WO2022080305A1 - Anticorps anti-ptdss2 - Google Patents

Anticorps anti-ptdss2 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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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.

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Abstract

La présente invention concerne, entre autres, un anticorps qui se lie spécifiquement à PTDSS2, un réactif de détection de PTDSS2 contenant un anticorps anti-PTDSS2, et une composition pour l'examen ou un réactif le pour diagnostic de maladies liées à l'expression de PTDSS2, ladite composition ou ledit réactif contenant l'anticorps anti-PTDSS2.　La présente invention concerne, entre autres, un anticorps, un fragment de liaison à l'antigène de celui-ci, ou un anticorps chimérique de celui-ci qui se lie spécifiquement à PTDSS2 humain ou un peptide partiel de celui-ci ; une composition contenant l'anticorps ; etc.
PCT/JP2021/037546 2020-10-13 2021-10-11 Anticorps anti-ptdss2 WO2022080305A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016148115A1 (fr) * 2015-03-16 2016-09-22 第一三共株式会社 Procédé pour prévoir la sensibilité à un inhibiteur de la phosphatidylsérine synthase 1
WO2020179859A1 (fr) * 2019-03-06 2020-09-10 第一三共株式会社 Dérivé de pyrrolopyrazole

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016148115A1 (fr) * 2015-03-16 2016-09-22 第一三共株式会社 Procédé pour prévoir la sensibilité à un inhibiteur de la phosphatidylsérine synthase 1
WO2020179859A1 (fr) * 2019-03-06 2020-09-10 第一三共株式会社 Dérivé de pyrrolopyrazole

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Anti-PTDSS2 monoclonal antibody (DCABH13098)", CD CREATIVE DIAGNOSTICS, 1 January 2019 (2019-01-01), pages 1 - 2, XP055920947 *
ANONYMOUS: "Anti-PTDSS2 monoclonal antibody, clone FQS25774 (DCABH-7680)", CD CREATIVE DIAGNOSTICS, 1 January 2019 (2019-01-01), pages 1 - 2, XP055920951 *
ANONYMOUS: "Anti-PTDSS2 polyclonal antibody (CABT-BL5464)", CD CREATIVE DIAGNOSTICS, 1 January 2019 (2019-01-01), pages 1 - 2, XP055920955 *
SENEVIRATNE AYESH K.; XU MINGJING; HENAO JUAN J. ARISTIZABAL; FAJARDO VAL A.; HAO ZHENYUE; VOISIN VERONIQUE; XU G. WEI; HURREN ROS: "The Mitochondrial Transacylase, Tafazzin, Regulates for AML Stemness by Modulating Intracellular Levels of Phospholipids", CELL STEM CELL, vol. 24, no. 4, 1 January 1900 (1900-01-01), AMSTERDAM, NL , pages 621, XP085654759, ISSN: 1934-5909, DOI: 10.1016/j.stem.2019.02.020 *

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