Antibody binding to colibactin-producing escherichia coli bacterium

Abstract

Provided are a means and method for specifically detecting a colibactin-producing Escherichia coli bacterium, and a means and method for specifically controlling a colibactin-producing bacterium.　A colibactin-producing E. coli bacterium in a sample is detected using an antibody specifically binding to a colibactin-producing E. coli bacterium. A colibactin-producing bacterium is specifically controlled using an antibody specifically binding to a colibactin-producing E. coli bacterium.

Description

colibactin-producing Escherichia coli-binding antibody

[0001] The present invention relates to an antibody that specifically binds to colibactin-producing Escherichia coli.

[0002] Colibactin induces double-strand breaks and interstrand crosslinks in host DNA, leading to genetic mutations in the host (Non-Patent Document 1). The presence of Escherichia coli strains carrying a gene cluster involved in colibactin biosynthesis, known as the clb cluster or pks cluster, is known to be involved in the development of colorectal cancer (Non-Patent Document 2). The colibactin-producing (clb+) subgroup of E. coli, classified as the commensal B2 phylogenetic group, inhabits the human colon and is thought to be involved in the development of colorectal cancer as a result of symbiosis. Upon contact between clb+ strains and eukaryotic cells, colibactin is thought to induce cell cycle arrest in the G2 / M phase and / or megakaryocyte formation. It has been reported that the detection rate of clb cluster genes in biopsy samples from colorectal cancer patients is 67%, higher than the 21-27% found in healthy individuals. To more accurately understand the role of clb+ strains in colorectal cancer development, more detailed and accurate knowledge of the chemical and biological properties of colibactin is needed.

[0003] Colibactin is a genotoxin secreted by bacteria belonging to the Enterobacteriaceae family. These bacteria are known to possess a 54-kilobase genomic island encoding a polyketide synthase (PKS), a nonribosomal peptide synthetase (NRPS), and a PKS-NRPS hybrid megasynthetase, all of which are required for colibactin biosynthesis (Non-Patent Document 3). The gene cluster involved in colibactin production contains genes for various accessory enzymes, one of which is the peptidase ClbP. Based on its crystal structure and other findings, ClbP has been suggested to activate precolibactin, a colibactin precursor, to produce a genotoxic end product (Non-Patent Documents 4 and 5). Patent Document 1 discloses a high-throughput method for measuring colibactin-producing bacteria in clinical samples using a fluorescent probe specifically activated by ClbP. This fluorescent probe is also useful for identifying high colibactin-producing strains from Escherichia coli.

[0004] Colibactin is believed to have a highly unstable structure, and due to this instability, its chemical structure has not been fully elucidated. Non-Patent Documents 6 and 7 disclose the structure of N-myristoyl-D-asparagine (N-myr-Asn), a colibactin prodrug motif, and a method for detecting it by LC-MS. To date, research on precolibactin (colibactin precursor) has been conducted using clb+ strains generated by genetic mutation, and various chemical structures have been identified as precolibactin candidate substances. However, these substances have not been isolated from wild-type clb+ E. coli strains, and it is unclear whether they actually exist as precolibactin in colibactin-producing bacteria present in the human large intestine.

[0005] International Publication No. 2019 / 044736

[0006] Nougayrede, JP et al., Science Vol. 313 (5788), pp. 848-851 (2006)Putze, J. et al., Infect. Immun. Vol. 77 (11), pp. 4696-4703 (2009)Homburg, S. et al., FEMS Microbiol. Lett. Vol. 275 (2), pp. 255-262 (2007)Dubois, D. et al, J. Biol. Chem. Vol. 286 (41), pp. 35562-35570 (2011)Brotherton, CA and Balskus, EP, J. Am. Chem. Soc. Vol. 135 (9), pp. 3359-3362 (2013)Bian, X. et al., Chembiochem. Vol. 14 (10), 1194-1197 (2013)Brotherton CA et al., Org. Lett., Vol.17, pp. 1545-1548 (2015)

[0007] As mentioned above, Patent Document 1 discloses a method for identifying colibactin-producing E. coli using a fluorescent probe, but this method requires a step of culturing candidate E. coli in the presence of the probe. A simpler method for identifying colibactin-producing E. coli is desired. Furthermore, a method for specifically controlling colibactin-producing bacteria has not yet been developed, and its development is also desired.

[0008] An object of the present invention is to provide means and methods for specifically detecting colibactin-producing Escherichia coli, and means and methods for specifically controlling colibactin-producing bacteria.

[0009] The present invention provides the following: [1] An antibody that specifically binds to colibactin-producing Escherichia coli. [2] The antibody according to [1], which has binding activity to at least one of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 39 and a polypeptide having the amino acid sequence set forth in SEQ ID NO: 40. [3] The antibody according to [1], which is a monoclonal antibody. [4] The antibody according to [3], which is an IgM. [5] The antibody according to [3], which comprises a polypeptide of (a) or (a') below: (a) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; or (a') a polypeptide comprising the amino acid sequence of the polypeptide according to (a) as a partial sequence. [6] The antibody described in [5], further comprising the following polypeptides (b) or (b') and (c) or (c'): (b) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2; (b') a polypeptide containing the amino acid sequence of the polypeptide described in (b) as a partial sequence; (c) a polypeptide having the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3; (c') a polypeptide containing the amino acid sequence of the polypeptide described in (c) as a partial sequence. [7] The antibody described in [5], further comprising the following polypeptides (d) or (d') and (e) or (e'): (d) a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (d') a polypeptide containing the amino acid sequence of the polypeptide described in (d) as a partial sequence; (e) a polypeptide having the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5; (e') a polypeptide containing the amino acid sequence of the polypeptide described in (e) as a partial sequence.[8] The antibody according to [5], further comprising the following polypeptides (f) or (f') and (g) or (g'): (f) a polypeptide having the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6; (f') a polypeptide comprising the amino acid sequence of the polypeptide shown in (f) as a partial sequence; (g) a polypeptide having the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7; (g') a polypeptide comprising the amino acid sequence of the polypeptide shown in (g) as a partial sequence. [9] The antibody according to [5], further comprising the following polypeptides (h) or (h') and (i) or (i'): (h) a polypeptide having the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8; (h') a polypeptide comprising the amino acid sequence of the polypeptide shown in (h) as a partial sequence. (i) a polypeptide having the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 9; (i') a polypeptide comprising the amino acid sequence of the polypeptide described in (i) as a partial sequence.

[10] The antibody according to [3], which has a light chain variable region including homology determining regions (CDRs) 1 to 3 shown in the following (a1) to (a3): (a1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10 or the amino acid sequence shown in SEQ ID NO: 10 in which one or two amino acids have been deleted, substituted or added; (a2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11; and (a3) ​​CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence shown in SEQ ID NO: 12 in which one or two amino acids have been deleted, substituted or added.

[11] A light chain variable region comprising CDR1 to CDR3 shown in the following (b1) to (b3): ​​(b1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13 or the amino acid sequence shown in SEQ ID NO: 13 with one or two amino acids deleted, substituted or added; (b2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14; and (b3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15 or the amino acid sequence shown in SEQ ID NO: 15 with one or two amino acids deleted, substituted or added; and a heavy chain variable region comprising CDR1 to CDR3 shown in the following (c1) to (c3): (c1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20 or the amino acid sequence shown in SEQ ID NO: 20 with one or two amino acids deleted, substituted or added; (c2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 21 or the amino acid sequence shown in SEQ ID NO: 21 with one or two amino acids deleted, substituted or added; and (c3) The antibody according to [3], comprising a CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 22.

[12] A light chain variable region comprising CDR1 to CDR3 shown in the following (d1) to (d3): (d1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13 or the amino acid sequence shown in SEQ ID NO: 13 with one or two amino acids deleted, substituted or added; (d2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14; and (d3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15 or the amino acid sequence shown in SEQ ID NO: 15 with one or two amino acids deleted, substituted or added; and a heavy chain variable region comprising CDR1 to CDR3 shown in the following (e1) to (e3): (e1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20 or the amino acid sequence shown in SEQ ID NO: 20 with one or two amino acids deleted, substituted or added; (e2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 with one or two amino acids deleted, substituted or added; and (e3) The antibody according to [3], comprising a CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 22.

[13] A light chain variable region comprising CDR1 to CDR3 shown in the following (f1) to (f3): (f1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16 or the amino acid sequence shown in SEQ ID NO: 16 with one or two amino acids deleted, substituted or added; (f2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17; and (f3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18 or the amino acid sequence shown in SEQ ID NO: 18 with one or two amino acids deleted, substituted or added; and a heavy chain variable region comprising CDR1 to CDR3 shown in the following (g1) to (g3): (g1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 with one or two amino acids deleted, substituted or added; (g2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 25 or the amino acid sequence shown in SEQ ID NO: 25 with one or two amino acids deleted, substituted or added; and (g3) The antibody according to [3], comprising a CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 26.

[14] A light chain variable region comprising CDR1 to CDR3 shown in the following (h1) to (h3): (h1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16 or the amino acid sequence shown in SEQ ID NO: 16 with one or two amino acids deleted, substituted or added; (h2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17; and (h3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 19 or the amino acid sequence shown in SEQ ID NO: 19 with one or two amino acids deleted, substituted or added; and a heavy chain variable region comprising CDR1 to CDR3 shown in the following (i1) to (i3): (i1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 27 or the amino acid sequence shown in SEQ ID NO: 27 with one or two amino acids deleted, substituted or added; (i2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 28 or the amino acid sequence shown in SEQ ID NO: 28 with one or two amino acids deleted, substituted or added; and (i3) The antibody according to [3], comprising a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence set forth in SEQ ID NO: 29.

[15] A method for detecting colibactin-producing E. coli in a sample, comprising the step of contacting the sample with the antibody according to any one of [1] to

[14] .

[16] The method according to

[15] , wherein the sample is a sample removed from a subject.

[17] The method according to

[16] , wherein the subject is a human.

[18] A composition comprising the antibody according to any one of [1] to

[14] .

[19] The composition according to

[18] , which is for detecting colibactin-producing E. coli.

[20] The composition according to

[18] , which is a pharmaceutical composition.

[21] A method for treating or preventing colorectal cancer, comprising administering the antibody according to any one of [1] to

[13] to a subject. This specification incorporates the disclosures of Japanese Patent Application No. 2022-079941, from which the present application claims priority.

[0010] According to the present invention, it is possible to provide means and methods for specifically detecting colibactin-producing E. coli, as well as means and methods for specifically controlling colibactin-producing bacteria.

[0011] 1 is a photograph showing the results of Western blotting of colibactin-producing E. coli-50 protein using a monoclonal antibody derived from the δ strain in Example 5. 2 is a photograph showing the results of epitope mapping in Example 5. 3 is a photograph showing the results of Western blotting of colibactin-producing E. coli-50 protein using an antibody before and after peptide competition reaction in Example 5.

[0012] 1. Antibodies that specifically bind to colibactin-producing E. coli A first embodiment of the present invention is an antibody that specifically binds to colibactin-producing E. coli (hereinafter also referred to as "colibactin-producing bacteria"). More specifically, the antibody of this embodiment binds to colibactin-producing bacteria but does not bind to E. coli that does not produce colibactin.

[0013] Conventionally, no antibodies that directly bind to colibactin-producing bacteria have been obtained, and colibactin-producing bacteria have been detected by reacting a substrate with a colibactin-producing enzyme, as shown in Patent Document 1. The present inventors immunized mice with a bacterial cell suspension of Escherichia coli 50 (high colibactin-producing bacterium (Hirayama, Y. et al., Org. Lett. Vol. 21, No. 12, pp. 4490-4494 (2019)) (hereinafter also referred to as "E. coli-50"), a high colibactin-producing bacterium, using this as an immunogen, and then appropriately screened samples obtained from the mice, thereby successfully obtaining antibodies that specifically bind to colibactin-producing strains.

[0014] As used herein, the term "antibody" refers to a protein that specifically binds to a specific substance (antigen). In the present invention, the antigen is a substance derived from colibactin-producing E. coli. The antibody is not particularly limited as long as it binds to colibactin-producing E. coli and not to E. coli that does not produce colibactin. It may be a polyclonal or monoclonal antibody, with monoclonal antibodies being preferred. The isotype of the antibody is not particularly limited and may be any of IgM, IgD, IgG, IgA, IgE, etc., with immunoglobulin M (IgM) being particularly preferred. The animal from which the antibody is derived is not particularly limited and may be, for example, mouse, rat, rabbit, goat, cow, pig, sheep, dog, cat, monkey, camel, alpaca, bird, fish, etc. Preferably, the antibody is derived from a mouse. Note that, as used herein, the term "antibody" does not necessarily refer to an intact antibody, and may refer to Fab, Fab', F(ab') ２ Alternatively, the antibody may be a single-domain antibody. Single-domain antibodies are also called single-chain antibodies or nanobodies (registered trademark), and are antibodies consisting of the variable region of an antibody (immunoglobulin) composed only of a heavy chain, and known examples include VHHs found in mammals of the Camelidae family and VNARs found in sharks.

[0015] The antibody of this embodiment preferably has binding activity to at least one of the polypeptides having the following amino acid sequences, and more preferably has binding activity to both of the polypeptides having the following amino acid sequences: KRLLFMIKSV (SEQ ID NO: 39) YTAVVKKSS (SEQ ID NO: 40) As used herein, the term "having binding activity" of an antibody to a polypeptide refers to a significantly higher binding amount of the antibody to the polypeptide of interest compared to a polypeptide having any amino acid sequence, and / or a higher binding strength to the polypeptide of interest compared to a polypeptide having any amino acid sequence. The binding amount of the antibody referred to here is not particularly limited, and can be measured, for example, by immunoblotting, ELISA, etc. Furthermore, the binding strength of the antibody is not particularly limited, and can be measured, for example, using an instrument such as Biacore (registered trademark).

[0016] SEQ ID NOs: 39 and 40 are specifically partial sequences of E. coli PapA. The present inventors constructed four monoclonal antibodies that react with colibactin-producing E. coli but not with E. coli that does not produce colibactin. When proteins derived from colibactin-producing E. coli were subjected to Western blotting using these antibodies, ladder-like bands were observed, suggesting that these antibodies react with proteins having a repeat sequence. Next, the present inventors focused on PapA, a protein that constitutes the repeat structure of pili, among E. coli proteins, and performed epitope mapping of the four antibodies based on the amino acid sequence of PapA (SEQ ID NO: 42). As a result, it was confirmed that all four antibodies had strong binding activity to polypeptides having the amino acid sequences represented by SEQ ID NOs: 39 and 40.

[0017] 1-1 First Aspect Hereinafter, this embodiment will be described by giving specific aspects of the present embodiment, but it is not intended to limit the present embodiment to the following aspects. The first aspect of this embodiment is a monoclonal IgM antibody and has the following characteristics. a light chain variable region 1 comprising the polypeptide of (a) or (a') below: (a) a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence which has 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; or (a') a polypeptide comprising the amino acid sequence of the polypeptide set forth in (a) as a partial sequence; and a light chain variable region 2 comprising the polypeptide of (b) or (b') below: (b) an amino acid sequence set forth in SEQ ID NO: 2 or a polypeptide having 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; (b') A polypeptide comprising the amino acid sequence of the polypeptide described in (b) as a partial sequence; and having a heavy chain variable region comprising the polypeptide of (c) or (c') below: (c) A polypeptide having the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3; (c') A polypeptide comprising the amino acid sequence of the polypeptide described in (c) as a partial sequence.

[0018] The first aspect of this embodiment preferably has the following features in addition to or instead of the above features: CDR1 to CDR3 in the light chain variable region 1 are represented by the following (a1) to (a3): (a1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10 or the amino acid sequence shown in SEQ ID NO: 10 with one or two amino acids deleted, substituted or added; (a2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11; and (a3) ​​CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence shown in SEQ ID NO: 12 with one or two amino acids deleted, substituted or added.

[0019] The first aspect of this embodiment preferably has the following features in addition to or instead of the above features: CDR1 to 3 in the light chain variable region 2 are represented by the following (b1) to (b3): ​​(b1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13 or the amino acid sequence shown in SEQ ID NO: 13 with one or two amino acids deleted, substituted or added; (b2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14; and (b3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15 or the amino acid sequence shown in SEQ ID NO: 15 with one or two amino acids deleted, substituted or added; and CDR1 to 3 in the heavy chain variable region are represented by the following (c1) to (c3): (c1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20 or the amino acid sequence shown in SEQ ID NO: 20 with one or two amino acids deleted, substituted or added; (c2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence obtained by deleting, substituting, or adding one or two amino acids from the amino acid sequence shown in SEQ ID NO: 21; and (c3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence obtained by deleting, substituting, or adding one or two amino acids from the amino acid sequence shown in SEQ ID NO: 22.

[0020] 1-2 Second Aspect The second aspect of this embodiment is a monoclonal IgM antibody, which has the following characteristics: a polypeptide having a light chain variable region 1 comprising the polypeptide of (a) or (a') below: (a) having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence which has 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1; or (a') a polypeptide comprising the amino acid sequence of the polypeptide of (a) as a partial sequence; and a light chain variable region 2 comprising the polypeptide of (d) or (d') below: (d) having the amino acid sequence of SEQ ID NO: 4 or a polypeptide having 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 4; (d') A polypeptide comprising the amino acid sequence of the polypeptide described in (d) as a partial sequence; and having a heavy chain variable region comprising the polypeptide of (e) or (e') below: (e) A polypeptide having the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 5; (e') A polypeptide comprising the amino acid sequence of the polypeptide described in (e) as a partial sequence.

[0021] The second aspect of this embodiment preferably has the following features in addition to or instead of the above features: CDR1 to CDR3 in the light chain variable region 1 are represented by the following (a1) to (a3): (a1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10 or the amino acid sequence shown in SEQ ID NO: 10 with one or two amino acids deleted, substituted or added; (a2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11; and (a3) ​​CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence shown in SEQ ID NO: 12 with one or two amino acids deleted, substituted or added.

[0022] The second aspect of this embodiment preferably has the following features in addition to or instead of the above features: CDR1 to 3 in the light chain variable region 2 are represented by the following (d1) to (d3): (d1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13 or the amino acid sequence shown in SEQ ID NO: 13 with one or two amino acids deleted, substituted or added; (d2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14; and (d3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15 or the amino acid sequence shown in SEQ ID NO: 15 with one or two amino acids deleted, substituted or added; and CDR1 to 3 in the heavy chain variable region are represented by the following (e1) to (e3): (e1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20 or the amino acid sequence shown in SEQ ID NO: 20 with one or two amino acids deleted, substituted or added; (e2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence obtained by deleting, substituting, or adding one or two amino acids from the amino acid sequence shown in SEQ ID NO: 23; and (e3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence obtained by deleting, substituting, or adding one or two amino acids from the amino acid sequence shown in SEQ ID NO: 22.

[0023] 1-3 Third Aspect The third aspect of this embodiment is a monoclonal IgM antibody, which has the following characteristics: a polypeptide having a light chain variable region 1 comprising the polypeptide of (a) or (a') below: (a) having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence which has 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1; or (a') a polypeptide comprising the amino acid sequence of the polypeptide of (a) as a partial sequence; and a light chain variable region 2 comprising the polypeptide of (f) or (f') below: (f) having the amino acid sequence of SEQ ID NO: 6 or a polypeptide having 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 6; (f') A polypeptide comprising the amino acid sequence of the polypeptide described in (f) as a partial sequence; and having a heavy chain variable region comprising the polypeptide of (g) or (g') below: (g) A polypeptide having the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 7; (g') A polypeptide comprising the amino acid sequence of the polypeptide described in (g) as a partial sequence.

[0024] The third aspect of this embodiment preferably has the following features in addition to or instead of the above features: CDR1 to CDR3 in the light chain variable region 1 are represented by the following (a1) to (a3): (a1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10 or the amino acid sequence shown in SEQ ID NO: 10 with one or two amino acids deleted, substituted or added; (a2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11; and (a3) ​​CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence shown in SEQ ID NO: 12 with one or two amino acids deleted, substituted or added.

[0025] The third aspect of this embodiment preferably has the following features in addition to or instead of the above features: CDR1 to 3 in the light chain variable region 2 are represented by the following (f1) to (f3): (f1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16 or the amino acid sequence shown in SEQ ID NO: 16 with one or two amino acids deleted, substituted or added; (f2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17; and (f3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18 or the amino acid sequence shown in SEQ ID NO: 18 with one or two amino acids deleted, substituted or added; and CDR1 to 3 in the heavy chain variable region are represented by the following (g1) to (g3): (g1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 with one or two amino acids deleted, substituted or added; (g2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence obtained by deleting, substituting, or adding one or two amino acids from the amino acid sequence shown in SEQ ID NO: 25; and (g3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence obtained by deleting, substituting, or adding one or two amino acids from the amino acid sequence shown in SEQ ID NO: 26.

[0026] 1-4 Fourth Aspect The fourth aspect of this embodiment is a monoclonal IgM antibody, which has the following characteristics: a polypeptide having a light chain variable region 1 comprising the polypeptide of (a) or (a') below: (a) having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence which has 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1; or (a') a polypeptide comprising the amino acid sequence of the polypeptide of (a) as a partial sequence; and a light chain variable region 2 comprising the polypeptide of (h) or (h') below: (h) having the amino acid sequence of SEQ ID NO: 8 or a polypeptide having 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 8; (h') A polypeptide comprising the amino acid sequence of the polypeptide described in (h) as a partial sequence; and having a heavy chain variable region comprising the polypeptide of (i) or (i') below: (i) A polypeptide having the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having 80% or more, preferably 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 9; (i') A polypeptide comprising the amino acid sequence of the polypeptide described in (i) as a partial sequence.

[0027] The fourth aspect of this embodiment preferably has the following features in addition to or instead of the above features: CDR1 to CDR3 in the light chain variable region 1 are represented by the following (a1) to (a3): (a1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10 or the amino acid sequence shown in SEQ ID NO: 10 with one or two amino acids deleted, substituted or added; (a2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11; and (a3) ​​CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence shown in SEQ ID NO: 12 with one or two amino acids deleted, substituted or added.

[0028] The fourth aspect of this embodiment preferably has the following features in addition to or instead of the above features: CDR1 to 3 in the light chain variable region 2 are represented by the following (h1) to (h3): (h1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16 or the amino acid sequence shown in SEQ ID NO: 16 with one or two amino acids deleted, substituted or added; (h2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17; and (h3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 19 or the amino acid sequence shown in SEQ ID NO: 19 with one or two amino acids deleted, substituted or added; and CDR1 to 3 in the heavy chain variable region are represented by the following (i1) to (i3): (i1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 27 or the amino acid sequence shown in SEQ ID NO: 27 with one or two amino acids deleted, substituted or added; (i2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence obtained by deleting, substituting, or adding one or two amino acids from the amino acid sequence shown in SEQ ID NO: 28; and (i3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence obtained by deleting, substituting, or adding one or two amino acids from the amino acid sequence shown in SEQ ID NO: 29.

[0029] 2. Method for detecting colibactin-producing E. coli A second embodiment of the present invention is a method for detecting colibactin-producing E. coli in a sample, comprising the step of contacting the sample with at least one antibody described in the section "1. Antibodies that specifically bind to colibactin-producing E. coli."

[0030] The method of this embodiment can assist in diagnosing a subject's colon cancer or risk of developing colon cancer by detecting colibactin-producing bacteria in vitro in a sample taken from a subject, particularly a sample taken from a subject suspected of having colon cancer or suspected of being at high risk of developing colon cancer.

[0031] As used herein, the term "subject" includes humans, primates including chimpanzees, pet animals such as dogs and cats, livestock animals such as cows, pigs, horses, sheep, and goats, rodents such as mice and rats, and mammals kept in zoos. The subject herein is preferably a human. As used herein, the term "sample" refers to a sample taken from a subject, such as blood (e.g., whole blood, serum, plasma), urine, feces, milk, tissue or cell extract, nasal discharge, saliva, or a mixture thereof. Preferably, the sample is a fecal sample.

[0032] Specifically, the method of this embodiment is a method for immunoassay, comprising a step of contacting a sample taken from a subject with an antibody that binds to colibactin-producing bacteria. Examples of such immunoassays include direct competitive assays, indirect competitive assays, and sandwich assays. Examples of such immunoassays include chemiluminescent enzyme immunoassays (CLEIA), chemiluminescent immunoassays (CLIA), turbidimetric immunoassays (TIA), enzyme immunoassays (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassays (RIA), latex agglutination assays, fluorescent immunoassays (FIA), and immunochromatography. The principles and specific techniques of the above immunoassays are well known to those skilled in the art.

[0033] In the method of this embodiment, the detection of colibactin-producing bacteria may be qualitative or quantitative. A qualitative method may involve, for example, comparing a signal obtained from a sample with a preset cutoff to determine the presence or absence of colibactin-producing bacteria in the sample. A quantitative method may involve performing an immunoassay on a standard solution containing a known amount of antigen in the same manner as for the sample, and then comparing the signal intensities obtained from the sample and the standard solution to calculate the amount of antigen in the sample. Furthermore, the method of this embodiment may include the steps of detecting the antigen in the standard solution and calculating the amount of colibactin-producing bacteria in the sample from the detection results of the standard solution.

[0034] In the method of this embodiment, if colibactin-producing bacteria are qualitatively determined to be "positive" or if the quantitative value exceeds the determination criterion, it can be supported to determine that the subject from whom the sample was taken is infected with E. coli or is at high risk of infection.

[0035] 3. Composition for detecting colibactin-producing bacteria The present invention encompasses compositions comprising at least one antibody described in Section "1. Antibodies that specifically bind to colibactin-producing E. coli." A third embodiment of the present invention is a composition for detecting colibactin-producing E. coli, comprising at least one antibody described in Section "1. Antibodies that specifically bind to colibactin-producing E. coli."

[0036] The composition of this embodiment comprises at least one type of antibody selected from the antibodies of the first embodiment of the present invention. The composition of this embodiment can be used to carry out the method described in Section "2. Method for detecting colibactin-producing E. coli," and can be used to detect colibactin-producing bacteria in a sample. In particular, by using the composition to detect colibactin-producing bacteria in a sample taken from a subject suspected of having colorectal cancer or suspected of being at high risk of having colorectal cancer, it becomes possible to assist in diagnosing the subject's colorectal cancer or risk of having colorectal cancer. The "sample" and "subject" referred to here are as described in Section "2. Method for detecting colibactin-producing E. coli."

[0037] The composition of this embodiment can be used alone or in combination with other reagent solutions, equipment, etc. as a reagent or kit for detecting colibactin-producing bacteria. Such a reagent or kit is for immunoassays that use antibodies that specifically bind to colibactin-producing bacteria. Examples of such immunoassays include direct competitive assays, indirect competitive assays, and sandwich assays. Furthermore, examples of such immunoassays include CLEIA, CLIA, TIA, EIA (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), RIA, latex agglutination assay, FIA, and immunochromatography. The principles and specific techniques of the above immunoassays are all well known to those skilled in the art.

[0038] The composition of this embodiment may contain, as necessary, a pH buffer such as phosphate buffer, Tris, MES, HEPES, or PIPES, a chelating agent such as EDTA, or a preservative. Furthermore, antibody stabilizers such as BSA, casein, or glycine may be contained. The composition of this embodiment preferably has a pH of 5 to 9, particularly 6 to 8, and even more preferably 6.5 to 7.5.

[0039] 4. Pharmaceutical Composition A fourth embodiment of this embodiment is a pharmaceutical composition comprising at least one antibody described in the section "1. Antibodies that specifically bind to colibactin-producing E. coli." The pharmaceutical composition of this embodiment is capable of specifically controlling colibactin-producing bacteria in a subject. As a result, the pharmaceutical composition of this embodiment can be used for the treatment and / or prevention of colorectal cancer.

[0040] The term "subject" as used herein refers to a subject to which the pharmaceutical composition is administered, and specifically includes humans, primates including chimpanzees, pet animals such as dogs and cats, livestock animals such as cows, pigs, horses, sheep and goats, rodents such as mice and rats, and mammals kept in zoos. Preferably, the subject is a human.

[0041] The route of administration of the pharmaceutical composition of this embodiment is not particularly limited, and may be any known route of administration such as oral, nasal, sublingual, subcutaneous, or intramuscular.

[0042] The pharmaceutical composition of this embodiment may contain, in addition to the antibody, a pharmaceutically acceptable carrier as needed. The term "pharmaceutically acceptable carrier" as used herein refers to additives commonly used in the pharmaceutical technology field. Examples include excipients, binders, disintegrants, fillers, emulsifiers, flow regulators, lubricants, etc.

[0043] Examples of excipients include sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides (more specifically, but not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, and cellulose), metal salts (e.g., sodium chloride, sodium or calcium phosphate, calcium sulfate, magnesium sulfate, calcium carbonate), citric acid, tartaric acid, glycine, low-, medium-, and high-molecular-weight polyethylene glycols (PEG), Pluronic®, kaolin, silicic acid, or combinations thereof.

[0044] Examples of binders include starch paste using corn, wheat, rice, or potato starch, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, and / or polyvinylpyrrolidone.

[0045] Examples of disintegrants include the above-mentioned starches, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium hydrogen carbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, or salts thereof.

[0046] Examples of the filler include the sugars and / or calcium phosphate (for example, tricalcium phosphate or calcium hydrogen phosphate).

[0047] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0048] Examples of flow regulators and lubricants include silicates, talc, stearates or polyethylene glycol.

[0049] Such carriers are primarily used to facilitate the formation of the dosage form and to maintain the dosage form and pharmacological effect, and may be used appropriately as needed. In addition to the above-mentioned additives, flavoring agents, solubilizing agents, suspending agents, diluents, surfactants, stabilizers, absorption enhancers, bulking agents, wetting agents, humectants, adsorbents, disintegration inhibitors, coating agents, coloring agents, preservatives, antioxidants, perfumes, flavoring agents, sweeteners, buffers, etc. may also be included as needed.

[0050] The pharmaceutical composition of this embodiment may also contain other drugs to the extent that the effect of the antibody is not lost. For example, in the case of an injection, it may contain a predetermined amount of other antibiotics.

[0051] The dosage form of the pharmaceutical composition of this embodiment is not particularly limited as long as it does not inactivate the antibody as an active ingredient and other additional active ingredients. For example, it may be any of liquid, solid, and semisolid. Specific dosage forms include oral dosage forms such as liquids, powders, granules, tablets, capsules, sublingual tablets, and lozenges, and parenteral dosage forms such as injections, suspensions, emulsions, eye drops, nasal drops, creams, ointments, plasters, patches, and suppositories.

[0052] The pharmaceutical composition of the present embodiment can be administered by any suitable method that does not inactivate the active ingredient contained therein, for example, orally or parenterally (for example, by injection, aerosol, topical application, eye drops, or nasal drops).

[0053] The pharmaceutical composition of this embodiment preferably contains the antibody in an amount that is effective in controlling colibactin-producing bacteria and is highly unlikely to cause serious side effects. The number of times the pharmaceutical composition of the present invention is administered is not particularly limited, as long as a sufficient effect in controlling colibactin-producing bacteria is obtained and serious side effects are not caused, but is preferably administered, for example, once every three days to five times a day, and particularly once to three times a day. The administration period of the pharmaceutical composition of this embodiment is not particularly limited, and can be, for example, three days to one month, and particularly one to two weeks.

[0054] The present specification also provides a method for producing the pharmaceutical composition of this embodiment. The method for producing the pharmaceutical composition disclosed herein is a method for producing the pharmaceutical composition of this embodiment using at least one antibody described in the section "1. Antibodies that specifically bind to colibactin-producing E. coli."

[0055] 5. Method for Treating or Preventing Colon Cancer A fifth embodiment of the present invention is a method for treating or preventing colon cancer using at least one antibody described in Section "1. Antibodies that specifically bind to colibactin-producing E. coli." More specifically, the method of this embodiment is characterized by comprising the step of administering to a subject the pharmaceutical composition described in Section "4. Pharmaceutical Composition."

[0056] The method of this embodiment includes, for example, an embodiment in which the antibody is orally or parenterally administered to a subject as a pharmaceutical composition or the like to kill colibactin-producing bacteria in the intestine, an embodiment in which the antibody labeled with a fluorescent dye is sprayed into the intestine of a subject under endoscopy, and the location of the fluorescently stained colibactin-producing bacteria is used as a therapeutic target (e.g., tumor resection), and the like.

[0057] Example 1 Preparation of Antibodies Binding to Colibactin-Producing Bacteria (1) Immunization Colibactin-producing strain 50 (high colibactin-producing bacterium (Hirayama, Y. et al., Org. Lett. Vol. 21, No. 12, pp. 4490-4494 (2019)) (hereinafter also referred to as "E. coli-50")) was cultured in LB liquid medium at 37°C for 20 hours. Bacterial cells were collected from the culture medium, washed twice with PBS(-), and then resuspended in PBS(-) at a concentration of 1 x 10 １０The E. coli cells were suspended at 100 μL / mL and heat-treated at 60°C for 60 minutes. 100 μL of the heated E. coli suspension was mixed with 100 μL of Sigma Adjuvant System (Catalog No. S6322, Sigma) to prepare a mixed solution, which was then subcutaneously injected in 50 μL in each leg into two mice (BALB / c, female, 8 weeks old). After two weeks, the mice were sacrificed, and the spleens and subpopliteal lymph nodes were collected.

[0058] (2) Cell fusion The collected spleen and popliteal lymph nodes were each cut in half, and the cells were pushed into a petri dish with deck tweezers and suspended in 5 mL of FBS(+). The cells were passed through a 100 μm cell strainer and then transferred to a 50 mL centrifuge tube. The petri dish and cell strainer were washed with 10 mL of FBS(+) and centrifuged (1000 rpm, 5 minutes, room temperature). The supernatant was discarded, and the pellet was tapped and suspended in FBS(+) for cell counting. Mouse myeloma P3U1 cells were also counted and 10 ８ The cells were suspended in FBS(+) to a ratio of 1000 cells / mL. P3U1 was added to a 50 mL centrifuge tube and suspended at a splenocyte / lymph node cell:P3U1 ratio of 3-4:1. After centrifugation (1000 rpm, 5 minutes, room temperature), the cells were resuspended in 30 mL of FBS(-) and centrifuged (1000 rpm, 5 minutes, room temperature). The supernatant was removed and the pellet was tapped. 1 mL of PEG was added at a rate of 1 mL / min and allowed to stand for 1 minute. 30 mL of FBS(-) was then added while shaking at a rate of 1 mL / min, and the remaining 20 mL was also slowly added. After centrifugation (1000 rpm, 5 minutes, room temperature), the supernatant was discarded and the pellet was tapped. The cells were suspended in HAT medium and dispensed at 200 μL per well into a 96-well microwell plate and incubated at 37°C and 5% CO ２ (Hereafter, cell culture was carried out under the same conditions.) The state of the cells was confirmed, and the cell culture supernatant was collected.

[0059] (3) Measurement of antibody titer by ELISA E. coli-50 and colibactin-negative E. coli #A (hereinafter also referred to as "#A") were cultured in LB liquid medium at 37°C for 20 hours. Here, #A is a strain confirmed to be colibactin-negative by the method described in Patent Document 1. After culture, the cells were washed three times with pure water, flash-frozen using liquid nitrogen, and then lyophilized. Each strain was suspended in PBS(-) (hereinafter referred to as T-PBS) supplemented with 0.05% Tween to a concentration of 0.5 mg / mL. 100 μL of the E. coli-50 or #A suspension was dispensed into each well of a 96-well microwell plate (Nunc Immunoplate, Catalog No. 439454) and allowed to stand at 4°C for at least one day for immobilization. After washing the plate three times with T-PBS, 1% BSA (dissolved in T-PBS) was added and the plate was left to stand at room temperature for 2 hours for blocking. This was then washed three times with T-PBS to obtain antigen-immobilized wells. A large number of antibody-producing cells (hybridomas) obtained by cell fusion in (2) above were cultured for one week. 100 μL or 50 μL of culture supernatant was added to each well and the plate was left to stand overnight at 4°C. After washing four times with T-PBS, 100 μL of 0.5 ng / mL HRP-labeled sheep anti-mouse IgG+IgM+IgA antibody was added and the plate was left to stand at 37°C for one hour. After washing four times with T-PBS, 100 μL of substrate solution (0.04% o-phenylenediamine) was added and the plate was left to stand at room temperature for 30 minutes in the dark. The reaction was stopped by adding 450 μL of 2N sulfuric acid, and the absorbance of each well at 490 nm was measured using a multiplate reader.

[0060] Seven samples of supernatants from the popliteal lymph nodes and 96 samples from the spleen had an absorbance of 0.09 or greater at 490 nm. These supernatants were subjected to limiting dilution to obtain monoclonal clones, yielding 753 cell lines. These cells were cultured and subjected to ELISA in the same manner as described above, yielding 641 positive cell lines. After limiting dilution and culture, four strains (α strain, β strain, γ strain, and δ strain) with particularly high absorbance differences (ΔABS) between the E. coli -50 plate and the #A plate were selected. All four strains were spleen-derived hybridomas.

[0061] Example 2: Examination of binding specificity of monoclonal antibodies to colibactin-producing E. coli The binding specificity of the four monoclonal antibodies obtained in Example 1 to colibactin-producing E. coli was examined. Solid-phase wells were prepared for E. coli-50, Nissle 1917 strain (a colibactin-positive strain), and four strains of E. coli obtained from clinical specimens, using the same method as described in Example 1(3). All four strains were confirmed to be colibactin-negative / positive by the method described in Patent Document 1.

[0062] The four hybridoma strains obtained in Example 1 and one hybridoma strain (nonspecific reaction strain) that tested positive for both E. coli -50 and #A were cultured for one week, and 100 μL of the culture supernatant was dispensed into six types of antigen-immobilized wells. ELISA was then performed in the same manner as described in Example 1(3). The ELISA was performed in duplicate. The ELISA results are shown in Table 1. Using an absorbance of 0.15 as the cutoff, with an absorbance of 0.15 or higher considered positive, the positive detection rates for each antibody were 75% (3 / 4 cases) for colibactin-positive E. coli and 0% (0 / 2 cases) for colibactin-negative E. coli. All monoclonal antibodies produced by the four hybridoma strains obtained were shown to bind to colibactin-positive E. coli with high specificity.

[0063]

[0064] Example 3: Determination of the homology-determining region (CDR) sequence of monoclonal antibodies. CDR sequence analysis was performed on the four monoclonal antibodies obtained in Example 1. CDR sequence analysis was performed by PGL LLC (Okazaki City, Aichi Prefecture). Specifically, the CDR sequences were determined through RNA extraction from each hybridoma, cDNA preparation, amplification of the variable region by PCR, cloning, and sequence analysis by capillary sequencing. All clones were confirmed to produce IgM antibodies. Two light chain sequences (L chain, κ) and one heavy chain sequence were detected in each clone. The amino acid and nucleic acid sequences of each chain obtained from the α, β, γ, and δ strains are shown in Tables 2, 3, 4, and 5, respectively.

[0065]

[0066]

[0067]

[0068]

[0069] The CDR sequences of the monoclonal antibodies of each strain are shown in Table 6.

[0070]

[0071] As described above, we have successfully obtained an antibody specific to colibactin-producing bacteria, and further succeeded in identifying the structure of the antibody, particularly the CDR structure.

[0072] Example 4: Immunohistochemical staining of colon tissue specimens from colon cancer patients using monoclonal antibodies. Immunohistochemical staining was performed on 10 tissue specimens from colon adenoma patients using an automated staining system (Roche Diagnostics, Benchmark XT). The detailed staining method is as follows: The specimens were deparaffinized and hydrated according to standard procedures. They were immersed in CC1 buffer (Roche Diagnostics) and heat-treated (60°C) for 60 minutes. The specimens were immersed in a primary antibody reaction solution prepared by diluting the δ cell supernatant obtained in Example 2 5-fold with PBS and incubated at room temperature for 32 minutes. Subsequently, secondary antibody and substrate reactions were performed according to standard procedures. Furthermore, the specimens were washed using UltraWash (Roche Diagnostics) and then immersed in HEMATOXYLIN II (Roche Diagnostics) for nuclear staining. After washing with UltraWash (Roche Diagnostics), Bluing Reagent was added dropwise. The specimen was removed from the automatic stainer, and the reaction buffer was washed off with a neutral detergent, followed by washing with purified water. The specimen was dehydrated and permeabilized according to standard methods, and then embedded in paraffin.

[0073] The intensity of immunohistochemical staining of the adenoma and non-tumor areas in each case was visually evaluated under a microscope. The evaluation results are shown in Table 7.

[0074]

[0075] Example 5: Determination of the epitope of the monoclonal antibody (1) Western blotting. After suspending colibactin-producing E. coli E. coli-50 and colibactin-non-producing E. coli E. coli #A in PBS and sonicating, the cells were loaded onto an acrylamide gel at a protein content of 0.3 to 5.0 μg, and SDS-PAGE was performed. The gel-like protein was transferred to a membrane, and Western blotting was performed according to standard methods using 10 ng / mL of δ strain monoclonal antibody (cell culture supernatant diluted 150,000-fold with PBS) as the primary antibody and HRP-labeled goat anti-mouse IgM heavy chain antibody (diluted 10,000-fold) as the secondary antibody. Immunostar® LD (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as the substrate. The results of the Western blotting are shown in Figure 1. E. Since the bands of E. coli strain-50 were ladder-like, it was speculated that the antibody reacted with a protein having a repeating structure.

[0076] (2) Production of Recombinant PapA and Western Blotting The repeat structure of E. coli was assumed to be the repeat structure of PapA in fimbriae, and recombinant PapA was produced to confirm its reactivity with the antibody. The papA gene of E. coli-50 (SEQ ID NO: 41, Table 8) was inserted downstream of the T7 promoter of the pET21c vector and introduced into E. coli BC21(DE3). E. coli BC21(DE3) itself is known to not contain the papA gene. The E. coli was cultured in a medium containing 0.1 mM IPTG, and the culture was centrifuged to obtain a cell pellet. The cells were suspended in PBS and disrupted by sonication. SDS-PAGE and CBB staining were performed according to standard methods to confirm the production of a PapA protein of approximately 20 kDa. Next, Western blotting was performed according to standard methods using 10 ng / mL of the δ strain monoclonal antibody as the primary antibody and an HRP-labeled goat anti-mouse IgM heavy chain antibody as the secondary antibody, as in (1). As a result, two strongly colored bands of approximately 20 kDa and approximately 16 kDa were confirmed. This confirmed that the δ strain monoclonal antibody reacts with PapA.

[0077]

[0078] (3) Epitope Mapping Using ResPep SL (Intavis), a total of 179 polypeptides containing 12 amino acid residues from the amino acid sequence of E. coli-50 PapA (SEQ ID NO: 42, Table 9) were synthesized on a membrane, shifting one amino acid residue from the N-terminus (Figure 2). Partial peptides of PapA were immobilized on spots A1 to F29 on the membrane shown in Figure 2. After washing the membrane with 0.05% Tween-PBS, the membrane was blocked with 4% BSA-PBS for 2 hours at room temperature. The membrane was immersed in a 10 ng / mL solution of δ strain monoclonal antibody in PBS as the primary antibody and incubated overnight. After washing with 0.05% Tween-PBS, the membrane was incubated for 2 hours in PBS containing a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM heavy chain secondary antibody as the secondary antibody. After washing with 0.05% Tween-PBS, the cells were reacted with a substrate solution of ImmunoStar (registered trademark) LD to develop color, and photographed with Luminograph I (ATTO).

[0079]

[0080] 2 is a photograph of the membrane after color development. The sequences of the peptides immobilized on the spots A1 to A4, D4 to D10, and F3 to F15 where color development was observed are shown in Table 10 below.

[0081]

[0082] Based on the structure of the peptides immobilized on the spots that showed particularly high reactivity with the δ strain monoclonal antibody, the antibody epitopes were presumed to be the following two polypeptides: KRLLFMIKSV (SEQ ID NO: 39) and YTAVVKKSS (SEQ ID NO: 40).

[0083] (4) Peptide Competition Test Peptide KRLLFMIKSV (SEQ ID NO: 39, hereinafter referred to as "peptide KR") and peptide YTAVVKKSS (SEQ ID NO: 40, hereinafter referred to as "peptide YT") were synthesized using ResPep SL (Intavis). E. coli strain-50 was heat-treated at 60°C for 60 minutes and then lyophilized according to a standard method to obtain a lyophilized extract. Peptide KR, peptide YT, a mixture of peptide KR and peptide YT, and the lyophilized extract of E. coli strain-50 were each reacted in a 10 ng / mL δ strain monoclonal antibody / PBS solution at room temperature for 3 hours.

[0084] E. coli-50 was suspended in PBS and sonicated, then loaded onto an acrylamide gel at 1 μg of protein, and subjected to SDS-PAGE. The gel-like protein was transferred to a membrane, and the membrane was reacted with a 10 ng / mL δ strain monoclonal antibody solution and an antibody solution prepared by reacting δ strain monoclonal antibody with various peptides or lyophilized extract of E. coli-50 strain with δ strain monoclonal antibody. Western blotting was performed according to standard methods using an HRP-labeled goat anti-mouse IgM heavy chain antibody (diluted 10,000 times) as the secondary antibody. Immunostar® LD (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as the substrate.

[0085] Figure 3 shows a photograph of the Western blot. Antibodies that were not subjected to competitive reactions showed a strong reaction with the peptide on the membrane. Antibodies reacted with either peptide KR or peptide YT also showed a strong reaction with the peptide on the membrane. In contrast, antibodies reacted with a mixture of peptide KR and peptide YT or a freeze-dried extract of E. coli strain-50 showed almost no reaction with the peptide on the membrane. These results indicated that the epitopes of the δ strain monoclonal antibody are the two peptides, peptide KR and peptide YT. Similar tests were performed with other monoclonal antibodies derived from α, β, and γ strains, and similar results were obtained, indicating that the epitopes of these monoclonal antibodies are nearly identical.

[0086] The present invention relates to an antibody that is useful for determining the presence or absence of and / or the presence or absence of the risk of developing a disease associated with colibactin-producing E. coli, such as colon cancer, and for treating and / or preventing the disease, and can be used in the industries of clinical test reagents, pharmaceuticals, etc. All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

1. An antibody that specifically binds to colibactin-producing E. coli.

2. The antibody according to claim 1, having binding activity to at least one of a polypeptide having the amino acid sequence represented by SEQ ID NO: 39 and a polypeptide having the amino acid sequence represented by SEQ ID NO:

40.

3. The antibody according to claim 1, which is a monoclonal antibody.

4. The antibody according to claim 3, which is IgM or IgG.

5. The antibody according to claim 4, comprising the polypeptide of (a) or (a') below: (a) A polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1; or (a') A polypeptide containing the amino acid sequence of the polypeptide described in (a) as a partial sequence.

6. Furthermore, the antibody according to claim 5 comprises the polypeptides of (b) or (b') and (c) or (c'): (b) A polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2; (b') A polypeptide containing the amino acid sequence of the polypeptide described in (b) as a partial sequence; (c) A polypeptide having the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3; A polypeptide containing the amino acid sequence of the polypeptide described in (c')(c) as a partial sequence.

7. Furthermore, the antibody according to claim 5 comprises the polypeptides (d) or (d') and (e) or (e') below: (d) A polypeptide having the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4; Polypeptides containing the amino acid sequence of the polypeptide described in (d')(d) as a partial sequence; (e) Polypeptides having the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5; A polypeptide containing the amino acid sequence of the polypeptide described in (e')(e) as a partial sequence.

8. Furthermore, the antibody according to claim 5 comprises the following polypeptides: (f) or (f') and (g) or (g'): (f) A polypeptide having the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6; Polypeptides containing the amino acid sequence of the polypeptide described in (f')(f) as a partial sequence; (g) A polypeptide having the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7; A polypeptide containing the amino acid sequence of the polypeptide described in (g')(g) as a partial sequence.

9. Furthermore, the antibody according to claim 5 comprises the following polypeptides: (h) or (h') and (i) or (i'): (h) A polypeptide having the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8; A polypeptide containing the amino acid sequence of the polypeptide described in (h')(h) as a partial sequence. (i) Polypeptides having the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9; A polypeptide containing the amino acid sequence of the polypeptide described in (i')(i) as a partial sequence.

10. The antibody according to claim 3, comprising a light chain variable region including homology-determining regions (CDRs) 1 to 3 shown below (a1) to (a3): (a1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 10; (a2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11; and (a3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO:

12.

11. The light chain variable region including CDR1-3 as shown in (b1) to (b3) below: (b1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 13; (b2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14; and (b3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 15; The heavy chain variable region including CDR1-3 as shown in (c1) to (c3) below: (c1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 20; (c2) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 21 or the amino acid sequence shown in SEQ ID NO: 21 in which one or two amino acids are deleted, substituted or added; and CDR2 consisting of the above; (c3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 22; The antibody according to claim 3, comprising:

12. The light chain variable region including CDR1-3 as shown in (d1) to (d3) below: (d1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 13; (d2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14; and (d3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 15; The heavy chain variable region including CDR1-3 as shown in (e1) to (e3) below: (e1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 20; (e2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence in which one or two amino acids are deleted, substituted or added from the amino acid sequence shown in SEQ ID NO: 23; and (e3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 22; The antibody according to claim 3, comprising:

13. The light chain variable region including CDR1-3 as shown in (f1) to (f3) below: (f1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 16; (f2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17; and (f3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 18; The heavy chain variable region including CDR1-3 as shown in (g1) to (g3) below: (g1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 24; (g2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 25; and (g3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 26; The antibody according to claim 3, comprising:

14. The light chain variable region including CDR1-3 as shown in (h1)-(h3) below: (h1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 16; (h2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17; and (h3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 19; The heavy chain variable region including CDR1-3 as shown in (i1) to (i3) below: (i1) CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 27; (i2) CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence in which one or two amino acids are deleted, substituted or added from the amino acid sequence shown in SEQ ID NO: 28; and (i3) CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence in which one or two amino acids are deleted, substituted, or added from the amino acid sequence shown in SEQ ID NO: 29; The antibody according to claim 3, comprising:

15. A method for detecting colibactin-producing Escherichia coli in a sample, comprising the step of contacting the sample with an antibody according to any one of claims 1 to 14.

16. The method according to claim 15, wherein the sample is a sample taken from the subject.

17. The method according to claim 16, wherein the subject is a human.

18. A composition comprising the antibody described in any one of claims 1 to 14.

19. The composition according to claim 18 for detecting colibactin-producing Escherichia coli.

20. The composition according to claim 18, which is a pharmaceutical composition.