documents 1 and 2). ). PTDSS1 and PTDSS2 have about 28% in common in amino acid sequences (Non-Patent Documents 3 and 4).
　ＰＴＤＳＳ１およびＰＴＤＳＳ２は合成致死の関係にあることが明らかになっている（特許文献１）。従って、ＰＴＤＳＳ１を阻害する治療は、ＰＴＤＳＳ２の機能抑制が生じているがん（以下、「ＰＴＤＳＳ２機能抑制型がん」と表記することもある）の治療のための有望なアプローチになる。 It has been clarified that PTDSS1 and PTDSS2 have a synthetic lethal relationship (Patent Document 1). Therefore, treatment that inhibits PTDSS1 is a promising approach for the treatment of cancers in which PTDSS2 function is suppressed (hereinafter, may be referred to as "PTDSS2 function-suppressing cancer").
　ＰＴＤＳＳ２機能抑制型がんの類型としては、がん組織中においてＰＴＤＳＳ２の発現が他のがんに比べて少ない場合や、全く発現していない場合、更に遺伝子変異等により機能が低下したり、機能を失っている場合を挙げることができる。 As a type of PTDSS2 function-suppressing cancer, when the expression of PTDSS2 in the cancer tissue is lower than that of other cancers, or when it is not expressed at all, the function is further reduced due to gene mutation or the like, or the function is reduced. Can be mentioned if you have lost.
　前述のように、ＰＴＤＳＳ１とＰＴＤＳＳ２は合成致死の関係にあり、ＰＴＤＳＳ１を阻害する治療の対象としてＰＴＤＳＳ２機能抑制型がんを見出す必要がある。がん組織中におけるＰＴＤＳＳ２の発現の有無や発現量を評価できる方法としてＰＴＤＳＳ２に対する抗体を用いた免疫組織化学的染色法を挙げることができる。免疫組織化学的染色法を用いることにより、ＰＴＤＳＳ２を発現しないがんや発現量が少ないがんを検出することが可能となる。 As mentioned above, PTDSS1 and PTDSS2 have a synthetic lethal relationship, and it is necessary to find a PTDSS2 function-suppressing cancer as a target for treatment that inhibits PTDSS1. An immunohistochemical staining method using an antibody against PTDSS2 can be mentioned as a method for evaluating the presence or absence of PTDSS2 expression in cancer tissue and the expression level. By using the immunohistochemical staining method, it becomes possible to detect cancers that do not express PTDSS2 or cancers that have a low expression level.
　従来、ホルマリン固定された組織中のヒトＰＴＤＳＳ２を検出できるとされるポリクローナル抗体は知られているが、検出感度や反応特異性が充分ではなく、均一な品質の抗体を提供するには問題があった。また、ＰＴＤＳＳ２の免疫組織化学染色に有用なモノクローナル抗体は知られていなかった。 Conventionally, polyclonal antibodies that can detect human PTDSS2 in formalin-fixed tissues are known, but the detection sensitivity and reaction specificity are not sufficient, and there is a problem in providing an antibody of uniform quality. rice field. In addition, no monoclonal antibody useful for immunohistochemical staining of PTDSS2 was known.
国際公開ＷＯ２０１６／１４８１１５パンフレットInternational release WO2016 / 148115 Pamphlet
　本発明の一つの課題は、ＰＴＤＳＳ２に特異的に結合する抗体を提供することである。 One object of the present invention is to provide an antibody that specifically binds to PTDSS2.
　本発明の他の一つの課題は抗ＰＴＤＳＳ２抗体を含むことからなるＰＴＤＳＳ２検出用試薬を提供することである。本発明の他の一つの課題は、抗ＰＴＤＳＳ２抗体を含むことからなる、ＰＴＤＳＳ２の発現と関連する疾患の診断用試薬または検査用組成物等を提供することである。 Another object of the present invention is to provide a reagent for detecting PTDSS2, which comprises an anti-PTDSS2 antibody. Another object of the present invention is to provide a diagnostic reagent or a test composition for a disease associated with the expression of PTDSS2, which comprises an anti-PTDSS2 antibody.
　また、本発明の課題には、該抗体が有するアミノ酸配列をコードするヌクレオチド、該ヌクレオチドが挿入されたベクター、該ヌクレオチドまたはベクターが導入された細胞、該細胞を培養する工程を含む該抗体の製造方法等が含まれる。 Further, the subject of the present invention includes a nucleotide encoding the amino acid sequence of the antibody, a vector into which the nucleotide is inserted, a cell into which the nucleotide or the vector has been introduced, and a step of culturing the antibody. Methods etc. are included.
　発明者らは上記課題を解決するために鋭意、検討を行い、新規な抗ＰＴＤＳＳ２抗体を創出し、該抗体を用いてＰＴＤＳＳ２を検出できることを見出し、本発明を完成させた。すなわち、本発明は、以下の発明を包含する。
（１）ヒトＰＴＤＳＳ２またはその部分ペプチドに特異的に結合する抗体または当該抗体の抗原結合性断片。
（１－１）抗体がモノクローナル抗体である、（1）に記載の抗体または当該抗体の抗原結合性断片。
（２）ヒトＰＴＤＳＳ２の部分ペプチドが配列表の配列番号1に記載のアミノ酸配列からなるペプチドである、（1）に記載の抗体または当該抗体の抗原結合性断片。
（３）ヒトＰＴＤＳＳ２またはその部分ペプチドへの結合に対して、配列番号２のアミノ酸番号２０乃至４５１に示されるアミノ酸配列からなる重鎖および配列番号１０のアミノ酸番号２３乃至２３９に示されるアミノ酸配列からなる軽鎖を有する抗体と競合阻害活性を有する抗体または当該抗体の抗原結合性断片。
（４）重鎖配列が、配列番号５に示されるアミノ酸配列からなるＣＤＲＨ１、配列番号６に示されるアミノ酸配列からなるＣＤＲＨ２、並びに、配列番号７に示されるアミノ酸配列からなるＣＤＲＨ３を有する可変領域を含み；
軽鎖配列が配列番号１３に示されるアミノ酸配列からなるＣＤＲＬ１、配列番号１４に示されるアミノ酸配列からなるＣＤＲＬ２、および配列番号１５に示されるアミノ酸配列からなるＣＤＲＬ３を有する可変領域を含む、（１）乃至（３）のいずれか1項に記載の抗体または当該抗体の抗原結合性断片。
（５）配列番号３に示されるアミノ酸配列からなる重鎖可変領域および、配列番号１１に示される軽鎖可変領域を含むことからなる、（１）乃至（４）のいずれか１項に記載の抗体または当該抗体の抗原結合性断片。
（６）配列番号２のアミノ酸番号２０乃至４５１またはアミノ酸番号２０乃至４５０に示されるアミノ酸配列を含むことからなる重鎖、および、配列番号１０のアミノ酸番号２３乃至２３９に示されるアミノ酸配列を含むことからなる軽鎖からなる（１）乃至（５）のいずれか１項に記載の抗体または当該抗体の抗原結合性断片。
（７）キメラ抗体であることを特徴とする（１）乃至（５）のいずれか１項に記載の抗体または当該抗体の抗原結合性断片。
（８）定常領域がウサギ抗体由来であることを特徴とする、（７）に記載のキメラ抗体。
（９）ヒト化されていることを特徴とする、（１）乃至（５）のいずれか１項に記載の抗体または当該抗体の抗原結合性断片。
（１０）Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’およびＦｖからなる群から選択される、（１）乃至（９）のいずれか１項に記載の抗体の抗原結合性断片。
（１１）ｓｃＦｖであることを特徴とする、（１）乃至（１０）のいずれか１項に記載の抗体。
（１２）（１）乃至（１１）のいずれか１項に記載の抗体または当該抗体の抗原結合性断片を含む組成物。
（１３）（１）乃至（１１）のいずれか１項に記載の抗体または当該抗体の抗原結合性断片を含み、パラフィン包埋処理した後脱パラフィン処理した組織標本中のＰＴＤＳＳ２の検出または測定方法に使用される、（１２）に記載の組成物。
（１４）（１）乃至（１１）のいずれか１項に記載の抗体または該抗体の抗原結合性断片と被検標本を接触させる工程を含む、標本中のＰＴＤＳＳ２の検出または測定方法に使用される、（１２）または（１３）に記載の組成物。上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。
（１５）ＰＴＤＳＳ２の検出または測定方法が、被検標本においてＰＴＤＳＳ２が検出もしくは測定されたか、または被検標本におけるＰＴＤＳＳ２の発現量が事前に決定された基準と同等かまたはそれより高い場合、該被検標本を陽性と判定し、被検標本においてＰＴＤＳＳ２が検出もしくは測定されなかったか、または被検標本におけるＰＴＤＳＳ２の発現量が事前に決定された基準と同等かまたはそれより低い場合、該被検標本を陰性と判定する工程を含む、（１３）または（１４）に記載の組成物。上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。
（１６）ＰＴＤＳＳ２陰性疾患（がん）の検査または診断方法に使用される、（１２）乃至（１５）のいずれか１項に記載の組成物。
（１７）（１）乃至（１１）のいずれか１項に記載の抗体または該抗体の抗原結合性断片をコードするポリヌクレオチド。
（１８）（１７）記載のポリヌクレオチドを含むベクター。
（１９）（１７）記載のポリヌクレオチドまたは（１８）記載のベクターを含む細胞。
（２０）下記の工程（ａ）および（ｂ）を含む、（１）乃至（１１）のいずれか１項に記載の抗体または該抗体の抗原結合性断片の製造方法：
（ａ）（１９）記載の細胞を培養する工程；
（ｂ）工程（ａ）の培養物からモノクローナル抗体または該抗体の抗原結合性断片を回収する工程。
（２１）被検標本を用い、該被検標本中におけるＰＴＤＳＳ２の有無を検出し（該検出には、（１）乃至（１１）のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ２が陰性と判定された被験者を、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療への応答性があると判定することを含む、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療への応答性を予測する方法。上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。
（２２）被検標本を用いてＰＴＤＳＳ２が陰性と判定された被験者を（該判定には、（１）乃至（１１）のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療への応答性があると判定することを含む、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療への応答性を予測する方法。上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。
（２３）被検標本を用い、該被検標本中におけるＰＴＤＳＳ２の有無を検出し（該検出には、（１）乃至（１１）のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ２が陰性と判定された被験者を、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療の対象として選別することを含む、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療の対象を選別する方法。上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。
（２４）被検標本を用いてＰＴＤＳＳ２が陰性と判定された被験者を（該判定には、（１）乃至（１１）のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療の対象として選別することを含む、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療の対象を選別する方法。上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。
（２５）被検標本を用い、該被検標本中におけるＰＴＤＳＳ２の有無を検出し（該検出には、（１）乃至（１１）のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ２が陰性と判定された被験者に対し、ＰＴＤＳＳ１阻害剤を投与することを含む、ＰＴＤＳＳ２陰性疾患（がん）の治療方法。上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。
（２６）被検標本を用いてＰＴＤＳＳ２の機能抑制が検出された被験者に対し（該検出には、（１）乃至（１１）のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ１阻害剤を投与することを含む、ＰＴＤＳＳ２陰性疾患（がん）の治療方法。上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。 The inventors have diligently studied to solve the above problems, created a new anti-PTDSS2 antibody, and found that PTDSS2 can be detected using the antibody, and completed the present invention. That is, the present invention includes the following inventions.
(1) An antibody that specifically binds to human PTDSS2 or a partial peptide thereof, or an antigen-binding fragment of the antibody.
(1-1) The antibody according to (1) or an antigen-binding fragment of the antibody, wherein the antibody is a monoclonal antibody.
(2) The antibody according to (1) or an antigen-binding fragment of the antibody, wherein the partial peptide of human PTDSS2 is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing.
(3) From the heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 451 of SEQ ID NO: 2 and the amino acid sequences shown in amino acid numbers 23 to 239 of SEQ ID NO: 10 for binding to human PTDSS2 or its partial peptide. An antibody having competitive inhibitory activity with an antibody having a light chain or an antigen-binding fragment of the antibody.
(4) A variable region in which the heavy chain sequence has CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 6, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 7. Including;
(1) The light chain sequence comprises a variable region having CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 13, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 15. (1). The antibody according to any one of (3) to (3) or an antigen-binding fragment of the antibody.
(5) The item according to any one of (1) to (4), which comprises a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 11. An antibody or antigen-binding fragment of the antibody.
(6) Containing a heavy chain consisting of amino acid numbers 20 to 451 of SEQ ID NO: 2 or amino acid sequences shown in amino acid numbers 20 to 450, and an amino acid sequence shown in amino acid numbers 23 to 239 of SEQ ID NO: 10. The antibody according to any one of (1) to (5), which comprises a light chain composed of a light chain, or an antigen-binding fragment of the antibody.
(7) The antibody according to any one of (1) to (5), which is a chimeric antibody, or an antigen-binding fragment of the antibody.
(8) The chimeric antibody according to (7), wherein the constant region is derived from a rabbit antibody.
(9) The antibody according to any one of (1) to (5) or an antigen-binding fragment of the antibody, which is characterized by being humanized.
(10) The antigen-binding fragment of the antibody according to any one of (1) to (9), which is selected from the group consisting of Fab, F (ab') 2, Fab'and Fv.
(11) The antibody according to any one of (1) to (10), which is scFv.
(12) A composition comprising the antibody according to any one of (1) to (11) or an antigen-binding fragment of the antibody.
(13) A method for detecting or measuring PTDSS2 in a tissue specimen containing the antibody according to any one of (1) to (11) or an antigen-binding fragment of the antibody and subjected to paraffin embedding treatment and then deparaffin treatment. The composition according to (12), which is used in.
(14) Used in a method for detecting or measuring PTDSS2 in a specimen, which comprises a step of contacting the test specimen with the antibody according to any one of (1) to (11) or an antigen-binding fragment of the antibody. , (12) or (13). The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
(15) If the method for detecting or measuring PTDSS2 is such that PTDSS2 is detected or measured in the test specimen, or the expression level of PTDSS2 in the test specimen is equal to or higher than a predetermined criterion, the subject is said to be the subject. If the test sample is judged to be positive and PTDSS2 is not detected or measured in the test sample, or if the expression level of PTDSS2 in the test sample is equal to or lower than a predetermined criterion, the test sample is concerned. The composition according to (13) or (14), which comprises a step of determining negative. The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
(16) The composition according to any one of (12) to (15), which is used for a test or diagnostic method for a PTDSS2-negative disease (cancer).
(17) A polynucleotide encoding the antibody according to any one of (1) to (11) or an antigen-binding fragment of the antibody.
(18) A vector containing the polynucleotide according to (17).
(19) A cell containing the polynucleotide according to (17) or the vector according to (18).
(20) The method for producing an antibody or an antigen-binding fragment of the antibody according to any one of (1) to (11), which comprises the following steps (a) and (b):
(A) The step of culturing the cells according to (19);
(B) A step of recovering a monoclonal antibody or an antigen-binding fragment of the antibody from the culture of step (a).
(21) Using the test sample, the presence or absence of PTDSS2 in the test sample is detected (for the detection, the antibody according to any one of (1) to (11) or the antigen binding property of the antibody. PTDSS2-negative disease (using a fragment), a PTDSS2-negative disease due to a PTDSS1 inhibitor, including determining that a subject determined to be PTDSS2 negative is responsive to treatment of a PTDSS2-negative disease (cancer) with a PTDSS1 inhibitor. How to predict the responsiveness to treatment. The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
(22) A subject whose PTDSS2 is determined to be negative using the test specimen (for the determination, the antibody according to any one of (1) to (11) or an antigen-binding fragment of the antibody is used. ), A method for predicting the responsiveness of a PTDSS1 inhibitor to the treatment of a PTDSS2-negative disease (cancer), which comprises determining that the PTDSS1 inhibitor is responsive to the treatment of a PTDSS2-negative disease (cancer). The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
(23) Using the test sample, the presence or absence of PTDSS2 in the test sample is detected (for the detection, the antibody according to any one of (1) to (11) or the antigen binding property of the antibody. Treatment of PTDSS2-negative disease (cancer) with PTDSS1 inhibitor, including selecting subjects who are determined to be negative for PTDSS2 (using fragments) for treatment of PTDSS2-negative disease (cancer) with PTDSS1 inhibitor. How to select the target of. The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
(24) A subject whose PTDSS2 is determined to be negative using the test specimen (for the determination, the antibody according to any one of (1) to (11) or an antigen-binding fragment of the antibody is used. ), A method for selecting a target for treatment of a PTDSS2-negative disease (cancer) with a PTDSS1 inhibitor, which comprises selecting the target for treatment of a PTDSS2-negative disease (cancer) with a PTDSS1 inhibitor. The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
(25) Using the test specimen, the presence or absence of PTDSS2 in the test specimen is detected (for the detection, the antibody according to any one of (1) to (11) or the antigen binding property of the antibody. A method for treating a PTDSS2-negative disease (cancer), which comprises administering a PTDSS1 inhibitor to a subject who is determined to be negative for PTDSS2 (using a fragment). The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
(26) The antibody according to any one of (1) to (11) or an antigen-binding fragment of the antibody was detected in a subject in which the suppression of PTDSS2 function was detected using the test specimen. A method for treating a PTDSS2-negative disease (cancer), which comprises administering a PTDSS1 inhibitor. The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
401-P-17 重鎖アミノ酸配列401-P-17 Heavy chain amino acid sequence 401-P-17 重鎖可変領域アミノ酸配列401-P-17 Heavy chain variable region amino acid sequence 401-P-17重鎖定常領域アミノ酸配列401-P-17 Heavy chain constant region amino acid sequence CDRH1アミノ酸配列CDRH1 amino acid sequence CDRH2アミノ酸配列CDRH2 amino acid sequence CDRH3アミノ酸配列CDRH3 amino acid sequence 401-P-17 軽鎖アミノ酸配列401-P-17 Light chain amino acid sequence 401-P-17軽鎖可変領域アミノ酸配列401-P-17 Light chain variable region amino acid sequence 401-P-17軽鎖定常領域アミノ酸配列401-P-17 Light chain constant region amino acid sequence 401-P-17　CDRL1アミノ酸配列401-P-17 CDRL1 amino acid sequence 401-P-17　CDRL2アミノ酸配列401-P-17 CDRL2 amino acid sequence 401-P-17　CDRL3アミノ酸配列401-P-17 CDRL3 amino acid sequence Ａ３７５細胞（野生型，　ＷＴ；　ｈｅｔｅｒｏｌｏｇｕｓ　ＫＯ，　Ｈｅｔｅｒｏ；　Ｈｏｍｏｌｏｇｕｓ　ＫＯ，Ｈｏｍｏ）におけるＰＴＤＳＳ２の免疫染色像を示す。図１３－１はウサギ抗ＰＴＤＳＳ２モノクローナル抗体の染色像を示している。The immunostaining image of PTDSS2 in A375 cells (wild type, WT; heterologus KO, Hetero; Homologus KO, Homo) is shown. FIG. 13-1 shows a stained image of a rabbit anti-PTDSS2 monoclonal antibody. Ａ３７５細胞（野生型，　ＷＴ；　ｈｅｔｅｒｏｌｏｇｕｓ　ＫＯ，　Ｈｅｔｅｒｏ；　Ｈｏｍｏｌｏｇｕｓ　ＫＯ，Ｈｏｍｏ）におけるＰＴＤＳＳ２の免疫染色像を示す。図１３－２はウサギ抗ＰＴＤＳＳ２モノクローナル抗体の染色像を示している。The immunostaining image of PTDSS2 in A375 cells (wild type, WT; heterologus KO, Hetero; Homologus KO, Homo) is shown. FIG. 13-2 shows a stained image of the rabbit anti-PTDSS2 monoclonal antibody. ヒト乳がん組織におけるＰＴＤＳＳ２の免疫染色像を示す。図１４－１はウサギ抗ＰＴＤＳＳ２モノクローナル抗体の染色像と陰性対照としてＮｏｒｍａｌ　Ｒａｂｂｉｔ　ＩｇＧの染色像を示している。The immunostaining image of PTDSS2 in the human breast cancer tissue is shown. FIG. 14-1 shows a stained image of a rabbit anti-PTDSS2 monoclonal antibody and a stained image of Normal Rabbit IgG as a negative control. ヒト乳がん組織におけるＰＴＤＳＳ２の免疫染色像を示す。図１４－２はウサギ抗ＰＴＤＳＳ２モノクローナル抗体の染色像と陰性対照としてＮｏｒｍａｌ　Ｒａｂｂｉｔ　ＩｇＧの染色像を示している。The immunostaining image of PTDSS2 in the human breast cancer tissue is shown. FIG. 14-2 shows a stained image of a rabbit anti-PTDSS2 monoclonal antibody and a stained image of Normal Rabbit IgG as a negative control.
　１．定義
　本発明において、「遺伝子」とは、蛋白質のアミノ酸配列をコードする塩基配列が含まれるヌクレオチドまたはその相補鎖を意味し、例えば、蛋白質のアミノ酸配列をコードする塩基配列が含まれるヌクレオチドまたはその相補鎖であるポリヌクレオチド、オリゴヌクレオチド、ＤＮＡ、ｍＲＮＡ、ｃＤＮＡ、ｃＲＮＡ等は「遺伝子」の意味に含まれる。かかる遺伝子は一本鎖、二本鎖または三本鎖以上のヌクレオチドであり、ＤＮＡ鎖とＲＮＡ鎖の会合体、一本のヌクレオチド鎖上にリボヌクレオチド（ＲＮＡ）とデオキシリボヌクレオチド（ＤＮＡ）が混在するものおよびそのようなヌクレオチド鎖を含む二本鎖または三本鎖以上のヌクレオチドも「遺伝子」の意味に含まれる。本発明の「ＰＴＤＳＳ２遺伝子」としては、例えば、ＰＴＤＳＳ２蛋白質のアミノ酸配列をコードする塩基配列が含まれるＤＮＡ、ｍＲＮＡ、ｃＤＮＡ、ｃＲＮＡ等を挙げることができる。 1. 1. Definition In the present invention, the "gene" means a nucleotide containing a base sequence encoding a protein's amino acid sequence or a complementary strand thereof, and for example, a nucleotide containing a base sequence encoding a protein's amino acid sequence or its complement. Strands such as polynucleotides, oligonucleotides, DNAs, mRNAs, cDNAs, and cRNAs are included in the meaning of "gene". Such genes are single-stranded, double-stranded or triple-stranded or higher nucleotides, which are aggregates of DNA strands and RNA strands, in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed on one nucleotide strand. Nucleotides and double-stranded or triple-stranded or higher nucleotides containing such nucleotide chains are also included in the meaning of "gene". Examples of the "PTDSS2 gene" of the present invention include DNA, mRNA, cDNA, cRNA and the like containing a base sequence encoding the amino acid sequence of the PTDSS2 protein.
　本発明において、「ヌクレオチド」と「核酸」は同義であり、例えば、ＤＮＡ、ＲＮＡ、プローブ、オリゴヌクレオチド、ポリヌクレオチド、プライマー等も「ヌクレオチド」の意味に含まれる。かかるヌクレオチドは一本鎖、二本鎖または三本以上の鎖からなるヌクレオチドであり、ＤＮＡ鎖とＲＮＡ鎖の会合体、一本のヌクレオチド鎖上にリボヌクレオチド（ＲＮＡ）とデオキシリボヌクレオチド（ＤＮＡ）が混在するものおよびそのようなヌクレオチド鎖を含む二本鎖または三本以上の鎖の会合体も「ヌクレオチド」の意味に含まれる。 In the present invention, "nucleotide" and "nucleic acid" are synonymous, and for example, DNA, RNA, probe, oligonucleotide, polynucleotide, primer and the like are also included in the meaning of "nucleotide". Such a nucleotide is a nucleotide consisting of a single strand, a double strand, or three or more strands, and an aggregate of a DNA strand and an RNA strand, a ribonucleotide (RNA) and a deoxyribonucleotide (DNA) on the single nucleotide strand. Consolidations and aggregates of double or three or more strands containing such nucleotide chains are also included in the meaning of "nucleotide".
　本発明において、「ポリペプチド」、「ペプチド」および「蛋白質」は同義である。 In the present invention, "polypeptide", "peptide" and "protein" are synonymous.
　本発明において、「抗原」を「免疫原」の意味に用いることがある。 In the present invention, "antigen" may be used to mean "immunogen".
　本発明において、「細胞」には、動物個体に由来する各種細胞、継代培養細胞、初代培養細胞、細胞株、組換え細胞および微生物等も含まれる。 In the present invention, the "cell" also includes various cells derived from individual animals, subcultured cells, primary cultured cells, cell lines, recombinant cells, microorganisms and the like.
　本発明においては、ＰＴＤＳＳ２を認識する抗体を、それぞれ「抗ＰＴＤＳＳ２抗体」と表記することがある。かかる抗体には、ポリクローナル抗体、モノクローナル抗体、キメラ化抗体、ウサギ化抗体、ヒト化抗体、ヒト抗体等が含まれる。 In the present invention, the antibody that recognizes PTDSS2 may be referred to as "anti-PTDSS2 antibody". Such antibodies include polyclonal antibodies, monoclonal antibodies, chimerized antibodies, rabbitized antibodies, humanized antibodies, human antibodies and the like.
　本発明における「抗体の機能断片」とは、元の抗体が奏する機能の少なくとも一部を奏する抗体断片を意味する。「抗体の機能断片」としては、例えば、Ｆａｂ、Ｆ（ａｂ’）２、ｓｃＦｖ、Ｆａｂ’、一本鎖免疫グロブリン等を挙げることができるが、それらに限定されるものではない。かかる抗体の機能断片は、抗体蛋白質の全長分子をパパイン、ペプシン等の酵素で処理することによって得られたものに加え、組換え遺伝子を用いて適当な宿主細胞において産生された組換え蛋白質であってもよい。 The "functional fragment of an antibody" in the present invention means an antibody fragment that plays at least a part of the function of the original antibody. Examples of the "functional fragment of the antibody" include, but are not limited to, Fab, F (ab') 2, scFv, Fab', single-chain immunoglobulin and the like. The functional fragment of such an antibody is a recombinant protein produced in a suitable host cell using a recombinant gene in addition to the one obtained by treating the full-length molecule of the antibody protein with an enzyme such as papain or pepsin. May be.
　本発明において、抗体が結合する「部位」、すなわち抗体が認識する「部位」とは、抗体が結合または認識する抗原上の部分ペプチドまたは部分高次構造を意味する。本発明においては、かかる部位のことをエピトープ、抗体の結合部位とも呼ぶ。本発明の抗ＰＴＤＳＳ２抗体が結合または認識するＰＴＤＳＳ２蛋白質上の部位としては、ＰＴＤＳＳ２蛋白質上の部分ペプチドまたは部分高次構造等を例示することができる。 In the present invention, the "site" to which the antibody binds, that is, the "site" recognized by the antibody means a partial peptide or a partial higher-order structure on the antigen to which the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or an antibody binding site. Examples of the site on the PTDSS2 protein to which the anti-PTDSS2 antibody of the present invention binds or recognizes include a partial peptide or a partial higher-order structure on the PTDSS2 protein.
　抗体分子の重鎖および軽鎖にはそれぞれ３箇所の相補性決定領域（ＣＤＲ：Ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ　ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ）があることが知られている。相補性決定領域は、超可変領域（ｈｙｐｅｒｖａｒｉａｂｌｅ　ｄｏｍａｉｎ）とも呼ばれ、抗体の重鎖および軽鎖の可変領域内にあって、一次構造の変異性が特に高い部位であり、重鎖および軽鎖のポリペプチド鎖の一次構造上において、通常、それぞれ３ヶ所に分離している。本発明においては、抗体の相補性決定領域について、重鎖の相補性決定領域を重鎖アミノ酸配列のアミノ末端側からＣＤＲＨ１、ＣＤＲＨ２、ＣＤＲＨ３と表記し、軽鎖の相補性決定領域を軽鎖アミノ酸配列のアミノ末端側からＣＤＲＬ１、ＣＤＲＬ２、ＣＤＲＬ３と表記する。これらの部位は立体構造の上で相互に近接し、結合する抗原に対する特異性を決定している。 It is known that the heavy chain and the light chain of the antibody molecule each have three complementarity determining regions (CDRs). Complementarity determining regions, also called hypervariable domains, are sites within the variable regions of the heavy and light chains of an antibody that are particularly highly variable in primary structure and of the heavy and light chains. On the primary structure of the polypeptide chain, they are usually separated into three locations. In the present invention, the complementarity determining regions of an antibody are referred to as CDRH1, CDRH2, and CDRH3 from the amino-terminal side of the heavy chain amino acid sequence, and the complementarity determining regions of the light chain are represented by light chain amino acids. From the amino terminal side of the sequence, they are referred to as CDRL1, CDRL2, and CDRL3. These sites are close to each other on the three-dimensional structure and determine the specificity for the antigen to which they bind.
　本発明において、「抗体変異体」とは、元の抗体が有するアミノ酸配列においてアミノ酸が置換、欠失、付加および／または挿入（以下、「変異」と総称する）してなるアミノ酸配列を有し、且つ本発明のＰＴＤＳＳ２蛋白質に結合するポリペプチドを意味する。かかる抗体変異体における変異アミノ酸の数は、１乃至２個、１乃至３個、１乃至４個、１乃至５個、１乃至６個、１乃至７個、１乃至８個、１乃至９個、１乃至１０個、１乃至１２個、１乃至１５個、１乃至２０個、１乃至２５個、１乃至３０個、１乃至４０個または１乃至５０個である。かかる抗体変異体も本発明の「抗体」に包含される。 In the present invention, the "antibody variant" has an amino acid sequence in which an amino acid is substituted, deleted, added and / or inserted (hereinafter collectively referred to as "mutation") in the amino acid sequence of the original antibody. And, it means a polypeptide that binds to the PTDSS2 protein of the present invention. The number of mutant amino acids in such antibody variants is 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9. 1 to 10 pieces, 1 to 12 pieces, 1 to 15 pieces, 1 to 20 pieces, 1 to 25 pieces, 1 to 30 pieces, 1 to 40 pieces or 1 to 50 pieces. Such antibody variants are also included in the "antibodies" of the present invention.
　本発明において、「１乃至数個」における「数個」とは、３乃至１０個を指す。 In the present invention, "several pieces" in "1 to several pieces" means 3 to 10 pieces.
　本発明の抗体が奏する活性・性質としては、例えば、生物的活性、理化学的性質等を挙げることができ、具体的には、各種生物活性、抗原やエピトープに対する結合活性、製造や保存時における安定性、熱安定性等を挙げることができる。 Examples of the activity / property exhibited by the antibody of the present invention include biological activity, physicochemical property, etc. Specifically, various biological activities, binding activity to antigens and epitopes, stability during production and storage. Properties, thermal stability, etc. can be mentioned.
　本発明において、「ストリンジェントな条件下でハイブリダイズする」とは、５×ＳＳＣを含む溶液中で６５℃にてハイブリダイゼーションを行い、ついで２×ＳＳＣ－０．１％ＳＤＳを含む水溶液中で６５℃にて２０分間、０．５×ＳＳＣ－０．１％ＳＤＳを含む水溶液中で６５℃にて２０分間、ならびに、０．２×ＳＳＣ－０．１％ＳＤＳを含む水溶液中で６５℃にて２０分間、それぞれ洗浄する条件またはそれと同等の条件でハイブリダイズすることを意味する。ＳＳＣとは１５０ｍＭＮａＣｌ－１５ｍＭクエン酸ナトリウムの水溶液であり、ｎ×ＳＳＣはｎ倍濃度のＳＳＣを意味する。 In the present invention, "hybridizing under stringent conditions" means that hybridization is performed at 65 ° C. in a solution containing 5 × SSC, and then in an aqueous solution containing 2 × SSC-0.1% SDS. 65 ° C. for 20 minutes at 65 ° C. in an aqueous solution containing 0.5 × SSC-0.1% SDS and 65 ° C. in an aqueous solution containing 0.2 × SSC-0.1% SDS. It means that the cells are hybridized under the conditions of washing or equivalent for 20 minutes. SSC is an aqueous solution of 150 mM NaCl-15 mM sodium citrate, and n × SSC means n times the concentration of SSC.
　本発明において「細胞傷害」とは、何らかの形で、細胞に病理的な変化をもたらすことを指し、直接的な外傷にとどまらず、ＤＮＡの切断や塩基の二量体の形成、染色体の切断、細胞分裂装置の損傷、各種酵素活性の低下などあらゆる細胞の構造や機能上の損傷を意味する。 In the present invention, "cell injury" refers to causing pathological changes in cells in some way, and is not limited to direct trauma, but also DNA cleavage, base dimer formation, chromosomal cleavage, etc. It means damage to the structure and function of all cells, such as damage to the cell division device and decreased activity of various enzymes.
　本発明において「細胞傷害活性」とは上記細胞傷害を引き起こすことを意味する。本発明において「抗体依存性細胞傷害活性」とは、「ａｎｔｉｂｏｄｙ　ｄｅｐｅｎｄｅｎｔ　ｃｅｌｌｕｌａｒ　ｃｙｔｏｔｏｘｉｃｉｔｙ（ＡＤＣＣ）活性」を指し、ＮＫ細胞が抗体を介して腫瘍細胞等の標的細胞を傷害する作用活性を意味する。 In the present invention, "cytotoxic activity" means to cause the above-mentioned cytotoxicity. In the present invention, "antibody-dependent cellular cytotoxicity (ADCC) activity" refers to "antibody dependent cellular cytotoxicity (ADCC) activity", and means an action activity in which NK cells damage target cells such as tumor cells via an antibody.
　本発明において、用語「腫瘍」および「がん」は交換可能に使用される。また、本発明において、腫瘍、悪性腫瘍、がん、悪性新生物、がん腫、肉腫等を総称して、「腫瘍」または「がん」と表現する場合がある。 In the present invention, the terms "tumor" and "cancer" are used interchangeably. Further, in the present invention, tumors, malignant tumors, cancers, malignant neoplasms, cancers, sarcomas and the like may be collectively referred to as "tumors" or "cancers".
　本発明において「免疫組織化学（ｉｍｍｕｎｏｈｉｓｔｏｃｈｅｍｉｓｔｒｙ：ＩＨＣ）」とは組織標本中の抗原を検出する組織学的（組織化学的）手法を意味し、「免疫抗体法」と同義であり、「免疫染色（ｉｍｍｕｎｏｓｔａｉｎｉｎｇ）」も同じ意味に用いられる。 In the present invention, "immunohistochemistry (IHC)" means a histological (histochemical) method for detecting an antigen in a tissue sample, and is synonymous with "immunohistochemical method" and is synonymous with "immunostaining (immunostaining (iHC)". "Immunostaining)" is also used interchangeably.
　本発明において「変性型ＰＴＤＳＳ２」とは、ホルマリンで固定した標本中のＰＴＤＳＳ２分子を意味する。ホルマリンで固定した後にパラフィン処理および脱パラフィン処理した標本中のＰＴＤＳＳ２分子も「変性型ＰＴＤＳＳ２」という。 In the present invention, "modified PTDSS2" means a PTDSS2 molecule in a specimen fixed with formalin. The PTDSS2 molecules in the specimens that have been fixed with formalin and then paraffinized and deparaffinized are also referred to as "modified PTDSS2".
　本発明において「非変性型ＰＴＤＳＳ２」とは、ホルマリンで固定していないサンプル中のＰＴＤＳＳ２を意味する。ホルマリンで固定していない標本中のＰＴＤＳＳ２分子も「非変性型ＰＴＤＳＳ２」という。 In the present invention, "non-denatured PTDSS2" means PTDSS2 in a sample not fixed with formalin. The PTDSS2 molecule in the specimen not fixed with formalin is also called "non-denatured PTDSS2".
　本発明において、「被検標本」とは、被験者由来の生物学的試料を意味する。 In the present invention, the "test specimen" means a biological sample derived from a subject.
　本発明において「被験者」とは、ＰＴＤＳＳ１阻害剤に対する応答性を予測する方法による検査を受けるヒトおよびヒト以外の哺乳動物を意味する。例えば、ＰＴＤＳＳ１阻害剤による治療効果が期待される疾患に罹患したヒトおよびヒト以外の哺乳動物を意味する（好ましくは、ヒトである）。ＰＴＤＳＳ１阻害剤による治療効果が期待される疾患として、ＰＴＤＳＳ１阻害剤による治療効果が期待される疾患であれば特に限定されないが、好ましくはがんを例示できる。ヒト以外の哺乳動物は、哺乳動物として類別される生物であればいかなる生物であってもよく、例えばモルモット、ラット、マウス、ウサギ、ブタ、ヒツジ、ウシ、サル、等を含む。本発明における「被験者」として、がんを疑われたヒトおよびヒト以外の哺乳動物、およびがんと診断されたヒトおよびヒト以外の哺乳動物を好ましく例示できる。 In the present invention, the "subject" means a human and a non-human mammal undergoing a test by a method for predicting responsiveness to a PTDSS1 inhibitor. For example, it means humans and non-human mammals suffering from a disease that is expected to have a therapeutic effect by a PTDSS1 inhibitor (preferably humans). The disease in which the therapeutic effect of the PTDSS1 inhibitor is expected is not particularly limited as long as it is a disease in which the therapeutic effect of the PTDSS1 inhibitor is expected, but cancer can be preferably exemplified. The non-human mammal may be any organism as long as it is classified as a mammal, and includes, for example, guinea pigs, rats, mice, rabbits, pigs, sheep, cows, monkeys, and the like. As the "subject" in the present invention, humans and non-human mammals suspected of having cancer, and humans and non-human mammals diagnosed with cancer can be preferably exemplified.
　本発明において「生物学的試料」は、個体から単離された組織、液体、細胞、またはそれらの２種以上の混合物をいい、例えば腫瘍生検、髄液、胸腔内液、腹腔内液、リンパ液、皮膚切片、血液、尿、糞便、痰、呼吸器、腸管、尿生殖器管、唾液、乳、消化器官、またはこれらから採取された細胞を挙げることができるが、特に限定されない。「生物学的試料」は、例えば、がんの治療目的で行われた手術の際に得られた切除組織の一部、がんを疑われた対象者から生検等によって採取された組織の一部、血液あるいは胸腔内液や腹腔内液に由来する細胞を好ましく例示できる。 In the present invention, a "biological sample" refers to a tissue, fluid, cell, or a mixture of two or more thereof isolated from an individual, for example, tumor biopsy, spinal fluid, intrathoracic fluid, intraperitoneal fluid, etc. Examples thereof include, but are not limited to, lymph, skin sections, blood, urine, feces, sputum, respiratory organs, intestinal tract, urogenital tract, saliva, milk, digestive organs, or cells collected from these. A "biological sample" is, for example, a part of excised tissue obtained during surgery performed for the purpose of treating cancer, or tissue collected by biopsy from a subject suspected of having cancer. Cells derived in part from blood or intrathoracic or intraperitoneal fluid can be preferably exemplified.
　生物学的試料は、個体から単離された組織、液体、細胞、またはそれらの２種以上の混合物等から調製したタンパク質抽出液や核酸抽出液であっても良い。タンパク質抽出液や核酸抽出液の調製は、自体公知のタンパク質調製法や核酸調製法を利用して実施できる。 The biological sample may be a protein extract or a nucleic acid extract prepared from a tissue, a liquid, a cell, or a mixture of two or more kinds thereof isolated from an individual. The protein extract and the nucleic acid extract can be prepared by using a protein preparation method or a nucleic acid preparation method known per se.
　生物学的試料は、ＰＴＤＳＳ１阻害剤による治療を行う前に採取された生物学的試料が好ましい。このような生物学的試料を使用することにより、ＰＴＤＳＳ１阻害剤による治療を実施する前にＰＴＤＳＳ１阻害剤に対する感受性予測が可能になり、その結果、被験者に対してＰＴＤＳＳ１阻害剤を含む治療を適用するか否かの判定、すなわち、ＰＴＤＳＳ１阻害剤を含む治療を適用する被験者の選別を実施することができる。 The biological sample is preferably a biological sample collected before treatment with a PTDSS1 inhibitor. The use of such biological samples makes it possible to predict susceptibility to PTDSS1 inhibitors prior to treatment with PTDSS1 inhibitors, and as a result, apply treatments containing PTDSS1 inhibitors to subjects. It is possible to determine whether or not the subject is to be treated, that is, to select a subject to which the treatment containing the PTDSS1 inhibitor is applied.
　本発明において、ホスファチジルセリンシンターゼ１（ＰＴＤＳＳ１）とは、ＰＴＤＳＳ１遺伝子によりコードされるタンパク質である。ヒトＰＴＤＳＳ１遺伝子はＮＣＢＩにＧｅｎｅ　ＩＤ：　９７９１，　ＲｅｆＳＥＱ：　ａｃｃｅｓｓｉｏｎ　ＮＭ＿０１４７５４．２（タンパク質：ＲｅｆＳｅｑ　ＮＰ＿０５５５６９．１）として登録されている。 In the present invention, phosphatidylserine synthase 1 (PTDSS1) is a protein encoded by the PTDSS1 gene. The human PTDSS1 gene is registered in NCBI as Gene ID: 9791, RefSEQ: accession NM_014754.2 (protein: RefSeq NP_055569.1).
　本発明において、ホスファチジルセリンシンターゼ２（ＰＴＤＳＳ２）とは、ＰＴＤＳＳ２遺伝子によりコードされるタンパク質である。ヒトＰＴＤＳＳ２遺伝子はＮＣＢＩにＧｅｎｅ　ＩＤ：　８１４９０，　ＲｅｆＳＥＱ　：ａｃｃｅｓｓｉｏｎ　ＮＭ＿０３０７８３．１＿（タンパク質：ＲｅｆＳｅｑ　ＮＰ＿１１０４１０．１）として登録されている。 In the present invention, phosphatidylserine synthase 2 (PTDSS2) is a protein encoded by the PTDSS2 gene. The human PTDSS2 gene is registered in NCBI as Gene ID: 81490, RefSEQ: accession NM_030783.1_ (protein: RefSeq NP_110410.1).
　２．ＰＴＤＳＳ２
　本発明で用いるＰＴＤＳＳ２は、ヒト、ヒト以外の哺乳動物（例えば、モルモット、ラット、マウス、ウサギ、ブタ、ヒツジ、ウシ、サルなど）あるいはニワトリのＴ細胞あるいは肥満細胞から直接精製して使用するか、あるいは上記の細胞の細胞膜画分を調製して使用することができ、また、ＰＴＤＳＳ２をｉｎ　ｖｉｔｒｏにて合成する、あるいは遺伝子操作により宿主細胞に産生させることによって得ることができる。遺伝子操作では、具体的には、ＰＴＤＳＳ２　ｃＤＮＡを発現可能なベクターに組み込んだ後、転写と翻訳に必要な酵素、基質およびエネルギー物質を含む溶液中で合成する、あるいは他の原核生物、または真核生物の宿主細胞を形質転換させることによってＰＴＤＳＳ２を発現させることにより、該蛋白質を得ることが出来る。 2. 2. PTDSS2
Whether PTDSS2 used in the present invention is directly purified from humans, non-human mammals (eg, guinea pigs, rats, mice, rabbits, pigs, sheep, cows, monkeys, etc.) or chicken T cells or mast cells. Alternatively, the cell membrane fraction of the above cells can be prepared and used, or can be obtained by synthesizing PTDSS2 in vitro or by genetically manipulating it in a host cell. In genetic manipulation, specifically, the PTDSS2 cDNA is incorporated into an expressible vector and then synthesized in a solution containing the enzymes, substrates and energy substances required for transcription and translation, or other prokaryotes, or eukaryotes. The protein can be obtained by expressing PTDSS2 by transforming the host cell of an organism.
　ヒトＰＴＤＳＳ２のｃＤＮＡのヌクレオチド配列は、上記ヒトＰＴＤＳＳ２遺伝子に基づくものである。 The nucleotide sequence of the cDNA of human PTDSS2 is based on the above-mentioned human PTDSS2 gene.
　ＰＴＤＳＳ２のｃＤＮＡは例えば、ＰＴＤＳＳ２のｍＲＮＡを発現している臓器のｃＤＮＡライブラリーまたはヒト細胞から抽出したゲノムＤＮＡを鋳型として、ＰＴＤＳＳ２のｃＤＮＡを特異的に増幅するプライマーを用いてポリメラーゼ連鎖反応（以下「ＰＣＲ」という）（Ｓａｉｋｉ，Ｒ．　Ｋ．，ｅｔ　ａｌ．，Ｓｃｉｅｎｃｅ，（１９８８）２３９，４８７－４９１）を行なう、いわゆるＰＣＲ法により取得することができる。 The cDNA of PTDSS2 is, for example, a polymerase chain reaction using a cDNA library of an organ expressing the mRNA of PTDSS2 or a genomic DNA extracted from human cells as a template and a primer that specifically amplifies the cDNA of PTDSS2 (hereinafter referred to as "" It can be obtained by the so-called PCR method in which (referred to as PCR) (Saiki, R.K., et al., Science, (1988) 239, 487-491) is performed.
　なお、ヒトＰＴＤＳＳ２をコードするヌクレオチド配列と相補的なヌクレオチド配列からなるポリヌクレオチドとストリンジェントな条件でハイブリダイズし、かつ、ＰＴＤＳＳ２と同等の生物活性を有する蛋白質をコードするポリヌクレオチドもＰＴＤＳＳ２のｃＤＮＡに含まれる。さらに、ヒトＰＴＤＳＳ２遺伝子座から転写されるスプライシングバリアントまたはこれにストリンジェントな条件でハイブリダイズするポリヌクレオチドであって、かつ、ＰＴＤＳＳ２と同等の生物活性を有する蛋白質をコードするポリヌクレオチドもＰＴＤＳＳ２のｃＤＮＡに含まれる。 In addition, a polynucleotide encoding a protein having a biological activity equivalent to that of PTDSS2, which hybridizes with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence encoding human PTDSS2 under stringent conditions, is also included in the cDNA of PTDSS2. included. Furthermore, a splicing variant transcribed from the human PTDSS2 locus or a polynucleotide that hybridizes to the splicing variant under stringent conditions and encodes a protein having the same biological activity as PTDSS2 is also included in the cDNA of PTDSS2. included.
　また、ヒトＰＴＤＳＳ２のアミノ酸配列において、１個、２もしくは３個または４もしくは５個のアミノ酸が置換、欠失、または付加されたアミノ酸配列からなり、ＰＴＤＳＳ２と同等の生物活性を有する蛋白質もＰＴＤＳＳ２に含まれる。さらに、ヒトＰＴＤＳＳ２遺伝子座から転写されるスプライシングバリアントにコードされるアミノ酸配列または該アミノ酸配列において、１個、２もしくは３個または４もしくは５個のアミノ酸が置換、欠失、または付加されたアミノ酸配列からなり、かつ、ＰＴＤＳＳ２と同等の生物活性を有する蛋白質もＰＴＤＳＳ２に含まれる。 In addition, a protein consisting of an amino acid sequence in which one, two, three, or four or five amino acids are substituted, deleted, or added in the amino acid sequence of human PTDSS2 and having the same biological activity as PTDSS2 is also added to PTDSS2. included. In addition, an amino acid sequence encoded by a splicing variant transcribed from the human PTDSS2 locus or an amino acid sequence in which one, two or three or four or five amino acids have been substituted, deleted or added. PTDSS2 also contains a protein consisting of PTDSS2 and having the same biological activity as PTDSS2.
　３．抗ＰＴＤＳＳ２抗体の製造
　本発明のＰＴＤＳＳ２に対する抗体は、常法を用いて、ＰＴＤＳＳ２またはＰＴＤＳＳ２のアミノ酸配列から選択される任意のポリペプチドを動物に免疫し、生体内に産生される抗体を採取、精製することによって得ることができる。抗原となるＰＴＤＳＳ２の生物種はヒトに限定されず、サル、マウス、ラット等のヒト以外の動物に由来するＰＴＤＳＳ２を動物に免疫することもできる。この場合には、取得された異種ＰＴＤＳＳ２に結合する抗体とヒトＰＴＤＳＳ２との交差性を試験することによって、ヒトの疾患に適用可能な抗体を選別できる。なお、抗原となるＰＴＤＳＳ２はＰＴＤＳＳ２遺伝子を遺伝子操作により宿主細胞に産生させることによっても得ることができる。具体的には、ＰＴＤＳＳ２遺伝子を発現可能なベクターを作製し、これを宿主細胞に導入して該遺伝子を発現させ、発現したＰＴＤＳＳ２を精製すればよい。 3. 3. Production of Anti-PTDSS2 Antibody The antibody against PTDSS2 of the present invention is obtained by immunizing an animal with an arbitrary polypeptide selected from the amino acid sequence of PTDSS2 or PTDSS2 using a conventional method, and collecting and purifying the antibody produced in vivo. Can be obtained by doing. The species of PTDSS2 as an antigen is not limited to humans, and PTDSS2 derived from non-human animals such as monkeys, mice, and rats can be immunized against animals. In this case, by testing the crossing property between the obtained antibody that binds to the heterologous PTDSS2 and human PTDSS2, an antibody applicable to human diseases can be selected. The PTDSS2 as an antigen can also be obtained by causing the host cell to produce the PTDSS2 gene by genetic engineering. Specifically, a vector capable of expressing the PTDSS2 gene may be prepared, introduced into a host cell to express the gene, and the expressed PTDSS2 may be purified.
　また、公知の方法（例えば、Ｋｏｈｌｅｒ　ａｎｄ　Ｍｉｌｓｔｅｉｎ，Ｎａｔｕｒｅ　（１９７５）２５６，ｐ．４９５－４９７、Ｋｅｎｎｅｔ，Ｒ．ｅｄ．，Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ，ｐ．３６５－３６７，Ｐｌｅｎｕｍ　Ｐｒｅｓｓ，Ｎ．Ｙ．（１９８０））に従って、ＰＴＤＳＳ２に対する抗体を産生する抗体産生細胞とミエローマ細胞とを融合させることによりハイブリドーマを樹立し、モノクローナル抗体を得ることもできる。このような方法の具体的な例は、国際公開第ＷＯ０９／４８０７２号パンフレット（２００９年４月１６日公開）および第ＷＯ１０／１１７０１１号（２０１０年１０月１４日公開）パンフレットに記載されている。 Also, known methods (eg, Kohler and Milstein, Nature (1975) 256, p.495-497, Kennet, R.ed., Monoclonal Antibodies, p.365-367, Plenum Press, NY (1980)). ), By fusing an antibody-producing cell that produces an antibody against PTDSS2 with a myeloma cell, a hybridoma can be established and a monoclonal antibody can be obtained. Specific examples of such methods are described in Pamphlets WO 09/48072 (published April 16, 2009) and Pamphlets WO 10/110171 (published October 14, 2010).
　このようにして樹立された抗ＰＴＤＳＳ２抗体の実例としては、４０１－Ｐ－１７抗体を挙げることができる。４０１－Ｐ－１７抗体の重鎖ヌクレオチド配列は配列表の配列番号８に記載され、ヌクレオチド番号１－５７番がシグナル配列、５８－３８４番が重鎖可変領域、３８５－１３５６番が重鎖定常領域を示している。ＩＭＧＴ（ｈｔｔｐ：／／ｗｗｗ．ｉｍｇｔ．ｏｒｇ／）により決定したＣＤＲの配列は配列番号８の１３０－１５３番がＣＤＲＨ１、２０５－２２５番がＣＤＲＨ２、３３６－３５１番がＣＤＲＨ３を示している。４０１－Ｐ－１７抗体の重鎖アミノ酸配列は、配列表の配列番号２および図１に記載され、アミノ酸番号１－１９番がシグナル配列、２０－１２８番が重鎖可変領域、１２９－４５１番が重鎖定常領域を示している。ＩＭＧＴ（ｈｔｔｐ：／／ｗｗｗ．ｉｍｇｔ．ｏｒｇ／）により決定したＣＤＲのアミノ酸配列は配列番号２のアミノ酸番号４４－５１番がＣＤＲＨ１、６９－７５番がＣＤＲＨ２、１１２－１１６番がＣＤＲＨ３を示している。また、４０１－Ｐ－１７抗体の重鎖可変領域のアミノ酸配列は配列表の配列番号３および図２に記載されている。４０１－Ｐ－１７抗体の重鎖定常領域のアミノ酸配列は配列表の配列番号４および図３に記載されている。４０１－Ｐ－１７抗体のＣＤＲＨ１のアミノ酸配列は配列表の配列番号５および図４に記載されている。４０１－Ｐ－１７抗体のＣＤＲＨ２のアミノ酸配列は配列表の配列番号６および図５に記載されている。４０１－Ｐ－１７抗体のＣＤＲＨ３のアミノ酸配列は配列表の配列番号７および図６に記載されている。 As an example of the anti-PTDSS2 antibody thus established, the 401-P-17 antibody can be mentioned. The heavy chain nucleotide sequence of the 401-P-17 antibody is described in SEQ ID NO: 8 of the sequence listing, nucleotide number 1-57 is the signal sequence, 58-384 is the heavy chain variable region, and 385-1356 is the heavy chain constant. Shows the area. As for the sequence of CDR determined by IMGT (http://www.imgt.org/), SEQ ID NO: 8: 130-153 indicates CDRH1, 205-225 indicates CDRH2, and 336-351 indicates CDRH3. The heavy chain amino acid sequence of the 401-P-17 antibody is shown in SEQ ID NO: 2 and FIG. 1 of the sequence listing, where amino acid numbers 1-19 are the signal sequence, 20-128 are the heavy chain variable region, and 129-451. Indicates the heavy chain constant region. The amino acid sequence of CDR determined by IMGT (http://www.imgt.org/) indicates that amino acid numbers 44-51 of SEQ ID NO: 2 indicate CDRH1, 69-75 indicates CDRH2, and 112-116 indicate CDRH3. There is. The amino acid sequence of the heavy chain variable region of the 401-P-17 antibody is shown in SEQ ID NO: 3 and FIG. 2 in the sequence listing. The amino acid sequence of the heavy chain constant region of the 401-P-17 antibody is shown in SEQ ID NO: 4 and FIG. 3 of the sequence listing. The amino acid sequence of CDRH1 of the 401-P-17 antibody is shown in SEQ ID NO: 5 and FIG. 4 of the sequence listing. The amino acid sequence of CDRH2 of the 401-P-17 antibody is shown in SEQ ID NO: 6 and FIG. 5 of the sequence listing. The amino acid sequence of CDRH3 of the 401-P-17 antibody is shown in SEQ ID NO: 7 and FIG. 6 of the sequence listing.
　４０１－Ｐ－１７抗体の軽鎖ヌクレオチド配列は配列表の配列番号１６に記載され、ヌクレオチド番号１－６６番がシグナル配列、６７－４０５番が軽鎖可変領域、４０６－７２０番が軽鎖定常領域を示している。ＩＭＧＴ（ｈｔｔｐ：／／ｗｗｗ．ｉｍｇｔ．ｏｒｇ／）により決定したＣＤＲの配列は配列番号１６の１４５－１６８番がＣＤＲＬ１、２２０－２２８番がＣＤＲＬ２、３３７－３７５番がＣＤＲＬ３を示している。４０１－Ｐ－１７抗体の軽鎖アミノ酸配列は、配列表の配列番号１０および図７に記載され、アミノ酸番号１－２２番がシグナル配列、２３－１３５番が軽鎖可変領域、１３６－２３９番が軽鎖定常領域を示している。ＩＭＧＴ（ｈｔｔｐ：／／ｗｗｗ．ｉｍｇｔ．ｏｒｇ／）により決定したＣＤＲのアミノ酸配列は配列番号１０のアミノ酸番号４９－５６番がＣＤＲＬ１、７４－７６番がＣＤＲＬ２、１１３－１２５がＣＤＲＬ３を示している。また、４０１－Ｐ－１７抗体の軽鎖可変領域のアミノ酸配列は配列表の配列番号１１および図８に記載されている。４０１－Ｐ－１７抗体の軽鎖定常領域のアミノ酸配列は配列表の配列番号１２および図９に記載されている。４０１－Ｐ－１７抗体のＣＤＲＬ１のアミノ酸配列は配列表の配列番号１３および図１０に記載されている。４０１－Ｐ－１７抗体のＣＤＲＬ２のアミノ酸配列は配列表の配列番号１４および図１１に記載されている。４０１－Ｐ－１７抗体のＣＤＲＬ３のアミノ酸配列は配列表の配列番号１５および図１２に記載されている。 The light chain nucleotide sequence of the 401-P-17 antibody is set forth in SEQ ID NO: 16 of the sequence listing, nucleotide number 1-66 is the signal sequence, 67-405 is the light chain variable region, and 406-720 is the light chain constant. Shows the area. As for the sequence of CDR determined by IMGT (http://www.imgt.org/), SEQ ID NO: 16: 145-168 indicates CDRL1, 220-228 indicates CDRL2, and 337-375 indicates CDRL3. The light chain amino acid sequence of the 401-P-17 antibody is shown in SEQ ID NO: 10 and FIG. 7 of the sequence listing, where amino acid numbers 1-22 are the signal sequence, 23-135 is the light chain variable region, 136-239. Indicates the light chain constant region. As for the amino acid sequence of CDR determined by IMGT (http://www.imgt.org/), amino acid numbers 49-56 of SEQ ID NO: 10 indicate CDRL1, 74-76 indicates CDRL2, and 113-125 indicate CDRL3. .. The amino acid sequence of the light chain variable region of the 401-P-17 antibody is shown in SEQ ID NO: 11 and FIG. 8 of the sequence listing. The amino acid sequence of the light chain constant region of the 401-P-17 antibody is shown in SEQ ID NO: 12 and FIG. 9 of the sequence listing. The amino acid sequence of CDRL1 of the 401-P-17 antibody is set forth in SEQ ID NO: 13 and FIG. 10 of the sequence listing. The amino acid sequence of CDRL2 of the 401-P-17 antibody is set forth in SEQ ID NO: 14 and FIG. 11 of the sequence listing. The amino acid sequence of CDRL3 of the 401-P-17 antibody is set forth in SEQ ID NO: 15 and FIG. 12 of the sequence listing.
　本発明の抗体はＰＴＤＳＳ２蛋白質を特異的に認識する。言い換えれば、本発明の好適な抗体はＰＴＤＳＳ２蛋白質に特異的に結合する。 The antibody of the present invention specifically recognizes the PTDSS2 protein. In other words, the preferred antibody of the invention specifically binds to the PTDSS2 protein.
　ある態様において、好適な抗体は、非変性型のヒトＰＴＤＳＳ２、および、ホルマリンで固定した標本中の変性型ヒトＰＴＤＳＳ２の両方に特異的に結合する。また、より好適な抗体は、非変性型のヒトＰＴＤＳＳ２、および、ホルマリンで固定した標本中の変性型ヒトＰＴＤＳＳ２の両方に特異的に結合し、かつ他のＰＴＤＳＳファミリーに特異的に結合しない抗体等を挙げることができるが、それらに限定されるものではない。 In some embodiments, the suitable antibody specifically binds to both the non-denatured human PTDSS2 and the denatured human PTDSS2 in a formalin-fixed specimen. Further, more suitable antibodies include antibodies that specifically bind to both non-denatured human PTDSS2 and denatured human PTDSS2 in formalin-fixed specimens, but do not specifically bind to other PTDSS families. However, it is not limited to them.
　本発明の抗体には、上記のＰＴＤＳＳ２に対するモノクローナル抗体に加え、異種抗原性を低下させること等を目的として人為的に改変した遺伝子組換え型抗体、例えば、キメラ（Ｃｈｉｍｅｒｉｃ）抗体、ヒト化（Ｈｕｍａｎｉｚｅｄ）抗体、またはマウス化抗体等も含まれる。これらの抗体は、既知の方法を用いて製造することができる。 In addition to the above-mentioned monoclonal antibody against PTDSS2, the antibody of the present invention includes a recombinant antibody artificially modified for the purpose of reducing heterologous antigenicity, for example, a Chimeric antibody, a humanized antibody. ) Antibodies, mouse antibodies, etc. are also included. These antibodies can be produced using known methods.
　キメラ抗体としては、抗体の可変領域と定常領域が互いに異種である抗体、例えばマウスまたはラット由来抗体の可変領域をヒト由来の定常領域に接合したキメラ抗体を挙げることができる（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．，８１，６８５１－６８５５，（１９８４）参照）。 Examples of the chimeric antibody include an antibody in which the variable region and the constant region of the antibody are different from each other, for example, a chimeric antibody in which the variable region of a mouse or rat-derived antibody is bonded to the constant region of human origin (Proc. Natl. Acad). .Sci. USA, 81, 6851-6855, (1984)).
　本発明の抗体には、前記抗体のＣＤＲを改変した抗体が含まれる。これらの抗体は、既知の方法を用いて製造することができる。 The antibody of the present invention includes an antibody obtained by modifying the CDR of the antibody. These antibodies can be produced using known methods.
　なお、哺乳類培養細胞で生産される抗体の重鎖のカルボキシル末端のリシン残基が欠失することが知られており（Ｊｏｕｒｎａｌ　ｏｆ　Ｃｈｒｏｍａｔｏｇｒａｐｈｙ　Ａ，７０５：１２９－１３４（１９９５））、また、同じく重鎖カルボキシル末端のグリシン、リシンの２アミノ酸残基が欠失し、新たにカルボキシル末端に位置するプロリン残基がアミド化されることが知られている（Ａｎａｌｙｔｉｃａｌ　Ｂｉｏｃｈｅｍｉｓｔｒｙ，３６０：７５－８３（２００７））。しかし、これらの重鎖配列の欠失および修飾は、抗体の抗原結合能およびエフェクター機能（補体の活性化や抗体依存性細胞傷害作用など）には影響を及ぼさない。従って、本発明には当該修飾を受けた抗体も含まれ、重鎖カルボキシル末端において１または２つのアミノ酸が欠失した欠失体、およびアミド化された当該欠失体（例えば、カルボキシル末端部位のプロリン残基がアミド化された重鎖）等を挙げることができる。但し、抗原結合能およびエフェクター機能が保たれている限り、本発明に係る抗体の重鎖のカルボキシル末端の欠失体は上記の種類に限定されない。本発明に係る抗体を構成する２本の重鎖は、完全長および上記の欠失体からなる群から選択される重鎖のいずれか一種であっても良いし、いずれか二種を組み合わせたものであっても良い。各欠失体の量比は本発明に係る抗体を産生する哺乳類培養細胞の種類および培養条件に影響を受け得るが、本発明に係る抗体の主成分としては２本の重鎖の双方でカルボキシル末端の１つのアミノ酸残基が欠失している場合を挙げることができる。 It is known that the glycine residue at the carboxyl terminus of the heavy chain of the antibody produced in cultured mammalian cells is deleted (Journal of Chromatografy A, 705: 129-134 (1995)), and the same weight. It is known that the two amino acid residues of glycine and lysine at the chain carboxyl terminus are deleted, and the proline residue located at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83 (2007)). ). However, deletions and modifications of these heavy chain sequences do not affect the antigen-binding ability and effector function of the antibody (such as complement activation and antibody-dependent cellular cytotoxicity). Therefore, the present invention also includes the modified antibody, which is a deletion in which one or two amino acids are deleted at the heavy chain carboxyl terminus, and the amidated deletion (for example, at the carboxyl terminus site). A heavy chain in which a proline residue is amidated) and the like can be mentioned. However, as long as the antigen-binding ability and the effector function are maintained, the deletion of the carboxyl terminus of the heavy chain of the antibody according to the present invention is not limited to the above types. The two heavy chains constituting the antibody according to the present invention may be any one of the heavy chains selected from the group consisting of the full length and the above-mentioned deletion product, or a combination of the two heavy chains. It may be a thing. The amount ratio of each deletion substance may be affected by the type of cultured mammalian cells producing the antibody according to the present invention and the culture conditions, but the main component of the antibody according to the present invention is carboxyl in both of the two heavy chains. The case where one amino acid residue at the terminal is deleted can be mentioned.
　以上の方法によって得られた抗体は、抗原に対する結合性を評価し、好適な抗体を選抜することができる。抗体の性質を比較する際の別の指標の一例としては、抗体の安定性を挙げることができる。示差走査カロリメトリー（ＤＳＣ）は、蛋白の相対的構造安定性の良い指標となる熱変性中点（Ｔｍ）を素早く、また正確に測定することができる方法である。ＤＳＣを用いてＴｍ値を測定し、その値を比較することによって、熱安定性の違いを比較することができる。抗体の保存安定性は、抗体の熱安定性とある程度の相関を示すことが知られており（Ｌｏｒｉ　Ｂｕｒｔｏｎ，ｅｔ．　ａｌ．，Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｄｅｖｅｌｏｐｍｅｎｔ　ａｎｄ　Ｔｅｃｈｎｏｌｏｇｙ（２００７）１２，ｐ．２６５－２７３）、熱安定性を指標に、好適な抗体を選抜することができる。抗体を選抜するための他の指標としては、適切な宿主細胞における収量が高いこと、および水溶液中での凝集性が低いことを挙げることができる。例えば収量の最も高い抗体が最も高い熱安定性を示すとは限らないので、以上に述べた指標に基づいて総合的に判断して、最も適した抗体を選抜する必要がある。 The antibody obtained by the above method can evaluate the binding property to the antigen and select a suitable antibody. As another example of an index for comparing the properties of an antibody, the stability of the antibody can be mentioned. Differential scanning calorimetry (DSC) is a method that can quickly and accurately measure the heat denaturation midpoint (Tm), which is a good indicator of the relative structural stability of proteins. By measuring the Tm value using DSC and comparing the values, the difference in thermal stability can be compared. It is known that the storage stability of an antibody shows a certain degree of correlation with the thermal stability of the antibody (Lori Burton, et. Al., Pharmaceutical Development and Technology (2007) 12, p.265-273). Suitable antibodies can be selected using the stability as an index. Other indicators for selecting antibodies include high yields in suitable host cells and low cohesiveness in aqueous solution. For example, the antibody with the highest yield does not always show the highest thermal stability, so it is necessary to make a comprehensive judgment based on the above-mentioned indicators and select the most suitable antibody.
　抗体遺伝子を一旦単離した後、適当な宿主に導入して抗体を作製する場合には、適当な宿主と発現ベクターの組み合わせを使用することができる。抗体遺伝子の具体例としては、本明細書に記載された抗体の重鎖配列をコードする遺伝子、および軽鎖配列をコードする遺伝子を組み合わせたものを挙げることができる。宿主細胞を形質転換する際には、重鎖配列遺伝子と軽鎖配列遺伝子は、同一の発現ベクターに挿入されていることが可能であり、又別々の発現ベクターに挿入されていることも可能である。真核細胞を宿主として使用する場合、動物細胞、植物細胞、真核微生物を用いることができる。動物細胞としては、哺乳類細胞、例えば、ヒト児腎細胞２９３（ＨＥＫ２９３）、サルの細胞であるＣＯＳ細胞（Ｇｌｕｚｍａｎ，Ｙ．Ｃｅｌｌ（１９８１）２３，ｐ．１７５－１８２、ＡＴＣＣ　ＣＲＬ－１６５０）、マウス線維芽細胞ＮＩＨ３Ｔ３（ＡＴＣＣ　Ｎｏ．ＣＲＬ－１６５８）やチャイニーズ・ハムスター卵巣細胞（ＣＨＯ細胞、ＡＴＣＣ　ＣＣＬ－６１）のジヒドロ葉酸還元酵素欠損株（Ｕｒｌａｕｂ，Ｇ．ａｎｄ　Ｃｈａｓｉｎ，Ｌ．Ａ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．（１９８０）７７，ｐ．４１２６－４２２０）を挙げることができる。また、原核細胞を使用する場合は、例えば、大腸菌、枯草菌を挙げることができる。これらの細胞に目的とする抗体遺伝子を形質転換により導入し、形質転換された細胞をｉｎ　ｖｉｔｒｏで培養することにより抗体が得られる。以上の培養法においては抗体の配列によって収量が異なる場合があり、同等な結合活性を持つ抗体の中から収量を指標に医薬としての生産が容易なものを選別することが可能である。 When an antibody gene is once isolated and then introduced into an appropriate host to prepare an antibody, a combination of an appropriate host and an expression vector can be used. Specific examples of the antibody gene include a combination of a gene encoding a heavy chain sequence of an antibody described herein and a gene encoding a light chain sequence. When transforming a host cell, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or can be inserted into different expression vectors. be. When using eukaryotic cells as a host, animal cells, plant cells, and eukaryotic microorganisms can be used. Examples of animal cells include mammalian cells such as human infant kidney cells 293 (HEK293), monkey cells COS cells (Gluzman, Y. Cell (1981) 23, p.175-182, ATCC CRL-1650), and mice. Dihydrofolate reductase-deficient strains of fibroblastic NIH3T3 (ATCC No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chain, LA Proc. Natl. Acad. Sci. USA (1980) 77, p. 4126-4220) can be mentioned. When prokaryotic cells are used, for example, Escherichia coli and Bacillus subtilis can be mentioned. Antibodies can be obtained by introducing the target antibody gene into these cells by transformation and culturing the transformed cells in vitro. In the above culture method, the yield may differ depending on the sequence of the antibody, and it is possible to select an antibody having the same binding activity that is easy to produce as a pharmaceutical by using the yield as an index.
　本発明の抗体のアイソタイプとしての制限はなく、例えばＩｇＧ（ＩｇＧ１，ＩｇＧ２，ＩｇＧ３，ＩｇＧ４）、ＩｇＭ、ＩｇＡ（ＩｇＡ１，ＩｇＡ２）、ＩｇＤあるいはＩｇＥ等を挙げることができるが、好ましくはＩｇＧまたはＩｇＭ、さらに好ましくはＩｇＧ１またはＩｇＧ２を挙げることができる。 The isotype of the antibody of the present invention is not limited, and examples thereof include IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, IgE, etc., but IgG or IgM is preferable. More preferably, IgG1 or IgG2 can be mentioned.
　また本発明の抗体は、抗体の抗原結合部を有する抗体の抗原結合性断片またはその修飾物であってもよい。抗体をパパイン、ペプシン等の蛋白質分解酵素で処理するか、あるいは抗体遺伝子を遺伝子工学的手法によって改変し適当な培養細胞において発現させることによって、該抗体の断片を得ることができる。このような抗体断片のうちで、抗体全長分子の持つ機能の全てまたは一部を保持している断片を抗体の抗原結合性断片と呼ぶことができる。抗体の機能としては、一般的には抗原結合活性、抗原の活性を中和する活性、抗原の活性を増強する活性、抗体依存性細胞傷害活性、補体依存性細胞傷害活性および補体依存性細胞性細胞傷害活性を挙げることができる。本発明における抗体の抗原結合性断片が保持する機能は、ＰＴＤＳＳ２に対する結合活性である。 Further, the antibody of the present invention may be an antigen-binding fragment of an antibody having an antigen-binding portion of the antibody or a modified product thereof. Fragments of the antibody can be obtained by treating the antibody with a proteolytic enzyme such as papain or pepsin, or by modifying the antibody gene by a genetic engineering technique and expressing it in a suitable cultured cell. Among such antibody fragments, a fragment that retains all or part of the functions of the full-length antibody molecule can be referred to as an antigen-binding fragment of the antibody. The functions of antibodies are generally antigen-binding activity, activity that neutralizes antigen activity, activity that enhances antigen activity, antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and complement-dependent cytotoxicity. Cell-mediated cytotoxic activity can be mentioned. The function retained by the antigen-binding fragment of the antibody in the present invention is the binding activity to PTDSS2.
　例えば、抗体の断片としては、Ｆａｂ、Ｆ（ａｂ’）２、Ｆｖ、または重鎖および軽鎖のＦｖを適当なリンカーで連結させたシングルチェインＦｖ（ｓｃＦｖ）、ｄｉａｂｏｄｙ（ｄｉａｂｏｄｉｅｓ）、線状抗体、および抗体断片より形成された多特異性抗体などを挙げることができる。また、Ｆ（ａｂ’）２を還元条件下で処理した抗体の可変領域の一価の断片であるＦａｂ’も抗体の断片に含まれる。 For example, antibody fragments include Fab, F (ab') 2, Fv, or single chain Fv (scFv), diabody (diabodies), linear antibodies in which heavy and light chain Fv are linked with an appropriate linker. , And a multispecific antibody formed from an antibody fragment. In addition, Fab', which is a monovalent fragment of the variable region of the antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the antibody fragment.
　得られた抗体は、均一にまで精製することができる。抗体の分離、精製は通常の蛋白質で使用されている分離、精製方法を使用すればよい。例えばカラムクロマトグラフィー、フィルター濾過、限外濾過、塩析、透析、調製用ポリアクリルアミドゲル電気泳動、等電点電気泳動等を適宜選択、組み合わせれば、抗体を分離、精製することができる（Ｓｔｒａｔｅｇｉｅｓ　ｆｏｒ　Ｐｒｏｔｅｉｎ　Ｐｕｒｉｆｉｃａｔｉｏｎ　ａｎｄ　Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｃｏｕｒｓｅ　Ｍａｎｕａｌ，Ｄａｎｉｅｌ　Ｒ．Ｍａｒｓｈａｋ　ｅｔ　ａｌ．ｅｄｓ．，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９９６）；Ａｎｔｉｂｏｄｉｅｓ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ．Ｅｄ　Ｈａｒｌｏｗ　ａｎｄ　Ｄａｖｉｄ　Ｌａｎｅ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ（１９８８））が、これらに限定されるものではない。 The obtained antibody can be purified uniformly. For the separation and purification of the antibody, the separation and purification methods used for ordinary proteins may be used. For example, by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, polyacrylamide gel electrophoresis for preparation, isoelectric point electrophoresis, etc., antibodies can be separated and purified (Strategies). for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R.Marshak et al.eds, Cold Spring Harbor Laboratory Press (1996); Antibodies:. A Laboratory Manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)) However, it is not limited to these.
　クロマトグラフィーとしては、アフィニティークロマトグラフィー、イオン交換クロマトグラフィー、疎水性クロマトグラフィー、ゲル濾過クロマトグラフィー、逆相クロマトグラフィー、吸着クロマトグラフィー等を挙げることができる。これらのクロマトグラフィーは、ＨＰＬＣやＦＰＬＣ等の液体クロマトグラフィーを用いて行うことができる。アフィニティークロマトグラフィーに用いるカラムとしては、プロテインＡカラム、プロテインＧカラムを挙げることができる。例えばプロテインＡカラムを用いたカラムとして、Ｈｙｐｅｒ　Ｄ，ＰＯＲＯＳ，Ｓｅｐｈａｒｏｓｅ　Ｆ．Ｆ．（ファルマシア）等を挙げることができる。また、抗原を固定化した担体を用いて、抗原への結合性を利用して抗体を精製することも可能である。 Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography and the like. These chromatographies can be performed using liquid chromatography such as HPLC or FPLC. Examples of the column used for affinity chromatography include a protein A column and a protein G column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose F. F. (Pharmacia) and the like. It is also possible to purify the antibody by utilizing the binding property to the antigen by using the carrier on which the antigen is immobilized.
　４．診断用組成物
　本発明は抗ＰＴＤＳＳ２抗体もしくはその機能断片またはその修飾体を含む検査用または診断用組成物（以下、まとめて「診断用組成物」という）をも提供する。 4. Diagnostic Composition The present invention also provides a test or diagnostic composition (hereinafter collectively referred to as "diagnostic composition") containing an anti-PTDSS2 antibody or a functional fragment thereof or a modified product thereof.
　本発明の診断用組成物は、検体中でのＰＴＤＳＳ２の発現の有無を検出することができ、さらには検体中でのＰＴＤＳＳ２の発現量を評価することができる。したがって、本発明の診断用組成物はＰＴＤＳＳ１阻害剤による合成致死に基づく治療の対象となるＰＴＤＳＳ２が発現していないかあるいは発現量の少ないがんや肉腫の検出に有用である。 The diagnostic composition of the present invention can detect the presence or absence of expression of PTDSS2 in a sample, and can further evaluate the expression level of PTDSS2 in a sample. Therefore, the diagnostic composition of the present invention is useful for detecting cancer or sarcoma in which PTDSS2, which is a target of synthetic lethal treatment with a PTDSS1 inhibitor, is not expressed or the expression level is low.
　本発明において検査または診断には、例えば、罹患の有無の判定、ＰＴＤＳＳ１阻害剤等の医薬組成物による薬物治療の効果の測定または判定、薬物治療以外の治療の効果の測定または判定、再発リスクの測定、再発の有無の判定等が含まれるが、検査または診断であればそれらに限定されるものではない。 In the present invention, the test or diagnosis includes, for example, determination of the presence or absence of illness, measurement or determination of the effect of drug treatment with a pharmaceutical composition such as PTDSS1 inhibitor, measurement or determination of the effect of treatment other than drug treatment, and risk of recurrence. It includes, but is not limited to, measurement, determination of the presence or absence of recurrence, etc., as long as it is an examination or diagnosis.
　かかる診断用組成物には、ｐＨ緩衝剤、浸透圧調節剤、塩類、安定化剤、防腐剤、顕色剤、増感剤、凝集防止剤等を含有せしめることができる。 The diagnostic composition may contain a pH buffer, an osmotic pressure regulator, salts, a stabilizer, a preservative, a color developer, a sensitizer, an anti-aggregation agent and the like.
　本発明はがんなどＰＴＤＳＳ２に関わる疾患の検査方法または診断方法、該疾患の診断用組成物を調製するための本発明の抗体の使用、該疾患の検査または診断のための本発明の抗体の使用、をも提供する。本発明の抗体を含む検査または診断用キットも本発明に含まれる。 The present invention relates to a method for testing or diagnosing a disease related to PTDSS2 such as cancer, the use of the antibody of the present invention for preparing a composition for diagnosing the disease, and the antibody of the present invention for testing or diagnosing the disease. Use, also provide. Test or diagnostic kits comprising the antibodies of the invention are also included in the invention.
　本発明の診断用組成物を用いた検査または診断の方法としてはサンドウィッチＥＬＩＳＡが望ましいが、通常のＥＬＩＳＡ法やＲＩＡ法、ＥＬＩＳＰＯＴ（Ｅｎｚｙｍｅ－Ｌｉｎｋｅｄ　ＩｍｍｕｎｏＳｐｏｔ）法、ドットブロット法、オクタロニー法、ＣＩＥ（Ｃｏｕｎｔｅｒｉｍｍｕｎｏｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ）法、ＣＬＩＡ（Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ　ｉｍｍｕｎｏ　ａｓｓａｙ）、ＦＣＭ（Ｆｌｏｗ　Ｃｙｔｏｍｅｔｒｙ）などの抗体を利用した検出方法が利用可能である。抗体の標識法としてはビオチンのほか、ＨＲＰ、アルカリフォスファターゼ、ＦＩＴＣやＡＬＥＸＡなどの発蛍光団、放射性同位元素などのラベルなど生化学的解析に実施可能な標識法が利用できる。酵素標識を利用した検出にはＴＭＢ（３，３’，５，５’－ｔｅｔｒａｍｅｔｈｙｌｂｅｎｚｉｄｉｎｅ）、ＢＣＩＰ（５－ｂｒｏｍｏ－４－ｃｈｌｏｒｏ－３－ｉｎｄｏｌｙｌ　ｐｈｏｓｐｈａｔｅ）、ρ－ＮＰＰ（ρ－ｎｉｔｒｏｐｈｅｎｙｌ　ｐｈｏｓｐｈａｔｅ）、ＯＰＤ（ｏ－Ｐｈｅｎｙｌｅｎｅｄｉａｍｉｎｅ）、ＡＢＴＳ（３－Ｅｔｈｙｌｂｅｎｚｏｔｈｉａｚｏｌｉｎｅ－６－ｓｕｌｆｏｎｉｃ　ａｃｉｄ）、ＳｕｐｅｒＳｉｇｎａｌ　ＥＬＩＳＡ　Ｐｉｃｏ　Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ　Ｓｕｂｓｔｒａｔｅ（サーモフィッシャーサイエンティフィック社）などの発色基質やＱｕａｎｔａＢｌｕ（登録商標）　Ｆｌｕｏｒｏｇｅｎｉｃ　Ｐｅｒｏｘｉｄａｓｅ　Ｓｕｂｓｔｒａｔｅ（サーモフィッシャーサイエンティフィック社）蛍光基質のほか、化学発光基質を用いることができる。本測定には、ヒトまたは非ヒト動物由来の試料に加え、組換え蛋白質等の人工的な処理を加えた試料をも供することができる。生物個体由来の被検試料としては、例えば、血液、関節液、腹水、リンパ液、脳脊髄液、組織ホモジネート上清、組織切片等を挙げることができるが、それらに限定されるものではない。 Sandwich ELISA is desirable as a method of examination or diagnosis using the diagnostic composition of the present invention, but ordinary ELISA method, RIA method, ELISA (Enzyme-Linked ImmunoSpot) method, dot blot method, octalony method, CIE (Counterimneuometry). ) Method, a detection method using an antibody such as CLIA (Chemiluminescence immunoassay) or FCM (Flow Cytometry) can be used. As the antibody labeling method, in addition to biotin, a labeling method that can be carried out for biochemical analysis such as HRP, alkaline phosphatase, fluorescent groups such as FITC and ALEXA, and labels such as radioactive isotopes can be used. For detection using enzyme labeling, TMB (3,3', 5,5'-tetramethylbenzidine), BCIP (5-blomo-4-chlor-3-indolyl phosphate), ρ-NPP (ρ-nitrophenyl phosphate), OPD (O-Phylenediamine), ABTS (3-Ethylbenzothiazoline-6-sulfonic acid), SuperSignal ELISA PicoChemilluminescent Substrate Substrate (Thermo-Fischer Scientific Substrate) Substrate (Thermo-Fischer Scientific Substrate) In addition to the fluorescent substrate, a chemically luminescent substrate can be used. In addition to samples derived from humans or non-human animals, samples that have been artificially treated with recombinant proteins or the like can also be used for this measurement. Examples of the test sample derived from an individual organism include, but are not limited to, blood, joint fluid, ascites, lymph fluid, cerebrospinal fluid, tissue homogenate supernatant, tissue section, and the like.
　本発明の抗体を含む検査または診断用のサンドウィッチＥＬＩＳＡキットには、コントロール（ＰＴＤＳＳ２由来ペプチドの標準液）、発色試薬、希釈用緩衝液、固相用抗体、検出用抗体、ならびに洗浄液等が含まれてよい。抗原に結合した抗体量を測定する方法としては、吸光法、蛍光法、発光法、ＲＩ（Ｒａｄｉｏｉｓｏｔｏｐｅ）法等が好適に適用され、測定には、吸光プレートリーダー、蛍光プレートリーダー、発光プレートリーダー、ＲＩ液体シンチレーションカウンター等が好適に使用される。 The test or diagnostic sandwich ELISA kit containing the antibody of the present invention contains a control (standard solution of PTDSS2-derived peptide), a coloring reagent, a buffer solution for dilution, an antibody for a solid phase, an antibody for detection, a washing solution, and the like. It's okay. As a method for measuring the amount of antibody bound to an antigen, an absorption method, a fluorescence method, a luminescence method, an RI (Radioisotope) method or the like is preferably applied, and for the measurement, an absorption plate reader, a fluorescence plate reader, a luminescence plate reader, etc. An RI liquid scintillation counter or the like is preferably used.
　前記の免疫組織学的試験だけでなく、試料中の細胞、組織または臓器もしくはその一部から、常法に従って可溶化した蛋白を調製し、当該可溶化蛋白に標識化抗体を反応させることにより可溶化蛋白中のＰＴＤＳＳ２の存否を確認するウエスタンブロッティング法やドットブロット法においても用いることができる。対象となる被検試料としては、血液等の体液中に含まれる、細胞、血中循環腫瘍細胞、がん細胞を含む様々な細胞が分泌するエクソソームなどから調製した可溶化蛋白質が含まれるが、それらに限定されるものではない。 Not only the above-mentioned immunohistochemical test, but also possible by preparing a solubilized protein from cells, tissues or organs or a part thereof in a sample according to a conventional method and reacting the solubilized protein with a labeled antibody. It can also be used in a Western blotting method or a dot blotting method for confirming the presence or absence of PTDSS2 in a lysed protein. The target test samples include solubilized proteins prepared from cells, circulating tumor cells in the blood, exosomes secreted by various cells including cancer cells, etc. contained in body fluids such as blood. It is not limited to them.
　本発明は免疫組織化学（ｉｍｍｕｎｏｈｉｓｔｏｃｈｅｍｉｓｔｒｙ：ＩＨＣ）の分析に有用な抗体、その機能断片およびその修飾体、ならびにそれらを含む組成物を提供する。かかる組成物も本発明の「診断用組成物」に包含される。 The present invention provides an antibody useful for analysis of immunohistochemistry (IHC), a functional fragment thereof and a modified product thereof, and a composition containing them. Such compositions are also included in the "diagnostic composition" of the present invention.
　免疫組織化学は、組織切片を抗原に結合する抗体（一次抗体）と反応させ、抗原に結合した一次抗体を検出する手法であれば特に限定されない。
組織切片は、好適にはホルマリン固定後にパラフィン包埋処理する。パラフィン包埋後、薄切した組織切片を脱パラフィン処理した後、抗原賦活処理および非特異的反応抑制処理を行う。抗原賦活処理の方法としては、加熱処理、プロテアーゼ等による酵素処理を例示することができ、好適には加熱処理である。加熱処理の条件としては、通常、温度９０乃至１１０℃、ｐＨ８乃至１０、処理時間２０乃至６０分の範囲が好適である。ｐＨの調整にはＴｒｉｓ－ＥＤＴＡ緩衝液（例えば、１ｍＭのＥＤＴＡを含有する１０ｍＭトリス緩衝液）等を使用することができる。非特異的反応抑制処理としては、発色に使用する酵素と同様または類似の触媒活性を有する内因性の酵素を不活性化する方法が通常用いられる。ペルオキシダーゼ反応により発色させる場合、予め組織中に存在する内因性のペルオキシダーゼをＨ２Ｏ２等で阻害することが好ましい。Ｈ２Ｏ２の溶媒は水、メタノール等を使用することができ、Ｈ２Ｏ２の濃度は０．１乃至３％、好適には０．３乃至３％である。Ｈ２Ｏ２溶液にはアジ化ナトリウムを添加することができる。また、血清やカゼインによりブロッキングする方法も非特異的反応抑制処理として使用することができる。血清やカゼインは、一次抗体反応の前に組織を処理することができるが、１次抗体を希釈する溶媒に含有せしめることもできる。 Immunohistochemistry is not particularly limited as long as it is a method of reacting a tissue section with an antibody (primary antibody) that binds to an antigen and detecting the primary antibody bound to the antigen.
Tissue sections are preferably paraffin-embedded after being fixed in formalin. After embedding in paraffin, the sliced tissue sections are deparaffinized, and then subjected to antigen activation treatment and non-specific reaction suppression treatment. As a method of antigen activation treatment, heat treatment, enzyme treatment with a protease or the like can be exemplified, and heat treatment is preferable. As the conditions for the heat treatment, a temperature range of 90 to 110 ° C., a pH of 8 to 10, and a treatment time of 20 to 60 minutes are usually suitable. Tris-EDTA buffer (for example, 10 mM Tris buffer containing 1 mM EDTA) or the like can be used to adjust the pH. As the non-specific reaction suppression treatment, a method of inactivating an endogenous enzyme having the same or similar catalytic activity as the enzyme used for color development is usually used. When the color is developed by the peroxidase reaction, it is preferable to inhibit the endogenous peroxidase existing in the tissue in advance with H2O2 or the like. Water, methanol or the like can be used as the solvent for H 2 O 2 , and the concentration of H 2 O 2 is 0.1 to 3%, preferably 0.3 to 3%. Sodium azide can be added to the H2O2 solution . In addition, a method of blocking with serum or casein can also be used as a non-specific reaction suppression treatment. Serum and casein can be treated with tissue prior to the primary antibody reaction, but can also be contained in a solvent that dilutes the primary antibody.
　一次抗体の反応条件は特に限定されないが、温度４乃至５０℃、好適には２０乃至３７℃、より好適には２４℃である。反応時間は５分乃至一昼夜、好適には１０分乃至４時間、より好適には３０分乃至１時間である。 The reaction conditions of the primary antibody are not particularly limited, but the temperature is 4 to 50 ° C, preferably 20 to 37 ° C, and more preferably 24 ° C. The reaction time is 5 minutes to one day and night, preferably 10 minutes to 4 hours, and more preferably 30 minutes to 1 hour.
　一次抗体の検出には、好適には、可視化することができ且つ一次抗体に結合する抗体（二次抗体）を用いることができる。二次抗体自体に結合する抗体（三次抗体）を用いて３回以上の反応を行う場合もある。二次抗体または三次抗体の可視化法としては、ペルオキシダーゼやアルカリフォスファターゼ等の酵素をそれらの抗体に結合させるか、またはそれらの抗体にビオチン等を付加して前記酵素を結合させたストレプトアビジン等と結合させ、それらの酵素に対応する発色基質を反応させる方法を好適に使用することができる。酵素を二次抗体または三次抗体に結合させる方法としては、デキストリンポリマーやアミノ酸ポリマーに前記酵素と二次抗体を多数結合させた試薬を用いる方法（ポリマー法）を例示することができる。ビオチン化二次抗体およびペルオキシダーゼ標識したストレプトアビジンを反応させる方法（ＬＳＡＢ法）では、発色基質としてＤＡＢ等を用いることができる。また、蛍光色素などで標識された二次抗体を使用することもできる。蛍光標識二次抗体で処理した場合、処理後に蛍光顕微鏡を用いて陽性細胞を検出する。 For the detection of the primary antibody, an antibody that can be visualized and binds to the primary antibody (secondary antibody) can be preferably used. In some cases, an antibody that binds to the secondary antibody itself (tertiary antibody) is used to perform the reaction three or more times. As a method for visualizing a secondary antibody or a tertiary antibody, an enzyme such as peroxidase or alkaline phosphatase is bound to those antibodies, or biotin or the like is added to those antibodies to bind to streptavidin or the like to which the enzyme is bound. A method of reacting with a color-developing substrate corresponding to those enzymes can be preferably used. As a method for binding an enzyme to a secondary antibody or a tertiary antibody, a method using a reagent in which a large number of the enzyme and the secondary antibody are bound to a dextrin polymer or an amino acid polymer can be exemplified (polymer method). In the method of reacting a biotinylated secondary antibody and peroxidase-labeled streptavidin (LSAB method), DAB or the like can be used as a color-developing substrate. Further, a secondary antibody labeled with a fluorescent dye or the like can also be used. When treated with a fluorescently labeled secondary antibody, positive cells are detected using a fluorescence microscope after the treatment.
　スメア法では、摘出細胞をガラスに塗布または遠心分離機で分離し、細胞成分と液性成分に分け、細胞成分について免疫染色を行う。すなわち、細胞成分をスライドガラス上に塗布し、エタノール液または１０％ホルマリン液などで固定した後、組織切片と同様の免疫染色を行うことができる。 In the smear method, the excised cells are coated on glass or separated by a centrifuge, separated into cell components and humoral components, and immunostaining is performed on the cell components. That is, after applying the cell component on a slide glass and fixing it with an ethanol solution or a 10% formalin solution, immunostaining similar to that of a tissue section can be performed.
　凍結包埋法では、摘出組織をＯＣＴコンパウンド等での包埋後に液体窒素等で急速凍結し、クリオスタットで薄切することでスライド標本を作製する。この標本を１０％ホルマリンやエタノール液などで固定した後、組織切片と同様の免疫染色を行うことができる。 In the freeze embedding method, the excised tissue is embedded in OCT compound or the like, then rapidly frozen in liquid nitrogen or the like, and sliced with a cryostat to prepare a slide specimen. After fixing this specimen with 10% formalin or ethanol solution, immunostaining similar to that of a tissue section can be performed.
　免疫組織化学に係る操作は、反応液、反応条件、洗浄回数等をプログラムして免疫装置に組み込み、自動化して行なうことができる。 Operations related to immunohistochemistry can be performed automatically by programming the reaction solution, reaction conditions, number of washings, etc., incorporating them into the immune device.
　画像診断の場合は、薬学的に許容可能な放射性核種や発光体を抗体にラベルし、被験者に該抗体を投与し、ＰＥＴ／ＣＴなどの画像診断技術を使用して画像を取り、ＰＴＤＳＳ２の存在を判定または検査することができる。 In the case of diagnostic imaging, an antibody is labeled with a pharmaceutically acceptable radionuclide or luminescent material, the antibody is administered to the subject, an image is taken using diagnostic imaging techniques such as PET / CT, and the presence of PTDSS2 is present. Can be determined or inspected.
　本発明の診断用組成物に含まれる抗体、その機能断片またはその修飾体は、好適にはＰＴＤＳＳ２に結合する抗体、すなわちＰＴＤＳＳ２選択性を有する抗体、その機能断片またはその修飾体である。 The antibody contained in the diagnostic composition of the present invention, a functional fragment thereof or a modified product thereof is preferably an antibody that binds to PTDSS2, that is, an antibody having PTDSS2 selectivity, a functional fragment thereof or a modified product thereof.
　本発明は被検サンプル中のヒトＰＴＤＳＳ２を検出または測定する方法を提供する。 The present invention provides a method for detecting or measuring human PTDSS2 in a test sample.
　これらの検出または測定方法には、本発明の診断用組成物を使用することができる。かかる測定方法および診断用組成物は、ヒトＰＴＤＳＳ２陽性あるいは陰性がんの診断用または検査用としても本発明に含まれる。 The diagnostic composition of the present invention can be used for these detection or measurement methods. Such a measuring method and a diagnostic composition are also included in the present invention for diagnostic or testing for human PTDSS2-positive or negative cancer.
　本発明には、ＰＴＤＳＳ１阻害剤の治療対象としてＰＴＤＳＳ２の発現のないがんを有する患者を同定する方法も包含される。かかる同定方法においては、該個体由来サンプル中のヒトＰＴＤＳＳ２を測定し、該サンプル中に、ヒトＰＴＤＳＳ２の陰性が検出されたか、または、健常個体由来サンプル中に検出されたヒトＰＴＤＳＳ２の量と比較してより少ない、または、まったく陰性であることが検出された場合に、該個体を陰性と判定し、ＰＴＤＳＳ１阻害剤が投与される個体と同定することができる。 The present invention also includes a method for identifying a patient having cancer in which PTDSS2 is not expressed as a therapeutic target of a PTDSS1 inhibitor. In such an identification method, human PTDSS2 in the sample derived from the individual is measured and compared with the amount of human PTDSS2 detected in the sample, either negative of human PTDSS2 or detected in the sample derived from a healthy individual. If less or no negative is detected, the individual can be determined to be negative and identified as an individual to which the PTDSS1 inhibitor is administered.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　当該方法には、本発明の診断用組成物を使用することができる。 The diagnostic composition of the present invention can be used in the method.
　本発明において、ＰＴＤＳＳ１阻害剤等の医薬組成物は、その一態様において、かかる同定方法において陰性と判定された個体に投与され得る。 In the present invention, a pharmaceutical composition such as a PTDSS1 inhibitor can be administered to an individual determined to be negative by such an identification method in one embodiment thereof.
　５．試薬
　本発明の抗体もしくはその抗原結合性断片またはその修飾体は、試薬としても有用である。かかる試薬は、上述の検査または診断用、研究用およびその他の用途で使用される。 5. Reagent The antibody of the present invention, an antigen-binding fragment thereof, or a modified product thereof is also useful as a reagent. Such reagents are used for the above-mentioned tests or diagnostics, research and other uses.
　６．ＰＴＤＳＳ１阻害剤
　本発明のＰＴＤＳＳ１阻害剤としては特に制限はなく、例えば、合成低分子化合物ライブラリー、遺伝子ライブラリーの発現産物、ペプチドライブラリー、siRNA、抗体、細菌放出物質、細胞（微生物、植物細胞、動物細胞）の抽出液および培養上清、精製または部分精製ポリペプチド、海洋生物、植物または動物由来の抽出物、ランダムファージペプチドディスプレイライブラリーを由来とするＰＴＤＳＳ１阻害剤が挙げられる。ＰＴＤＳＳ１阻害剤としては、例えば、ＷＯ／２０２０／１７９８５９や、ＷＯ２０１６／１４８１１５、Ｂｉｏｃｈｅｍ．　Ｊ．，　４１８，　４２１－４２９　（２００９）に記載のＰＴＤＳＳ１阻害剤が挙げられる。 6. PTDSS1 inhibitor The PTDSS1 inhibitor of the present invention is not particularly limited, and is, for example, a synthetic small molecule compound library, an expression product of a gene library, a peptide library, siRNA, an antibody, a bacterial release substance, a cell (microorganism, plant cell). , Animal cell) extracts and culture supernatants, purified or partially purified polypeptides, marine organisms, plant or animal derived extracts, PTDSS1 inhibitors derived from random phage peptide display libraries. Examples of the PTDSS1 inhibitor include WO / 2020/179859, WO2016 / 148115, and Biochem. J. , 418, 421-429 (2009).
　ＰＴＤＳＳ１阻害剤は、薬学上許容される担体と混合し、公知の製剤学的方法で製剤化することにより、医薬品とすることができる。薬学上許容される担体としては、例えば、滅菌水や生理食塩水、植物油、溶剤、基剤、乳化剤、懸濁剤、界面活性剤、安定剤、香味剤、芳香剤、賦形剤、ベヒクル、防腐剤、結合剤、希釈剤、等張化剤、無痛化剤、増量剤、崩壊剤、緩衝剤、コーティング剤、滑沢剤、着色剤、甘味剤、粘稠剤、矯味矯臭剤、溶解補助剤あるいはその他の添加剤等が挙げられるが、これらに制限されない。 The PTDSS1 inhibitor can be made into a pharmaceutical product by mixing it with a pharmaceutically acceptable carrier and formulating it by a known pharmaceutical method. Pharmaceutically acceptable carriers include, for example, sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, fragrances, excipients, vehicles, etc. Preservatives, binders, diluents, tonics, soothing agents, bulking agents, disintegrants, buffers, coatings, lubricants, colorants, sweeteners, thickeners, flavoring agents, dissolution aids Examples include, but are not limited to, agents or other additives.
　以下、本発明を実施例により具体的に説明するが、本発明はこれらに限定されるものではない。なお、下記実施例において遺伝子操作に関する各操作は特に明示がない限り、「モレキュラークローニング（Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ）」（Ｓａｍｂｒｏｏｋ，Ｊ．，Ｆｒｉｔｓｃｈ，Ｅ．Ｆ．およびＭａｎｉａｔｉｓ，Ｔ．著，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓより１９８９年に発刊）に記載の方法により行うか、または、市販の試薬やキットを用いる場合には市販品の指示書に従って使用した。また、本明細書において、特に記載のない試薬、溶媒および出発材料は、市販の供給源から容易に入手可能である。 Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto. In the following examples, unless otherwise specified, each operation related to gene manipulation is "Molecular Cloning" (Sambrook, J., Fritsch, EF and Maniatis, T., Cold Spring Harbor Laboratory Press. (Published in 1989)), or when a commercially available reagent or kit was used, it was used according to the instruction manual of the commercially available product. Also, reagents, solvents and starting materials not specifically described herein are readily available from commercially available sources.
　実施例１：ウサギ抗ヒトＰＴＤＳＳ２抗体の作製
　１）－１　ＰＴＤＳＳ２部分ペプチドの合成
ヒトＰＴＤＳＳ２の４４９から４８７番目までのアミノ酸配列のＮ末端側にシステインを付加したペプチド（合成ペプチド配列：　
　［Ｈ］ＣＱＮＫＤＤＱＧＳＴＶＧＮＧＤＱＨＰＬＧＬＤＥＤＬＬＧＰＧＶＡＥＧＥＧＡＰＴＰＮ［ＯＨ］）（配列番号：１）
をペプチド合成機を用いて常法に従い合成した。この合成ペプチドをキャリアタンパク質であるＫＬＨ（スカシ貝ヘモシアニン）またはＢＳＡ（ウシ血清アルブミン）に結合させた。次以降の工程で、ＫＬＨに結合させたものを免疫抗原として、ＢＳＡに結合させたものをスクリーニング抗原として用いた。 Example 1: Preparation of rabbit anti-human PTDSS2 antibody 1) -1 Synthesis of PTDSS2 partial peptide A peptide in which cysteine is added to the N-terminal side of the amino acid sequences 449 to 487 of human PTDSS2 (synthetic peptide sequence:
[H] CQNKDDQGSTVGNGDQHPLGLDEDLLGPGVAEGEGAPTPN [OH]) (SEQ ID NO: 1)
Was synthesized according to a conventional method using a peptide synthesizer. This synthetic peptide was bound to the carrier protein KLH (keyhole limpet hemocyanin) or BSA (bovine serum albumin). In the following steps, the one bound to KLH was used as an immune antigen, and the one bound to BSA was used as a screening antigen.
　１）－２　免疫試験
　抗体産生用の免疫動物は１３週齢のＪＷ／ＣＳＫウサギのメス（日本エスエルシー社）を使用した。アジュバントとしてＴｉｔｅｒＭａｘ　Ｇｏｌｄ（ＴｉｔｅｒＭａｘ社、ＵＳＡ）を用い、上記１）－１で作製した免疫抗原１　ｍＬとアジュバント１　ｍＬを混合しエマルジョンを作製し、免疫動物の背部皮下４か所へ等量ずつ注射した。２週間に１度、同様に注射を繰り返した後、初回免疫日から４９日目にラビットの全採血を行った後、脾臓を採取した。血液からはＰＢＭＣ（末梢血リンパ球）を分離・回収し、脾臓からはリンパ球を回収した。回収したＰＢＭＣおよび脾臓リンパ球はそれぞれ凍結保護液に懸濁し、－８０℃以下で冷凍保管した。 1) -2 Immune test A 13-week-old JW / CSK rabbit female (Japan SLC Corporation) was used as an immune animal for antibody production. Using TitterMax Gold (TitterMax, USA) as an adjuvant, 1 mL of the immune antigen prepared in 1) -1 above and 1 mL of the adjuvant are mixed to prepare an emulsion, and the same amount is injected subcutaneously into 4 places on the back of the immune animal. bottom. After repeating the same injection once every two weeks, the entire blood of Rabbit was collected 49 days after the first immunization day, and then the spleen was collected. PBMC (peripheral blood lymphocytes) were isolated and collected from the blood, and lymphocytes were collected from the spleen. The recovered PBMC and spleen lymphocytes were each suspended in a cryoprotective solution and stored frozen at -80 ° C or lower.
　１）－３　シングルセルピッキング試験
　上記１）－１で作製したスクリーニング抗原を１０ｍＭリン酸バッファー（ｐＨ７．０）で３　μｇ／ｍＬ，１　ｍＬに調製し、マイクロチャンバー（アズワン社、内径Φ２０　μｍ）へ添加し、３７℃で１時間静置し反応させた。マイクロチャンバーから溶液を除去後、１０％ＦＢＳ含有ＲＰＭＩ－１６４０　（Ｗａｋｏ社、フェノールレッド含無）を１ｍＬ添加し室温で１０分間静置反応させた。マイクロチャンバーから溶液を除去後、上記回収したリンパ球　２×１０５個を播種した後、Ｇｏａｔ　ａｎｔｉ－Ｒａｂｂｉｔ　ＩｇＧ　（Ｈ＋Ｌ）　Ｈｉｇｈｌｙ　Ｃｒｏｓｓ－Ａｄｓｏｒｂｅｄ　Ｓｅｃｏｎｄａｒｙ　Ａｎｔｉｂｏｄｙ，　Ａｌｅｘａ　Ｆｌｕｏｒ　４８８（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ社、Ａ１１０３４）を１０％ＦＢＳ含有ＲＰＭＩ－１６４０で５００倍希釈した溶液１ｍＬを添加し、３７℃、５％ＣＯ２の条件下で４５分間静置培養させた。マイクロチャンバーから溶液を除去後、１０％ＦＢＳ含有ＲＰＭＩ－１６４０で洗浄し、ＡＳＯＮＥ　Ｃｅｌｌ　Ｐｉｃｋｉｎｇ　Ｓｙｓｔｅｍ（アズワン社）を用いて、陽性シグナルが得られたウェルのリンパ球をそれぞれ回収した。 1) -3 Single cell picking test The screening antigen prepared in 1) -1 above was prepared in 3 μg / mL, 1 mL with a 10 mM phosphate buffer (pH 7.0), and microchamber (AS ONE, inner diameter Φ20 μm). Was added to the mixture, and the mixture was allowed to stand at 37 ° C. for 1 hour for reaction. After removing the solution from the microchamber, 1 mL of RPMI-1640 containing 10% FBS (Wako, without phenol red) was added, and the mixture was allowed to stand at room temperature for 10 minutes. After removing the solution from the microchamber, the above-mentioned collected 2 × 10 5 lymphocytes were seeded, and then Goat anti-Labbit IgG (H + L) Highly Cross-Absorbed Secondary Antibody, Alexa Fluor 488 (Thermo10A10F). 1 mL of a solution diluted 500-fold with RPMI-1640 containing% FBS was added, and the cells were allowed to stand for 45 minutes under the conditions of 37 ° C. and 5% CO 2 . After removing the solution from the microchamber, the solution was washed with RPMI-1640 containing 10% FBS, and lymphocytes from wells with positive signals were collected using AS ONE Cell Picking System (AS ONE Corporation).
　１）－４　抗体遺伝子取得
　ＳｕｐｅｒＳｃｒｉｐｔＴＭ　ＩＩＩ　Ｏｎｅ－Ｓｔｅｐ　ＲＴ－ＰＣＲ　Ｓｙｓｔｅｍ　ｗｉｔｈ　ＰｌａｔｉｎｕｍＴＭＴａｑ　Ｈｉｇｈ　Ｆｉｄｅｌｉｔｙ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ社、１２５７４０３０）のＥｎｚｙｍｅ　ＭｉｘとＲＮＡ分解酵素阻害剤であるＲＮａｓｉｎ（Ｐｒｏｍｅｇａ社）を混合した溶液を用いて上記回収したリンパ球を溶解し、ＲＴ－ＰＣＲ法およびＰＣＲ法により、特異的プライマーを用いて重鎖および軽鎖の可変領域のＤＮＡサンプルをそれぞれ増幅した。 1) -4 Antibody gene acquisition SuperScript TM III One-Step RT-PCR System with Platinum TM Taq High Fidelity DNA Polymerase (Thermo Fisher, 12574030) Enzyme-degrading Enzyme (Thermo Fisher, 12574030) Enzyme The collected lymphocytes were lysed using the above-mentioned solution, and RNA samples in the variable regions of the heavy chain and the light chain were amplified by the RT-PCR method and the PCR method using specific primers, respectively.
　１）－５　プラスミド作製
　重鎖または軽鎖の抗体遺伝子を含む各ＰＣＲ産物をｐＣＥＣ３．１ベクター（ＣＡＧプロモーターおよびラビット抗体遺伝子の定常領域配列を予め付加したベクター）に挿入した。抗体遺伝子を含むｐＣＥＣ３．１プラスミドサンプルをＥ．　ｃｏｌｉ　ＪＭ１０９　Ｃｏｍｐｅｔｅｎｔ　Ｃｅｌｌｓ（ＴＡＫＡＲＡ社、＃９０５２）に形質転換しアンピシリン含有ＬＢ寒天上で３７℃、一晩インキュベートした後、コロニーＰＣＲ法によりプラスミドサンプルに遺伝子が挿入されていることを確認した。その後、ＧｅｎＥｌｕｔｅ（登録商標）　Ｐｌａｓｍｉｄ　Ｍｉｎｉｐｒｅｐ　Ｋｉｔ（ＳＩＧＭＡ社）を用いてプラスミドサンプルを精製し、ＨＥＫ２９３細胞へポリエチレンイミンを用いて形質移入手順に従って導入した。３７℃、５％ＣＯ２の条件下で４８時間静置培養し、抗体が分泌された培養上清を回収した。培養上清を用いて以下に記載のＥＬＩＳＡ試験を行い、ＥＬＩＳＡ試験での陽性シグナルを確認し、プラスミドサンプルに目的の抗体遺伝子が挿入されていることを確認した。 1) -5 Preparation of plasmid Each PCR product containing a heavy chain or light chain antibody gene was inserted into a pCEC3.1 vector (a vector to which a constant region sequence of a CAG promoter and a rabbit antibody gene was preliminarily added). A pCEC3.1 plasmid sample containing the antibody gene was prepared with E. coli. After transforming into coli JM109 Competent Cells (TAKARA, # 9052) and incubating on LB agar containing ampicillin at 37 ° C. overnight, it was confirmed that the gene was inserted into the plasmid sample by the colony PCR method. Then, a plasmid sample was purified using a GenElute (registered trademark) Prismid Miniprep Kit (SIGMA) and introduced into HEK293 cells using polyethyleneimine according to the transfection procedure. The cells were statically cultured for 48 hours under the conditions of 37 ° C. and 5% CO 2 , and the culture supernatant secreted by the antibody was collected. The ELISA test described below was performed using the culture supernatant, and a positive signal in the ELISA test was confirmed, and it was confirmed that the antibody gene of interest was inserted into the plasmid sample.
　１）－６　ＥＬＩＳＡ試験
　９６－ｗｅｌｌ　Ｃｌｅａｒ　Ｒｏｕｎｄ　Ｂｏｔｔｏｍ　Ｐｏｌｙｖｉｎｙｌ　Ｃｈｌｏｒｉｄｅ　（ＰＶＣ）　Ｎｏｔ　Ｔｒｅａｔｅｄ　Ｍｉｃｒｏｐｌａｔｅ（ｃｏｓｔａｒ社、　２７９７）に１０ｍＭリン酸バッファー（ｐＨ７．０）で１　μｇ／ｍＬに希釈したスクリーニング抗原を５０　μＬ／ｗｅｌｌで添加し、３７℃で１時間静置反応させた。その後、溶液を除去し、１％ＢＳＡ含有ＰＢＳを１００　μＬ／ｗｅｌｌで添加し、４度で一晩静置反応させた。翌日、溶液を除去し、ＰＢＳを３００　μＬ／ｗｅｌｌ添加および溶液の除去を３回繰り返した洗浄を行い、上記回収した培養上清を５０　μＬ／ｗｅｌｌで添加し、室温で１時間、静置反応させた。溶液を除去しＰＢＳにより洗浄を３回行い、Ｇｏａｔ　Ａｎｔｉ－Ｒａｂｂｉｔ　ＩｇＧ　Ｈ＆Ｌ（ＨＲＰ）　ｐｒｅａｄｓｏｒｂｅｄ（ａｂｃａｍ社、ａｂ９７０８０）を１％ＢＳＡ含有ＰＢＳで１万倍希釈した溶液を５０　μＬ／ｗｅｌｌで添加し、室温で３０分間静置反応させた後、溶液を除去しＰＢＳにより洗浄を３回行い、ＴＭＢＺ溶液とＨ２Ｏ２溶液を混合した発色液を５０　μＬ／ｗｅｌｌで添加し、室温で３分間反応させた後、リン酸溶液を５０　μＬ／ｗｅｌｌで添加し、反応を停止させた。プレートリーダーを用いて４５０　ｎｍの吸光度を測定した。ヒトＰＴＤＳＳ２の４４９から４８７番目までのアミノ酸配列とは相同性のないペプチド（配列番号１８：［ＡＣ］ＲＲＧＥＲＲＤＡＧＧＰＲＰＥＳＰＶＰＡＧＲＡＳＬＥＥＰＰＤＧＰＳＡＧＱＡＴＧＰＧＥＧＲＲＣ［ＯＨ］）
をキャリアタンパク質であるＢＳＡに結合させたものをスクリーニング抗原のネガティブコントロールとして用意し、上記同条件で同時に試験を行い、ネガティブコントロールを固相化したプレートでは反応せず、目的のスクリーニング抗原に特異的に反応したクローンを陽性と判断した。 1) -6 ELISA test 96-well Clear Round Bottom Polyvinyl Chloride (PVC) Not Treated Microplate (Costar, 2977) diluted with 10 mM phosphate buffer (pH 7.0) to 1 μg / mL. It was added by well and allowed to stand at 37 ° C. for 1 hour. Then, the solution was removed, PBS containing 1% BSA was added at 100 μL / well, and the reaction was allowed to stand overnight at 4 degrees. The next day, the solution was removed, and washing was performed by repeating the addition of PBS at 300 μL / well and the removal of the solution three times, and the collected culture supernatant was added at 50 μL / well, and the reaction was allowed to stand at room temperature for 1 hour. I let you. The solution was removed and washed with PBS three times, and a solution of Goat Anti-Rabbit IgG H & L (HRP) preadsorbed (abcam, ab97080) diluted 10,000 times with PBS containing 1% BSA was added at 50 μL / well. After allowing to stand for 30 minutes at room temperature, the solution is removed, washed with PBS three times, a color - developing solution containing a mixture of TMBZ solution and H2O2 solution is added at 50 μL / well, and the reaction is carried out at room temperature for 3 minutes. After that, a phosphate solution was added at 50 μL / well to stop the reaction. Absorbance at 450 nm was measured using a plate reader. Peptide not homologous to the amino acid sequence from 449 to 487 of human PTDSS2 (SEQ ID NO: 18: [AC] RRGERRDAGGPRPESPVPAGRASLEEPPDGPSAGQATGPGEGRRC [OH])
Was prepared as a negative control for the screening antigen, and the test was performed simultaneously under the same conditions as above. The negative control did not react on the plate immobilized on the solid phase, and was specific to the target screening antigen. The clone that reacted with was judged to be positive.
　その結果、陽性クローンとしてクローン４０１－Ｐ－１７が取得された。 As a result, clone 401-P-17 was obtained as a positive clone.
　１）－７　抗体精製
　上記１）－５で作製したプラスミドサンプルをＨＥＫ２９３細胞へポリエチレンイミンを用いて形質移入手順に従って導入し、３７℃、５％ＣＯ２の条件下で１０日間培養し、抗体が分泌された培養上清を回収した。培養上清をｒＰｒｏｔｅｉｎ　Ａ　Ｓｅｐｈａｒｏｓｅ　Ｆａｓｔ　Ｆｌｏｗ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ社、１７－１２７９－０１）のカラムに通し、抗体を担体へ吸着させ、ＴＢＳで担体を洗浄後、Ｇｅｎｔｌｅ　Ａｇ／Ａｂ　Ｅｌｕｔｉｏｎ　ｂｕｆｆｅｒ，　ｐＨ６．６（Ｔｈｅｒｍｏ社、２１０２７）で溶出を行った。Ａｍｉｃｏｎ　Ｕｌｔｒａ－４　Ｃｅｎｔｒｉｆｕｇａｌ　Ｆｉｌｔｅｒ　Ｄｅｖｉｃｅｓ　５０Ｋ　ｄｅｖｉｃｅ（Ｍｉｌｌｉｐｏｒｅ社、ＵＦＣ８０３０２４）を用いてＰＢＳへバッファー置換を行い、抗体濃度の測定はＰｉｅｒｃｅＴＭ　ＢＣＡ　Ｐｒｏｔｅｉｎ　Ａｓｓａｙ　Ｋｉｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ社、２３２２５）を用い、測定した。 1) -7 Antibody purification The plasmid sample prepared in 1) -5 above was introduced into HEK293 cells using polyethyleneimine according to the transfection procedure, and cultured under the conditions of 37 ° C. and 5% CO 2 for 10 days to obtain the antibody. The secreted culture supernatant was collected. The culture supernatant was passed through a column of rProtein A Sepharose Fast Flow (GE Healthcare, 17-1279-01), the antibody was adsorbed on the carrier, the carrier was washed with TBS, and then Gentle Ag / Ab Elution buffer, pH 6.6 (. Elution was performed by Thermo, 21027). Buffer substitution was performed on PBS using the American Ultra-4 Centrifugal Filter Devices 50K device (Millipore, UFC803024), and the antibody concentration was measured using Pierce TM BCA Protein Assay Kit (Thermo 32).
　１）－８　クローン４０１－Ｐ－１７由来の抗ＰＴＤＳＳ２抗体である４０１－Ｐ－１７抗体の配列解析
　クローン４０１－Ｐ－１７のシークエンス解析を実施し、重鎖および軽鎖のシグナル配列と可変領域のｃＤＮＡのヌクレオチド配列を取得した。４０１－Ｐ－１７抗体の重鎖ヌクレオチド配列は配列表の配列番号８に記載され、ヌクレオチド番号１－５７番がシグナル配列、５８－３８４番が重鎖可変領域、３８５－１３５６番が重鎖定常領域を示している。ＩＭＧＴ（ｈｔｔｐ：／／ｗｗｗ．ｉｍｇｔ．ｏｒｇ／）により決定したＣＤＲの配列は配列番号８の１３０－１５３番がＣＤＲＨ１、２０５－２２５番がＣＤＲＨ２、３３６－３５１番がＣＤＲＨ３を示している。４０１－Ｐ－１７抗体の重鎖アミノ酸配列は、配列表の配列番号２および図１に記載され、アミノ酸番号１－１９番がシグナル配列、２０－１２８番が重鎖可変領域、１２９－４５１番が重鎖定常領域を示している。ＩＭＧＴ（ｈｔｔｐ：／／ｗｗｗ．ｉｍｇｔ．ｏｒｇ／）により決定したＣＤＲのアミノ酸配列は配列番号２のアミノ酸番号４４－５１番がＣＤＲＨ１、６９－７５番がＣＤＲＨ２、１１２－１１６番がＣＤＲＨ３を示している。また、４０１－Ｐ－１７抗体の重鎖可変領域のアミノ酸配列は配列表の配列番号３および図２に記載されている。４０１－Ｐ－１７抗体の重鎖定常領域のアミノ酸配列は配列表の配列番号４および図３に記載されている。４０１－Ｐ－１７抗体のＣＤＲＨ１のアミノ酸配列は配列表の配列番号５および図４に記載されている。４０１－Ｐ－１７抗体のＣＤＲＨ２のアミノ酸配列は配列表の配列番号６および図５に記載されている。４０１－Ｐ－１７抗体のＣＤＲＨ３のアミノ酸配列は配列表の配列番号７および図６に記載されている。 1) -8 Sequence analysis of 401-P-17 antibody, which is an anti-PTDSS2 antibody derived from clone 401-P-17, sequence analysis of clone 401-P-17 was performed, and the signal sequences and variable regions of heavy and light chains were performed. The nucleotide sequence of the cDNA was obtained. The heavy chain nucleotide sequence of the 401-P-17 antibody is described in SEQ ID NO: 8 of the sequence listing, nucleotide number 1-57 is the signal sequence, 58-384 is the heavy chain variable region, and 385-1356 is the heavy chain constant. Shows the area. As for the sequence of CDR determined by IMGT (http://www.imgt.org/), SEQ ID NO: 8: 130-153 indicates CDRH1, 205-225 indicates CDRH2, and 336-351 indicates CDRH3. The heavy chain amino acid sequence of the 401-P-17 antibody is shown in SEQ ID NO: 2 and FIG. 1 of the sequence listing, where amino acid numbers 1-19 are the signal sequence, 20-128 are the heavy chain variable region, and 129-451. Indicates the heavy chain constant region. The amino acid sequence of CDR determined by IMGT (http://www.imgt.org/) indicates that amino acid numbers 44-51 of SEQ ID NO: 2 indicate CDRH1, 69-75 indicates CDRH2, and 112-116 indicate CDRH3. There is. The amino acid sequence of the heavy chain variable region of the 401-P-17 antibody is shown in SEQ ID NO: 3 and FIG. 2 in the sequence listing. The amino acid sequence of the heavy chain constant region of the 401-P-17 antibody is shown in SEQ ID NO: 4 and FIG. 3 of the sequence listing. The amino acid sequence of CDRH1 of the 401-P-17 antibody is shown in SEQ ID NO: 5 and FIG. 4 of the sequence listing. The amino acid sequence of CDRH2 of the 401-P-17 antibody is shown in SEQ ID NO: 6 and FIG. 5 of the sequence listing. The amino acid sequence of CDRH3 of the 401-P-17 antibody is shown in SEQ ID NO: 7 and FIG. 6 of the sequence listing.
　４０１－Ｐ－１７抗体の軽鎖ヌクレオチド配列は配列表の配列番号１６に記載され、ヌクレオチド番号１－６６番がシグナル配列、６７－４０５番が軽鎖可変領域、４０６－７２０番が軽鎖定常領域を示している。ＩＭＧＴ（ｈｔｔｐ：／／ｗｗｗ．ｉｍｇｔ．ｏｒｇ／）により決定したＣＤＲの配列は配列番号１６の１４５－１６８番がＣＤＲＬ１、２２０－２２８番がＣＤＲＬ２、３３７－３７５番がＣＤＲＬ３を示している。４０１－Ｐ－１７抗体の軽鎖アミノ酸配列は、配列表の配列番号１０および図７に記載され、アミノ酸番号１－２２番がシグナル配列、２３－１３５番が軽鎖可変領域、１３６－２３９番が軽鎖定常領域を示している。ＩＭＧＴ（ｈｔｔｐ：／／ｗｗｗ．ｉｍｇｔ．ｏｒｇ／）により決定したＣＤＲのアミノ酸配列は配列番号１０のアミノ酸番号４９－５６番がＣＤＲＬ１、７４－７６番がＣＤＲＬ２、１１３－１２５がＣＤＲＬ３を示している。また、４０１－Ｐ－１７抗体の軽鎖可変領域のアミノ酸配列は配列表の配列番号１１および図８に記載されている。４０１－Ｐ－１７抗体の軽鎖定常領域のアミノ酸配列は配列表の配列番号１２および図９に記載されている。４０１－Ｐ－１７抗体のＣＤＲＬ１のアミノ酸配列は配列表の配列番号１３および図１０に記載されている。４０１－Ｐ－１７抗体のＣＤＲＬ２のアミノ酸配列は配列表の配列番号１４および図１１に記載されている。４０１－Ｐ－１７抗体のＣＤＲＬ３のアミノ酸配列は配列表の配列番号１５および図１２に記載されている。 The light chain nucleotide sequence of the 401-P-17 antibody is set forth in SEQ ID NO: 16 of the sequence listing, nucleotide number 1-66 is the signal sequence, 67-405 is the light chain variable region, and 406-720 is the light chain constant. Shows the area. As for the sequence of CDR determined by IMGT (http://www.imgt.org/), SEQ ID NO: 16: 145-168 indicates CDRL1, 220-228 indicates CDRL2, and 337-375 indicates CDRL3. The light chain amino acid sequence of the 401-P-17 antibody is shown in SEQ ID NO: 10 and FIG. 7 of the sequence listing, where amino acid numbers 1-22 are the signal sequence, 23-135 is the light chain variable region, 136-239. Indicates the light chain constant region. As for the amino acid sequence of CDR determined by IMGT (http://www.imgt.org/), amino acid numbers 49-56 of SEQ ID NO: 10 indicate CDRL1, 74-76 indicates CDRL2, and 113-125 indicate CDRL3. .. The amino acid sequence of the light chain variable region of the 401-P-17 antibody is shown in SEQ ID NO: 11 and FIG. 8 of the sequence listing. The amino acid sequence of the light chain constant region of the 401-P-17 antibody is shown in SEQ ID NO: 12 and FIG. 9 of the sequence listing. The amino acid sequence of CDRL1 of the 401-P-17 antibody is set forth in SEQ ID NO: 13 and FIG. 10 of the sequence listing. The amino acid sequence of CDRL2 of the 401-P-17 antibody is set forth in SEQ ID NO: 14 and FIG. 11 of the sequence listing. The amino acid sequence of CDRL3 of the 401-P-17 antibody is set forth in SEQ ID NO: 15 and FIG. 12 of the sequence listing.
　実施例２：ウサギ抗ヒトＰＴＤＳＳ２抗体のＩＨＣ適性評価
　２）－１　ＰＴＤＳＳ２の発現を有する野生型Ａ３７５細胞とＰＴＤＳＳ２遺伝子をＫｎｏｃｋｏｕｔしたＡ３７５細胞を用いたＰＴＤＳＳ２の染色性評価
　２）－１－１　セルブロックの作製
　Ａ３７５細胞は、ＰＴＤＳＳ２遺伝子をｋｎｏｃｋｏｕｔし、ｈｅｔｅｒｏｌｏｇｕｓまたはｈｏｍｏｌｏｇｕｓに遺伝子を不活化した細胞を選択した。このＰＴＤＳＳ２遺伝子の野生型（ｗｉｌｄ　ｔｙｐｅ）、ｈｅｔｅｒｏｌｏｇｕｓまたはｈｏｍｏｌｏｇｕｓ　ｋｎｏｃｋｏｕｔの３型のＡ３７５細胞のペレットをホルマリン固定後にパラフィン包埋ブロックとした。 Example 2: IHC suitability evaluation of rabbit anti-human PTDSS2 antibody 2) -1 Stainability evaluation of PTDSS2 using wild-type A375 cells expressing PTDSS2 and A375 cells knocked out from the PTDSS2 gene 2) -1-1 Cell block Preparation of A375 cells were selected by knocking out the PTDSS2 gene and inactivating the gene in heterologus or homologus. Pellets of A375 cells of the wild type (wild type), heterologus or homologus knockout of the PTDSS2 gene were fixed with formalin and then used as a paraffin-embedded block.
　２）－１－２　ＰＴＤＳＳ２をＫｎｏｃｋｏｕｔしたＡ３７５細胞の免疫染色
　１）－８で調製したウサギモノクローナル抗ＰＴＤＳＳ２抗体（４０１－Ｐ－１７抗体）の染色性を検討した。脱パラフィン、抗原賦活およびＩＨＣは、自動免疫染色装置であるＢｅｎｃｈＭａｒｋ　Ｕｌｔｒａ（Ｖｅｎｔａｎａ社製）を用いた。脱パラフィン後、抗原賦活は抗原賦活液ＵＬＴＲＡ　ＣＣ１（Ｖｅｎｔａｎａ社製）を用いて、１００℃で５６分間実施した。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒ（Ｖｅｎｔａｎａ社製）で洗浄した後、ＯｐｔｉＶｉｅｗ　Ｐｅｒｏｘｉｄａｓｅ　Ｉｎｈｉｂｉｔｏｒ（Ｖｅｎｔａｎａ社製）を加え４分間インキュベートした。一次抗体４０１－Ｐ－１７をＲｅａｃｔｉｏｎ　ＢｕｆｆｅｒをＡｎｔｉｂｏｄｙ　ｄｉｌｕｅｎｔｚ（Ｃａｔ．　Ｎｏ．　２５１－０１８、Ｖｅｎｔａｎａ社製）で表２に記載の濃度に希釈し、３７℃で６０分間反応させた。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＯｐｔｉＶｉｅｗ　ＨＱ　Ｕｎｉｖｅｒｓａｌ　ＬｉｎｋｅｒまたはＤＩＳＣＯＶＥＲＹ　ａｎｔｉ－Ｒａｂｂｉｔ　ＨＱ（Ｖｅｎｔａｎａ社製）を加え８分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＯｐｔｉＶｉｅｗ　ＨＲＰ　Ｍｕｌｔｉｍｅｒ（Ｖｅｎｔａｎａ社製）を加え８分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＯｐｔｉＶｉｅｗ　Ｈ２Ｏ２とＯｐｔｉＶｉｅｒ　ＤＡＢ（Ｖｅｎｔａｎａ社製）を加え８分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＯｐｔｉＶｉｅｗ　Ｃｏｐｐｅｒ（Ｖｅｎｔａｎａ社製）を加え４分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＨＥＭＡＴＯＸＹＬＩＮ　ＩＩ（Ｖｅｎｔａｎａ社製）を加え８分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＢＬＵＩＮＧ　ＲＥＡＧＥＮＴ（Ｖｅｎｔａｎａ社製）を加え４分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒおよびイオン交換水で洗浄した後、マリノール（武藤化学）で封入した。 2) -1-2 Immunostaining of A375 cells knocked out of PTDSS2 1) The stainability of the rabbit monoclonal anti-PTDSS2 antibody (401-P-17 antibody) prepared in -8 was examined. For deparaffinization, antigen activation and IHC, an automated immunostaining apparatus, BenchMark Ultra (manufactured by Ventana) was used. After deparaffinization, antigen activation was carried out at 100 ° C. for 56 minutes using an antigen activation solution ULTRA CC1 (manufactured by Ventana). After washing with Reaction Buffer (manufactured by Ventana), OptiView Peroxidase Inhibitor (manufactured by Ventana) was added and incubated for 4 minutes. The primary antibody 401-P-17 was diluted with Reaction Buffer with Antibody diluentz (Cat. No. 251-018, manufactured by Ventana) to the concentration shown in Table 2, and reacted at 37 ° C. for 60 minutes. After washing with Reaction Buffer, OptiView HQ Universal Linker or DISCOVERY anti-Rabbit HQ (manufactured by Ventana) was added and incubated for 8 minutes. After washing with Reaction Buffer, OptiView HRP Multimer (manufactured by Ventana) was added and incubated for 8 minutes. After washing with Reaction Buffer, OptiView H2O2 and OptiVier DAB (manufactured by Ventana) were added and incubated for 8 minutes. After washing with Reaction Buffer, OptiView Copper (manufactured by Ventana) was added and incubated for 4 minutes. After washing with Reaction Buffer, HEMATOXYLIN II (manufactured by Ventana) was added and incubated for 8 minutes. After washing with Reaction Buffer, BLUING REAGENT (manufactured by Ventana) was added and incubated for 4 minutes. After washing with Reaction Buffer and ion-exchanged water, the cells were sealed with Marinol (Muto Kagaku).
　抗ＰＴＤＳＳ２抗体（４０１－Ｐ－１７抗体）を用いた染色結果を図１３－１および図１３－２に示す。また、図１３－１および図１３－２における染色評価結果を表２に示す。一次抗体濃度として、１、２．５、５ならびに１０　μｇ／ｍＬの４段階の濃度で染色した。ＷＴとＨｅｔｅｒｏは陽性を呈したが、ＷＴがＨｅｔｅｒｏに比較して強い染色強度を示した。Ｈｏｍｏでは、すべて陰性と判断されたが、５および１０　μｇ／ｍＬにおいてかすかな背景染色が認められた。したがって、２．５　μｇ／ｍＬ以下では十分に特異的な染色性が確認された。ＷＴにおいて１　μｇ／ｍＬでも十分な染色強度が確認された。 The results of staining using the anti-PTDSS2 antibody (401-P-17 antibody) are shown in FIGS. 13-1 and 13-2. Table 2 shows the results of dyeing evaluation in FIGS. 13-1 and 13-2. The primary antibody concentration was 1, 2.5, 5 and 10 μg / mL, which were stained in four stages. WT and Hetero were positive, but WT showed stronger staining intensity compared to Hetero. In Homo, all were judged to be negative, but faint background staining was observed at 5 and 10 μg / mL. Therefore, it was confirmed that the stainability was sufficiently specific at 2.5 μg / mL or less. In WT, sufficient staining intensity was confirmed even at 1 μg / mL.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　＊　ＷＴにおける染色強度より弱い．　＊＊　かすかに細胞が染色されている．非特異的染色と判断。 * Weaker than the staining intensity in WT. ** The cells are faintly stained. Judged as non-specific staining.
　２）－２　ヒト腫瘍組織切片の免疫染色
　２）－２－１　ヒト乳がん組織アレイの染色
　臨床検体における抗ＰＴＤＳＳ２抗体の染色性をＢＲ２０８１１、Ｂｒｅａｓｔ　ｃａｎｃｅｒ　ｔｉｓｓｕｅ　ａｒｒａｙ、１０４　ｃａｓｅｓ（ＵＳ　Ｂｉｏｍａｘ社製）を用いて検討した。 2) -2 Immunostaining of human tumor tissue sections 2) -2-1 Staining of human breast cancer tissue array The stainability of anti-PTDSS2 antibody in clinical specimens was determined using BR20811, Breast cancer tissue array, 104 cases (manufactured by US Biomax). I examined it.
　ウサギモノクローナル抗ＰＴＤＳＳ２抗体（クローン４０１－Ｐ－１７）の染色性を検討した。脱パラフィン、抗原賦活およびＩＨＣは、自動免疫染色装置であるＢｅｎｃｈＭａｒｋ　Ｕｌｔｒａ（Ｖｅｎｔａｎａ社製）を用いた。脱パラフィン後、抗原賦活は抗原賦活液ＵＬＴＲＡ　ＣＣ１（Ｖｅｎｔａｎａ社製）を用いて、１００℃で５６分間実施した。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒ（Ｖｅｎｔａｎａ社製）で洗浄した後、ＯｐｔｉＶｉｅｗ　Ｐｅｒｏｘｉｄａｓｅ　Ｉｎｈｉｂｉｔｏｒ（Ｖｅｎｔａｎａ社製）を加え４分間インキュベートした。一次抗体４０１－Ｐ－１７をＲｅａｃｔｉｏｎ　ＢｕｆｆｅｒをＡｎｔｉｂｏｄｙ　ｄｉｌｕｅｎｔｚ（Ｃａｔ．　Ｎｏ．　２５１－０１８、Ｖｅｎｔａｎａ社製）で１　μｇ／ｍＬの濃度に希釈し、３７℃で６０分間反応させた。陰性対照として、隣接切片に一次抗体４０１－Ｐ－１７の代わりにＮｏｒｍａｌ　Ｒａｂｂｉｔ　ＩｇＧ　（ｃａｔ．＃２７２９，　ＣＳＴ）を同様に反応させた。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＯｐｔｉＶｉｅｗ　ＨＱ　Ｕｎｉｖｅｒｓａｌ　ＬｉｎｋｅｒまたはＤＩＳＣＯＶＥＲＹ　ａｎｔｉ－Ｒａｂｂｉｔ　ＨＱ（Ｖｅｎｔａｎａ社製）を加え８分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＯｐｔｉＶｉｅｗ　ＨＲＰ　Ｍｕｌｔｉｍｅｒ（Ｖｅｎｔａｎａ社製）を加え８分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＯｐｔｉＶｉｅｗ　Ｈ２Ｏ２とＯｐｔｉＶｉｅｒ　ＤＡＢ（Ｖｅｎｔａｎａ社製）を加え８分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＯｐｔｉＶｉｅｗ　Ｃｏｐｐｅｒ（Ｖｅｎｔａｎａ社製）を加え４分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＨＥＭＡＴＯＸＹＬＩＮ　ＩＩ（Ｖｅｎｔａｎａ社製）を加え８分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒで洗浄した後、ＢＬＵＩＮＧ　ＲＥＡＧＥＮＴ（Ｖｅｎｔａｎａ社製）を加え４分間インキュベートした。Ｒｅａｃｔｉｏｎ　Ｂｕｆｆｅｒおよびイオン交換水で洗浄した後、マリノール（武藤化学）で封入した。 The stainability of the rabbit monoclonal anti-PTDSS2 antibody (clone 401-P-17) was examined. For deparaffinization, antigen activation and IHC, an automatic immunostaining apparatus, BenchMark Ultra (manufactured by Ventana) was used. After deparaffinization, antigen activation was carried out at 100 ° C. for 56 minutes using an antigen activation solution ULTRA CC1 (manufactured by Ventana). After washing with Reaction Buffer (manufactured by Ventana), OptiView Peroxidase Inhibitor (manufactured by Ventana) was added and incubated for 4 minutes. The primary antibody 401-P-17 was diluted with Reaction Buffer with Antibody diluentz (Cat. No. 251-018, manufactured by Ventana) to a concentration of 1 μg / mL, and reacted at 37 ° C. for 60 minutes. As a negative control, adjacent sections were similarly reacted with Normal Rabbit IgG (cat. # 2729, CST) instead of the primary antibody 401-P-17. After washing with Reaction Buffer, OptiView HQ Universal Linker or DISCOVERY anti-Labbit HQ (manufactured by Ventana) was added and incubated for 8 minutes. After washing with Reaction Buffer, OptiView HRP Multimer (manufactured by Ventana) was added and incubated for 8 minutes. After washing with Reaction Buffer, OptiView H2O2 and OptiVier DAB (manufactured by Ventana) were added and incubated for 8 minutes. After washing with Reaction Buffer, OptiView Copper (manufactured by Ventana) was added and incubated for 4 minutes. After washing with Reaction Buffer, HEMATOXYLIN II (manufactured by Ventana) was added and incubated for 8 minutes. After washing with Reaction Buffer, BLUING REAGENT (manufactured by Ventana) was added and incubated for 4 minutes. After washing with Reaction Buffer and ion-exchanged water, the cells were sealed with Marinol (Muto Kagaku).
　抗ＰＴＤＳＳ２抗体（ｃｌｏｎｅ　４０１－Ｐ－１７）を用いた染色結果を図１４－１および図１４－２に示す。また、図１４－１および図１４－２における染色評価結果を表３に示す。Ｂ４とＦ９は、腫瘍が強い陽性を呈した。Ｊ４では、腫瘍は微弱陽性を呈した。Ｅ５では、腫瘍は陰性を示した。なお、陰性対照として一次抗体にＮｏｒｍａｌ　Ｒａｂｂｉｔ　ＩｇＧをおいた切片では、非特異的染色も含めてわずかな染色性を示すものはなかった。以上のことから、ヒトの腫瘍組織切片においても、ＰＴＤＳＳ２発現の陽性または陰性を識別できることが確認された。表３に示すヒト乳がん組織におけるＰＴＤＳＳ２の発現(Breast tissue tumor array, BR20811, US Biomax)では、いずれのコアにおいても間質の細胞が染色されている。 The results of staining using the anti-PTDSS2 antibody (clone 401-P-17) are shown in FIGS. 14-1 and 14-2. Table 3 shows the results of dyeing evaluation in FIGS. 14-1 and 14-2. B4 and F9 showed strong positive tumors. At J4, the tumor was weakly positive. At E5, the tumor was negative. In addition, in the section in which Normal Rabbit IgG was added to the primary antibody as a negative control, none showed slight staining property including non-specific staining. From the above, it was confirmed that positive or negative PTDSS2 expression can be discriminated even in human tumor tissue sections. In the expression of PTDSS2 (Breast tissue tumor array, BR20811, US Biomax) in human breast cancer tissue shown in Table 3, interstitial cells were stained in all cores.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　本発明の提供する抗ＰＴＤＳＳ２抗体を用いることによりＰＴＤＳＳ２の発現と関連する疾患の検査または診断が可能となる。また、本発明の抗ＰＴＤＳＳ２抗体を含むことからなる、ＰＴＤＳＳ２の発現と関連する疾患の検査または診断用キット等を提供することができる。 By using the anti-PTDSS2 antibody provided by the present invention, it is possible to test or diagnose a disease related to the expression of PTDSS2. In addition, it is possible to provide a kit for testing or diagnosis of a disease related to the expression of PTDSS2, which comprises the anti-PTDSS2 antibody of the present invention.
配列番号１８：ネガティブコントロール SEQ ID NO: 18: Negative control

Claims (26)

  1. 　ヒトＰＴＤＳＳ２またはその部分ペプチドに特異的に結合する抗体または当該抗体の抗原結合性断片。 An antibody that specifically binds to human PTDSS2 or a partial peptide thereof, or an antigen-binding fragment of the antibody.
  2. 　ヒトＰＴＤＳＳ２の部分ペプチドが配列表の配列番号1に記載のアミノ酸配列からなるペプチドである、請求項１に記載の抗体または当該抗体の抗原結合性断片。 The antibody according to claim 1 or an antigen-binding fragment of the antibody, wherein the partial peptide of human PTDSS2 is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing.
  3. 　ヒトＰＴＤＳＳ２またはその部分ペプチドへの結合に対して、配列番号２のアミノ酸番号２０乃至４５１に示されるアミノ酸配列からなる重鎖および配列番号１０のアミノ酸番号２３乃至２３９に示されるアミノ酸配列からなる軽鎖を有する抗体と競合阻害活性を有する抗体または当該抗体の抗原結合性断片。 For binding to human PTDSS2 or a partial peptide thereof, a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 451 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequences shown in amino acid numbers 23 to 239 of SEQ ID NO: 10. An antibody having competitive inhibitory activity with an antibody having the same or an antigen-binding fragment of the antibody.
  4. 　重鎖配列が、配列番号５に示されるアミノ酸配列からなるＣＤＲＨ１、配列番号６に示されるアミノ酸配列からなるＣＤＲＨ２、並びに、配列番号７に示されるアミノ酸配列からなるＣＤＲＨ３を有する可変領域を含み；
    軽鎖配列が配列番号１３に示されるアミノ酸配列からなるＣＤＲＬ１、配列番号１４に示されるアミノ酸配列からなるＣＤＲＬ２、および配列番号１５に示されるアミノ酸配列からなるＣＤＲＬ３を有する可変領域を含む、請求項１乃至３のいずれか1項に記載の抗体または当該抗体の抗原結合性断片。     The heavy chain sequence comprises a variable region having CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 6, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 7.
    1. 3. The antibody according to any one of 3 or an antigen-binding fragment of the antibody.
  5. 　配列番号３に示されるアミノ酸配列からなる重鎖可変領域および、配列番号１１に示される軽鎖可変領域を含むことからなる、請求項１乃至４のいずれか１項に記載の抗体または当該抗体の抗原結合性断片。 The antibody according to any one of claims 1 to 4, or the antibody thereof, which comprises a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 11. Antigen-binding fragment.
  6. 　配列番号２のアミノ酸番号２０乃至４５１またはアミノ酸番号２０乃至４５０に示されるアミノ酸配列を含むことからなる重鎖、および、配列番号１０のアミノ酸番号２３乃至２３９に示されるアミノ酸配列を含むことからなる軽鎖からなる請求項１乃至５のいずれか１項に記載の抗体または当該抗体の抗原結合性断片。 A heavy chain comprising the amino acid sequences set forth in amino acid numbers 20 to 451 or amino acids 20 to 450 of SEQ ID NO: 2 and a light chain comprising the amino acid sequences set forth in amino acid numbers 23 to 239 of SEQ ID NO: 10. The antibody according to any one of claims 1 to 5, which comprises a chain, or an antigen-binding fragment of the antibody.
  7. 　キメラ抗体であることを特徴とする請求項１乃至５のいずれか１項に記載の抗体または当該抗体の抗原結合性断片。 The antibody according to any one of claims 1 to 5, which is a chimeric antibody, or an antigen-binding fragment of the antibody.
  8. 　定常領域がウサギ抗体由来であることを特徴とする、請求項７に記載のキメラ抗体。 The chimeric antibody according to claim 7, wherein the constant region is derived from a rabbit antibody.
  9. 　ヒト化されていることを特徴とする、請求項１乃至５のいずれか１項に記載の抗体または当該抗体の抗原結合性断片。 The antibody according to any one of claims 1 to 5, which is characterized by being humanized, or an antigen-binding fragment of the antibody.
  10. 　Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’およびＦｖからなる群から選択される、請求項１乃至９のいずれか１項に記載の抗体の抗原結合性断片。 The antigen-binding fragment of the antibody according to any one of claims 1 to 9, which is selected from the group consisting of Fab, F (ab') 2, Fab'and Fv.
  11. 　ｓｃＦｖであることを特徴とする、請求項１乃至１０のいずれか１項に記載の抗体。 The antibody according to any one of claims 1 to 10, which is characterized by being scFv.
  12. 　請求項１乃至１１のいずれか１項に記載の抗体または当該抗体の抗原結合性断片を含む組成物。 A composition comprising the antibody according to any one of claims 1 to 11 or an antigen-binding fragment of the antibody.
  13. 　請求項１乃至１１のいずれか１項に記載の抗体または当該抗体の抗原結合性断片を含み、パラフィン包埋処理した後脱パラフィン処理した組織標本中のＰＴＤＳＳ２の検出または測定方法に使用される、請求項１２に記載の組成物。 A method for detecting or measuring PTDSS2 in a tissue specimen containing the antibody according to any one of claims 1 to 11 or an antigen-binding fragment of the antibody and subjected to paraffin embedding treatment and then deparaffin treatment. The composition according to claim 12.
  14. 　請求項１乃至１１のいずれか１項に記載の抗体または該抗体の抗原結合性断片と被検標本を接触させる工程を含む、標本中のＰＴＤＳＳ２の検出または測定方法に使用される、請求項１２または請求項１３に記載の組成物。
    　上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。     12. A method for detecting or measuring PTDSS2 in a specimen, which comprises a step of contacting a test specimen with the antibody according to any one of claims 1 to 11 or an antigen-binding fragment of the antibody. Alternatively, the composition according to claim 13.
    The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  15. 　ＰＴＤＳＳ２の検出または測定方法が、被検標本においてＰＴＤＳＳ２が検出もしくは測定されたか、または被検標本におけるＰＴＤＳＳ２の発現量が事前に決定された基準と同等かまたはそれより高い場合、該被検標本を陽性と判定し、被検標本においてＰＴＤＳＳ２が検出もしくは測定されなかったか、または被検標本におけるＰＴＤＳＳ２の発現量が事前に決定された基準と同等かまたはそれより低い場合、該被検標本を陰性と判定する工程を含む、請求項１３または１４に記載の組成物。
    　上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。     If the method for detecting or measuring PTDSS2 is such that PTDSS2 is detected or measured in the test specimen, or the expression level of PTDSS2 in the test specimen is equal to or higher than a predetermined criterion, the test specimen is used. If PTDSS2 is not detected or measured in the test specimen, or if the expression level of PTDSS2 in the test specimen is equal to or lower than the predetermined criterion, the test specimen is regarded as negative. The composition according to claim 13 or 14, comprising the step of determining.
    The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  16. 　ＰＴＤＳＳ２陰性疾患（がん）の検査または診断方法に使用される、請求項１２乃至１５のいずれか１項に記載の組成物。 The composition according to any one of claims 12 to 15, which is used for a method for testing or diagnosing a PTDSS2-negative disease (cancer).
  17. 　請求項１乃至１１のいずれか１項に記載の抗体または該抗体の抗原結合性断片をコードするポリヌクレオチド。 A polynucleotide encoding the antibody according to any one of claims 1 to 11 or an antigen-binding fragment of the antibody.
  18. 　請求項１７に記載のポリヌクレオチドを含むベクター。 A vector containing the polynucleotide according to claim 17.
  19. 　請求項１７に記載のポリヌクレオチドまたは請求項１８に記載のベクターを含む細胞。 A cell containing the polynucleotide according to claim 17 or the vector according to claim 18.
  20. 　下記の工程（ａ）および（ｂ）を含む、請求項１乃至１１のいずれか１項に記載の抗体または該抗体の抗原結合性断片の製造方法：
    　（ａ）請求項１９記載の細胞を培養する工程；
    　（ｂ）工程（ａ）の培養物からモノクローナル抗体または該抗体の抗原結合性断片を回収する工程。     The method for producing an antibody or an antigen-binding fragment of the antibody according to any one of claims 1 to 11, which comprises the following steps (a) and (b):
    (A) The step of culturing the cells according to claim 19.
    (B) A step of recovering a monoclonal antibody or an antigen-binding fragment of the antibody from the culture of step (a).
  21. 　被検標本を用い、該被検標本中におけるＰＴＤＳＳ２の有無を検出し（該検出には、請求項１乃至１１のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ２が陰性と判定された被験者を、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療への応答性があると判定することを含む、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療への応答性を予測する方法。
    　上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。     The presence or absence of PTDSS2 in the test sample is detected using the test sample (the antibody according to any one of claims 1 to 11 or an antigen-binding fragment of the antibody is used for the detection). To the treatment of PTDSS2-negative disease (cancer) with PTDSS1 inhibitor, including determining that subjects who are determined to be negative for PTDSS2 are responsive to the treatment of PTDSS2-negative disease (cancer) with PTDSS1 inhibitor. How to predict the responsiveness of.
    The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  22. 　被検標本を用いてＰＴＤＳＳ２が陰性と判定された被験者を（該判定には、請求項１乃至１１のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療への応答性があると判定することを含む、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療への応答性を予測する方法。
    上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。     A subject whose PTDSS2 was determined to be negative using the test specimen (the antibody according to any one of claims 1 to 11 or an antigen-binding fragment of the antibody is used for the determination) is used as a PTDSS1 inhibitor. A method for predicting the responsiveness of a PTDSS1 inhibitor to the treatment of a PTDSS2-negative disease (cancer), which comprises determining that the PTDSS2-negative disease (cancer) is responsive to the treatment.
    The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  23. 　被検標本を用い、該被検標本中におけるＰＴＤＳＳ２の有無を検出し（該検出には、請求項１乃至１１のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ２が陰性と判定された被験者を、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療の対象として選別することを含む、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療の対象を選別する方法。
    　上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。     The presence or absence of PTDSS2 in the test sample is detected using the test sample (the antibody according to any one of claims 1 to 11 or an antigen-binding fragment of the antibody is used for the detection). Selection of subjects for treatment of PTDSS2-negative disease (cancer) with PTDSS1 inhibitor, including selection of subjects tested negative for PTDSS2 as targets for treatment of PTDSS2-negative disease (cancer) with PTDSS1 inhibitor. Method.
    The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  24. 　被検標本を用いてＰＴＤＳＳ２が陰性と判定された被験者を（該判定には、請求項１乃至１１のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療の対象として選別することを含む、ＰＴＤＳＳ１阻害剤によるＰＴＤＳＳ２陰性疾患（がん）の治療の対象を選別する方法。
    　上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。     A subject whose PTDSS2 was determined to be negative using the test specimen (the antibody according to any one of claims 1 to 11 or an antigen-binding fragment of the antibody is used for the determination) is used as a PTDSS1 inhibitor. A method of selecting a target for treatment of a PTDSS2-negative disease (cancer) with a PTDSS1 inhibitor, which comprises selecting the target for treatment of a PTDSS2-negative disease (cancer).
    The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  25. 　被検標本を用い、該被検標本中におけるＰＴＤＳＳ２の有無を検出し（該検出には、請求項１乃至１１のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ２が陰性と判定された被験者に対し、ＰＴＤＳＳ１阻害剤を投与することを含む、ＰＴＤＳＳ２陰性疾患（がん）の治療方法。
    　上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。     The presence or absence of PTDSS2 in the test specimen is detected using the test specimen (the antibody according to any one of claims 1 to 11 or an antigen-binding fragment of the antibody is used for the detection). A method for treating a PTDSS2-negative disease (cancer), which comprises administering a PTDSS1 inhibitor to a subject who is determined to be negative for PTDSS2.
    The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
  26. 　被検標本を用いてＰＴＤＳＳ２の機能抑制が検出された被験者に対し（該検出には、請求項１乃至１１のいずれか１項に記載の抗体または該抗体の抗原結合性断片を用いる）、ＰＴＤＳＳ１阻害剤を投与することを含む、ＰＴＤＳＳ２陰性疾患（がん）の治療方法。
    　上記標本は、パラフィン包埋処理した後脱パラフィン処理した組織標本をいう。     For a subject in which functional suppression of PTDSS2 was detected using a test specimen (the antibody according to any one of claims 1 to 11 or an antigen-binding fragment of the antibody is used for the detection), PTDSS1 A method for treating a PTDSS2-negative disease (cancer), which comprises administering an inhibitor.
    The above-mentioned specimen refers to a tissue specimen that has been subjected to paraffin embedding treatment and then deparaffinization treatment.
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