WO2022075485A1 - Protéine modifiée de type collagène et son utilisation - Google Patents

Protéine modifiée de type collagène et son utilisation Download PDF

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WO2022075485A1
WO2022075485A1 PCT/JP2021/037556 JP2021037556W WO2022075485A1 WO 2022075485 A1 WO2022075485 A1 WO 2022075485A1 JP 2021037556 W JP2021037556 W JP 2021037556W WO 2022075485 A1 WO2022075485 A1 WO 2022075485A1
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amino acid
human
collagen
protein
acid sequence
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渉 西江
翔子 眞井
洋輔 眞井
健太郎 泉
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国立大学法人北海道大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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

Definitions

  • the present invention comprises a collagen-like modified protein useful for diagnosing drug-induced bullous pemphigoid, a method and kit for detecting human autoantibodies using the protein, and drug-induced bullous pemphigoid using the protein. Regarding methods and kits for making a diagnosis of.
  • BP Bullous pemphigoid
  • BP180 XVII type collagen, also called BPAG2
  • BPAG2 XVII type collagen, also called BPAG2
  • Most anti-BP180 autoantibodies are known to target the 16th non-collagen region ( NC16A region, non-collagenous (NC) 16th A region) located in the extracellular domain of BP180.
  • DPP-4 dipeptidyl peptidase-IV
  • DPP4i-BP dipeptidyl peptidase-IV
  • DPP4i-BP autoantibody this autoantibody
  • Autoantibodies targeting NC16A of BP180 can be detected by ELISA or CLEIA method (Non-Patent Document 2), and tests using this method are covered by insurance in Japan.
  • this method cannot detect anti-BP180 autoantibodies, typically DPP4i-BP autoantibodies, that target regions other than NC16A in the extracellular domain of BP180.
  • the present inventors succeeded in producing a full-length BP180 recombinant protein by optimizing the method for purifying a membrane protein, and further established an ELISA method using the full-length BP180 (Non-Patent Document 1). Although the DPP4i-BP autoantibody can be detected by using the full-length BP180, the anti-BP180-NC16A antibody is also detected at the same time, and it is difficult to selectively detect the DPP4i-BP autoantibody.
  • BP180 is a homotrimer in which three monomer polypeptides having a molecular weight of 180 kDa are associated with each other, and is a giant protein having a molecular weight of 540 kDa.
  • the extracellular domain has a triple helix structure called a collagen spiral structure. It is not easy to make an antigenic protein containing the extracellular domain of BP180.
  • the present inventors have an amino acid sequence including a specific amino acid sequence involved in the formation of a trimer of type II transmembrane collagen and a partial amino acid sequence of the extracellular domain of BP180 located on the C-terminal side thereof.
  • Collagen-like modified proteins can selectively detect anti-BP180 autoantibodies targeting regions other than NC16A in the extracellular domain of BP180, and further identify non-NC16A BP180 extracellular domains in drug-induced BP patients.
  • Item 1 The trimeric region corresponding to the coiled coil arrangement of human type II transmembrane collagen and the 7th collagen from the 11th non-collagen region (NC11) of human BP180 located on the C-terminal side of the trimeric region.
  • a human that includes a region corresponding to the region (COL7) and / or a region corresponding to the 7th non-collagen region (NC7) to the 4th collagen region (COL4), but targeting the NC16A region of human BP180. Collagen-like modified protein that does not bind to self-antibodies.
  • Item 2. The protein according to Item 1, wherein the human type II transmembrane collagen is human XIII type collagen.
  • the amino acid sequence of the region corresponding to NC11 to COL7 of human BP180 is an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2, and human BP180.
  • the amino acid sequence of the region corresponding to NC7 to COL4 in No. 1 to Item 1 to 3 is an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3.
  • Item 5 The protein according to any one of the above.
  • Item 6. The protein according to any one of Items 1 to 4, which comprises the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 21.
  • Item 6. Item 6.
  • Item 7. A nucleic acid encoding a protein specified in any one of Items 1 to 5.
  • Item 8. An expression vector containing the nucleic acid specified in Item 7.
  • Item 9. A host cell containing the nucleic acid specified in Item 7 or the expression vector specified in Item 8.
  • Human BP180 includes a step of contacting a sample sample collected from a subject with the protein specified in any one of Items 1 to 6, and a step of detecting the binding between the antibody in the sample sample and the protein.
  • a method for detecting a human autoantibody targeting the region from NC11 to COL7 and / or a human autoantibody targeting the region from NC7 to COL4 of human BP180. Item 11. Item 6. The method according to Item 10, wherein the sample is a blood sample.
  • Item 12. Target the region from NC11 to COL7 of human BP180 and / or the region from NC7 to COL4 of human BP180, which comprises the protein specified in any one of Items 1 to 6.
  • a kit for detecting human autoantibodies. Item 13.
  • Drug property which comprises a step of contacting a sample sample collected from a subject with a protein specified in any one of Items 1 to 6 and a step of detecting a binding between an antibody in the sample sample and the protein. How to collect data for the diagnosis of bullous pemphigoid.
  • Item 14. Item 3. The method according to Item 13, wherein the sample is a blood sample.
  • Item 15. A kit for diagnosing drug-induced bullous pemphigoid, which comprises the protein specified in any one of Items 1 to 6.
  • the anti-BP180 autoantibodies targeting the region from NC11 to COL7 of BP180 and the anti-BP180 autoantibodies targeting the region from NC7 to COL4 Antibodies can be selectively detected, and drug-induced BP can be distinguished from drug-independent BP.
  • the first aspect of the present invention is a trimeric region corresponding to the coiled coil arrangement of human type II transmembrane collagen, and NC11 to COL7 of human BP180 located on the C-terminal side of the trimeric region.
  • collagen-like modified proteins comprising corresponding regions and / or regions corresponding to NC7 to COL4, but not binding to human autoantibodies targeting NC16A of human BP180.
  • Trimerized region type II transmembrane collagen is expressed in many different tissues and cells and protects the host from epithelial and neural cell adhesion and epithelial-mesenchymal interactions during morphogenesis. It is involved in a wide range of biological functions.
  • Type II transmembrane collagen has XIII type collagen, BP180 (XVII type collagen), XXIII type collagen, XXV type collagen, MARCO (macrophage receptor with collagenous structures) which is a class A macrophage scavenger receptor-like protein, and collagen structure. Macrophagee receptor), SRCL (scavenger receptor with C-type lectin), which is a scavenger receptor having C-type lectin, ectodisplacin A and the like.
  • the coiled coil sequences (called coiled-coil sequences, coiled-coil heptad repeats, coiled-coil oligomerization domain, N-terminal heptad repeats, etc.) existing on the N-terminal side of the type II transmembrane collagen extracellular region are HPPHPPP (H).
  • HPPHPPP HPPHPPP
  • the coiled coil arrangement of a typical human type II transmembrane collagen is shown in the table below.
  • the coiled coil sequence preferably used in the present invention is the coiled coil sequence of human XIII type collagen (SEQ ID NO: 1).
  • the extracellular domain of type XIII collagen includes three collagen regions (COL1 to COL3 from the N-terminal side to the C-terminal side) and four non-collagen regions (NC1 from the N-terminal side to the C-terminal side). ⁇ NC4) exist alternately, and the coiled coil arrangement exists in NC1.
  • the amino acid sequence (SEQ ID NO: 9) of human XIII type collagen ⁇ 1 chain (COL13A1) registered as NP_001123575.1 in the Reference Sequence Database of National Center for Biotechnology Information (NCBI) the coiled coil sequence of XIII type collagen is 65. It is present at the amino acid sequence site at the 85th position.
  • BP180 BP180 is a transmembrane collagen, also called XVII type collagen, in which three monomers having a molecular weight of 180 kDa are associated to form a homotrimer.
  • the extracellular domain of BP180 includes 15 collagen regions (COL1 to COL15 from the C-terminal side to the N-terminal side) and 16 non-collagen regions (NC1 to NC16 from the C-terminal side to the N-terminal side). ) And alternately exist.
  • BP180 amino acid sequence and the base sequence of the gene encoding it are known, and are registered as NP_000485.3 (amino acid sequence) and NM_000494.4 (cDNA base sequence) in the Reference Sequence Database of the National Center for Biotechnology Information (NCBI), respectively. ing.
  • the amino acid sequence of BP180 is shown in SEQ ID NO: 10, and the base sequence of the cDNA encoding it is shown in SEQ ID NO: 11.
  • the region of BP180 from NC11 to COL7 (NC11-COL7) and the region of BP180 from NC7 to COL4 (NC7-COL4) are used.
  • the amino acid sequence of BP180 is the amino acid sequence shown in SEQ ID NO: 10
  • the position of NC11-COL7 is 982-1160
  • the position of NC7-COL4 is 1161-1279.
  • the amino acid sequences 982 to 1160 of SEQ ID NO: 10 are shown in SEQ ID NO: 2
  • the amino acid sequences 1161 to 1279 are shown in SEQ ID NO: 3, respectively.
  • Collagen-like modified protein contains a trimerized region, and further corresponds to a region corresponding to NC11-COL7 and / or NC7-COL4 of BP180 on the C-terminal side of the trimerized region. Includes area.
  • the collagen-like modified protein of this embodiment does not bind to human autoantibodies targeting NC16A of BP180.
  • the collagen-like modified protein of this embodiment is a sequence recognized by a human autoantibody present in NC16A of BP180 (typically, the amino acid sequence shown in SEQ ID NO: 12; D. Zillikens et al., J Invest Dermatol: 109,573-9) is not included.
  • the collagen-like modified protein does not contain the amino acid sequence of NC16A of BP180 (SEQ ID NO: 13).
  • BP180 does not bind to a human autoantibody that targets NC16A means that the binding affinity of the antibody to collagen-like modified protein is similar to that of other antigens other than NC16A. , It does not preclude that the antibody non-specifically binds to the collagen-like modified protein.
  • the trimerization region corresponds to the coiled coil arrangement of type II transmembrane collagen.
  • the equivalent to the coiled-coil sequence of type II transmembrane collagen means that it consists of the amino acid sequence of the coiled-coil sequence of type II transmembrane collagen, or is a functionally equivalent variation in which the amino acid sequence of the coiled-coil sequence is modified. It means that it consists of an amino acid sequence.
  • NC11-COL7 of BP180 consists of the natural amino acid sequence of NC11-COL7 or a functionally equivalent mutant amino acid obtained by modifying the natural amino acid sequence of NC11-COL7. It means that it consists of an array.
  • equivalent to NC7-COL4 means that it consists of a natural amino acid sequence of NC7-COL4 or a functionally equivalent mutant amino acid sequence obtained by modifying the natural amino acid sequence of NC7-COL4. ..
  • the functionally equivalent mutant amino acid sequence is an amino acid sequence obtained by modifying the natural amino acid sequence of the coiled coil sequence, which retains the ability to form a homotrimer in a collagen-like modified protein.
  • An example of a mutant amino acid sequence is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97%, 98% or 99 with the natural amino acid sequence of a coiled coil sequence. It is an amino acid sequence having a sequence identity of% or more.
  • Another example is an amino acid sequence in which one or two amino acid residues have been deleted, substituted or added in the natural amino acid sequence of a coiled coil sequence.
  • mutant amino acid sequences are humans that retain the ability to form homotrimers when contained in collagen-like modified proteins and that target NC11-COL7 of BP180. It is an amino acid sequence obtained by modifying the natural amino acid sequence of NC11-COL7, which retains the ability to bind to an autoantibody.
  • An example of such an amino acid sequence is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97%, 98 with the natural amino acid sequence of NC11-COL7. % Or an amino acid sequence having 99% or more sequence identity.
  • Yet another example is the deletion of 1-18, preferably 1-12, more preferably 1-8, and even more preferably 1-4 amino acid residues in the natural amino acid sequence of NC11-COL7. , Substituted or added amino acid sequence.
  • mutant amino acid sequences are humans that retain the ability to form homotrimers when contained in collagen-like modified proteins and that target NC7-COL4 in BP180.
  • An example of such an amino acid sequence is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97%, 98 with the natural amino acid sequence of NC7-COL4.
  • Yet another example is the deletion of 1-12, preferably 1-9, more preferably 1-6, even more preferably 1-3 amino acid residues in the natural amino acid sequence of NC7-COL4. , Substituted or added amino acid sequence.
  • Substitutions are preferably so-called conservative substitutions, such as glycine (Gly) and proline (Pro), glycine and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamine acid ( Amino acids such as Glu) and glutamine (Gln), aspartic acid (Asp) and aspartin (Asn), cysteine (Cys) and threonine (Thr), alanine and serine (Ser) or alanine, lysine (Lys) and arginine (Arg).
  • conservative substitutions such as glycine (Gly) and proline (Pro), glycine and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamine acid ( Amino acids such as Glu) and glutamine (Gln), aspartic acid (Asp) and aspartin (Asn), cysteine
  • Amino acid sequence identity is expressed as the ratio of the number of identical amino acid residues to the alignment length, and the alignment of two amino acid sequences to be compared is performed according to a conventional method so that the number of identical amino acid residues is the largest. Will be. Sequence identity can be determined by any method known to those of skill in the art using a sequence comparison program such as BLAST.
  • the region corresponding to NC11-COL7 and the region corresponding to NC7-COL4 are arranged on the C-terminal side of the trimerization region.
  • This configuration allows collagen-like modified proteins to form trimers.
  • the region corresponding to NC11-COL7 and the region corresponding to NC7-COL4 may contain only one or both. When both are included, the order of the region corresponding to NC11-COL7 and the region corresponding to NC7-COL4 does not matter as long as they are arranged on the C-terminal side of the trimerization region. Further, one region corresponding to NC11-COL7 and one region corresponding to NC7-COL4 may be included or a plurality of regions may be included.
  • the collagen-like modified protein of this embodiment retains the ability to form homotrimers and binds to human autoantibodies targeting NC11-COL7 of BP180 and / or targets NC7-COL4 of BP180. Unless it retains the ability to bind to human autoantibodies and does not bind to human autoantibodies that target NC16A of BP180, it is a trimerization region, a region corresponding to NC11-COL7, and a region corresponding to NC7-COL4, respectively. It can contain any additional amino acid sequence other than the amino acid sequence of.
  • the additional amino acid sequence may be located at the N-terminus or C-terminus of the modified protein, or between each region contained in the modified protein (eg, the trimerization region and the region corresponding to NC11-COL7). It may be located between the trimerization region and the region corresponding to NC7-COL4).
  • additional amino acid sequences are amino acid sequences derived from type XIII collagen other than the coiled-coil sequence, for example, the amino acid sequences 1 to 64 of SEQ ID NO: 9, the amino acid sequences 86 to 217 of SEQ ID NO: 9. The amino acid sequences from the 700th to the 717th of SEQ ID NO: 9 can be mentioned.
  • additional amino acid sequences are amino acid sequences corresponding to restriction enzyme recognition sequences, tag sequences such as His tags, GST tags, HA tags, FLAG tags, and amino acid sequences of GFP and other fluorescent proteins.
  • collagen-like modified proteins are the trimerized region corresponding to the coiled coil sequence of human XIII type collagen and the region corresponding to one NC11-COL7 or NC7-COL4 of BP180 located on the C-terminal side thereof. It is a protein containing a region corresponding to.
  • the protein comprises the amino acid sequence of SEQ ID NO: 1 or a variant amino acid sequence thereof and the amino acid sequence of SEQ ID NO: 2 or a variant amino acid sequence thereof, or the amino acid sequence of SEQ ID NO: 3 or a variant amino acid sequence thereof.
  • a more preferred example of a collagen-like modified protein is a protein in which the NC2 to COL3 region of human XIII type collagen (Pro 217 -Asp 699 shown in the lower part of FIG. 1) is replaced with NC11-COL7 or NC7-COL4 of BP180.
  • the protein comprises, in order from the N-terminus, the amino acid sequence from position 2 to 216 of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence from position 700 to 717 of SEQ ID NO: 9. Consists of.
  • the protein is composed of the amino acid sequences of SEQ ID NO: 9 from 2 to 216, the amino acid sequence of SEQ ID NO: 3, and the amino acids of SEQ ID NO: 9 from 700 to 717, in order from the N-terminal. It consists of an array.
  • the collagen-like modified protein is that the NC2 to COL3 region of human XIII collagen has one or more ends of either NC11-COL7 or NC7-COL4 of BP180 or both ends. It is a protein that has been replaced with a fragment to which an amino acid has been added.
  • the number of amino acids added is, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 to 2.
  • the protein has one or more amino acids added to the N-terminal to the 2nd to 216th amino acid sequences of SEQ ID NO: 9 and the N-terminal and C-terminal of the amino acid sequence of SEQ ID NO: 2.
  • the protein comprises one or more amino acids at the N-terminal to the N-terminal and C-terminal of the amino acid sequences 2 to 216 of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 3, in order from the N-terminal. It consists of an amino acid sequence to which is added and an amino acid sequence from position 700 to 717 of SEQ ID NO: 9.
  • the protein consists of the amino acid sequence set forth in SEQ ID NO: 19, and in a further specific embodiment, the protein consists of the amino acid sequence set forth in SEQ ID NO: 21.
  • Collagen-like modified proteins retain the ability to form homotrimers and bind to human autoantibodies targeting NC11-COL7 of BP180 and / or human autoantibodies targeting NC7-COL4 of BP180. It may be chemically modified as long as it retains its ability to bind to and does not bind to human autoantibodies targeting NC16A of BP180. Chemical modifications include, for example, acylation, prenylation, acetylation, phosphorylation, glycosylation, PEGylation.
  • the collagen-like modified protein of this embodiment binds to BP-related autoantibodies resulting from drug administration, particularly DPP4i-BP autoantibodies, but is present in many BP cases. Does not bind to autoantibodies targeting NC16A.
  • the collagen-like modified protein of this embodiment makes it possible to distinguish drug-induced BP patients, especially DPP4i-BP patients, from BP patients with autoantibodies targeting NC16A of BP180.
  • the collagen-like modified protein can be prepared by a method for producing a recombinant protein using Escherichia coli or other microorganisms, insect cells or animal cells as host cells, or a cell-free protein expression method. Construction of recombinant genes, introduction of expression vectors into host cells, expression of target proteins in host cells and other genetic engineering techniques are based on the instructions in the experimental operation manual that explains various gene recombination operations in detail. Can be done.
  • nucleic acid encoding collagen-like modified protein can be used.
  • the nucleic acid is DNA
  • it is preferably used in a form incorporated into an appropriate expression vector.
  • the expression vector carrying the DNA encoding the collagen-like modified protein may be in any form such as circular or linear.
  • the expression vector may have another base sequence in addition to the base sequence encoding the collagen-like modified protein. Examples of other base sequences are enhancer sequences, promoter sequences, ribosome binding sequences, base sequences used for the purpose of amplifying the number of copies, base sequences encoding peptides such as signal peptides and other polypeptides, and poly A addition.
  • a suitable synthetic DNA adapter is used to add translation initiation codons and translation termination codons to the DNA encoding the collagen-like modified protein, or to add or delete appropriate restriction enzyme cleavage sequences in the base sequence. It is also possible. These are within the scope of the work normally performed by those skilled in the art, and those skilled in the art can arbitrarily and easily process the DNA encoding the collagen-like modified protein.
  • an appropriate vector can be selected according to the host to be used, and in addition to the plasmid, bacteriophage, baculovirus, retrovirus, vaccinia virus, etc. can be selected. It is also possible to use various viruses.
  • the collagen-like modified protein can also be used by linking another appropriate expression promoter upstream of the base sequence encoding the collagen-like modified protein.
  • an expression promoter may be appropriately selected depending on the host, for example, if the host is an Escherichia bacterium, preferably Escherichia coli, the T7 promoter, lac promoter, trp promoter, ⁇ PL promoter, etc., and the host is Bacillus genus.
  • Bacteria, preferably B. subtilis include P43 promoter, vegI promoter, xylose-inducible promoter, tetracycline inducible promoter and the like.
  • the host is yeast, the PHO5 promoter, GAP promoter, ADH promoter, etc., and if the host is animal cells, the SV40-derived promoter, retrovirus promoter, cytomegalovirus (CMV) IE (immediate early). Examples thereof include a gene promoter, a metallothioneine promoter, a heat shock promoter, an SR ⁇ promoter and the like.
  • host cells include the genera Escherichia, Bacillus, Corynebacterium, Brevibacterium, Serratia, Pseudomonas, and Earthlover.
  • Bacteria such as (Arthrobacter), Erwinia, Methylobacterium and Rhodobacter, fungi such as Streptomyces, Zymomonas and Saccharomyces.
  • Insect cells such as silkworm, HEK293 cells, MEF cells, Vero cells, Hela cells, CHO cells, WI38 cells, BHK cells, COS-7 cells, MDCK cells, C127 cells, HKG cells and animal cells such as human kidney cell lines. Is also available.
  • transformation method for introducing an expression vector into a host cell examples include an electroporation method, an alkali metal method, a calcium phosphate precipitation method, a DEAE dextran method, a microinjection method, and a lipofection method.
  • the collagen-like modified protein can be obtained by culturing a transformed cell into which an expression vector has been introduced, expressing the polypeptide in the cell, recovering the target polypeptide from the cell or medium, and purifying the protein.
  • the transformed cells may be cultured according to a conventional method according to the properties of the host cell such as carbon assimilation and auxotrophy, the selection marker contained in the introduced recombinant gene, the promoter and the like.
  • Purification of collagen-like modified protein can be performed by appropriately selecting an appropriate method from the methods usually used for purification of protein. That is, various affinity chromatography such as salting out method, ultrafiltration method, isoelectric point precipitation method, gel filtration method, electrophoresis method, ion exchange chromatography, hydrophobic chromatography and antibody chromatography, chromatographic focusing method, adsorption.
  • An appropriate method may be appropriately selected from commonly used methods such as chromatography and reverse phase chromatography, and if necessary, purification may be performed in an appropriate order using an HPLC system or the like.
  • the collagen-like modified protein contains a tag sequence or other functional protein or an amino acid sequence of a polypeptide other than collagen
  • a purification method characteristic of the functional protein include histidine tags consisting of about 6 to 10 consecutive histidine residues and nickel-immobilized affinity chromatography, glutathione S-transferase (GST) and glutathione-immobilized affinity. Chromatography, FLAG tags, anti-FLAG antibodies and the like can be mentioned.
  • the collagen-like modified protein can be recovered by cleaving the purified fusion protein with an appropriate protease (thrombin, trypsin, etc.).
  • a cell-free synthesis method using a nucleic acid encoding a collagen-like modified protein is also one of the genetic engineering production methods.
  • Cell-free protein synthesis systems include systems that use cell extracts such as Escherichia coli, wheat germ, yeast, rabbit reticulocytes, insect cells, and cultured mammalian cells, and reconstituted types that are composed by combining factors necessary for protein synthesis. System can be mentioned.
  • the collagen-like modified protein is produced by an organic chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method) using a suitable commercially available peptide synthesizer.
  • an organic chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method) using a suitable commercially available peptide synthesizer.
  • the nucleic acids described above, especially the DNA integrated into the expression vector can be introduced into a suitable expression system using a suitable host cell selected from prokaryotes or eukaryotes. It is preferable to produce it.
  • the collagen-like modified protein is produced as a homotrimer by the method exemplified above, particularly by culturing and expressing transformed cells into which an expression vector has been introduced, and recovering and purifying the target protein from the cells or medium. can do.
  • Homotrimers can also be converted into monomers and used by placing them in the presence of SDS or other surfactants or high-concentration salts.
  • the present invention provides a nucleic acid encoding a collagen-like modified protein, an expression vector containing the nucleic acid, and the nucleic acid or a host cell containing the expression vector, as different embodiments.
  • Another aspect of the present invention is a step of contacting a sample sample collected from a subject with the collagen-like modified protein of the first aspect, and an antibody in the sample sample and the collagen-like modified protein.
  • the present invention relates to a method for detecting a human autoantibody targeting NC11-COL7 and / or a human autoantibody targeting NC7-COL4 of human BP180, which comprises a step of detecting the binding of.
  • the collagen-like modified protein is as described in the first aspect.
  • the detection of human autoantibodies can be performed by detecting the binding between the antibody and the collagen-like modified protein in the sample sample, which is caused by contacting the sample sample with the collagen-like modified protein of the first aspect.
  • the sample sample used here is a biological sample derived from humans, and examples thereof include skin tissue, skin cells, and body fluids (eg, blood, lymph, saliva, mucus, bone marrow, urine, semen, and ascitic fluid). Etc.), serum or plasma prepared from blood, etc. can be mentioned.
  • the biological sample may be used as it is collected, or may be used after pretreatment such as pulverization, homogenization, centrifugation, concentration, and dilution.
  • a particularly preferred sample sample is a blood sample, specifically blood, serum or plasma.
  • Detection of human autoantibodies can be performed by immunoassay utilizing antigen-antibody reaction.
  • immunoassay examples include ELISA, radioimmunoassay, immunoblotting, and immunochromatography.
  • a collagen-like modified protein is immobilized on a carrier, which is brought into contact with a sample sample to react with the collagen-like modified protein and an antibody in the sample sample, and then the antibody bound to the collagen-like modified protein is labeled. It can be carried out by detecting with the obtained human antibody-binding substance.
  • human antibody-binding substances include antibody-binding proteins capable of binding to human antibodies, particularly to the Fc region of human IgG, such as antibodies to human antibodies and protein G. ..
  • labeling compounds used for labeling human antibody-binding substances include fluorescent substances (eg, FITC, rhodamine, etc.), metal particles such as gold colloid, fluorescent microbeads such as Luminex (registered trademark, Luminex), and dye proteins. (Eg, phycoerythrin, phycocyanin, etc.), radioisotopes (eg, 3 H, 14 C, 32 P, 35 S, 125 I, 131 I, etc.), enzymes (eg, peroxidase, alkaline phosphatase, etc.), biotin, streptavidin, etc. Can be done.
  • fluorescent substances eg, FITC, rhodamine, etc.
  • metal particles such as gold colloid
  • fluorescent microbeads such as Luminex (registered trademark, Luminex)
  • dye proteins e.g, phycoerythrin, phycocyanin, etc.
  • radioisotopes eg, 3 H, 14
  • For conditions, washing, blocking treatment, detection of labeled compounds, etc. refer to the description of methods known or well known to those skilled in the art, for example, The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques (4TH), Elsevier. It can be carried out. This document is incorporated herein by reference in its entirety.
  • human autoantibodies targeting NC11-COL7 and / or NC7-COL4 of human BP180 can be detected.
  • a human autoantibody targeting NC11-COL7 can be detected, and the collagen-like modified protein is NC7 of BP180.
  • -Human autoantibodies targeting NC7-COL4 can be detected if they contain a region corresponding to COL4.
  • These human autoantibodies are drug-induced BP-related autoantibodies, especially DPP4i-BP autoantibodies.
  • the present invention comprises a step of contacting a sample sample collected from a subject with a collagen-like modified protein, and a step of detecting binding between an antibody in the sample sample and the collagen-like modified protein. It also provides a method of collecting data for the diagnosis of sex BP, especially for the diagnosis of DPP4i-BP. Further, in the present invention, the sample sample used in this data acquisition method, the collagen-like modified protein, and the method of using these are as described in the above-mentioned method for detecting human autoantibodies.
  • Kit The present invention is for detecting a human autoantibody targeting NC11-COL7 and / or a human autoantibody targeting NC7-COL4 of human BP180, which comprises at least a collagen-like modified protein according to the first aspect.
  • Kits, as well as kits for the diagnosis of drug-induced bullous pemphigoid, are provided as yet another embodiment.
  • the kit includes carriers such as plates used for detecting human autoantibodies that bind to the polypeptide in immunoassays, blocking solutions, washing solutions, human antibody-binding substances, color-developing substrates, and the like. Additional reagents may be included.
  • Example 1 Preparation of collagen-like modified protein (1) Preparation of expression vector cDNA encoding five types of collagen-like modified protein (collagen 13-BP180 swap protein 1 to 5) was synthesized using a DNA synthesizer.
  • the cDNA of swap protein 1 consists of FLAG tag (SEQ ID NO: 24), amino acid sequence from 2 to 121 of human XIII collagen isoform COL13A1 (SEQ ID NO: 9), and extracellular domain fragment of BP180 in order from the 5'end side. It encodes the amino acid sequence of Gly 567 -Ile 808 and the amino acid sequence of human XIII collagen isoform COL13A1 (SEQ ID NO: 9) from position 700 to 717.
  • the cDNAs of swap proteins 2 to 5 are, in order from the 5'end side, the FLAG tag (SEQ ID NO: 24), the amino acid sequences 2 to 216 of the human XIII collagen isoform COL13A1 (SEQ ID NO: 9), and the extracellular of BP180.
  • Amino acid sequence of domain fragment Val 809 -Ser 981 , Glu 982 -Ser 1160 , Tyr 1161 -Ser 1279 or Arg 1280 -Pro 1497 and amino acids 700-717 of human XIII collagen isoform COL13A1 (SEQ ID NO: 9). Encoding the array.
  • Expression vector A total of 5 types of collagen-like modified protein expression vectors (collagen 13-BP180 swap protein expression vector) by recombining the above cDNAs to the NheI-ApaI sites of pcDNA3.1 / Hyg (Invitrogen) or pcDNA5 / FRT (Invitrogen). was built.
  • Example 2 Immunoassay using human sample (1) Western blotting 17 patients diagnosed with DPP4i-BP (hereinafter referred to as DPP4i-BP patients) by the diagnosis of a dermatologist, no history of oral administration of DPP4i diagnosed with BP Serums were prepared from blood collected from each of 3 patients (hereinafter referred to as BP patients) and 3 healthy subjects, and used as sample samples. It was confirmed in advance that the sera of all DPP4i-BP patients used did not show responsiveness to NC16A.
  • DPP4i-BP patients Western blotting 17 patients diagnosed with DPP4i-BP (hereinafter referred to as DPP4i-BP patients) by the diagnosis of a dermatologist, no history of oral administration of DPP4i diagnosed with BP Serums were prepared from blood collected from each of 3 patients (hereinafter referred to as BP patients) and 3 healthy subjects, and used as sample samples. It was confirmed in advance that the sera of all DPP4i-BP patients used did not show responsiveness to NC16A.
  • Swap proteins 1 to 5 prepared in Example 1 are multiplied by 5 times with 5x sample buffer (4M Urea, 0.5M Tris-HCl pH 6.8, 0.0005% bromophenol blue, 10% SDS, 25% Glycerin, 0.05 M DTT). Diluted and subjected to SDS-PAGE on a gel containing 7% acrylamide.
  • HPR-labeled anti-mouse IgG Jackson # 115-036-006
  • HRP-labeled anti-human IgG Dako # P0214
  • TBS TBS containing 2% skim milk
  • Swap proteins 1 to 5 prepared in Example 1 were diluted with Carbonate buffer (50 mM CB pH 9.6) to a concentration of 0.4 ⁇ g / ml, and added to a 96-well plate (Nunc # 442404) at a concentration of 50 ⁇ l / well. After the reaction at 4 ° C. for 10 to 12 hours, the cells were washed 3 times with 200 ⁇ l of PBS, and Blocking buffer (Roche # 11112589001) was added at 90 ⁇ l / well. After reacting at room temperature for 2 hours, the cells were washed twice with 200 ⁇ l of PBS to prepare 5 types of 96-well plates for ELISA in which swap proteins 1 to 5 were immobilized.
  • Carbonate buffer 50 mM CB pH 9.6
  • Blocking buffer (Roche # 11112589001) was added at 90 ⁇ l / well. After reacting at room temperature for 2 hours, the cells were washed twice with 200 ⁇ l of PBS to prepare 5 types of 96
  • a sample sample diluted 101-fold with PBS or an anti-FLAG antibody (M2) diluted 1: 2,000 was added at a rate of 100 ⁇ l / well and reacted at room temperature for 1 hour.
  • HRP-labeled anti-human and anti-mouse IgG diluted 1: 10,000 with PBS were added at 100 ⁇ l / well each and reacted at room temperature for 1 hour. ..
  • a color reaction was carried out at room temperature for 12 minutes using TMB (NOVEX).
  • the color reaction was stopped with Stop solution (0.5N sulfuric acid), and the absorbance at wavelengths of 450 nm and 620 nm was measured with an absorptiometer (Sunrise, TECAN), and these differences were used as an index of the antigen-antibody reaction.
  • ELISA The result of ELISA is shown in Fig. 5.
  • Specimens of DPP4i-BP patients showed high reactivity among swap proteins 1 to 5, especially swap protein 4 containing NC7-COL4 of BP-180.
  • BP patients with skin diseases other than BP eczema dermatitis group, etc.
  • BP patients and DPP4i-BP patients' sera are used as sample samples, and one healthy person's serum is used as a sample.
  • ELISA using the above 7 types of plates was carried out in the same manner as in (2) above. The difference between the absorbance at a wavelength of 450 nm and the absorbance at a wavelength of 620 nm measured by ELISA was used as the OD value, and the ELISA index was calculated by the following formula.
  • the result of ELISA is shown in FIG.
  • the sera of DPP4i-BP patients were significantly more responsive to swap protein 3 containing NC11-COL7 and swap protein 4 containing NC7-COL4 of BP180 than the sera of non-BP and BP patients. Indicated.
  • the sera of all DPP4i-BP patients used did not show reactivity to NC16A.
  • SEQ ID NO: 1 Amino acid sequence of the coiled coil sequence of human XIII type collagen
  • SEQ ID NO: 2 Amino acid sequence of NC11-COL7 of human BP180 SEQ ID NO: 3
  • Amino acid sequence of coiled coil sequence of human XXIII type collagen SEQ ID NO: 5
  • SEQ ID NO: 6 Amino acid sequence of human MARCO coiled coil sequence
  • SEQ ID NO: 7 Amino acid sequence of human SRCL coiled coil sequence
  • SEQ ID NO: 8 Amino acid sequence of human ectodisplacin A coiled coil sequence No.
  • Amino Acid SEQ ID NO: 13 Amino Acid SEQ ID NO: 14 of NC16A region of human BP180 Base sequence of DNA encoding collagen-like modified protein (collagen 13-BP180 swap protein 1) containing COL15 of human BP180 SEQ ID NO: 15 COL15 of human BP180 Amino acid sequence number of collagen-like modified protein (collagen 13-BP180 swap protein 1) containing NC15-COL11-containing DNA base sequence number of DNA encoding collagen-like modified protein (collagen 13-BP180 swap protein 2) 17 Amino acid sequence of human BP180 NC15-COL11-containing collagen-like modified protein (collagen 13-BP180 swap protein 2) Amino acid sequence number 18 Human BP180 NC11-COL7-containing collagen-like modified protein (collagen 13-BP180 swap protein 3) Base sequence of encoding DNA SEQ ID NO: 19 Amino acid sequence of collagen-like modified protein containing NC11-COL7 of human BP180 (Collagen 13-BP180 swap protein 3) SEQ ID NO: 20 Collagen-

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Abstract

La présente invention concerne : une protéine modifiée de type collagène qui contient un domaine de trimérisation correspondant à la séquence à superhélice du collagène transmembranaire de type II humain et un domaine correspondant à NC11-COL7 de BP180 humain et/ou un domaine correspondant à NC7-COL4 de celui-ci, ledit(lesdits) domaine(s) étant situé(s) sur le côté à extrémité C-terminale du domaine de trimérisation, à condition que cette protéine modifiée de type collagène ne se lie pas à un auto-anticorps humain ciblant NC16A de BP180 humain ; une méthode et un kit pour détecter un auto-anticorps humain à l'aide de la protéine susmentionnée ; et une méthode et un kit pour diagnostiquer une pemphigoïde bulleuse induite par un médicament à l'aide de la protéine susmentionnée.
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