JP7789372B2 - Collagen-like modified proteins and uses thereof - Google Patents

Description

The present invention relates to a collagen-like modified protein useful for diagnosing drug-induced bullous pemphigoid, a method and kit for detecting human autoantibodies using said protein, and a method and kit for diagnosing drug-induced bullous pemphigoid using said protein.

Bullous pemphigoid (BP), one of the intractable diseases designated in Japan, is the most common autoimmune bullous disease and frequently occurs in elderly patients. Anti-BP180 autoantibodies, which target BP180 (type XVII collagen, also known as BPAG2), a transmembrane protein present between the epidermis and dermis, are detected in the blood of patients, and are thought to induce the onset of BP. Most anti-BP180 autoantibodies are known to target the 16th non-collagenous (NC) ^16th A region (NC16A region) in the extracellular domain of BP180.

In recent years, it has been discovered that dipeptidyl peptidase-IV (DPP-4) inhibitors, a drug used to treat diabetes, contribute to the development of BP. Autoantibodies that bind to BP180 have been detected in the blood of patients with BP who develop while taking DPP-4 inhibitors (hereafter referred to as DPP4i-BP). Unlike previously reported anti-BP180 autoantibodies that target NC16A, these autoantibodies (DPP4i-BP autoantibodies) have been shown to target regions of the extracellular domain of BP180 other than NC16A (Non-Patent Document 1).

Autoantibodies targeting NC16A of BP180 (anti-BP180-NC16A antibodies) can be detected by ELISA or CLEIA (Non-Patent Document 2), and tests using this method are covered by health insurance in Japan. However, these methods cannot detect anti-BP180 autoantibodies that target regions other than NC16A in the extracellular domain of BP180, typically DPP4i-BP autoantibodies.

The inventors succeeded in producing full-length BP180 recombinant protein by optimizing the membrane protein purification method and further established an ELISA method using full-length BP180 (Non-Patent Document 1). Using full-length BP180 makes it possible to detect DPP4i-BP autoantibodies, but anti-BP180-NC16A antibodies are also detected at the same time, making it difficult to selectively detect DPP4i-BP autoantibodies.

To selectively detect DPP4i-BP autoantibodies using immunological techniques, it is necessary to prepare an antigenic protein containing a region of the BP180 extracellular domain other than NC16A. However, BP180 is a homotrimer consisting of three 180 kDa monomeric polypeptides, making it a large protein with a molecular weight of 540 kDa. Furthermore, the extracellular domain has a triple helix structure known as the collagen helix. Therefore, it is not easy to prepare an antigenic protein containing the BP180 extracellular domain.

Izumi K, et al. J Invest. Dermatol., 136: 2201-10, 2016 Kobayashi M, et al. J Dermatol. Sci., 30: 224-232, 2002

The present invention aims to provide an antigenic protein that can be used to selectively detect anti-BP180 autoantibodies that target regions other than NC16A in the extracellular domain of BP180.

The inventors discovered that a collagen-like modified protein having an amino acid sequence including a specific amino acid sequence involved in the trimerization of type II transmembrane collagen and a partial amino acid sequence of the extracellular domain of BP180 located on its C-terminal side can selectively detect anti-BP180 autoantibodies that target regions other than NC16A in the extracellular domain of BP180, and further discovered that autoantibodies targeting specific regions other than NC16A in the extracellular domain of BP180 are detected in patients with drug-induced BP, but that such autoantibodies are rarely or not detected at all in patients with drug-unrelated BP or healthy individuals, and thus completed the following invention.

Item 1. A collagen-like modified protein comprising a trimerization domain corresponding to the coiled-coil sequence of human type II transmembrane collagen and a domain corresponding to the 11th non-collagen domain (NC11) to the 7th collagen domain (COL7) of human BP180, which is located C-terminal to the trimerization domain, and/or a domain corresponding to the 7th non-collagen domain (NC7) to the 4th collagen domain (COL4), wherein the collagen-like modified protein does not bind to a human autoantibody that targets the NC16A domain of human BP180.
Item 2. The protein according to Item 1, wherein the human type II transmembrane collagen is human type XIII collagen.
Item 3. The protein according to Item 1 or 2, wherein the amino acid sequence of the trimerization domain is the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.
Item 4. The protein according to any one of Items 1 to 3, wherein the amino acid sequence of a region corresponding to NC11 to COL7 of human BP180 is the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO:2, and the amino acid sequence of a region corresponding to NC7 to COL4 of human BP180 is the amino acid sequence set forth in SEQ ID NO:3 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO:3.
Item 5. The protein according to any one of Items 1 to 4, which consists of the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 21.
Item 6. The protein according to any one of Items 1 to 5, which forms a homotrimeric structure.
Item 7. A nucleic acid encoding a protein defined in any one of Items 1 to 5.
Item 8. An expression vector comprising the nucleic acid defined in Item 7.
Item 9. A host cell comprising the nucleic acid defined in Item 7 or the expression vector defined in Item 8.
Item 10. A method for detecting human autoantibodies targeting the region from NC11 to COL7 of human BP180 and/or human autoantibodies targeting the region from NC7 to COL4 of human BP180, the method comprising the steps of contacting a specimen sample collected from a subject with the protein defined in any one of Items 1 to 6, and detecting binding between an antibody in the specimen sample and the protein.
Item 11. The method according to Item 10, wherein the specimen sample is a blood sample.
Item 12. A kit for detecting human autoantibodies targeting the region from NC11 to COL7 of human BP180 and/or human autoantibodies targeting the region from NC7 to COL4 of human BP180, comprising the protein defined in any one of Items 1 to 6.
Item 13. A method for collecting data for diagnosing drug-induced bullous pemphigoid, comprising the steps of contacting a specimen sample collected from a subject with the protein defined in any one of Items 1 to 6, and detecting binding between an antibody in the specimen sample and the protein.
Item 14. The method according to Item 13, wherein the specimen sample is a blood sample.
Item 15. A kit for diagnosing drug-induced bullous pemphigoid, comprising the protein defined in any one of Items 1 to 6.

By using the collagen-like modified protein of the present invention, it is possible to selectively detect anti-BP180 autoantibodies, particularly those targeting the NC11 to COL7 region of BP180 and those targeting the NC7 to COL4 region, thereby making it possible to distinguish between drug-induced BP and drug-unrelated BP.

FIG. 1 shows the structures of BP180 and type XIII collagen (collagen 13). FIG. 1 shows the structure of the collagen 13-BP180 swap protein of Example 1, which is one embodiment of the present invention. FIG. 1 shows the results of Western blotting for swap protein 3 in Example 1 using sera from DPP4i-BP patients, BP patients, and healthy controls. FIG. 1 shows the results of Western blotting for swap protein 4 in Example 1 using sera from DPP4i-BP patients, BP patients, and healthy controls. FIG. 1 shows the results of ELISA for swap proteins 1 to 5 in Example 1 using sera from DPP4i-BP patients, BP patients, and healthy controls. FIG. 1 shows the results of ELISA for swap proteins 1 to 5, full-length BP180, and NC16A in Example 1 using sera from DPP4i-BP patients, BP patients, and non-BP patients. The ROC curve is plotted with DPP4i-BP as the dependent variable and the ELISA index of swap protein 4 as the explanatory variable.

The first aspect of the present invention relates to a collagen-like modified protein that comprises a trimerization domain corresponding to the coiled-coil sequence of human type II transmembrane collagen and a domain corresponding to NC11 to COL7 and/or NC7 to COL4 of human BP180 located C-terminal to the trimerization domain, but does not bind to human autoantibodies that target NC16A of human BP180.

trimerization region
Type II transmembrane collagens are expressed in many different tissues and cells and are involved in a wide range of biological functions, from epithelial and neural cell adhesion, epithelial-mesenchymal interactions during morphogenesis, to host defense against microorganisms. These include type XIII collagen, BP180 (type XVII collagen), type XXIII collagen, type XXV collagen, class A macrophage scavenger receptor-like protein MARCO (macrophage receptor with collagenous structures), scavenger receptor with C-type lectin (SRCL), and ectodysplasin A.

The N-terminal coiled-coil sequences (also known as coiled-coil heptad repeats, coiled-coil oligomerization domains, or N-terminal heptad repeats) present in the extracellular domains of type II transmembrane collagen contain a heptad repeat of HPPHPPP (H is a hydrophilic amino acid residue, P is a hydrophobic amino acid residue) and are involved in collagen trimerization (McAlinden A. et al., J. Biol. Chem. 278: 42200-42207, 2003; Snellman A. et al., J. Biol. Chem. 282: 14898-14905, 2007). The coiled-coil sequences of representative human type II transmembrane collagens are shown in the table below.

The coiled-coil sequence preferably utilized in the present invention is the coiled-coil sequence of human type XIII collagen (SEQ ID NO: 1). The extracellular domain of type XIII collagen is composed of three collagenous regions (COL1-COL3, N-terminal to C-terminal) alternating with four non-collagenous regions (NC1-NC4, N-terminal to C-terminal), with the coiled-coil sequence located in NC1. In the amino acid sequence (SEQ ID NO: 9) of human type XIII collagen α1 chain (COL13A1), registered as NP_001123575.1 in the Reference Sequence Database of the National Center for Biotechnology Information (NCBI), the coiled-coil sequence of type XIII collagen is located at amino acid positions 65 to 85.

BP180
BP180, also known as type XVII collagen, is a transmembrane collagen that forms a homotrimer consisting of three 180 kDa monomers. The extracellular domain of BP180 contains 15 collagenous regions (COL1-COL15, C-terminal to N-terminal) alternating with 16 non-collagenous regions (NC1-NC16, C-terminal to N-terminal). The amino acid sequence of BP180 and the nucleotide sequence of the gene encoding it are publicly known and are registered in the National Center for Biotechnology Information (NCBI) Reference Sequence Database as NP_000485.3 (amino acid sequence) and NM_000494.4 (cDNA nucleotide sequence), respectively. The amino acid sequence of BP180 is shown in SEQ ID NO: 10, and the nucleotide sequence of the cDNA encoding it is shown in SEQ ID NO: 11.

In the present invention, the region from NC11 to COL7 of BP180 (NC11-COL7) and the region from NC7 to COL4 of BP180 (NC7-COL4) are utilized. When the amino acid sequence of BP180 is the amino acid sequence shown in SEQ ID NO: 10, the positions of NC11-COL7 are from 982 to 1160, and the positions of NC7-COL4 are from 1161 to 1279. The amino acid sequence from 982 to 1160 of SEQ ID NO: 10 is shown in SEQ ID NO: 2, and the amino acid sequence from 1161 to 1279 is shown in SEQ ID NO: 3.

Collagen-like modified protein The collagen-like modified protein of this embodiment comprises a trimerization domain and further comprises a region corresponding to NC11-COL7 and/or NC7-COL4 of BP180 on the C-terminal side of the trimerization domain.

In addition, the collagen-like modified protein of this embodiment does not bind to human autoantibodies that target NC16A of BP180. In terms of amino acid sequence, the collagen-like modified protein of this embodiment does not contain a sequence recognized by human autoantibodies present in NC16A of BP180 (typically the amino acid sequence shown in SEQ ID NO: 12; see D. Zillikens et al., J Invest Dermatol: 109,573-9). Preferably, the collagen-like modified protein does not contain the amino acid sequence of NC16A of BP180 (SEQ ID NO: 13).

Note that "not binding to human autoantibodies targeting NC16A of BP180" means that the binding affinity of the antibody to the collagen-like modified protein is similar to the binding affinity to other antigens other than NC16A, and does not exclude the possibility that the antibody may bind nonspecifically to the collagen-like modified protein.

The trimerization region corresponds to the coiled-coil sequence of type II transmembrane collagen. "Corresponding to the coiled-coil sequence of type II transmembrane collagen" here means that it consists of the amino acid sequence of the coiled-coil sequence of type II transmembrane collagen, or that it consists of a functionally equivalent mutant amino acid sequence obtained by adding a mutation to the amino acid sequence of the coiled-coil sequence.

Similarly, in the present invention, "corresponding to NC11-COL7 of BP180" means that it consists of the naturally occurring amino acid sequence of NC11-COL7, or that it consists of a functionally equivalent mutant amino acid sequence obtained by adding a mutation to the naturally occurring amino acid sequence of NC11-COL7. Furthermore, "corresponding to NC7-COL4" means that it consists of the naturally occurring amino acid sequence of NC7-COL4, or that it consists of a functionally equivalent mutant amino acid sequence obtained by adding a mutation to the naturally occurring amino acid sequence of NC7-COL4.

With respect to a coiled-coil sequence, a functionally equivalent mutant amino acid sequence is an amino acid sequence in which a mutation has been added to the natural amino acid sequence of the coiled-coil sequence, while retaining the ability of the collagen-like modified protein to form a homotrimer. One example of a mutant amino acid sequence is an amino acid sequence that has at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 97%, 98%, or 99% or more sequence identity with the natural amino acid sequence of the coiled-coil sequence. 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 the coiled-coil sequence.

With respect to NC11-COL7, a functionally equivalent mutant amino acid sequence is an amino acid sequence obtained by adding mutations to the native amino acid sequence of NC11-COL7, which retains the ability to form homotrimers when included in a collagen-like modified protein and the ability to bind to the human autoantibody BP180 that targets NC11-COL7. An example of such an amino acid sequence is an amino acid sequence that shares at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 97%, 98%, or 99% or more sequence identity with the native amino acid sequence of NC11-COL7. Another example is an amino acid sequence in which 1 to 18, preferably 1 to 12, more preferably 1 to 8, and even more preferably 1 to 4 amino acid residues have been deleted, substituted, or added to the native amino acid sequence of NC11-COL7.

With respect to NC7-COL4, a functionally equivalent mutant amino acid sequence is a native amino acid sequence of NC7-COL4 that retains the ability to form homotrimers when incorporated into a collagen-like modified protein and retains the ability to bind to BP180 human autoantibodies that target NC7-COL4. An example of such an amino acid sequence is an amino acid sequence that shares at least 70% identity with the native amino acid sequence of NC7-COL4, preferably 80% identity, more preferably 90% identity, even more preferably 95% identity, and particularly preferably 97%, 98%, or 99% identity. Another example is an amino acid sequence in which 1 to 12, preferably 1 to 9, more preferably 1 to 6, and even more preferably 1 to 3 amino acid residues have been deleted, substituted, or added from the native amino acid sequence of NC7-COL4.

Substitutions are preferably conservative, and examples of such substitutions include substitutions between amino acids such as glycine (Gly) and proline (Pro), glycine and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln), aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), threonine and serine (Ser) or alanine, and lysine (Lys) and arginine (Arg).

Amino acid sequence identity is expressed as the ratio of the number of identical amino acid residues to the length of the alignment, and the alignment of the two amino acid sequences to be compared is performed according to standard methods to maximize the number of identical amino acid residues. Sequence identity can be determined by any method known to those skilled in the art, for example, using a sequence comparison program such as BLAST.

In the collagen-like modified protein, the region corresponding to NC11-COL7 and the region corresponding to NC7-COL4 are located on the C-terminal side of the trimerization region. This configuration enables the collagen-like modified protein to form trimers. The collagen-like modified protein may contain either or both of the region corresponding to NC11-COL7 and the region corresponding to NC7-COL4. If both are contained, the order of the region corresponding to NC11-COL7 and the region corresponding to NC7-COL4 does not matter, as long as they are located on the C-terminal side of the trimerization region. Furthermore, the region corresponding to NC11-COL7 and the region corresponding to NC7-COL4 may each be contained in one or more copies.

The collagen-like modified protein of this embodiment may contain any additional amino acid sequence other than the amino acid sequences of the trimerization region, the region corresponding to NC11-COL7, and the region corresponding to NC7-COL4, as long as it retains the ability to form homotrimers and binds to human autoantibodies targeting NC11-COL7 and/or NC7-COL4 of BP180, and does not bind to human autoantibodies targeting NC16A of BP180. The additional amino acid sequence may be located at the N-terminus or C-terminus of the modified protein, or between the respective regions contained in the modified protein (e.g., between the trimerization region and the region corresponding to NC11-COL7, or between the trimerization region and the region corresponding to NC7-COL4).

Examples of additional amino acid sequences are amino acid sequences derived from type XIII collagen other than the coiled-coil sequence, such as the amino acid sequence from positions 1 to 64 of SEQ ID NO: 9, the amino acid sequence from positions 86 to 217 of SEQ ID NO: 9, and the amino acid sequence from positions 700 to 717 of SEQ ID NO: 9. Other examples of additional amino acid sequences are amino acid sequences corresponding to restriction enzyme recognition sequences, tag sequences such as His tag, GST tag, HA tag, and FLAG tag, and the amino acid sequences of GFP and other fluorescent proteins.

A preferred example of a collagen-like modified protein is a protein comprising a trimerization region corresponding to the coiled-coil sequence of human type XIII collagen and a region corresponding to one NC11-COL7 or NC7-COL4 of BP180 located C-terminally thereto. In one embodiment, this protein comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof, and the amino acid sequence of SEQ ID NO: 2 or a variant thereof, or the amino acid sequence of SEQ ID NO: 3 or a variant thereof.

A more preferred example of a collagen-like modified protein is a protein in which the NC2 to COL3 region of human type XIII collagen (Pro ²¹⁷ -Asp ⁶⁹⁹ shown in the lower panel of Figure 1 ) is replaced with NC11-COL7 or NC7-COL4 of BP180. In one embodiment, this protein consists, from the N-terminus, of the amino acid sequence from positions 2 to 216 of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of positions 700 to 717 of SEQ ID NO: 9. In another embodiment, the protein consists, from the N-terminus, of the amino acid sequence from positions 2 to 216 of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 3, and the amino acid sequence of positions 700 to 717 of SEQ ID NO: 9.

Another preferred example of a collagen-like modified protein is a protein in which the NC2 to COL3 region of human type XIII collagen has been replaced with a fragment of BP180 in which one or more amino acids have been added to either or both ends of NC11-COL7 or NC7-COL4. The number of added amino acids is, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 to 2. In one embodiment, this protein consists, in order from the N-terminus, of the amino acid sequence from positions 2 to 216 of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been added to the N-terminus and C-terminus, and the amino acid sequence of positions 700 to 717 of SEQ ID NO: 9. In yet another embodiment, the protein consists, in order from the N-terminus, of the amino acid sequence from positions 2 to 216 of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 3 in which one or more amino acids have been added to the N-terminus and C-terminus, and the amino acid sequence of positions 700 to 717 of SEQ ID NO: 9. In a particular embodiment, the protein consists of the amino acid sequence set forth in SEQ ID NO:19, and in a further particular embodiment, the protein consists of the amino acid sequence set forth in SEQ ID NO:21.

The collagen-like modified protein may be chemically modified, as long as it retains the ability to form homotrimers, binds to human autoantibodies targeting NC11-COL7 of BP180 and/or binds to human autoantibodies targeting NC7-COL4 of BP180, and does not bind to human autoantibodies targeting NC16A of BP180. Chemical modifications include, for example, acylation, prenylation, acetylation, phosphorylation, glycosylation, and PEGylation.

As shown in the Examples below, the collagen-like modified protein of this embodiment binds to autoantibodies associated with drug-induced BP, particularly DPP4i-BP autoantibodies, but does not bind to autoantibodies targeting NC16A of BP180, which are present in many BP cases. Therefore, the collagen-like modified protein of this embodiment makes it possible to distinguish drug-induced BP patients, particularly DPP4i-BP patients, from BP patients with autoantibodies targeting NC16A of BP180.

Preparation of collagen-like modified proteinsCollagen- like modified proteins can be prepared by recombinant protein production methods using E. coli or other microorganisms, insect cells, or animal cells as host cells, or by cell-free protein expression methods.Construction of recombinant genes, introduction of expression vectors into host cells, expression of target proteins in host cells, and other genetic engineering techniques can be performed according to the instructions in experimental operation manuals that provide detailed explanations of various genetic engineering procedures.

For recombinant production of collagen-like modified proteins, nucleic acids encoding the collagen-like modified proteins can be used. When the nucleic acid is DNA, it is preferably used in the form of 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. Furthermore, the expression vector may contain other base sequences in addition to the base sequence encoding the collagen-like modified protein. Examples of other base sequences include enhancer sequences, promoter sequences, ribosome binding sequences, base sequences used to amplify copy number, base sequences encoding peptides such as signal peptides or other polypeptides, poly(A) addition sequences, splicing sequences, and base sequences of genes that serve as selection markers.

During genetic recombination, it is possible to use an appropriate synthetic DNA adapter to add a translation initiation codon or translation termination codon to the DNA encoding the collagen-like modified protein, or to add or delete an appropriate restriction enzyme cleavage sequence within the base sequence. These are within the scope of routine work performed by those skilled in the art, and they can easily process the DNA encoding the collagen-like modified protein as desired.

In addition, an appropriate vector can be selected as the expression vector carrying the DNA encoding the collagen-like modified protein depending on the host used, and in addition to plasmids, various viruses such as bacteriophages, baculoviruses, retroviruses, and vaccinia viruses can also be used.

The collagen-like modified protein can also be used by linking another appropriate expression promoter upstream of the nucleotide sequence encoding it. Such expression promoters can be selected appropriately depending on the host. For example, when the host is an Escherichia bacterium, preferably E. coli, examples of promoters include the T7 promoter, lac promoter, trp promoter, and λPL promoter. When the host is a Bacillus bacterium, preferably B. subtilis, examples of promoters include the P43 promoter, vegI promoter, xylose-inducible promoter, and tetracycline-inducible promoter. When the host is yeast, examples of promoters include the PHO5 promoter, GAP promoter, and ADH promoter. When the host is an animal cell, examples of promoters include SV40-derived promoters, retrovirus promoters, cytomegalovirus (CMV) IE (immediate early) gene promoters, metallothionein promoters, heat shock promoters, and SRα promoters.

Examples of host cells include bacteria such as those of the genera Escherichia, Bacillus, Corynebacterium, Brevibacterium, Serratia, Pseudomonas, Arthrobacter, Erwinia, Methylobacterium, and Rhodobacter, and fungi such as those of the genera Streptomyces, Zymomonas, and Saccharomyces. Additionally, insect cells such as silkworm cells, 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 can also be used.

Examples of transformation methods for introducing an expression vector into a host cell include electroporation, alkali metal precipitation, calcium phosphate precipitation, DEAE-dextran precipitation, microinjection, and lipofection.

Collagen-like modified proteins can be obtained by culturing transformed cells into which an expression vector has been introduced, expressing the polypeptide within the cells, and recovering and purifying the desired polypeptide from the cells or culture medium. Transformed cells can be cultured according to standard methods, depending on the properties of the host cells, such as carbon assimilation and auxotrophy, and the selection marker and promoter contained in the introduced recombinant gene.

The collagen-like modified protein can be purified by an appropriate method selected from among methods commonly used for protein purification. That is, an appropriate method can be selected from commonly used methods such as salting out, ultrafiltration, isoelectric precipitation, gel filtration, electrophoresis, ion exchange chromatography, various affinity chromatographies such as hydrophobic chromatography and antibody chromatography, chromatofocusing, adsorption chromatography, and reversed-phase chromatography, and purification can be performed in an appropriate order using an HPLC system or the like, if necessary.

Furthermore, when the collagen-like modified protein contains a tag sequence or the amino acid sequence of another functional protein or polypeptide other than collagen, it is preferable to employ a purification method characteristic of that functional protein. Examples of such functional proteins or peptides and their corresponding purification methods include a histidine tag consisting of approximately 6 to 10 consecutive histidine residues and nickel-immobilized affinity chromatography, glutathione S-transferase (GST) and glutathione-immobilized affinity chromatography, and a FLAG tag and anti-FLAG antibody. Furthermore, the collagen-like modified protein can be recovered by cleaving the purified fusion protein with an appropriate protease (thrombin, trypsin, etc.).

Another genetic engineering production method is cell-free synthesis using nucleic acids encoding collagen-like modified proteins. Cell-free protein synthesis systems include systems that use extracts from cells such as E. coli, wheat germ, yeast, rabbit reticulocytes, insect cells, and cultured mammalian cells, as well as reconstituted systems constructed by combining factors necessary for protein synthesis.

Collagen-like modified proteins can be produced using organic chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method) using a suitable commercially available peptide synthesizer, but they are preferably produced using genetic engineering techniques by introducing the above-mentioned nucleic acid, particularly DNA incorporated into an expression vector, into a suitable expression system using a suitable host cell selected from prokaryotes or eukaryotes.

Collagen-like modified proteins can be produced as homotrimers by the methods exemplified above, particularly by culturing transformed cells into which an expression vector has been introduced, and then recovering and purifying the target protein from the cells or culture medium. Homotrimers can also be converted to monomers for use by placing them in the presence of SDS or other detergents or high concentrations of salt.

As described above, the present invention provides, in separate aspects, a nucleic acid encoding a collagen-like modified protein, an expression vector containing the nucleic acid, and a host cell containing the nucleic acid or the expression vector.

Another aspect of the present invention relates to a method for detecting human autoantibodies targeting NC11-COL7 of human BP180 and/or human autoantibodies targeting NC7-COL4, the method comprising the steps of contacting a specimen sample collected from a subject with the collagen-like modified protein of the first aspect and detecting binding between antibodies in the specimen sample and the collagen-like modified protein, wherein the collagen-like modified protein is as described in the first aspect.

Human autoantibodies can be detected by detecting binding between antibodies in a sample and the collagen-like modified protein of the first embodiment, which occurs upon contacting the sample with the collagen-like modified protein. The sample used here is a biological sample derived from a human, such as skin tissue, skin cells, body fluids (e.g., blood, lymph, saliva, mucus, bone marrow fluid, urine, semen, peritoneal fluid, etc.), or serum or plasma prepared from blood. Biological samples may be used as collected or after pretreatment such as pulverization, homogenization, centrifugation, concentration, or dilution. A particularly preferred sample is a blood sample, specifically blood, serum, or plasma.

Human autoantibodies can be detected by immunoassays that utilize antigen-antibody reactions. Examples of immunoassays include ELISA, radioimmunoassay, immunoblotting, and immunochromatography.

An immunoassay can be performed, for example, by immobilizing a collagen-like modified protein on a carrier, contacting this with a sample to allow the collagen-like modified protein to react with antibodies in the sample, and then detecting antibodies bound to the collagen-like modified protein using a labeled human antibody-binding substance.

Examples of human antibody-binding substances include antibody-binding proteins that can bind to human antibodies, particularly those that can bind to the Fc region of human IgG, such as antibodies against human antibodies and protein G.

Examples of labeling compounds used to label human antibody-binding substances include fluorescent substances (e.g., FITC, rhodamine, etc.), metal particles such as gold colloids, fluorescent microbeads such as Luminex (registered trademark, Luminex Inc.), pigmented proteins (e.g., phycoerythrin, phycocyanin, etc.), radioisotopes (e.g., ^3H , ^14C , ^32P , 35S, ^125I , ^{131I ,} etc.), enzymes (e.g., peroxidase, alkaline phosphatase, etc.), biotin, and streptavidin.

The carrier and immobilization method for immobilizing collagen-like modified proteins, the labeling compound for labeling human antibody-binding substances and the labeling method using the same, and the immunoassay procedures using these, such as incubation conditions between the sample and carrier, washing, blocking treatment, and detection of the labeled compound, can be performed by methods known to those skilled in the art, for example, by referring to the descriptions in *The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques* (4TH), Elsevier. This document is incorporated herein by reference in its entirety.

Data Collection Method : The above-described method for detecting human autoantibodies can detect human autoantibodies targeting NC11-COL7 and/or NC7-COL4 of human BP180. Specifically, when a collagen-like modified protein contains a region corresponding to NC11-COL7 of BP180, human autoantibodies targeting NC11-COL7 can be detected. When a collagen-like modified protein contains a region corresponding to NC7-COL4 of BP180, human autoantibodies targeting NC7-COL4 can be detected. These human autoantibodies are autoantibodies associated with drug-induced BP, particularly DPP4i-BP autoantibodies. Therefore, data collected by performing this method on a specimen sample collected from a patient with BP can be useful for distinguishing whether the patient's BP is drug-induced or due to other causes. Thus, the present invention also provides a method for collecting data for diagnosing drug-induced BP, particularly DPP4i-BP, comprising the steps of contacting a specimen sample collected from a subject with a collagen-like modified protein and detecting binding of antibodies in the specimen sample to the collagen-like modified protein. Furthermore, the test sample, collagen-like modified protein, and method of using them used in this data collection method of the present invention are as explained in the above-mentioned method for detecting human autoantibodies.

In yet another aspect, the present invention provides a kit for detecting human autoantibodies targeting NC11-COL7 and/or NC7-COL4 of human BP180, and a kit for diagnosing drug-induced bullous pemphigoid, each kit comprising at least the collagen-like modified protein of the first aspect. In addition to the collagen-like modified protein, the kit may further comprise reagents, such as a support such as a plate, a blocking solution, a washing solution, a human antibody-binding substance, and a chromogenic substrate, used for immunoassay detection of human autoantibodies binding to the polypeptide.

The present invention will be described in further detail by the following examples, but the present invention is not limited to these examples.

Example 1: Preparation of collagen-like modified protein (1) Preparation of expression vector
cDNAs encoding five collagen-like modified proteins (collagen 13-BP180 swap proteins 1 to 5) were synthesized using a DNA synthesizer. The cDNA for swap protein 1 encodes, from the 5' end, a FLAG tag (SEQ ID NO: 24), the amino acid sequence from amino acids 2 to 121 of human type XIII collagen isoform COL13A1 (SEQ ID NO: 9), the amino acid sequence of the extracellular domain fragment of BP180 (Gly ⁵⁶⁷ -Ile ⁸⁰⁸⁾ , and the amino acid sequence from amino acids 700 to 717 of human type XIII collagen isoform COL13A1 (SEQ ID NO: 9). The cDNAs of swap proteins 2 to 5 encode, from the 5' end, a FLAG tag (SEQ ID NO: 24), the amino acid sequence from amino acids 2 to 216 of human type XIII collagen isoform COL13A1 (SEQ ID NO: 9), the amino acid sequence of the extracellular domain fragment of BP180 (Val ⁸⁰⁹ -Ser ⁹⁸¹ , Glu ⁹⁸² -Ser ¹¹⁶⁰ , Tyr ¹¹⁶¹ -Ser ¹²⁷⁹ , or Arg ¹²⁸⁰ -Pro ¹⁴⁹⁷ ), and the amino acid sequence from amino acids 700 to 717 of human type XIII collagen isoform COL13A1 (SEQ ID NO: 9). EcoRV and HindIII restriction sites were added in frame to the 5' and 3' ends of the nucleotide sequence encoding the BP180 extracellular domain fragment, respectively. Furthermore, an NheI restriction site was added to the 5' end of each cDNA, and an ApaI restriction site was added to the 3' end. The nucleotide sequences of swap proteins 1 to 5 excluding the restriction enzyme sites at the 5' and 3' ends and the sequences corresponding to the FLAG tag sequence are shown in SEQ ID NOs: 14, 16, 18, 20, and 22, and the amino acid sequences corresponding to these nucleotide sequences are shown in SEQ ID NOs: 15, 17, 19, 21, and 23.

A total of five collagen-like modified protein expression vectors (collagen 13-BP180 swap protein expression vectors) were constructed by recombining the above cDNAs into the NheI-ApaI sites of the expression vectors pcDNA3.1/Hyg (Invitrogen) or pcDNA5/FRT (Invitrogen).

(2) Generation of Cells Stably Expressing Collagen-like Modified Proteins. The collagen 13-BP180 swap protein expression vector prepared in (1) was transfected alone (when integrated into pcDNA3.1/Hyg) or together with pOG44 (Invitrogen) into Flp-In 293 cells (Invitrogen) cultured in DMEM medium containing 10% fetal bovine serum using Avalanche-Omni Transfection Reagent (EZ Biosystems). Hygromycin (Invitrogen) was added to the medium at 50 μg/ml 48 hours after transfection, and the cells were cultured for 10–14 days. Drug-resistant clones stably expressing collagen 13-BP180 swap protein were selected.

(3) Purification of collagen-like modified proteins. Cells stably expressing collagen 13-BP180 swap protein were cultured in a petri dish until confluent and washed twice with PBS (Nakarai). After adding cell lysis buffer (25 mM Tris, pH 7.5, 1% NP-40 (Nakarai), 10 mM EDTA, 1x protease inhibitor cocktail (Sigma #P8340-1ML)), the cells were gently shaken on ice for 30 minutes. The lysed cells were collected into an Eppendorf tube using a cell scraper and centrifuged at 14,000 rpm for 20 minutes at 4°C. The supernatant was then collected. PBS was added to the supernatant, mixed, and anti-DDDDK antibody magnetic beads (MBL #M185-11) were added. The mixture was then shaken at 4°C for 10–12 hours. The magnetic beads were then collected using a magnetic separator (Invitrogen) and washed with PBS containing 0.1% NP-40. Next, elution buffer (500 μg/ml FLAG peptide (Sigma #F3290-4MG), 0.1% NP-40, PBS) was added, and the mixture was shaken at 700 rpm for 15 minutes at room temperature. The eluted proteins were then collected using a magnetic separator to produce five collagen-like modified proteins (collagen 13-BP180 swap proteins 1 to 5, Figure 2).

Example 2 Immunoassay Using Human Samples (1) Western Blotting Serum was prepared from blood collected from 17 patients diagnosed with DPP4i-BP by a dermatologist (hereinafter referred to as DPP4i-BP patients), 3 patients diagnosed with BP but without a history of DPP4i administration (hereinafter referred to as BP patients), and 3 healthy controls. Serum from all DPP4i-BP patients was confirmed in advance to be non-reactive with NC16A.

Swap proteins 1 to 5 prepared in Example 1 were diluted 5-fold with 5x sample buffer (4M urea, 0.5M Tris-HCl pH 6.8, 0.0005% bromophenol blue, 10% SDS, 25% glycerin, 0.05M DTT) and subjected to SDS-PAGE on a 7% acrylamide-containing gel.

After electrophoresis, proteins in the SDS-PAGE gel were transferred to a nitrocellulose membrane (BioRad). Anti-FLAG antibody (M2, Sigma #F1804-1MG, diluted 1:2,000 in 2% skim milk in TBS) or test sample (diluted 1:200 in 2% skim milk in TBS) was added as the primary antibody and incubated for 1 hour at room temperature or 10–12 hours at 4°C. Secondary antibodies, HPR-conjugated anti-mouse IgG (Jackson #115-036-006) or HRP-conjugated anti-human IgG (Dako #P0214), diluted 1:5,000 in 2% skim milk in TBS, were then added and incubated for 30–60 minutes at room temperature. Color development was performed using Clarity Western ECL Substrate (BIO-RAD #170-5060), and chemiluminescence was detected using an LAS-4000mini (FUJIFILM).

The results of Western blot for swap protein 3 are shown in Figure 3, and the results of Western blot for swap protein 4 are shown in Figure 4. When anti-FLAG antibody was used as the primary antibody, protein bands were detected at positions corresponding to the molecular weight of the monomer and homotrimer of each swap protein (left images in Figures 3 and 4).

As shown in Figure 3, all 17 DPP4i-BP patients reacted to both the monomer and trimer of swap protein 3, which contains NC11-COL7 of BP180, and two BP patients reacted to both the monomer and trimer. Healthy controls did not react to either the monomer or trimer.

Furthermore, as shown in Figure 4, for swap protein 4, which contains NC7-COL4 of BP180, four DPP4i-BP patients reacted to the monomer, and seven DPP4i-BP patients reacted to the trimer. On the other hand, neither BP patients nor healthy controls reacted to either the monomer or trimer.

For swap proteins 1, 2, and 5, Western blot analysis using anti-FLAG antibody as the primary antibody detected bands at the molecular weights corresponding to the monomer and homotrimer. However, none of the specimens from DPP4i-BP patients, BP patients, or healthy controls showed any reactivity to the monomer or trimer.

(2) ELISA
Serum samples were prepared from blood collected from 15 DPP4i-BP patients, 5 BP patients, and 3 healthy controls. All DPP4i-BP patient sera were confirmed to be non-reactive with NC16A.

Swap proteins 1 to 5 prepared in Example 1 were diluted to a concentration of 0.4 μg/ml with carbonate buffer (50 mM CB pH 9.6) and added at 50 μl/well to a 96-well plate (Nunc #442404). After incubation at 4°C for 10 to 12 hours, the plate was washed three times with 200 μl of PBS, and 90 μl/well of blocking buffer (Roche #11112589001) was added. After incubation at room temperature for 2 hours, the plate was washed twice with 200 μl of PBS to prepare five 96-well ELISA plates with immobilized swap proteins 1 to 5.

100 μl/well of the primary antibody (sample) diluted 1:101 in PBS or anti-FLAG antibody (M2) diluted 1:2,000 in PBS was added and incubated at room temperature for 1 hour. After washing four times with 200 μl/well of PBS containing 0.05% Tween, 100 μl/well of HRP-labeled anti-human or anti-mouse IgG (1:10,000 diluted in PBS) was added as secondary antibodies and incubated at room temperature for 1 hour. After washing four times with 200 μl/well of PBS containing 0.05% Tween, a color reaction was carried out using TMB (NOVEX) for 12 minutes at room temperature. The color reaction was terminated with stop solution (0.5N sulfuric acid), and the absorbance at wavelengths of 450 nm and 620 nm was measured using an absorption spectrophotometer (Sunrise, TECAN). The difference between these values was used as an index of the antigen-antibody reaction.

The ELISA results are shown in Figure 5. The specimens from DPP4i-BP patients showed high reactivity to swap proteins 1 to 5, particularly swap protein 4, which contains NC7-COL4 of BP-180.

(3) ELISA with increased sample number
Five types of 96-well ELISA plates were prepared by immobilizing swap proteins 1 to 5 in the same manner as in (2) above. Additionally, 98-well ELISA plates were prepared by immobilizing NC16A recombinant protein using the method described in Kobayashi M, et al. J Dermatol. Sci., 30: 224-232, 2002, and 98-well ELISA plates were prepared by immobilizing full-length BP180 recombinant protein using the method described in Izumi K, et al. J Invest. Dermatol., 136: 2201-10, 2016.

ELISA was performed in the same manner as described above (2) using the seven types of plates, using serum samples from patients with skin diseases other than BP (such as eczema dermatitis) (hereinafter referred to as non-BP patients), BP patients, and DPP4i-BP patients as sample samples, and one serum sample from a healthy subject as a negative control.
The difference between the absorbance at a wavelength of 450 nm and the absorbance at a wavelength of 620 nm measured by ELISA was taken as the OD value, and the ELISA index was calculated using the following formula.
Formula: (OD value of each test sample - OD value of healthy subject sample) / (OD value of anti-FLAG antibody - OD value of healthy subject sample) x 100

The results of the ELISA are shown in Figure 6. Compared with the sera from non-BP and BP patients, the sera from DPP4i-BP patients showed significantly higher reactivity to swap protein 3 containing NC11-COL7 of BP180 and swap protein 4 containing NC7-COL4. Furthermore, none of the sera from DPP4i-BP patients used showed reactivity to NC16A.

Furthermore, using the ELISA index for swap protein 4, ROC analysis was performed on the datasets of non-DPP4iBP patients and DPP4i-BP patients, BP patients and DPP4i-BP patients, and non-DPP4i-BP patients (non-BP patients + BP patients) and DPP4i-BP patients, with DPP4i-BP as the objective variable and the ELISA index as the explanatory variable. The analysis was performed using GraphPad Prism 8 (MDF), and the AUC was calculated from the generated ROC curve. The AUC was 0.8 or higher for all datasets, confirming that the determination of DPP4i-BP by ELISA for swap protein 4 exhibits good accuracy.

Comparative Example: Preparation of BP180 Extracellular Domain Fragments. DNAs (SEQ ID NOS: 25, 27, 29, and 31) encoding the amino acid sequences of human BP180 (COL15, NC15-COL9, NC9-NC4, and NC4-NC1) (SEQ ID NOS: 26, 28, 30, and 32) were inserted into the BamHI-NotI site of the expression vector pGEX-6P-1 (Amersham #27-4597-01) to construct five E. coli expression vectors for the BP180 extracellular domain fragments. After transformation of the BL21 E. coli strain (GE Healthcare #27-1542-01) with these expression vectors, ampicillin-resistant clones were selected. Cell lysates were harvested from E. coli cells induced with the Overnight Expression Autoinduction System 1 (Novagen #71300) using B-PERII (Thermo Fisher Scientific #78243). Western blotting revealed that sufficient amounts of the target protein could not be identified from either the soluble or insoluble fractions, demonstrating the difficulty of expressing the fragmented regions of the BP180 extracellular domain in E. coli.

SEQ ID NO: 1: Amino acid sequence of the coiled-coil sequence of human type XIII collagen SEQ ID NO: 2: Amino acid sequence of NC11-COL7 of human BP180 SEQ ID NO: 3: Amino acid sequence of NC7-COL4 of human BP180 SEQ ID NO: 4: Amino acid sequence of the coiled-coil sequence of human type XXIII collagen SEQ ID NO: 5: Amino acid sequence of the coiled-coil sequence of human type XXV collagen SEQ ID NO: 6: Amino acid sequence of the coiled-coil sequence of human MARCO SEQ ID NO: 7: Amino acid sequence of the coiled-coil sequence of human SRCL SEQ ID NO: 8: Amino acid sequence of the coiled-coil sequence of human ectodysplasin A SEQ ID NO: 9: Amino acid sequence of human type XIII collagen α1 chain (COL13A1) SEQ ID NO: 10: Amino acid sequence of human BP180 SEQ ID NO: 11: Nucleotide sequence of cDNA encoding human BP180 SEQ ID NO: 12: Amino acid sequence recognized by human autoantibodies present in NC16A of human BP180 SEQ ID NO: 13: Amino acid sequence of the NC16A region of human BP180 SEQ ID NO: 14 Nucleotide sequence of DNA encoding a collagen-like modified protein containing COL15 of human BP180 (collagen 13-BP180 swap protein 1) SEQ ID NO: 15. Amino acid sequence of collagen-like modified protein containing COL15 of human BP180 (collagen 13-BP180 swap protein 1) SEQ ID NO: 16. Nucleotide sequence of DNA encoding a collagen-like modified protein containing NC15-COL11 of human BP180 (collagen 13-BP180 swap protein 2) SEQ ID NO: 17. Amino acid sequence of collagen-like modified protein containing NC15-COL11 of human BP180 (collagen 13-BP180 swap protein 2) SEQ ID NO: 18. Nucleotide sequence of DNA encoding a collagen-like modified protein containing NC11-COL7 of human BP180 (collagen 13-BP180 swap protein 3) 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 Nucleotide sequence of DNA encoding collagen-like modified protein containing NC7-COL4 of human BP180 (collagen 13-BP180 swap protein 4) SEQ ID NO: 21. Amino acid sequence of collagen-like modified protein containing NC7-COL4 of human BP180 (collagen 13-BP180 swap protein 4) SEQ ID NO: 22. Nucleotide sequence of DNA encoding collagen-like modified protein containing NC4-NC1 of human BP180 (collagen 13-BP180 swap protein 5) SEQ ID NO: 23. Amino acid sequence of collagen-like modified protein containing NC4-NC1 of human BP180 (collagen 13-BP180 swap protein 5) SEQ ID NO: 24. Amino acid sequence of FLAG tag SEQ ID NO: 25. Nucleotide sequence of artificially synthesized DNA encoding COL15 of human BP180 SEQ ID NO: 26. Artificially synthesized amino acid sequence of COL15 of human BP180 SEQ ID NO: 27. Nucleotide sequence of artificially synthesized DNA encoding NC15-COL9 of human BP180 SEQ ID NO: 28 SEQ ID NO: 29: Artificially synthesized amino acid sequence of NC15-COL9 of human BP180. SEQ ID NO: 30: Artificially synthesized amino acid sequence of NC9-NC4 of human BP180. SEQ ID NO: 31: Artificially synthesized DNA sequence of NC4-NC1 of human BP180. SEQ ID NO: 32: Artificially synthesized amino acid sequence of NC4-NC1 of human BP180.

Claims (14)

A collagen-like modified protein comprising a trimerization domain consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and a domain corresponding to the seventh non-collagen domain (NC7) to the fourth collagen domain (COL4) of human BP180, which is located C-terminal to the trimerization domain , but which does not bind to human autoantibodies that target the NC16A domain of human BP180. A collagen-like modified protein comprising a trimerization domain consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and a domain corresponding to NC7 to COL4 of human BP180 located on the C-terminal side of the trimerization domain, but not including the amino acid sequence of NC16A of human BP180 . The protein according to claim 1 or 2, wherein the amino acid sequence of the region corresponding to NC7 to COL4 of human BP180 is the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 . The protein according to any one of claims 1 to 3 , consisting of the amino acid sequence shown in SEQ ID NO:21. The protein according to any one of claims 1 to 4 , which forms a homotrimeric structure. A nucleic acid encoding a protein defined in any one of claims 1 to 4 . An expression vector comprising the nucleic acid defined in claim 6 . A host cell comprising a nucleic acid as defined in claim 6 or an expression vector as defined in claim 7 . A method for detecting a human autoantibody that targets the region from NC7 to COL4 of human BP180, comprising the steps of contacting a specimen sample collected from a subject with a protein defined in any one of claims 1 to 5 , and detecting binding between an antibody in the specimen sample and the protein. The method of claim 9 , wherein the specimen sample is a blood sample. A kit for detecting human autoantibodies targeting the region from NC7 to COL4 of human BP180, comprising the protein defined in any one of claims 1 to 5 . A method for collecting data for diagnosing drug-induced bullous pemphigoid, comprising the steps of contacting a specimen sample collected from a subject with a protein defined in any one of claims 1 to 5 , and detecting binding between an antibody in the specimen sample and the protein. The method of claim 12 , wherein the specimen sample is a blood sample. A kit for diagnosing drug-induced bullous pemphigoid, comprising a protein as defined in any one of claims 1 to 5 .