Anti-ToH1 antibody and method of use thereof

Abstract

This disclosure relates to anti-ToH1 antibodies, methods for producing anti-ToH1 antibodies, and methods for using anti-ToH1 antibodies for the diagnosis and treatment of microbial diseases and / or chronic inflammatory diseases, including microbial infections and microbial-associated chronic inflammatory conditions.

Description

[Technical Field]

[0001] Priority statement This application is based on U.S. Provisional Application No. 63 / 506,769, filed on 7 June 2023, and its entire contents are incorporated herein by reference for all purposes.

[0002] Description of federal government subsidies This invention was made with government support under DK114713 granted by the National Institutes of Health. The government has certain rights to this invention.

[0003] Sequence List The text of the computer-readable sequence listing submitted with this specification, titled "CCF_42114_601_SequenceListing," created on June 7, 2023, with a file size of 56,149 bytes, is incorporated herein by reference in its entirety.

[0004] This disclosure relates to an anti-ToH1 antibody and to a method of using it for the diagnosis and treatment of microbial diseases and / or chronic inflammatory diseases in subjects. [Background technology]

[0005] The acellular mucosal barrier lies above the colon and normally limits physical and biochemical contact between microorganisms and host epithelium. When this primary barrier fails, microorganisms adhere to intestinal epithelial cells (IEC), leading to microbial diseases. High mobility group box 1 (HMGB1) is a multifunctional protein produced by many different human cells in response to innate immune detection of microorganisms. HMGB1 deficiency is likely associated with several diseases in which microorganisms adhere to host tissues, including infectious diarrhea, colorectal cancer, and IBD. [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, what is needed is a method for treating or preventing microbial diseases and / or chronic inflammatory diseases, including those in subjects with HMGB1 deficiency. [Means for solving the problem]

[0007] (Summary of the invention) In some embodiments, this specification provides a method for treating or preventing microbial diseases and / or chronic inflammatory diseases in a subject, comprising the step of providing an antibody bound to a target (ToH1) of HGMB1 in the subject. The antibody may be any antibody described herein, including scFv / IgG antibodies and nanobodies (e.g., scFv / IgG antibodies F5, F11, G6, or nanobodies G2 (VHH-G2) and F7 (VHH-F7)) provided in the appended examples. In some embodiments, microbial diseases include microbial infections. For example, a microbial infection may be a bacterial infection, a viral infection, a fungal infection, and / or a protozoan infection. In some embodiments, a microbial infection is a bacterial infection. In some embodiments, chronic inflammatory diseases include microbial-associated chronic inflammatory diseases. In some embodiments, chronic inflammatory diseases are inflammatory bowel disease, rheumatoid arthritis, non-alcoholic fatty liver disease, type II diabetes mellitus, urinary tract infections, pneumonia, or sepsis. In some embodiments, chronic inflammatory diseases are inflammatory bowel disease.

[0008] In some embodiments, this specification provides a method for diagnosing a microbial disease and / or chronic inflammatory disease in a subject, comprising the steps of: determining the level of the HGMB1 target (ToH1) in a sample obtained from the subject; and determining that the subject has a microbial disease if the level of ToH1 in the sample is equal to or higher than a threshold. In some embodiments, the step of determining the level of ToH1 in the sample comprises contacting the sample obtained from the subject with an antibody that binds to the HGMB1 target (ToH1); and detecting the antibody in the sample. The antibody may be any antibody described herein, including scFv / IgG antibodies and nanobodies (e.g., scFv / IgG antibodies F5, F11, G6, or nanobodies G2 (VHH-G2) and F7 (VHH-F7)) provided in the appended examples. In some embodiments, a microbial disease includes a microbial infection. For example, a microbial infection may be a bacterial infection, a viral infection, a fungal infection, and / or a protozoan infection. In some embodiments, a microbial infection is a bacterial infection. In some embodiments, a chronic inflammatory disease includes a microbial-associated chronic inflammatory disease. In some embodiments, the chronic inflammatory disease is inflammatory bowel disease, rheumatoid arthritis, non-alcoholic fatty liver disease, type II diabetes mellitus, urinary tract infection, pneumonia, or sepsis. In some embodiments, the chronic inflammatory disease is inflammatory bowel disease.

[0009] In some embodiments, a method for generating antibodies that bind to a target of HMGB1 is provided herein. In some embodiments, a method for generating antibodies that bind to a target of HGMB1 (ToH1) is provided, comprising the steps of sequentially immunizing a host with two or more unique peptides, and isolating antibodies produced in response to immunization. In some embodiments, each of the two or more unique peptides has a [S / T]xExPx[I / V] motif, where x is a variable amino acid. In some embodiments, the two or more unique peptides include three unique peptides. In some embodiments, the two or more unique peptides include four unique peptides. In some embodiments, the four unique peptides include SxExPxI, SxExPxV, TxExPxI, and TxExPxV.

[0010] In some embodiments, anti-ToH1 antibodies are provided herein. In some embodiments, antibodies that bind to a target of HGMB1 (ToH1) are provided herein, comprising a heavy chain variable domain including complementarity-determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable domain including complementarity-determining regions LCDR1, LCDR2 and LCDR3.

[0011] In some embodiments, HCDR1, HCDR2, and HCDR3 each contain an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 11, 12, and 13, respectively, and LCDR1, LCDR2, and LCDR3 each contain an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 14, 15, and 16, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 each contain an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 17, 18, and 19, respectively, and LCDR1, LCDR2, and LCDR3 each contain an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 20, 21, and 22, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 each contain an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 23, 24, and 25, respectively, and LCDR1, LCDR2, and LCDR3 each contain an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 26, 27, and 28, respectively.

[0012] In some embodiments, HCDR1, HCDR2, and HCDR3 each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NOs: 11, 12, and 13, respectively, and LCDR1, LCDR2, and LCDR3 each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NOs: 14, 15, and 16, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NOs: 17, 18, and 19, respectively, and LCDR1, LCDR2, and LCDR3 each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NOs: 20, 21, and 22, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NOs: 23, 24, and 25, respectively, and LCDR1, LCDR2, and LCDR3 each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NOs: 26, 27, and 28, respectively.

[0013] In some embodiments, HCDR1, HCDR2, and HCDR3 include SEQ ID NOs: 11, 12, and 13, respectively, and LCDR1, LCDR2, and LCDR3 include SEQ ID NOs: 14, 15, and 16, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 include SEQ ID NOs: 17, 18, and 19, respectively, and LCDR1, LCDR2, and LCDR3 include SEQ ID NOs: 20, 21, and 22, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 include SEQ ID NOs: 23, 24, and 25, respectively, and LCDR1, LCDR2, and LCDR3 include SEQ ID NOs: 26, 27, and 28, respectively.

[0014] In some embodiments, the antibody includes a heavy chain variable domain containing a sequence having at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; and a light chain variable domain containing a sequence having at least 80% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In some embodiments, the antibody includes a heavy chain variable domain containing a sequence having at least 90% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; and a light chain variable domain containing a sequence having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In some embodiments, the antibody includes a heavy chain variable domain containing a sequence having at least 95% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; and a light chain variable domain containing a sequence having at least 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In some embodiments, the antibody includes a heavy chain variable domain containing SEQ ID NO: 1 and a light chain variable domain containing SEQ ID NO: 2; a heavy chain variable domain containing SEQ ID NO: 3 and a light chain variable domain containing SEQ ID NO: 4; or a heavy chain variable domain containing SEQ ID NO: 5 and a light chain variable domain containing SEQ ID NO: 6. In some embodiments, the antibody is a single-chain variable region fragment (scFv). In some embodiments, the antibody is an IgG antibody.

[0015] In some embodiments, the antibody is a nanobody. In some embodiments, the antibody is a nanobody having at least 80% identity with SEQ ID NO: 29 or SEQ ID NO: 30. In some embodiments, the antibody is a nanobody having at least 90% identity with SEQ ID NO: 29 or SEQ ID NO: 30. In some embodiments, the antibody is a nanobody having at least 95% identity with SEQ ID NO: 29 or SEQ ID NO: 30. In some embodiments, the antibody is a nanobody having at least 98% identity with SEQ ID NO: 29 or SEQ ID NO: 30. In some embodiments, the antibody is a nanobody containing the sequence of SEQ ID NO: 29 or SEQ ID NO: 30. In some embodiments, the antibody is a nanobody consisting of the sequence of SEQ ID NO: 29 or SEQ ID NO: 30. Further embodiments are described herein. [Brief explanation of the drawing]

[0016] [Figure 1A-1F]This indicates that HMGB1 is released into colonic mucus in response to the gut microbiota. Figure 1A shows immunostaining of HMGB1 (yellow) and bisbenzimide H33258 (Hoechst, blue) in Carnoy-fixed proximal colon sections from HMGB1WT and HMGB1ΔIEC mice. Arrows indicate the epithelial surface. (n=20). Figure 1B shows the HMGB1 concentration in colonic mucus from HMGB1WT and HMGB1ΔIEC mice, measured by ELISA. (n=6) Figure 1C shows immunoblot of HMGB1 in colonic mucus from HMGB1WT and HMGB1ΔIEC mice. (n=7) Figure 1D shows immunostaining of HMGB1 (yellow) and Hoechst (blue) in Carnoy-fixed proximal colon sections from SPF and GF C57BL / 6 mice. Arrows indicate the epithelial surface. (n=6) Figure 1E shows immunoblot of HMGB1 in mucosal scraping from SPF and GF C57BL / 6 mice. (n=4) Figure 1F shows HMGB1 concentrations in feces from SPF and GF C57BL / 6 mice, measured by ELISA. (n=6) Data are mean ± standard deviation. Significance determined by Student's two-tailed t-test. Each data point represents one individual mouse. Scale bar, 100 μm. Original magnification 400x. [Figure 2A-2B] This demonstrates that HMGB1 prevents bacterial invasion into the mucus layer inside the colon. Figure 2A shows fluorescence in situ hybridization (FISH) using EUB338 probe (purple) and Hoechst (blue) in Carnoy-fixed proximal colon sections from HMGB1WT and HMGB1ΔIEC mice. Arrows indicate the epithelial surface. Dotted lines are located on the epithelial surface and the inner margin of the microbial community. Straight lines indicate the distance between the host tissue and the microbial community. Figure 2B shows the measured distances between the epithelium and bacterial cells in the image shown in Figure 2A. Each data point is the average of five measurements for each individual mouse. [Figure 2C-2E]This demonstrates that HMGB1 prevents bacterial invasion into the mucus layer inside the colon. Figure 2C shows quantitative PCR of the bacterial 16S rRNA gene in 1 cm colon tissue from HMGB1WT and HMGB1ΔIEC mice (n=6). Figure 2D shows invasion of green fluorescent protein (GFP)-labeled Escherichia coli (SWW33) into mucus isolated from HMGB1WT or HMGB1ΔIEC mice (n=3, 3 repeats). Figure 2E shows the percentage of total GFP signal in mucus from HMGB1WT versus HMGB1ΔIEC mice in the image shown in (d) (n=3; 3 repeats). [Figure 2F-2H] This shows that HMGB1 prevents bacterial invasion into the mucus layer inside the colon. Figure 2F shows the appearance of GFP-labeled Escherichia coli (SWW33) exposed to buffer (control) or HMGB1 (n=3; 3 replicates). Figure 2G shows flow cytometry of aggregates in samples of GFP-labeled Escherichia coli (SWW33) exposed to buffer (control) or HMGB1 (n=3; 3 replicates). Figure 2H shows the appearance of Syto9-labeled microbiota from C57BL / 6 mice exposed to buffer (control) or HMGB1 labeled with AF647 (n=3; 3 replicates). Data are mean ± standard deviation. Significance determined by Student's two-tailed t-test. Each data point represents one individual mouse. Scale bar, 100 μm. Original magnification 400x. [Figure 3A-3D]Through evolutionarily conserved amino acid sequences, it is shown that HMGB1 binds to the bacterium, adhesin FimH, inactivates it, and regulates the expression of the bacterial adhesin FimH. Figure 3A shows the amino acid sequence similarity among the known human HMGB1 target proteins, beclin-1 and Atg5, and the bacterial FimH. The putative HMGB1 interaction motif is derived from the amino acid sequence similarity between beclin-1 and Atg5, and common amino acid substitutions. Figures 3B and 3C show the flow cytometry of rHMGB1 bound to Escherichia coli (BW25113) with FimH knocked out (ΔFimH), or ΔFimH Escherichia coli complemented with a plasmid encoding wild-type FimH (ΔFimHWT; TSETPRV (SEQ ID NO: 33)) or FimH with conserved residues of the putative motif mutated (ΔFimHMUT; ASATARA). Both the percentage of Escherichia coli positive for HMGB1 (b) and the amount of HMGB1 protein bound to each bacterium (mean fluorescence intensity (MFI)) (c) were evaluated. (n = 3; 3 replicates) Figure 3D shows the label transfer of Sulfo-SBED from HMGB1 to the recombinant FimH lectin domain (FimHLD). The recipient protein was wild-type FimHLD (WT; TSETPRV (SEQ ID NO: 33)), or FimHLD with conserved amino acid residues of the putative interaction motif mutated (mutant; ASATARA). Transfer was evaluated with or without mannose. (3 replicates). [Figure 3E-3G]We demonstrate that HMGB1, through its evolutionarily conserved amino acid sequence, binds to and inactivates the bacterial adhesin FimH, thereby regulating the expression of bacterial adhesin FimH. Figure 3E shows the Escherichia coli colony-forming units (CFUs) attached to Caco2 IEC as a percentage of the input. Escherichia coli were treated with buffer, rHMGB1, or mannose before being added to the IEC. (n=6; 3 repeats) Figure 3F shows the percentage of FimHLD protein bound to mannose-coated plates in the presence of increased amounts of rHMGB1. (n=3; 3 repeats) Figure 3G shows RBC agglutination by Escherichia coli (SWW33) expressing wild-type FimH (FimH;TSETPRV(SEQ ID NO:33)), FimH knockout (FimH:KO), or FimH with a mutated ToH1 sequence (FimH:ASATARA and FimH:AAAAAAA). The number of bacteria decreases from left to right (3 repeats). [Figure 3H-3K] We demonstrate that HMGB1 binds to and inactivates the bacterial adhesin FimH, thereby regulating its expression, through evolutionarily conserved amino acid sequences. Figure 3H shows immunoblots of FimH in mucus isolated from HMGB1WT and HMGB1ΔIEC mice (n=4). Figure 3I shows band densitometry quantification of the immunoblots represented by (h) (n=4). Figure 3J shows immunostaining of FimH in seven Carnoy-fixed proximal colon sections from HMGB1WT and HMGB1ΔIEC mice. Figure 3K shows quantification of FimH-positive bacteria in the images represented by (j). [Figure 3L-3N]Through an evolutionarily conserved amino acid sequence, it is shown that HMGB1 binds to the bacterial adhesin FimH, inactivates it, and regulates the expression of the bacterial adhesin FimH. Figure 3L shows an immunoblot of FimH in Escherichia coli (SWW33) exposed to increasing amounts of HMGB1 (3 replicates). Figure 3M shows a DNA switch region directional PCR determination of Fim gene expression in Escherichia coli (ΔFimH) treated with medium conditioned by IEC organoids derived from HMGB1ΔIEC mice (ΔIEC CM), HMGB1WT mice (WT CM), or ΔIEC CM supplemented with rHMGB1. Phase-on indicates a switch oriented towards the production of the Fim gene. ftsZ is used for normalization. Figure 3N shows the relative band density of Phase-on in (l). Data are mean ± standard deviation. Significance determined by Student's two-sided t-test of two-way ANOVA for paired comparisons or multiple comparisons. Each data point represents an individual mouse. [Figure 4A-4H]This shows that HMGB1 mucosal protection is impaired in ulcerative colitis. Figure 4A shows immunohistochemistry of HMGB1 in Carnoy-fixed sections of resected colon from non-IBD or UC patients (n=16). Figure 4B shows quantification of surface-related HMGB1 using the image shown in (a). Staining intensity reported as relative fluorescence units (RFU) per 1 μm2 (n=16). Figure 4C shows surface-related HMGB1 as reported in (b), graphed by inflammation severity. Figure 4D shows immunohistochemistry of FimH in Carnoy-fixed sections of resected colon from non-IBD or UC patients. Serial tissue sections from the same patient as shown in (a) (n=16). Figure 4E shows quantification of FimH-positive bacteria using the image shown in (d). Reported as the number of subjects per high-magnification field. (n=16) Figure 4F shows the quantification of FimH-positive bacteria reported in (e), which is graphed according to the severity of inflammation. Figure 4G shows surface HMGB1 and FimH-positive bacteria plotted for each patient. The size of the black circles corresponds to the severity of inflammation. White ellipses indicate the characteristics of the population for each group (non-IBD and UC). Figure 4H shows a two-way scatter plot of surface HMGB1 and FimH-positive bacteria for each patient, along with a approximation curve. The relationship between HMGB1 and FimH was captured by nonlinear regression. Data are mean ± standard deviation. Each data point represents one individual. The Mann-Whitney U test was used to compare HMGB1 and FimH in the non-IBD vs. UC group (two-group comparison), and the Kruskal-Wallis test was used to assess the difference in HMGB1 and FimH between inflammation groups (three-group comparison). [Figures 5A-5E]This shows that HMGB1 interacts with intestinal microorganisms but does not exhibit strong selective pressure on the overall microbial community. Figure 5A shows immunostaining of HMGB1 in Carnoy-fixed proximal colon sections from HMGB1WT mice. Firm plane optimized to capture intestinal microorganisms. Scale bar, 100 μm. Original magnification 400x. Arrows indicate the tips of intestinal microorganisms. Figure 5B shows the Shannon α diversity index of ASV abundance using DNA isolated from colonic mucosal smears from HMGB1WT and HMGB1ΔIEC mice. Figure 5C shows canonical correspondence analysis (CCA) for ASV abundance using DNA isolated from colonic mucosal smears from HMGB1WT and HMGB1ΔIEC mice. Figure 5D shows a dimensionality reduction plot used to characterize microbiome differences between indicated sites (feces and mucosa) in samples from HMGB1WT and HMGB1ΔIEC mice. R² was obtained from a permutational multivariate analysis of variance (ANOVA) with site as the principal variable. R² represents the difference between the composition of the microbiome at two sites in mice of each genotype (HMGB1WT and HMGB1ΔIEC), and the p-value indicates the significance of the difference in composition between the two sites. Figure 5E shows the mean proportion of statistically different bacterial strains using DNA isolated from colon mucosal smears from HMGB1WT and HMGB1ΔIEC mice. Each data point represents an individual mouse, except (d) where each mouse has one data point for feces and one data point for mucosal samples. [Figures 6A-6D]This shows that HMGB1 binds to Escherichia coli (BW25113) (green) that produces FimH containing the ToH1 sequence. Figure 6A shows immunofluorescence staining of HMGB1 (red) bound to Escherichia coli (BW25113) (green). Wild-type Escherichia coli (WT), Escherichia coli with FimH knockout (ΔFimH), or ΔFimH Escherichia coli supplemented with a plasmid carrying WT FimH (ΔFimHWT) or ΔFimH Escherichia coli supplemented with a plasmid carrying FimH with a mutated ToH1 (ΔFimHMut). Figure 6B shows flow cytometry of FimH expression on the surface of ΔFimH Escherichia coli supplemented with plasmids carrying either WT FimH (ΔFimHWT) or FimH with a ToH1 mutation (ΔFimHMut). Figure 6C shows the flow cytometry gating strategies for HMGB1 binding reported in Figures 3b and 3c. Figure 6D shows flow cytometry of rHMGB1 binding to Escherichia coli of the indicated strains. Flow cytometry gating strategies for HMGB1 binding reported in (e) and (d). Data are mean ± standard deviation. Significance determined by Student's two-tailed t-test. Each data point represents one biological replicate. [Figure 7] This paper presents bioinformative predictions of HMGB1 target sequences based on experimental data using a position-specific score matrix (http: / / slim.icr.ac.uk / pssmsearch / ). [Figure 8] This graph shows that the exemplary anti-ToH1 antibody F11 inhibits FimH binding to its target ligand, mannose. [Figure 9]This image shows anti-ToH1 staining of colon tissue from HMGB1-deficient mice using exemplary antibody F11 (red). The results indicate that the antibody is suitable for immunohistochemistry to identify ToH1-positive microorganisms that are very close to or attached to the intestinal surface. Blue represents host cell DNA, and green represents E-cadherin, which stains the IEC boundary. [Figure 10A] The binding of antibodies G6 (Figure 10A) and F5 (Figure 10B) to the ToH1 peptide, as confirmed by ELISA, is shown. [Figure 10B] The binding of antibodies G6 (Figure 10A) and F5 (Figure 10B) to the ToH1 peptide, as confirmed by ELISA, is shown. [Figure 11] The results of ToH1 peptide binding for the antibody VHH-G2, confirmed by ELISA, are shown. VHH-G2 is shown to bind to all ToH1 peptides. [Figure 12] ELISA shows VHH-G2 binding to ToH1-positive adhesins. The adhesins are pyrin (Streptococcus pneumoniae), Duffy receptor (Plasmodium vivax), hemagglutinin (influenza B), basic membrane protein B (BmpB, Borrelia burgdorferi), non-structural protein 1 (NSP-1, dengue virus), and variant-specific surface protein VSP4A1 (CRISP-90) (VSP4A1, Giardia intestinalis (Giardia lamblia)). [Figure 13] ELISA demonstrates the binding of VHH-G2 to Escherichia coli FimH. [Figure 14]This image shows VHH-G2 binding to Escherichia coli and aggregated Escherichia coli. Bacteria are labeled green. The scale bar is 50 μm. [Figure 15] This shows that VHH-G2 increases bacterial clearance by macrophages. The upper image shows the phase contrast of RAW267 macrophages and Escherichia coli. Scale bar: 20 μm. The lower bar graph shows the percentage change in bacterial concentration in the culture medium from the cells shown in the image, based on colony-forming units. [Figure 16] ELISA demonstrates the binding of VHH-G2 to human IL1R1. [Figure 17] This shows the binding of the antibody VHH-F7 to the ToH1 peptide. [Figure 18] ELISA shows VHH-F7 binding to ToH1-positive adhesins. The adhesins are pyrin (Streptococcus pneumoniae), Duffy receptor (Plasmodium vivax), hemagglutinin (influenza B), basement membrane protein B (BmpB, Borrelia burgdorferi), non-structural protein 1 (NSP-1, dengue virus), and variant-specific surface protein VSP4A1 (CRISP-90) (VSP4A1, Giardia intestinalis (Giardia lambria)). [Figure 19] ELISA demonstrates the binding of VHH-F7 to Escherichia coli FimH. [Figure 20A] This image shows VHH-F7 binding to Escherichia coli and aggregated Escherichia coli. Bacteria are labeled green. The scale bar is 50 μm. [Figure 20B] This image shows VHH-F7 binding to Staphylococcus aureus and agglutinated Staphylococcus aureus. Bacteria are labeled green. The scale bar is 50 μm. [Figure 20C]This shows VHH-F7 binding to bacteria from a complex community and the aggregation of bacteria from the complex community. Bacteria (labeled green) were isolated from the colon of B6 mice and treated with VHH-F7 in vitro. Scale bar is 50 μm. The bacterial communities used in this assay and the following assay consisted of several hundred bacterial species, and the identified components included Lachnospiraceae, Listeria, Escherichia-Shigella, Bifidobacterium, Lactobacillus, Streptococcus, and Staphylococcus (confirmed by 16S rRNA DNA sequencing). [Figure 21] This image demonstrates that VHH-F7 increases bacterial clearance by macrophages. The upper image shows the phase contrast between RAW267 macrophages and Escherichia coli. Scale bar: 20 μm. The lower bar graph shows the percentage change in bacterial concentration in the culture medium, derived from the cells shown in the image, by colony-forming units. [Figure 22A] The results of an adhesion inhibition assay show that VHH-F7 inhibits Escherichia coli binding to mannose (Figure 22A), Staphylococcus aureus binding to fibronectin (Figure 22B), and bacterial community binding to fibronectin (Figure 22C). [Figure 22B] The results of an adhesion inhibition assay show that VHH-F7 inhibits Escherichia coli binding to mannose (Figure 22A), Staphylococcus aureus binding to fibronectin (Figure 22B), and bacterial community binding to fibronectin (Figure 22C). [Figure 22C] The results of an adhesion inhibition assay show that VHH-F7 inhibits Escherichia coli binding to mannose (Figure 22A), Staphylococcus aureus binding to fibronectin (Figure 22B), and bacterial community binding to fibronectin (Figure 22C). [Figure 23A]ELISA shows that VHH-F7 binds to IL1R1 (mammalian ToH1-positive protein). Figure 23 shows that VHH-F7 inhibits IL1R1 signaling. [Figure 23B] ELISA shows that VHH-F7 binds to IL1R1 (mammalian ToH1-positive protein). Figure 23 shows that VHH-F7 inhibits IL1R1 signaling. [Modes for carrying out the invention]

[0017] definition To facilitate understanding of the present invention, numerous terms and phrases are defined below.

[0018] Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the embodiments described herein, although some preferred methods, compositions, apparatus, and materials are described herein. However, before describing the materials and methods herein, it should be understood that the invention is not limited to these, as certain molecules, compositions, methodologies, or protocols described herein may be modified according to normal experimentation and optimization. It should also be understood that the terminology used herein is for describing certain types or embodiments and is not intended to limit the scope of the embodiments described herein.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the invention. However, in the event of any conflict, the definitions provided herein shall prevail. Accordingly, the following definitions apply in the context of the embodiments described herein.

[0020] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the articles. For example, "element" means at least one element and may include more than one element.

[0021] The term "approximately" is used to provide flexibility to the endpoint of a numerical range by specifying that a given value may be "slightly above" or "slightly below" the endpoint without affecting the desired result.

[0022] The use herein of the terms “including,” “comprising,” or “having,” and their variations, is intended to encompass the elements and their equivalents that follow them, as well as any further elements. Where used herein, “and / or” means all possible combinations of one or more of the related, listed items, as well as the absence of any combination as interpreted in the options ("or").

[0023] The enumeration of value ranges in this specification is intended to function simply as a simplified notation, unless otherwise indicated herein, to refer to each individual value belonging to the range, and each individual value is incorporated herein as if they were each enumerated herein. For example, where a concentration range is stated as 1% to 50%, values ​​such as 2% to 40%, 10% to 30%, or 1% to 3% are intended to be explicitly stated herein. These are merely examples of what is particularly intended, and all possible combinations of numbers between the lowest and highest values ​​listed, as well as those including the lowest and highest values ​​listed, should be considered explicitly stated in this disclosure.

[0024] As used herein, the term “antibody” is used in its broadest sense and includes antibodies, antibody derivatives, and antibody fragments. Examples include monoclonal antibodies, monospecific antibodies, polyspecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies, including, but not limited to, birds (e.g., ducks or geese), sharks, whales, and mammals, including non-primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.) or non-human primates (e.g., monkeys, chimpanzees, etc.), recombinant antibodies, chimeric antibodies, single-chain Fv ("scFv"), single-chain antibodies, single-domain antibodies (e.g., nanobodies), Fab fragments, F(ab') fragments, F(ab')2 fragments, Fc fragments (e.g., Fc region of an antibody), disulfide-bonded Fv ("sdFv"), and anti-idiotype ("anti-Id") antibodies, dual-region antibodies (dual variable Examples include antibodies, bivariate (DVD) or triplicate (TVD) antibodies, and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an analyte-binding site. The immunoglobulin molecule may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0025] An antibody fragment is included in the term "antibody," but refers to a portion of an intact antibody that includes an antigen-binding site or variable region. In some embodiments, an antibody fragment does not include the constant heavy chain domain of the Fc region of an intact antibody (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fc fragments, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, bispecific antibodies, single-chain Fv(scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of the light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of the heavy chain variable region.

[0026] An antibody "derivative," as used herein, is included in the term "antibody," but has one or more modifications to its amino acid sequence compared to the parent antibody. Antibody derivatives may exhibit modified domain structures. Derivatives may also adapt to typical domain configurations found in natural antibodies, as well as amino acid sequences capable of specifically binding to a target (antigen). Typical examples of antibody derivatives are antibodies bound to other polypeptides, rearranged antibody domains, or antibody fragments. Derivatives may also include at least one further compound linked by covalent or non-covalent bonds.

[0027] In this specification, "CDR" refers to the "complementarity-determining region" within the antibody variable sequence. There are three CDRs in each of the variable regions of the heavy chain and the light chain. Originating from the N-terminus of the heavy chain or light chain, these regions are denoted as "CDR1," "CDR2," and "CDR3" for each variable region. The term "CDR set," as used herein, refers to a group of three CDRs present in a single variable region that binds to an antigen. Thus, an antigen-binding site may contain six CDRs, including the CDR sets derived from the heavy chain and the light chain variable regions, respectively. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) may be called a "molecular recognition unit." Crystallographic analysis of antigen-antibody complexes has shown that amino acid residues of CDRs form extensive contact with the bound antigen, with the most extensive contact being with heavy chain CDR3. Therefore, molecular recognition units can be a major factor in the specificity of the antigen-binding site. Generally, CDR residues are involved in antigen binding that has a direct and most substantial effect.

[0028] As used herein, the term “concurrent administration” means the administration of at least two drugs (e.g., proteins or peptides of the present invention) or therapies to a subject. In some embodiments, the concurrent administration of two or more drugs / therapies is simultaneous. In some embodiments, the first drug / therapy is administered before the second drug / therapy. Those skilled in the art will understand that the formulations and / or routes of administration of the various drugs / therapies used may differ. Appropriate dosages for concurrent administration can be readily determined by those skilled in the art. In some embodiments, when drugs / therapies are concurrently administered, each drug / therapy is administered at a lower dosage than the appropriate dosage for their single administration. Concurrent administration is therefore particularly desirable in embodiments where concurrent administration of drugs / therapies reduces the required dosage of known potentially harmful (e.g., toxic) drugs.

[0029] As used herein, the term “diagnosed” means recognition of a disease, its signs and symptoms (e.g., resistance to conventional therapies) or by means of genetic analysis, pathophysiological analysis, histological analysis, diagnostic assays (e.g., of the disease).

[0030] As used herein, the term “effective dose” means an amount of therapeutic agent (e.g., the protein or peptide of the present invention) sufficient to produce a beneficial or desired result. The effective dose may be administered in one or more doses, applications, or dosages and is not intended to be limited to a particular formulation or route of administration.

[0031] As used herein, the term “host cell” means any eukaryotic cell, whether in vitro or in vivo, such as mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells.

[0032] As used herein, the term "in vitro" refers to an artificial environment and any process or reaction occurring within it. Examples of in vitro environments include, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and any process or reaction occurring within it.

[0033] As used herein, the term “pharmaceutical composition” means a combination of an active agent and an inert or active carrier that makes the composition particularly suitable for use in vivo or ex vivo for diagnostic or therapeutic purposes.

[0034] As used herein, the term “pharmaceutically acceptable carrier” means any of the standard pharmaceutically acceptable carriers, such as phosphate-buffered saline, water, emulsions (e.g., oil-water emulsion or water-oil emulsion), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th edition, Mack Publ. Co., Easton, Pennsylvania

[1975] .

[0035] The term "sample" as used herein is used in its broadest sense. A sample may include cells, tissues or bodily fluids, fecal or stool samples, nucleic acids or polypeptides isolated from cells, etc.

[0036] As used herein, the term “subject” means an organism treated by the methods of the embodiments of the present invention. In some embodiments, the subject is a vertebrate. In some embodiments, the subject is a mammal (e.g., mouse, monkey, horse, cattle, pig, dog, cat, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a bird (e.g., chicken). In some embodiments, the subject is a reptile. In the context of the present invention, the term “subject” generally means an individual that is receiving or has received treatment (e.g., administration of the protein or peptide of the present invention and optionally one or more other agents) for a disease (e.g., an inflammatory disease) or other condition requiring treatment.

[0037] As used herein, the term “toxicity” means any detrimental or harmful effect on a cell or tissue compared to the same cell or tissue before administration of the toxin.

[0038] As used herein, the terms “treat,” “treatment,” and “treating” mean reducing the amount or severity of a particular condition, disease (e.g., a microbial disease) or its symptoms in an object currently exhibiting or suffering from a condition or disease state. The term does not necessarily imply complete treatment (e.g., complete elimination of the condition, disease or its symptoms). “Treatment” encompasses any administration or application of a treatment or technique for a disease (in mammals, e.g., humans), including inhibiting the disease, preventing its onset, mitigating the disease, causing regression, or restoring or repairing lost, deficient, or incomplete function; or stimulating an inadequate process.

[0039] As used herein, the term "pathogenicity," as in "microbial pathogenicity," refers to the degree of damage (e.g., disease level) caused by a microorganism (e.g., bacteria) to its host (e.g., subject). In some embodiments, microbial pathogenicity relates to its intrinsic pathogenic factors. Bacterial pathogenic factors are typically proteins or other molecules that enable the bacteria to cause disease. For example, pathogenic factors may be adhesion proteins or toxins.

[0040] Detailed description of the invention Adhesion is the first step in microbial disease, and blocking adhesion has the potential to prevent infection. High mobility group box 1 (HMGB1) is a multifunctional protein produced by IEC and other human cells in response to detection by the innate immune response of microorganisms. Experiments described herein show that HMGB1 binds to a specific amino acid motif, the target of HMGB1 (ToH1), found in numerous bacterial, fungal, viral, and protozoan proteins. Many of these proteins are expressed on the surface of microorganisms and are associated with microbial pathogenicity and the pathophysiology of human and animal diseases. Hereinafter, it is shown that HMGB1 bound to ToH1 prevents microbial adhesion proteins from binding to their carbohydrate targets on mammalian host cells and properly fixing bacteria. Failure of this defense is likely to be associated with several diseases in which microorganisms adhere to host tissues, including infectious diarrhea, colorectal cancer, and IBD. Therefore, ToH1 provides a novel molecular target for the diagnosis and treatment of diseases. This specification provides anti-ToH1 antibodies, methods for producing anti-ToH1 antibodies, and methods for diagnosing and treating microbial diseases and / or chronic inflammatory diseases using the same.

[0041] I. Anti-ToH1 antibody In some embodiments, antibodies that bind to ToH1, also referred to herein as anti-ToH1 antibodies, are provided herein. In some embodiments, antibodies that bind to a target (ToH1) of HGMB1 are provided herein, and the antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the antibody comprises a VH containing three CDRs, HCDR1, HCDR2, and HCDR3, and a VL containing three CDRs, LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR1, HCDR2, and HCDR3 each have amino acid sequences having at least 80% sequence identity with GFIFSNYG (SEQ ID NO: 11), ISGYNGQT (SEQ ID NO: 12), and ARQSIPYYMDV (SEQ ID NO: 13), respectively (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity). LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 80% sequence identity with QSLVHSNGNTY (SEQ ID NO: 14), RISNRLSGVPDRFS (SEQ ID NO: 15), and MQAKQFPVT (SEQ ID NO: 16), respectively (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity).

[0042] In some embodiments, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 80% sequence identity to GYTFTGYY (SEQ ID NO: 17), INPNSGGT (SEQ ID NO: 18), and ARDRGSGATRYGMDV (SEQ ID NO: 19), respectively (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity). Furthermore, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 80% sequence identity with SSDIGNYNY (SEQ ID NO: 20), DVTKRPSGVSNRLSGSKSGNT (SEQ ID NO: 21), and SSYTGRSSWV (SEQ ID NO: 22), respectively (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity).

[0043] In some embodiments, HCDR1, HCDR2, and HCDR3 are amino acid sequences having at least 80% sequence identity with GDSVSSNSAA (SEQ ID NO: 23), TYYRSKWYN (SEQ ID NO: 24), and ARRSTWGTFDY (SEQ ID NO: 25), respectively (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity). LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 80% sequence identity with QSVLYSSNNKNY (SEQ ID NO: 26), WASTRESGVPDRFS (SEQ ID NO: 27), and QQYYALPLT (SEQ ID NO: 28), respectively (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity).

[0044] In some embodiments, the anti-ToH1 antibody is a heavy chain variable containing a sequence having at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%). The antibody comprises a main (VH) and a light chain variable domain (VL) containing a sequence having at least 80% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity). In some embodiments, the antibody comprises a heavy chain variable domain containing a sequence having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, and a light chain variable domain containing a sequence having at least 90% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In some embodiments, the antibody includes a heavy chain variable domain containing a sequence having at least 95% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, and a light chain variable domain containing a sequence having at least 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

[0045] In some embodiments, the anti-ToH1 antibody has at least 80% sequence identity to SEQ ID NO: 1 (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity). The sequence comprises a chain variable domain and a light chain variable domain having at least 80% sequence identity with SEQ ID NO: 2 (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity). In some embodiments, the anti-ToH1 antibody has at least 80% sequence identity with SEQ ID NO: 3 (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity). The molecule includes a chain variable domain and a light chain variable domain having at least 80% sequence identity with SEQ ID NO: 4 (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity).In some embodiments, the anti-ToH1 antibody has at least 80% sequence identity with SEQ ID NO: 5 (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) The molecule includes a chain variable domain and a light chain variable domain having at least 80% sequence identity with SEQ ID NO: 6 (for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity).

[0046] In some embodiments, the antibody includes a heavy chain variable domain containing SEQ ID NO: 1 and a light chain variable domain containing SEQ ID NO: 2. In some embodiments, the antibody includes a heavy chain variable domain containing SEQ ID NO: 3 and a light chain variable domain containing SEQ ID NO: 4. In some embodiments, the antibody includes a heavy chain variable domain containing SEQ ID NO: 5 and a light chain variable domain containing SEQ ID NO: 6.

[0047] In some embodiments, VL and VH are linked by a linker. In some embodiments, the linker comprises a series of repeating glycine residues. In some embodiments, the linker comprises GGGGSGGGGSGGGGSGGGGAS (SEQ ID NO: 10).

[0048] In some embodiments, the anti-ToH1 antibody is a nanobody. A nanobody refers to an antibody fragment derived from heavy-chain only IgG antibodies found in camelids. Heavy-chain IgG (hcIgG) antibodies do not contain the CH1 region but retain a variable heavy-chain domain called "VHH" in heavy-chain only antibodies. Therefore, Fab fragments derived from camel antibodies are also referred to herein as "VHH". H Also known as "H," "single-domain antibody," or "nanobody," it refers to a fragment of a heavy-chain-only antibody consisting of a variable domain (or recombinant variable domain) of a heavy-chain-only antibody.

[0049] In some embodiments, the anti-ToH1 antibody is [ka] These are nanobodies that have at least 80% sequence identity.

[0050] In some embodiments, the anti-ToH1 antibody is a nanobody having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 29.

[0051] In some embodiments, the anti-ToH1 antibody is [ka] These are nanobodies that have at least 80% sequence identity.

[0052] In some embodiments, the anti-ToH1 antibody is a nanobody having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 30.

[0053] II. Methods for Antibody Production In some embodiments, methods for producing anti-ToH1 antibodies, including the anti-ToH1 antibodies described above, are provided herein. In some embodiments, methods for producing anti-ToH1 antibodies are provided, comprising the steps of sequentially immunizing a host with two or more unique peptides and isolating antibodies produced in response to immunization. In some embodiments, each of the two or more unique peptides has a [S / T]xExPx[I / V] motif, where x is a variable amino acid, where x can be any suitable amino acid. In some embodiments, the set of immobilized amino acids in each peptide is unique. In some embodiments, the variable amino acids of each unique peptide are the same. In some embodiments, the variable amino acids of each unique peptide are not the same. In some embodiments, each peptide differs from one another only at a single immobilized amino acid position. In some embodiments, the two or more unique peptides include three unique peptides. In some embodiments, the two or more unique peptides include four unique peptides. In some embodiments, two or more unique peptides are independently selected from SxExPxI, SxExPxV, TxExPxI, and TxExPxV. In some embodiments, the host receives vaccination with three or more of the following: SxExPxI, SxExPxV, TxExPxI, and TxExPxV. In some embodiments, the host receives vaccination with each of the following: SxExPxI, SxExPxV, TxExPxI, and TxExPxV.

[0054] In some embodiments, two or more unique peptides are independently selected from SAENPKI (SEQ ID NO: 42), SPEKPTV (SEQ ID NO: 36), TAEDPRI (SEQ ID NO: 41), and TSETPRV (SEQ ID NO: 33). In some embodiments, the host is immunized with three or more of SAENPKI (SEQ ID NO: 42), SPEKPTV (SEQ ID NO: 36), TAEDPRI (SEQ ID NO: 41), and TSETPRV (SEQ ID NO: 33). In some embodiments, the host is immunized with each of SAENPKI (SEQ ID NO: 42), SPEKPTV (SEQ ID NO: 36), TAEDPRI (SEQ ID NO: 41), and TSETPRV (SEQ ID NO: 33). The host may be immunized with two or more unique peptides in any appropriate order, with any appropriate interval between each immunization. In some embodiments, the host is immunized with one unique peptide on a predetermined day. In some embodiments, the host is immunized with more than one unique peptide on a predetermined day.

[0055] In some embodiments, the host is a genetically modified animal. In some embodiments, the host is a cell. In some embodiments, the host is a mammalian cell.

[0056] III. Diagnosis and Treatment Methods In some embodiments, anti-ToH1 antibodies, including the anti-ToH1 antibodies described herein, are used in the diagnosis, treatment, and / or prevention of microbial diseases and / or chronic inflammatory diseases. In some embodiments, the chronic inflammatory disease is a microbial-associated chronic inflammatory disease. In some embodiments, anti-ToH1 antibodies are used for the diagnosis and / or treatment of diseases caused or exacerbated by toxic microorganisms. In some embodiments, anti-ToH1 antibodies are used to treat or prevent microbial pathogenicity.

[0057] In some embodiments, methods for diagnosing microbial diseases and / or chronic inflammatory diseases in subjects are provided herein. In some embodiments, the method for diagnosing microbial diseases and / or chronic inflammatory diseases in subjects includes the steps of determining the level of HGMB1 target (ToH1) in a sample obtained from the subject, and determining that the subject has a microbial disease if the level of ToH1 in the sample is equal to or higher than a threshold. In some embodiments, the level of ToH1 in the sample is determined by performing an immunoassay (e.g., ELISA). In some embodiments, the step of determining the level of ToH1 in the sample includes contacting the sample obtained from the subject with an antibody that binds to HGMB1 target (ToH1), including an anti-ToH1 antibody as described herein, and detecting the antibody in the sample. In some embodiments, the anti-ToH1 antibody is labeled with a fluorescent label or tag, and the label can be detected by an appropriate assay to determine the level or amount of ToH1 in the sample. In some embodiments, the threshold is determined based on the level of ToH1 observed in a sample obtained from a subject that does not have a microbial infection. In some embodiments, the method further includes administering an appropriate treatment to a subject if the level of ToH1 in the sample indicates a microbial infection in the subject. In some embodiments, the treatment includes an anti-ToH1 antibody. In some embodiments, the treatment includes an antimicrobial agent such as an antibiotic, antiviral agent, antifungal agent, anti-inflammatory agent, or a combination thereof.

[0058] In some embodiments, methods are provided herein for treating or preventing microbial diseases and / or chronic inflammatory diseases in subjects, the methods comprising the step of providing subjects with antibodies that bind to the target (ToH1) of HGMB1. In some embodiments, subjects are determined to have a microbial disease by performing a diagnostic method provided herein, which comprises the steps of contacting a sample obtained from the subject with an anti-ToH1 antibody and measuring the antibody in the sample.

[0059] In some embodiments, the anti-ToH1 antibody is conjugated to a therapeutic agent (e.g., an antimicrobial agent, an anti-inflammatory agent, or other drug). In some embodiments, the anti-ToH1 antibody is administered in combination with a therapeutic agent (e.g., an antimicrobial agent, an anti-inflammatory agent, or other drug). In some embodiments, the anti-ToH1 antibody is formulated as a composition (e.g., a pharmaceutical composition) comprising the anti-ToH1 antibody and a suitable carrier (e.g., a pharmaceutically acceptable carrier). The antibody, or a composition containing the antibody, can be administered to a target by any suitable route. Administration may be topical (including mucosal delivery, including the eye, vagina, and rectum), pulmonary (e.g., inhalation or gas injection of powder or aerosol, including by nebulizer; intratracheal, intranasal, epithelial, and transdermal), oral, or parenteral. Parenteral administration may include intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration. In some embodiments, the antibody, or a composition containing the antibody, is administered parenterally (e.g., subcutaneous, intravenous).

[0060] The pharmaceutical compositions of the present invention may be provided in simple unit dosage forms, but may also be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of associating the active ingredient with a pharmaceutical carrier or excipient. Generally, formulations are prepared by uniformly and closely associating the active ingredient with a liquid carrier, a finely ground solid carrier, or both, and, if necessary, then shaping the product.

[0061] The compositions of the present invention may further contain other auxiliary elements conventionally found in pharmaceutical compositions. Therefore, for example, the compositions may contain further compatible and pharmaceutically active materials such as antipruritics, astringents, topical anesthetics, or other anti-inflammatory agents, or further materials useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials, when added, should not excessively interfere with the biological activity of the elements of the compositions of the present invention. The formulations may be stabilized and, if desired, mixed with auxiliary agents that do not adversely interact with the active ingredients of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0062] Medication depends on the severity and responsiveness of the disease state or condition being treated, over a course of treatment lasting several days to several months, or until a cure is achieved or a reduction in the disease state is achieved. In some embodiments, the treatment is administered in one or more courses, each course containing one or more doses per day over several days (e.g., 1, 2, 3, 4, 5, 6 days) or several weeks (e.g., 1, 2, or 3 weeks). In some embodiments, the courses of treatment are administered continuously (e.g., without interruption between courses), while in other embodiments, there are interruptions of one day or more, one week or more, or one month or more between courses. In some embodiments, the treatment is provided continuously or maintenanceally (e.g., multiple courses with or without interruption of indeterminate periods). The optimal medication schedule can be calculated from measurements of drug accumulation in the patient's body. The administering physician can easily determine the optimal dosage, method of administration, and frequency of repetition.

[0063] In some embodiments, the dosage is 0.01 μg to 100 g per kg of body weight and may be administered once or more times a day, week, month, or year. The treating physician can estimate the frequency of repeated administrations based on the measured residence time and concentration of the drug in body fluids or tissues.

[0064] In some embodiments, microbial diseases (e.g., diseases diagnosed, treated or prevented in a subject) include microbial infections. Microbial infections can be caused by any microorganism, including bacterial infections, viral infections, fungal infections and / or protist infections. In some embodiments, microbial infection is a bacterial infection. Non-limiting examples of exemplary infections include gastrointestinal infections (cholera, salmonellosis, Clostridium difficile infection, listeriosis), sexually transmitted infections (chlamydia, syphilis), meningococcal diseases, dermatological infections (staphylococcal infections), and pulmonary infections (pertussis, pneumonia).

[0065] In some embodiments, the chronic inflammatory disease is a microorganism-associated chronic inflammatory disease. In some embodiments, the chronic inflammatory disease is inflammatory bowel disease, rheumatoid arthritis, non-alcoholic fatty liver disease, type II diabetes mellitus, urinary tract infection, pneumonia, or sepsis. In some embodiments, the chronic inflammatory disease is inflammatory bowel disease. The term "inflammatory bowel disease" refers to disorders involving chronic inflammation of the tissues of the gastrointestinal tract, including Crohn's disease and ulcerative colitis (UC).

[0066] In some embodiments, the subject is a vertebrate. In some embodiments, the subject is a bird (e.g., poultry such as chickens), a reptile, or a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has a HMGB1 deficiency. [Examples]

[0067] The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting its scope.

[0068] [Example 1] method mouse All mice used in this study had a C57BL / 6 genetic background. Specific pathogen-free (SPF) mice were purchased from Jackson Labs. Germ-free (GF) mice were maintained under pure isolation conditions before euthanasia. Hmgb1fl / fl (WT) and Hmgb1fl / fl, Vil-CRE (ΔIEC) mice were created. Mice were maintained under SPF conditions, with the exception of GF mice. All animal experiments were performed at least twice, and in most cases, three times independently using mice that were matched for both age and sex. Lactate controls were used where possible. Mice were between 6 and 12 weeks of age, and both sexes were used for all experiments. Mice were housed in a 12-hour light / dark cycle and fed a standard diet.

[0069] bacterial culture All Escherichia coli strains were cultured under conditions that induced pili. Unless otherwise indicated, strains were passaged twice in nutrient broth at 37°C under static conditions, or once on nutrient agar plates incubated overnight at 37°C. The medium consisted of 0.5% Bacto peptone, 0.3% beef extract, 0.5% NaCl and 1.5% agar (if necessary), pH 6.8. The full-length BW25113 mutant construct FimH was PCR amplified from BW25113 and cloned into the pGex6-1 vector using restriction digestion cloning and the BamHI and XhoI restriction sites. Mutations in the ToH1 sequence in FimH were performed using two rounds of PCR. Mutations in the desired region were introduced in the first round of PCR. Two fragments were generated using primer pairs 1) FimH-For and FimH-Mut1 and 2) FimH-Rev and FimH-Mut2. These two fragments were gel-purified, combined, and used as templates for a second round of PCR. FimH-For and FimH-Rev were used in the second round of PCR. The products were gel-purified, digested with BamHI and XhoI, and cloned into pGex6-1 vectors digested with BamHI and XhoI. Wild-type and mutant plasmids were confirmed by the ability of transformed cells to grow on a selection antibiotic and by Sanger assay. Escherichia coli with FimH knocked out (ΔFimH) was transformed with either a plasmid containing wild-type FimH (ΔFimHWT) or a plasmid with a ToH1 mutation (ΔFimHMut). See Table 1 for oligonucleotide information.

[0070] [Table 1] JPEG2026521446000005.jpg40150

[0071] SWW33 Mutation Construct A homologous recombination arm of approximately 1 kb adjacent to the mutated / deleted sequence (FimH) was PCR-amplified from WT SWW33 genomic DNA. These two arms were then fused to the mutated sequence either by direct fusion or PCR. Using the TAKARA fusion kit, the PCR-generated fragments were ligated into a suicide plasmid containing the kanamycin-resistant cassette, R6K origin, RP4 mob, and sacB gene. This suicide plasmid was transformed into Escherichia coli S17-1λpir via electroporation, and then transformed into Escherichia coli SWW33 via conjugation. Successful conjugated organisms were obtained by plating the conjugation mixture onto LB agar plates supplemented with kanamycin and ampicillin. The transformed SWW33 strain possessed the ampicillin-resistant plasmid prior to conjugation. Next, the purified conjugated mutants were plated onto LSW61 agar plates supplemented with 10% sucrose to select successful knock-in mutants. These were then purified and confirmed by PCR, Sanger assay, and the ability to grow in the presence of ampicillin but not kanamycin. The composition of the LSW agar plate (per liter) was 10 g tryptone, 5 g yeast extract, 5 mL glycerol, 0.4 g NaCl, and 20 g agar. See the expanded Table 1 for oligonucleotide information.

[0072] immunostaining Mouse colon tissue was obtained from mice aged 8–12 weeks. Human colon samples were residual tissue from colectomies performed for clinical care of the disease (obtained under existing IRB conditions). Samples from mice without a history of inflammatory bowel disease were used as controls. All samples were fixed in methyl-Carnoy fixative (60% methanol, 30% chloroform, 10% glacial acetic acid) at 4°C for 3 hours, then transferred to 70% ethanol. Paraffin-embedded sections were deparaffinized in water, and the slides were subjected to antigen retrieval using a steamer in 10 mM sodium citrate, 0.05% Tween 20 pH 6.0 for 20 minutes, followed by cooling for 1 hour. These were blocked in serum-free protein blocks (Agilent, Dako, X0909) and stained with either Ulex Europaeus Agglutinin I (UEA I) conjugated with 10 mg / mL Dylight 594, 0.17 mg / mL anti-HMGB1 (Abcam), 0.874 mg / mL anti-FimH polyclonal antibody (custom antibody produced by Genscript), or 0.96 mg / mL anti-Muc2 (Novus). 1 mg / mL Alexa Fluor 647 donkey anti-rabbit IgG was used as a secondary, if necessary. The signal of the anti-FimH polyclonal antibody (custom antibody produced by Genscript) was amplified using Tyramide SuperBoost (Thermo Fisher Scientific). Slides were counterstained with 10 mg / mL bisbenzimide H33258 dissolved in TBS for 20 minutes in the dark at room temperature, and then covered with coverslips. For peptide inhibition staining, 0.85 mg / mL of human HMGB1 peptide (Abcam) or 9 ug / mL of full-length FimH protein (antigen for custom antibody generation, Genscript) was used. All immunofluorescence sections were analyzed using either a wide-field fluorescence microscope (Keyence BZ-800) or an inverted confocal microscope (Leica TCS-SP8-AOBS inverted microscope (Leica Microsystems, GmbH, Wetzlar, Germany) with a 40x / 1.25 oil objective lens).

[0073] HMGB1 Enzyme-Linked Immunoassay Samples used to measure HMGB1 protein levels in feces and colonic mucus were homogenized in cell lysis buffer (Cell Signaling) containing the cOmplete protease inhibitor (Roche) and 100 mM PMSF. After centrifugation at maximum speed for 15 minutes, the supernatant was collected and assayed for protein concentration via BCA (Thermo Scientific). Samples were analyzed using an HMGB1 detection kit according to the manufacturer's instructions for use (Chondrex).

[0074] Immunoblot analysis Bacterial and mucus samples were boiled in Remley buffer containing 10% β-mercaptoethanol. All samples were separated using NuPage Bis-Tris gel with MOPS or MES running buffer. Proteins were transferred to a PVDF membrane and probed overnight in 5% w / v skim milk powder Omniblock in 0.1% PBST or LI-COR Intercept PBS blocking buffer at 4°C with FimH (Sokurenko Lab), GAPDH (Cell Signaling), 0.17 mg / mL anti-HMGB1 (Abcam), 0.6 mg / mL mouse anti-FimH (Sokurenko (mouse sample) or custom antibody generated by Genscript (bacterial sample)), 0.1 mg / mL anti-GroEL (Enzo), or 680-labeled streptavidin. After incubation and washing, the samples were probed with 0.08 mg / mL HRP conjugate anti-mouse or anti-rabbit, 0.2 ug / mL 680RD streptavidin (LICOR), or 0.2 ug / mL 800RD (LI-COR) anti-mouse secondary antibody was added to the blot and incubated for 1 hour. After washing, the chemiluminescence signal was captured either by film or by using Bio-Rad ChemiDoc MP, and the fluorescence signal was detected using the LI-COR Odyssey CLX machine and LI-COR acquisition software.

[0075] Fluorescence in situ hybridization (FISH) for detecting bacteria Paraffin-embedded sections were deparaffinized in water. A permeabilization solution (5 mg / mL lysozyme, 0.05 M EDTA, 0.1 M Tris pH 7.4, PBS) was applied to the tissue and incubated at 37°C for 20 minutes, followed by washing with PBS. The slides were incubated in a hybridization oven at 46°C for 1 hour in a hybridization solution (0.9 M NaCl, 0.02 M Tris pH 7.4, 0.005 M EDTA, 1% v / v Triton X-100, 35% deionized formamide, 0.1% w / v BSA, water; preheated to 46°C). A universal bacterial probe (EUB338 modified with 5'ATTO 647N dye) was denatured for 2 minutes in a hybridization solution heated to 95°C. The slides were returned to a hybridization oven covered with 0.1 M probe in the hybridization solution at 46°C for 2.5 hours. Slides were washed three times in Stringency Wash Buffer 1 (2x SSC (preheated to 46°C, 300mM NaCl, 30mM sodium citrate, pH 7.0)). A secondary wash was performed three times at room temperature in Stringency Wash Buffer 2 (0.1x SSC (15mM NaCl, 1.5mM sodium citrate, pH 7.0)), followed by rinsing in PBS. Tissue was counterstained with 10 mg / mL bisbenzimide H33258 dissolved in TBS at room temperature in the dark for 20 minutes, and coverslips were placed over the tissues. The distance between bacteria and epithelium was determined using ImageJ analysis software.

[0076] Bacterial flow cytometry HMGB1 binding to Escherichia coli A bacterial preparation of 3.2 x 10⁸ (OD 0.4) Escherichia coli was prepared from an overnight agar culture and incubated at 37°C for 2 hours with 3 mM HMGB1 recombinant protein reconstituted in 1 mM DTT, 1 mM EDTA, and PBS buffer, containing either an HIS tag (Abclonal) or an hFc tag (Sino Biological). Cells were fixed with 4% PFA and blocked with 1% BSA in PBST for 1 hour. Unconjugated anti-HIS antibody (Genscript) (5.0 mg / mL) was added and incubated overnight at 4°C. The following day, cells were washed at room temperature for 1 hour in the added PBS and secondary antibody (0.25 mg / test PE-Cy7). Samples incubated with hFc-tagged HMGB1 were incubated at room temperature for 1 hour with PE anti-human IgG Fc recombinant antibody (0.25 mg / test). Finally, cells were stained with 1 mM SYTO9. Bacterial suspensions were analyzed using an Attune NxT flow cytometer (Invitrogen) along with FlowJo software (Tree Star).

[0077] HMGB1 binding to ToH1 A bacterial preparation of 3.2 x 10⁸ (OD 0.4) Escherichia coli was prepared from an overnight agar culture and incubated for 2 hours with 3 mM HMGB1 (R&D systems) diluted with protein block buffer (Dako). The bacteria were centrifuged, washed three times with PBS, fixed with 4% PFA for 10 minutes, and washed three more times with PBS. The samples were incubated overnight at 4°C with anti-FimH (Sokurenko) or rabbit anti-HMGB1 (Abcam). The samples were washed and 1:1500 Invitrogen Alexa a10040 was added. The samples were analyzed using Amnis (Luminex) Image Stream Mark II.

[0078] Intestinal epithelial cell adhesion assay Caco2BBe1 cells were seeded at a rate of 250,000 cells per well in 0.4 μm inserts of 6.5 mm Transwell (Corning) to form a polar monolayer membrane. 72 hours prior to the adhesion assay, 100 ug / mL kifunesin (Cayman Chemicals) was added to 1x DMEM + 10% FBS. Bacterial preparations grown from overnight vegetative agar cultures (1.6 x 10⁸ (OD 0.2) Escherichia coli individuals per well) were suspended in serum-free 1x DMEM and incubated at room temperature for 1 hour with 3 mM HMGB1 (R&D), 100 mM mannose (Sigma-Aldrich), or a buffer vehicle (1 mM DTT, 1 mM EDTA, PBS). The medium was removed from the apical end of confluent Caco2BBe1 cells, and the cells were washed three times with serum-free 1x DMEM. The wells were treated, and the cells were incubated at 37°C for 1 hour. The cells were washed three times by adding serum-free 1x DMEM and shaking the plate at 100 RPM for 1 minute between washes. The cells were removed by adding 0.1% Triton X-100 in PBS, incubated at room temperature for 5 minutes, then scraped and collected. Serial dilutions were performed and plated onto LB agar plates, incubated overnight at 37°C, and counted the following day. Serial dilutions of the added Escherichia coli were also plated and used to normalize the collected Escherichia coli.

[0079] qRT-PCR RNA isolation was performed from colonic mucosal swabs or bacterial lysates using TRIzol (Life Technologies) following a standard extraction procedure. Gene expression was determined using SYBR Green Master Mix (Bio-Rad) and the Roche LightCycler 480 System. See Table 1 for oligonucleotide information.

[0080] qPCR DNA isolation was performed from mucosal swabs or bacterial lysates using the DNeasy Powerlyzer Microbial kit according to the manufacturer's instructions. Quantitative PCR was performed using PowerUP SYBR Green Master Mix (Applied Biosystems) on a CFX96 Touch real-time PCR system. See Table 1 for oligonucleotide information.

[0081] red blood cell agglutination As previously described, a hemagglutination assay was performed. Briefly, serial dilutions of Escherichia coli were incubated with a 3% solution of washed red blood cells in a round-bottom 96-well plate. The wells were mixed by shaking the plate. The plate was incubated at room temperature and imaged 1 hour after shaking. For the HMGB1 inhibition assay, the minimum bacterial count required to achieve agglutination was used. For the inhibition assay, Escherichia coli was incubated with serial dilutions of HMGB1 recombinant protein (R&D systems) reconstituted in 1 mM DTT, 1 mM EDTA, and PBS buffer, and a 3% solution of washed red blood cells. The wells were mixed by shaking the plate. The plate was incubated at room temperature and imaged 1 hour after shaking.

[0082] Bacterial agglutination assay Escherichia coli aggregate Prior to experimental treatment, bacteria expressing green fluorescent protein (GFP) were allowed to grow statically by subculturing for two nights under antibiotic selection. The culture from the last night was adjusted to an OD of 0.4, and 100 mL was centrifuged at 5000 RPM for 5 minutes to form a pellet. The supernatant was discarded, and the cells were resuspended in TBS containing 10 mM CaCl2. The bacteria were treated with 3 mM recombinant HMGB1 (R&D systems) or buffer in a 50 mL reaction at 37°C for 2 hours. The cells were carefully distributed onto coverslips, covered with 0.15% agarose pads, and imaged using a fluorescence microscope. After fixation with 4% PFA, the remaining bacterial samples were analyzed using LSRFortessa (BD) with FlowJo software (Tree Star).

[0083] Colon community microbiota aggregation Colons from SPF C57BL / 6J mice were resected and the fillets were opened. Using an inoculation loop, the contents were scraped into a 1.5 mL centrifuge tube containing 1 mL of PBS. After centrifugation at 400 G for 5 minutes, the supernatant was stained using a 70 mm cell strainer. The pass-through fraction was washed twice with PBS and once with TBS containing 10 mM CaCl2 at 10,000 x G for 2 minutes. After the third wash, the sample was resuspended in 500 mL TBS / CaCl2. The sample was divided into 100 μL aliquots and centrifuged at 10,000 x G for 2 minutes. rHMGB1 (R&D systems) was fluorescently labeled with the AlexaFluor 647 labeling kit according to the manufacturer's protocol. The bacteria were resuspended in a buffer containing 3 mM labeled rHMGB1 or 1 mM SYTO 9 and incubated at 37 °C for 2 hours. The cells were carefully distributed onto coverslips, covered with a 0.15% agarose pad, and imaged under fluorescence.

[0084] Recombinant FimH lectin domain generation Recombinant FimH lectin domain protein was generated in HM125 Escherichia coli using a plasmid. The K12 FimH lectin domain was purified as previously described. FimH conjugated to mannose FimHLD (40 ug / mL) was incubated at room temperature for 1 hour with serial dilutions of HMGB1 recombinant protein (R&D systems). A mannose-coated 96-well plate was washed with 200 uL of 10 mM Tris + 0.05% tween pH 8.0, and then the sample was applied to the plate. The plate was sealed and incubated overnight at 4°C. The plate was washed four times with PBST (0.1%) and blocked at room temperature for 1 hour with 1% BSA in PBST (0.1%). The plate was then washed four times with PBST (0.1%), and the primary anti-HIS antibody was diluted to a concentration of 5 ug / mL in 0.1% BSA in PBST and added to the plate. This was incubated at room temperature for 1 hour. Next, the plate was washed four times with PBST (0.1%), and the secondary anti-mouse HRP antibody was added to the plate at a dilution of 0.16 ug / mL in 0.1% BSA in PBST. The plate was incubated at room temperature for 30 minutes. The plate was washed four times with PBST (0.1%), and TMB solution was added to the wells. The reaction was stopped with 0.5 M sulfuric acid, and the readings were obtained at 450 nm using a spectrophotometer.

[0085] Death of Escherichia cory by HMGB1 A bacterial preparation of 6.4 x 10⁸ (OD 0.8) Escherichia coli was prepared from a vegetative broth culture that had been passaged twice, and incubated with 3 μM HMGB1 recombinant protein (R&D systems) reconstituted in 1 mM DTT, 1 mM EDTA, and PBS buffer at 37°C for 2 hours. After incubation, the cells were serially diluted and plated on LB agar plates, and colonies were counted the day after incubation at 37°C.

[0086] FimH expression in Escherichia coli exposed to HMGB1 A bacterial preparation of 3.2 × 10⁸ (OD 0.4) Escherichia coli was prepared from an overnight agar culture and incubated at 37°C for 2 hours with 3 μM HMGB1 recombinant protein (R&D systems) reconstituted in 1 mM DTT, 1 mM EDTA, and PBS buffer. RNA was isolated using TRIzol (Life Technologies) according to the manufacturer's instructions. After two nights of passaging with shaking at 255 rmp in broth, an Escherichia coli preparation of 6.4 × 10⁸ cells / mL (OD 0.8) was prepared for a FIM-switched PCR assay. The bacteria were treated by standing at 37°C for 18 hours in a 1 / 100 dilution of conditioned organoid medium with or without HMGB1 (WT) or without (ΔIEC). Similar results were shown in pilot assays using media derived from small and large intestine organoids, and since the media contained few additives, additional assays were performed using small intestine organoids. Prior to passage, the conditioned organoid medium collected from the small intestinal organoids consisted of Advanced DMEM / F12 supplemented with 1x L-glutamine (Life Technologies), 10 mM HEPES buffer (Life Technologies), 1x penicillin and streptomycin (Life Technologies), 1x N2 supplement (Life Technologies), 1x B-27 Supplement Minus Vitamin A (Life Technologies), 50 ng / mL mouse epidermal growth factor (Peprotech), 100 ng / mL Noggin (Peprotech), 1 μM Jagged 1 (Anaspec), 10 nM Y-27632 (Cayman Chemical Company), and 100 ng / mL R-spongin 1 (Peprotech). This was concentrated using a 3 kDa MWCO filter insert with PBS as a diluent. Bacterial DNA was extracted using the DNeasy PowerLyzer Microbial kit according to the manufacturer's protocol. PCR amplification was performed as previously described.ImageJ software was used to normalize the band intensities of phase-ON and phase-OFF fmS PCR products relative to the fsZ amplified product. See Table 1 for oligonucleotide information.

[0087] Quantification of FimH-positive bacteria in IF images Image-Pro Plus 7.0 was used for image manipulation and measurement. Images were spatially corrected to micrometers and processed as intensity values. Background removal using the same criteria as the secondary antibody negative control was applied across the entire dataset. Semi-automatic counting was performed by manually setting the intensity range that overlapped with the image. Subjects and measurements were collected across five image fields per sample. Measurements for each subject were collected, classified, and described post-hoc. Two independent groups of people manually applied intensity ranges that had little deviation and produced similar results. The five image counts were averaged to express each sample in units of subjects per field. To measure the fluorescence intensity in images collected on a Leica TCS-SP8-AOBS inverted confocal microscope, surface-associated HMGB1 quantification in the IF Image Leica Application Suite (version 3.7.5 or higher) was used. The region of interest was plotted around representative surface epithelium and the maximized region. Average fluorescence values ​​(RFU / um^2) were collected for each image. In background removal from the secondary antibody, only the negative control was applied across the entire dataset. Each sample was represented using the mean of five imaging values. Two independent groups of individuals identified a representative region of interest (surface epithelium) and collected fluorescence values ​​for similar results.

[0088] Mouse mucus isolation As previously described, mucus isolation was performed. Mouse-derived mucosal scraped colon was resected, the fillet was opened, and the contents were washed away. 1 mL of PBS was added to the colon tissue in a 1.5 mL centrifuge tube and vortexed vigorously for 2 minutes. The tissue was removed, and the mucus-containing mixture was centrifuged at maximum speed for 5 minutes. The translucent mucus layer on top of the dark pellet was removed and resuspended in 1 mL of PBS. 20 mL was set aside for BCA. The remainder was centrifuged again, and the mucus-containing pellet was solubilized in Remley buffer for immunoblotting with sample buffer for ELISA.

[0089] Mucus penetration assay GFP-expressing bacteria were grown in broth overnight for two passages, allowing to stand to prepare a preparation of 8 × 10⁸ cells / mL (OD1.0). These Escherichia coli preparations were used to fill the middle channel of a chemotaxis μ-Slide (Ibidi). Mucus isolated from WT and DIEC was added to the reservoir on the opposite side of the same chamber and incubated at 37°C for 1 hour. Five representative images of fluorescent bacteria invading the mucus sample were captured along the proximal end of the main mucosa of the middle channel of the chamber. These assays were performed at least three times with different bacterial preparations. All image processing settings to reduce background signal were applied across the entire dataset. Percentage of relative fluorescence in the mucus was measured using ImageJ analysis software. After spatially calibrating the image set units to known micrometers, the region of interest was applied in each image, at the beginning of the main mucosal edge, along its entire length, and at a depth of approximately 300 mm; totaling approximately 170,000 μm2; or in the largest area common to all images. Integrated density values ​​were collected and divided by the area of ​​the region of interest (ROI) to account for possible variance when redrawing the ROI. These average fluorescence values ​​were averaged across five images and used to represent each sample in the chamber slide. Assuming that bacteria have an equal probability of migrating in either well, the average fluorescence of each well was compared to the other as the percentage of the total fluorescence in the reaction chamber. Two independent researchers identified the region of interest and collected fluorescence values ​​with similar results.

[0090] Sulfo-SBED label migration As previously described, a label transfer reaction was performed. Briefly, rHMGB1 (R&D Biosystems) was treated as a "bait protein" and reconstituted to 0.2 mg / mL in sterile PBS. Sulfo-SBED was calculated and added in a 3 molar excess of purified HMGB1. Sulfo-SBED was dissolved in DMSO to 10 mg / mL and added to rHMGB1. The reaction mixture was incubated at room temperature for 1 hour, protected from light. The labeled protein was then added to a desalting column (Thermo Fisher No. 89849) equilibrated with 50 mM HEPES, 150 mM NaCl; pH 7.3 to remove excess crosslinker, leaving the labeled purified HMGB1 in the appropriate reaction buffer. Both sulfo-SBED-labeled HMGB1 and unlabeled FimHLD were incubated at room temperature for 2 hours, protected from light to ensure interaction. The molar ratio of FimH:HMGB1 used was 2.2 mM FimH:4 mM HMGB1, and the reaction was carried out in 50 ml of water. To crosslink the sample, the reactant was transferred to a clear 96-well polypropylene low-binding plate placed 5 cm from a UV light source (Stratalinker) and exposed to 538 nm wavelength on ice for 8 minutes. The crosslinked reactant was transferred to a new tube to which DTT sample buffer was added to a final DTT concentration of 100 mM to complete the disulfide bond reduction and transfer the biotin label. The sample was heated to 70°C for 10 minutes and analyzed by immunoblotting.

[0091] Microbiome research Microbiome sampling: Littermates of both sexes and genotypes were separated into genotype-based groups at weaning. Soiled bedding from both genotypes of mice was collected, mixed, and distributed throughout the cages at the time of separation. At 20 weeks of age, fresh fecal samples were collected, and the mice were sacrificed for mucosal swab collection. Samples were submitted to the Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory for 16S rRNA analysis.

[0092] Bioinformatics: Individual fastq files without non-biological nucleotides were processed using the Divisive Amplicon Denoising Algorithm (DADA) pipeline. The output of the dada2 pipeline (a table of amplicon sequence variant features (ASV table)) was processed for α and β diversity analysis using the phyloseq and microbimeSeq (http: / / www.github.com / umerijaz / microbiomeSeq) packages in R. Alpha diversity estimates were measured within group categories using the estimate richness function of the phyloseq package. Multidimensional scaling (MDS, i.e., PCoA) was performed between groups using the Bray-Curtis dissimilarity matrix and visualized using the ggplot2 package. Where necessary, the inventors adjusted for multiple comparisons using the BH FDR method, while performing multiple tests for taxonomic abundance between groups. Analysis of variances between groups for α diversity was performed. Multivariate analysis of variance (PERMANOVA) for sorting was performed on all principal coordinates obtained during PCoA. Linear regression (parametric) and Wilcoxon (nonparametric) were performed using R-based functions for ASV abundance versus metadata variability levels (e.g., dietary composition).

[0093] result HMGB1 is released into colonic mucus in response to the gut microbiota. HMGB1 is highly concentrated in the luminal side of the mouse colon, closely associated with the epithelium in the mucus layer (Figure 1a, Figure 5). In littermate ΔIEC mice, HMGB1 is absent in the IEC body and greatly reduced in the mucus, suggesting that IEC is the primary source of HMGB1 in colonic mucus (Figure 1b, c). HMGB1 protein is similarly detectable in IEC from germ-free (GF) C57BL / 6 mice, and its levels appear to be only moderately reduced compared to specific pathogen-free (SPF) C57BL / 6 mice (Figure 1d, e). However, since HMGB1 staining was absent on the colonic surface and HMGB1 protein could not be detected in the feces of GF mice, the presence of HMGB1 in the intestinal lumen was microbiome-dependent (Figure 1d, f). Since GF mice produce little colonic mucus, HMGB1 was assayed in fecal samples. In summary, these data suggest that HMGB1 is released from the IEC into colonic mucus in response to the gut microbiota.

[0094] HMGB1 prevents bacteria from entering the mucus layer on the inside of the colon. The presence of HMGB1 in the intestinal lumen suggests that it may affect the gut microbiota; therefore, the effects of HMGB1 on the composition and behavior of the gut bacterial community were next evaluated. A normal colon boasts a diverse and abundant microbial community physically separated from the epithelial surface by an inner mucus barrier. In mice lacking mucosal HMGB1, this physical separation is essentially lost, and the microbial community becomes extremely close to the host tissue, along with an increase in host tissue-associated bacterial DNA (Figure 2a, b, c). The change in microbial biogeography was not due to the decrease in mucus production in ΔIEC mice. In the intestinal lumen, HMGB1 labels microorganisms, and by this, we evaluated whether HMGB1 directly affects the microbiota (Figure 5). The mucosa-associated microbial community did not differ between WT and ΔIEC mice (Figure 5b, c). In particular, the taxonomic distinctions normally present between feces and mucosal-associated bacteria were reduced in ΔIEC mice, strengthening the idea that HMGB1 is a factor in the exclusion of the microbiome from the inner mucus layer (Figure 5D). However, taxonomic differences in mucosal-associated bacteria between mouse genotypes were most recognized at the strain level, suggesting that HMGB1 does not exert strong selective pressure on intestinal bacteria (Figure 5E). Loss of HMGB1 may allow normally symbiotic microorganisms to invade the colonic mucus. Mucus is thought to act primarily as a physical antimicrobial barrier in the colon. Mucin 2 (Muc2), the most abundant protein in intestinal mucus, is a large, heavily glycosylated protein that oligomerizes to form a dense network structure that blocks bacterial movement. Furthermore, the oligosaccharides bound to Muc2 are the same oligosaccharides to which bacterial adhesins bind on the surface of host cells, and therefore they act as decoy adhesin sites, inhibiting movement through the mucus. Microbial invasion into mucus was limited when HMGB1 was present in the mucus (Figure 2d, e). Escherichia coli was selected for these studies because it is a well-characterized gut symbiont associated with colitis in animal models and in human inflammatory bowel disease (IBD) patients. Exposure to HMGB1 also caused aggregation of Escherichia coli and the complex microflora (Figure 2f, g, h).These findings suggest that HMGB1 traps bacteria invading colonic mucus, blocking them from reaching their adhesion targets. Microorganisms that come into contact with HMGB1 aggregate, preventing their movement through the mucus and their interaction with the colonic epithelial surface.

[0095] HMGB1 binds to and inactivates the bacterial adhesin FimH through an evolutionarily conserved amino acid sequence. Observations that HMGB1 directly binds to gut microbiota in vivo and in vitro led to the hypothesis that HMGB1 targets one or more proteins expressed on the surface of bacteria. In previous studies, amino acid sequences common among mammalian protein beclin-1 identified autophagy protein 5 (Atg5) targeted by cytoplasmic HMGB1 during microbial stress (Zhu / Messer, JCI, 2015). Searching the PROSITE database using motifs derived from this sequence yielded numerous hits among known or predicted bacterial adhesins, including FimH, a component of type 1 pili (T1F) adhesion, and perhaps the most characterized bacterial adhesin (Figure 3a). FimH is a phase-inducible pathogenic factor carried by Enterobacteriaceae, including Escherichia coli, and has been shown to be associated with infectious diarrhea, urinary tract infections, extraintestinal infections, colorectal cancer, and inflammatory bowel disease. FimH expression in Escherichia coli is low in the symbiotic state, but is highly expressed by pathogenic Escherichia coli strains or when symbiotic strains become pathogenic. HMGB1 interaction with the conserved amino acid motif target (ToH1) of HMGB1 was confirmed in FimH expressed by Escherichia coli. Recombinant human HMGB1 (rHMGB1) bound to Escherichia coli expressing WT FimH, but the number of HMGB1-positive bacteria and the amount of HMGB1 bound to each individual bacterium were significantly lower when cells lacked FimH (ΔFimH) or expressed FimH with a mutated ToH1 (ΔFimHMUT) (Figure 3b, c, Figure 6a-6d). A label transfer assay was then employed to obtain data on direct protein-protein interactions between HMGB1 and the IEC-binding lectin domain (FimHLD) of FimH. The label migrated to WT rFimHLD, but not to rFimHLD with mutated ToH1, indicating that HMGB1 binds to FimH via ToH1 (Figure 3d).The presence of mannose, a ligand bound by FimH for T1F adhesion, did not alter the efficiency of label transfer from rHMGB1 to WT rFimH. This, along with publicly available structural data showing that ToH1 is distinct from the mannose binding site in FimH, indicates that HMGB1 does not compete with mannose for binding to FimH. Autotransfer of the label from rHMGB1 to itself has also been observed in the WT rFimHLD reaction, supporting the formation of oligomers, most likely dimers, when HMGB1 interacts with FimH. This is consistent with HMGB1's ability to bind to more than one FimH on bacterial cells and induce aggregation.

[0096] Next, we investigated whether HMGB1 could limit Escherichia / Coli adhesion to host cells. rHMGB1 inhibited Escherichia / Coli adhesion to IEC at a level comparable to mannose and inhibited erythrocyte (RBC) aggregation in a dose-dependent manner (Figure 3e). rHMGB1 also inhibited the binding between rFimHLD and mannose, indicating that the reduced adhesion was due to a direct interaction between HMGB1 and FimH (Figure 3f). Mutations in ToH1 in FimH significantly impaired RBC aggregation by Escherichia / Coli, implicitly suggesting that this sequence is necessary for the adhesin function of this protein (Figure 3g).

[0097] HMGB1 regulates the expression of type 1 ciliary adhesion mechanism in Escherichia coli. In mice lacking IEC HMGB1, FimH protein levels were higher in colonic mucus and tissue than in wild littermates (Figure 3h, i, j, k). This is consistent with data showing an increase in the total number of bacteria on the epithelial surface in ΔIEC mice. However, the increase in FimH was obtained by an increase in the number of bacteria expressing the same amount of FimH, an increase in FimH expression in each bacterium without a change in bacterial number, or both. No difference in the relative abundance of Escherichia coli or Enterobacteriaceae between mouse genotypes was observed by 16s rRNA gene sequencing, and the effect of HMGB1 on Escherichia coli colony-forming units was not identified in in vitro assays. Therefore, it was considered that HMGB1 could regulate bacterial expression of FimH. Exposure to rHMGB1 dose-dependently reduced FimH expression by symbiotic Escherichia coli (Figure 3l). To determine whether HMGB1 causes Escherichia coli to turn off FimH expression or prevents bacteria from turning it on, we used a FimE knockout (ΔFimE) strain of Escherichia coli. The genes required for type 1 pili are located in a single operon regulated by a DNA switch region. The switch between pili-generating (FimON) and non-generating (FimOFF) states is regulated by two recombinases, FimB and FimE. When FimE is knocked out, bacteria can switch to FimON, but the switching to FimOFF is impaired, providing a relative "counter" for switching to FimON. Medium conditioned with WT primary intestinal epithelial cells suppressed FimON compared to medium conditioned with ΔIEC mouse-derived cells (Figure 3m, n). Furthermore, the addition of HMGB1 to ΔIEC-conditioned medium suppressed FimON as well as WT medium. Therefore, these combined results indicate that HMGB1 in the environment binds to its evolutionarily conserved target sequence (ToH1) in FimH, inhibiting FimH's binding to its target ligand, mannose, thereby impairing T1F adhesion to host cells and suppressing the expression of this adhesion mechanism.

[0098] Next, we investigated whether mucosal defenses released from IECs (environmental vascular tissue) are impaired in IBD patients. IBD has two main subtypes: Crohn's disease and ulcerative colitis. Ulcerative colitis affects only the colon and is thought to result from tissue damage initiated on the luminal side of the epithelium. Surface-associated HMGB1 was generally readily recognizable in resected colonic tissue from non-IBD patients, while tissue from UC patients generally had very low levels of HMGB1 with a mottled appearance (Figure 4a, b and Table 2). Surface-associated HMGB1 was lowest in patients with severe inflammation and absent in areas without IECs (Figure 4c). FimH assays were performed on serial tissue sections from the same patients, and a greater number of FimH-positive bacteria were observed in tissue from UC patients (Figure 4d, e). However, FimH was not associated with the severity of inflammation (Figure 4f). When surface HMGB1 and FimH were plotted from the same patients, UC patients grouped together with low HMGB1 and high FimH, while non-IBD patients grouped together with higher HMGB1 and lower FimH (Figure 4g). Mathematical modeling of the relationship between HMGB1 and FimH showed that the number of FimH-positive bacteria in tissues was HMGB1-dependent, and that FimH levels increased as HMGB1 decreased (Figure 4h). Therefore, UC is characterized by a failure of HMGB1 defense accompanied by a simultaneous increase in tissue-associated bacteria expressing the HMGB1 target protein FimH.

[0099] [Table 2]

[0100] High mobility box 1 (HMGB1) is an abundant and eccentrically expressed protein with intracellular and extracellular functions in many different cell types. Here, HMGB1 is shown to be an active component of the frontline mucosal barrier defense in the colon, with direct and indirect effects that limit the pathogenicity of the gut microbiota. Cross-border protein-protein interactions between mammalian HMGB1 and bacterial FimH directly limit bacterial pathogenicity by inactivating adhesion via FimH. T1F gene expression is also repressed by HMGB1, suggesting that bacteria such as Escherichia coli modulate phase or pathogenicity in response to HMGB1. In the studies described herein, the relationship between low levels of HMGB1 and high levels of FimH-positive bacteria in mouse models and resected colon tissue from ulcerative colitis patients was noted.

[0101] Ulcerative colitis has long been associated with adherent microorganisms, but no single pathogen has been consistently identified across studies. Herein, we suggest that HMGB1, a component of host defense, is absent in UC patients, allowing bacteria to adhere to host tissues by activating adhesion mechanisms normally suppressed by HMGB1. This data provides an explanation for why Escherichia coli and potentially other bacteria adhere to intestinal tissue in UC. The molecular target of HMGB1 in mucosal host defense is a small, evolutionarily conserved amino acid sequence found in many different types of bacterial adhesins. Similar to ligands that activate pattern recognition receptors, ToH1 appears to be widely utilized, pathogenic, and difficult to modify without loss of function. Since FimH and T1F adhesion are well-characterized, Escherichia coli FimH was used as a typical ToH1-positive protein in the mechanistic study described herein. The ToH1 sequence is identical and present in FimH derived from all Escherichia coli genomes examined, including Escherichia coli that cause infectious diarrhea, strains associated with chronic urinary tract infections, extraintestinal pathogenic Escherichia coli, and IBD-related adhesive and invasive Escherichia coli. The discovery of ToH1 in FimH has significant therapeutic implications because T1F adhesion is a preferred system for Escherichia coli, and therefore preventing T1F adhesion provides a new therapeutic strategy for diseases caused by this organism. Bacterial adhesion mechanisms are a high-value therapeutic target for almost all bacterial diseases because blocking adhesion in disease models and patients prevents tissue damage, inflammation, and immune activation.

[0102] [Example 2] Adhesion is the first step in microbial disease, and blocking adhesion has the potential to prevent infection. HMGB1 binding to ToH1 prevents microbial adhesion proteins from binding to their carbohydrate targets on mammalian host cells and fixing bacteria in place. Failure of this defense appears to be associated with several diseases in which Escherichia coli adheres to the intestinal epithelium, including infectious diarrhea, colorectal cancer, and IBD. Furthermore, HMGB1 inhibition of the ToH1 sequence and adhesion function is not limited to specific types of microorganisms and therefore has potential applications for bacteria, viruses, fungi, and protists. Thus, ToH1 provides a novel molecular target for disease diagnosis and treatment.

[0103] ToH1 is a seven-amino acid sequence found in surface-expressed adhesins derived from bacteria, viruses, fungi, and protists. This sequence is also present in mammalian proteins, mostly intracellularly. Therefore, therapeutically targeting ToH1 may also have anti-inflammatory functions. Table 3 lists exemplary, non-limiting organisms / pathogens that have adhesins containing ToH1.

[0104] [Table 3]

[0105] This specification provides an antibody that mimics the function of HMGB1. The antibody blocks adhesion and is useful for the treatment or prevention of microbial diseases, including both infectious and chronic inflammatory diseases caused by microorganisms. It is further shown that the antibody provided herein binds to ToH1 at the mammalian IL-1 receptor 1 (IL1R1) and blocks pro-inflammatory signaling (e.g., inhibits signaling through IL1R). Since adhesins containing the ToH1 sequence are not normally expressed or are expressed only at low levels in symbiotic microorganisms and are unlikely to be associated with healthy tissue, the antibody provided herein can also be used to diagnose microbial diseases.

[0106] Experiments were conducted to determine the HMGB1 target sequence. A series of sequences and ToH1-positive proteins were identified for HMGB1 inhibition of binding to target carbohydrates in plate assays. A position-specific score matrix (http: / / slim.icr.ac.uk / pssmsearch / ) was used to generate bioinformatics predictions of the HMGB1 target sequence based on experimental data. The results are shown in Figure 7. The numbers shown in the figure reflect the probability of a given residue being at that position. Analysis of this sequence in publicly available protein crystal structures suggests that the sequence determines the surface-exposed structure, and that sequence similarity can predict structural similarity.

[0107] Antibodies targeting ToH1 in FimH were obtained via phage display. ToH1-binding scFv were identified using a human phage display library. A proprietary Proteogenix human naive library was used (https: / / us.proteogenix.science / antibody-production / phage-display-services / #). To maximize diversity, 368 human donors from five different ethnic groups were used, resulting in 5.37 x 10⁶ samples. 10 We obtained scFv and Fab samples. First, we screened the library against the ToH peptide derived from Escherichia coli FimH (TSETPRV (SEQ ID NO: 33)). The hits from the initial screening were then screened against the functional Escherichia coli FimH lectin domain protein.

[0108] Three reads (scFv) were identified and sequenced. The sequences are provided below.

[0109] Antibody sequence: >F5(259AAs * , 27.51kDa * pI:9.18 * ) [ka]

[0110] Features: Signal peptide: [1:22] F5-scFv: [23:273] scFv linker: [141:161] Six His with linker: [274:281] * Theoretical value calculated without signal peptide

[0111] F5, VH, putative CDRs shown in bold

Chem.

[0112] F5, VL, putative CDRs shown in bold

Chem.

[0113] >F11 (261 AAs * , 27.25 kDa * , pI: 9.21 * )

Chem.

[0114] Features: Signal peptide: [1:22] F11-scFv: [23:275] scFv linker: [145:165] Six His with linker: [276:283] * Theoretical value calculated without signal peptide<000053I>

[0115] F11[[ID=S2]] F11, VH, putative CDRs shown in bold

Chem.

[0116] F11, VL, putative CDRs shown in bold

Chem.

[0117] >G6(263AAs * , 28.25kDa * pI:8.80 * ) [ka]

[0118] Characteristics: Signal peptide: [1:22] G6-scFv: [23:277] scFv linker: [144:164] Six His with linker: [278:285] * Theoretical value calculated without signal peptide

[0119] G6, VH, estimated CDR shown in bold [ka]

[0120] G6, VL, estimated CDR shown in bold [ka]

[0121] scFv was chemically synthesized and optimized for expression in Escherichia coli (with a signal peptide at the N-terminus for periplasm targeting and a 6His tag at the C-terminus). Antibody characterization shows that antibody F11 recognizes Escherichia coli FimH at the ToH site and exhibits blocking activity in the initial in vitro study. Exemplary results demonstrating inhibition of FimH binding to the target ligand, mannose, are shown in Figure 8. Antibody F11 was incubated with FimH, and FimH binding to mannose was measured via ELISA. NC is a negative control (detection region only). F11 dose-dependently inhibited FimH binding to mannose. Furthermore, antibody characterization shows that the anti-ToH1 antibody is suitable for diagnostic purposes, as demonstrated by the successful immunofluorescence labeling of colon tissue from HMGB1-deficient mice using the exemplary antibody F11. The results suggest that this antibody is suitable for immunostaining to identify ToH1-positive microorganisms that are very close to or attached to the intestinal surface.

[0122] Table 4 shows the binding of F5, F11, and G6 to Escherichia coli FimH. Phage display sequences of F5, F11, and G6 were used. The FimH protein was unloaded and mannose was loaded. All F5, G6, and F11 sequences were shown to recognize Escherichia coli FimH.

[0123] [Table 4]

[0124] Antibody binding was further confirmed by ELISA. The results are shown in Figures 10A (G6) and 10B (F5). The ToH1 peptide sequences shown are: FimH (Escherichia coli, TSETPRV (SEQ ID NO: 33)), Asa1 (Enterococcus faecalis, TKENPFV (SEQ ID NO: 39)), OmpA (Bacteroides, SFELPTI (SEQ ID NO: 35)), LptF (Escherichia coli, SPEKPTV (SEQ ID NO: 36)), BmpC (Borrelia burgdorferi, SYERPDI (SEQ ID NO: 40)), and HyR3 (Candida albicans, TFEPPVV (SEQ ID NO: 38)).

[0125] [Example 3] Antibodies broadly targeting ToH1 were obtained through a serial peptide injection method. Antigen design and immunization were developed for broader reactivity to short / universal target sequences. The goal of the immunization strategy was to produce reactivity only to the "fixed" amino acids in the motif (named amino acids). Two strategies were employed.

[0126] Strategy 1: Sequential immunization with one of the "fixed amino acids" and four different peptides. For this strategy, peptides following the motif shown in the table below are used, where "x" represents a variable amino acid, and the fixed amino acid is identified by the appropriate specific amino acid. The motif identified in the table below follows the format [S / T]xExPx[I / V], where "x" represents a variable amino acid.

[0127] [Table 5]

[0128] In the initial study, antibodies produced through this method were shown to possess FimH blocking activity. These were polyclonal rabbit antibodies. Monoclonal / recombinant antibodies with blocking activity can also be developed using this strategy.

[0129] <Strategy 2>: A sequential vaccination strategy was designed using a combination of experimentally confirmed ToH1 (target of HMGB1) epitopes and epitopes with enriched residues, based on the combination of bioinformatics analysis outputs, comparison with naturally identified sequences, and optimization of charged residues aimed at maximizing the assumption of antibody response binding.

[0130] We used peptides according to the motif [S / T]xExPx[I / V], each having a specific amino acid tested at each variable peptide position (each "x").

[0131] For vaccination, peptides having the formula identified below may be used. SAEN / DPR / KI SAEN / DPR / KV TAEN / DPR / KI TAEN / DPR / KV

[0132] The first residue is a fixed residue, either S or T. The second residue is a variable amino acid. For some peptides, the second residue is designed to be alpha (A), which allows for the "death" of the second residue, preventing it from participating in the binding process. Alpha is neutral and does not affect the binding. The third residue is the fixed residue, E. The fourth residue is a variable amino acid. For some peptides, the fourth residue is designed to be either N or D. Either N or D is an acceptable residue, adding similar hydrophilicity and having good prevalence based on the sequence logo. The fourth residue cannot "die" and become A because it would make the peptide excessively hydrophobic. The fifth residue is a fixed residue, P. The sixth residue is a variable amino acid. For some peptides, the sixth residue is designed to be either R or K. Either R or K is an acceptable residue, adding similar hydrophilicity and having some prevalence based on the sequence logo. The sixth residue cannot "die" and become A because it would make the peptide excessively hydrophobic. The seventh residue is a fixed residue, either I or V.

[0133] All combinations were presumed to be very similar in terms of appropriateness and likelihood of success.

[0134] The specific peptide sequence used for the injection was as follows: < injection#> 1 TSETPRV(Sequence ID 33) 2 SAENPKI (Sequence ID 42) 3 TAEDPRI (Sequence ID 41) 4 SPEKPTV (Sequence ID 36)

[0135] After injection, antibodies were isolated from the host and tested for binding to ToH1 peptide and ToH1-positive protein.

[0136] All publications and patents referenced in the above specification are incorporated herein by reference. Various modifications and changes to the methods and systems described of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described form for carrying out the invention, which will be apparent to those skilled in the art, are intended to fall within the scope of the following claims.

[0137] [Example 4] Nanobody Nanobodies were derived from animals immunized with a novel strategy for generating anti-ToH1 antibodies (as described, e.g., in Example 3). Following immunization, lymphocytes were collected and used to construct a phage display library of antibody sequences. This library was screened for ToH1 peptide conjugates, and then screened again for hits for binding to ToH1-positive proteins.

[0138] The cDNAs of two VHHs identified via phage display were chemically synthesized, optimized for mammalian expression in CHO cells and for C-terminal Avi-tagged 6His-tagged proteins, and then subcloned into the mammalian cell expression vector pTXs1. The resulting, expected corresponding proteins are shown below. The VHHs were then re-tested for binding to ToH1-positive proteins.

[0139] >G2-VHH 164 amino acids (also called VHH-G2) [ka]

[0140] Features: Signal peptide:[1:19] G2-VHH:[20:141] Avi tag:[142:156] Linker-accompanied 6His tags:[157:164]

[0141] >F7-VHH 164 amino acids (also called VHH-F7) [ka]

[0142] Features: Signal peptide:[1:19] F7-VHH:[20:141] Avi tag:[142:156] Linker-accompanied 6His tags:[157:164]

[0143] The binding activity of VHH-G2 was evaluated. The results are shown in Figures 11 to 16. VHH-G2 was shown to bind to all ToH1 peptides (Figure 11) and to ToH1-positive adhesins (Figure 12). VHH-G2 was also shown to bind to Escherichia coli FimH (Figure 13) and to agglutinate Escherichia coli (Figure 14). VHH-G2 was also shown to increase bacterial clearance by macrophages (Figure 15). VHH-G2 was also shown to bind to human IL1R1 (Figure 16), suggesting that VHH-G2 binds to IL1R1 and inhibits pro-inflammatory signaling.

[0144] The binding activity of VHH-F7 was evaluated. The results are shown in Figures 16 to 22. VHH-F7 was shown to bind to the ToH1 peptide (Figure 17) and to ToH1-positive adhesins (Figure 18). VHH-F7 was also shown to bind to Escherichia coli FiMH (Figure 19). VHH-F7 was also shown to bind to and agglutinate multiple bacterial species, including Escherichia coli (Figure 20A), Staphylococcus aureus (Figure 20B), and bacteria from complex communities (Figure 20C). VHH-F7 was also shown to increase bacterial clearance by macrophages (Figure 21).

[0145] An adhesion inhibition assay was performed to show that VHH-F7 inhibits the binding of Escherichia coli to mannose (Figure 22A), the binding of Staphylococcus aureus to fibronectin (Figure 22B), and the binding of bacterial communities to fibronectin (Figure 22C).

[0146] ELISA demonstrated that VHH-F7 binds to IL1R1 (mammalian ToH1-positive protein) (Figure 23A) and inhibits IL1R signaling (Figure 23B).

[0147] method Escherichia coli agglutination assay SWW33-GFP Escherichia coli was grown overnight in nutrient broth at 37°C. After overnight growth, the culture was rediluted to 0.8 OD. 200 μl of 0.8 OD culture was added to a new Eppendorf tube. The bacteria were pelletized at 5000 RPM for 5 minutes and then resuspended in 100 μl of TBS containing 10 mM CaCl2. This 100 μl was then divided in half, with 50 μl used as a control and the other 50 μl used for VHH F7 treatment. VHH F7 was added to 50 μl of the reaction mixture to a final concentration of 10 μM. After treatment, the reaction mixture was allowed to agglutinate at 37°C for 2 hours. Then, 3 μl of the reaction mixture was placed on a rectangular coverslip, sandwiched between 0.15% agarose pads, and imaged with a fluorescence microscope.

[0148] Staphylococcus aureus agglutination assay Staphylococcus aureus was grown overnight in BHI broth at 37°C. After overnight growth, the culture was rediluted to 0.8 OD. Biotium Cf® Dye lectin conjugate wheat germ agglutinin CF® 488A was added to the culture and incubated at 37°C for 30 minutes to enable fluorescent labeling of the bacteria. 200 μl of 0.8 OD culture was added to a new Eppendorf tube. The bacteria were pelletized at 5000 RPM for 5 minutes and then resuspended in 100 μl of TBS containing 10 mM CaCl2. This 100 μl was then divided in half, with 50 μl used as a control and the other 50 μl used for VHH F7 treatment. VHH F7 was added to 50 μl of the reaction mixture to a final concentration of 10 μM. After treatment, the reaction mixture was allowed to agglutinate at 37°C for 2 hours. Next, the 3 µl reactant was placed on a rectangular coverslip, sandwiched between 0.15% agarose pads, and imaged using a fluorescence microscope.

[0149] B6 Microbiota Colonic Community Aggregation Assay Colons derived from SPF C57BL.6J mice were resected from newly euthanized mice between 8 and 12 weeks of age, and the sections were opened along the long axis. Using an inoculation loop, the contents were scraped onto 1.5 mL of Eppendorf in 1 mL of PBS. The contents were centrifuged at 400 XG for 5 minutes; the supernatant was stained using a 70 μm cell strainer. The pass-through fraction was washed twice with PBS and once with TBS containing 10 mM CaCl2 at 10,000 XG for 2 minutes. After the third wash, the samples were resuspended in 500 μL of TBS, 10 mM CaCl2, and CellBrite 488 membrane staining dye, and incubated at 37°C for 30 minutes to allow for bacterial fluorescence labeling. The samples were divided into 100 μL aliquots and centrifuged at 10,000 XG for 2 minutes. The final, washed, and labeled B6 microbiota pellet was resuspended in 100 µl TBS containing 10 mM CaCl2. This 100 µl was then divided in half, with 50 µl used as a control and the other 50 µl used for VHH F7 treatment. VHH F7 was added to the 50 µl reactant to a final concentration of 10 µM. After treatment, the reactant was allowed to agglutinate at 37°C for 2 hours. Then, 3 µl of the reactant was placed on a rectangular coverslip, sandwiched between 0.15% agarose pads, and imaged with a fluorescence microscope.

[0150] Escherichia coli phagocytic assay Wild-type and HMGB1 KO RAW264.7 macrophages were plated at 250,000 cells / mL in poly-L-lysine coated glass-bottom dishes and incubated overnight in DMEM + 10% FBS at 37°C and 5% CO2. SWW33-GFP Escherichia coli was grown overnight in nutrient broth at 37°C. The following day, macrophages were inoculated with SWW33-GFP Escherichia coli at a final OD of 0.8 (bacterial input: 64 × 10^8 cells) and treated with 10 μM VHH F7 / G2 or control. The treated bacteria were then incubated with macrophages at 37°C for 1 hour and then imaged under a bright-field microscope. Phagocytosis was observed on the surface of the bacteria as they were captured and engulfed. Inhibition of Escherichia coli binding to mannose (adhesion inhibition assay)

[0151] SWW33-GFP Escherichia coli was grown overnight in nutrient broth at 37°C. After overnight growth, the culture was rediluted to 0.8 OD. 200 μl of 0.8 OD culture was added to a new Eppendorf tube. The bacteria were treated with a dilution series of VHH F7 (0-10 μM) and incubated at 37°C for 2 hours. After incubation, the treated bacteria were carefully placed on mannose-coated plates, sealed, and incubated at 37°C for 1 hour, repeating this process three times. After 1 hour of incubation, the bacteria were decanted from the plates, and the plates were gently rinsed twice with 200 μl / well PBS. After the final washing step, the number of mannose-bound bacteria was measured by spectrophotometer reading at 485 / 538 (Ex / Em).

[0152] Inhibition of Staphylococcus aureus binding to fibronectin (adhesion inhibition assay) Staphylococcus aureus was grown overnight in BHI broth at 37°C. After overnight growth, the culture was rediluted to 0.8 OD and labeled with Biotium Cf® Dye lectin conjugate wheat germ aglutinin CF® 488A for 30 minutes at 37°C. 200 μl of 0.8 OD labeled culture was added to a new Eppendorf tube. The bacteria were treated with a dilution series of VHH F7 (0-10 μM) and incubated at 37°C for 2 hours. After incubation, the treated bacteria were carefully placed on fibronectin-coated plates, sealed, and incubated at 37°C for 1 hour, repeating this process three times. After 1 hour of incubation, the bacteria were decanted from the plates, and the plates were gently rinsed twice with 200 μl / well PBS. After the final washing step, the plates were measured using a spectrophotometer at 485 / 538 (Ex / Em) to determine the number of bacteria bound to fibronectin.

[0153] Inhibition of B6 microbial community binding to fibronectin (adhesion inhibition assay) Colons derived from SPF C57BL.6J mice were resected from newly euthanized mice between 8 and 12 weeks of age, and the sections were opened along the long axis. Using an inoculation loop, the contents were scraped onto 1.5 mL of Eppendorf tube containing 1 mL of PBS. The contents were centrifuged at 400 XG for 5 minutes; the supernatant was stained using a 70 μm cell strainer. The pass-through fraction was washed twice with PBS and once with TBS containing 10 mM CaCl2 at 10,000 XG for 2 minutes. After the third wash, the samples were resuspended in 500 μL of TBS, 10 mM CaCl2, and CellBrite 488 membrane staining dye, and incubated at 37°C for 30 minutes to enable fluorescent labeling of bacteria. 200 μl of OD 0.8 labeled cultures were added to a new Eppendorf tube. Bacteria were treated with a dilution series of VHH F7 (0-10 μM) and incubated at 37°C for 2 hours. After incubation, the treated bacteria were carefully placed on fibronectin-coated plates, sealed, and incubated at 37°C for 1 hour, repeating this process three times. After 1 hour of incubation, the bacteria were decanted from the plates, and the plates were gently rinsed twice with 200 nl / well PBS. After the final washing step, the number of fibronectin-bound bacteria was measured by spectrophotometering, reading at 485 / 538 (Ex / Em).

[0154] Inhibition of IL1R1 signaling using HEK-Blue® IL-1R reporter cells. HEK-Blue® IL-1R reporter cells were grown to confluence in selective medium according to the manufacturer's instructions. Selected HEK-Blue cells were rinsed twice with preheated PBS and then isolated in PBS at 37°C for 2-3 minutes. The cells were gently tapped in the flask to loosen, then rinsed with preheated test medium and resuspended at 280,000 cells / mL. 180 μl of HEK-Blue cell suspension (approximately 50,000 cells) was added to a 96-well flat-bottom plate, and the plate was incubated overnight at 37°C and 5% CO2. The following day, reporter cells were treated with 10 μM VHH F7 for 2 hours and then stimulated with IL1-β for a further 2 hours. After stimulation, Quanti-Blue solution was prepared according to the manufacturer's protocol. 180 μl of Quanti-Blue solution was added to each well of a new 96-well flat-bottom plate. Each plate was plated with 20 µl of HEK-Blue-treated supernatant in three repetitions. The plates were then sealed and incubated at 37°C for 1 hour before determining the SEAP level by spectrophotometer at 620–655 nm.

[0155] Antibody binding to FimH Wells of a highly bound 96-well microtiter plate were coated overnight with FimH protein (2 μM) at 4°C. Various concentrations of antibody were added to the coated wells and incubated at room temperature for 1 hour. Then, at 450 nm (OD), the wells were exposed to light. 450 Antibodies bound to the FimH protein were detected using anti-Avi tagged antibodies.

[0156] Antibody binding to IL1R1 The wells of a highly bound 96-well microtiter plate were coated overnight with IL1R1 protein (1 μM) (4°C). Various concentrations of antibody were added to predetermined wells of the highly bound 96-well microtiter plate and incubated at room temperature for 1 hour. Then, at 450 nm (OD), the antibodies were incubated. 450 Antibodies bound to the IL1R1 protein were detected using anti-Avi tagged antibodies.

Claims

1. An antibody that binds to the target of HGMB1 (ToH1), a) Heavy chain variable domains including complementarity-determining regions HCDR1, HCDR2 and HCDR3, and b) Light chain variable domains including complementarity-determining regions LCDR1, LCDR2, and LCDR3 Includes, i. HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 80% sequence identity with SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 80% sequence identity with SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; ii. HCDR1, HCDR2, and HCDR3 each contain an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 each contain an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; or iii. HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 80% sequence identity with SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, and LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 80% sequence identity with SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, antibody.

2. a) HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; b) HCDR1, HCDR2, and HCDR3 each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 each contain an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; or c) HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, and LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, The antibody according to claim 1.

3. a) HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 95% sequence identity with SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 95% sequence identity with SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; b) HCDR1, HCDR2, and HCDR3 each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; or c) HCDR1, HCDR2, and HCDR3 each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, and LCDR1, LCDR2, and LCDR3 each contain an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, The antibody according to claim 1.

4. a) HCDR1, HCDR2, and HCDR3 each include SEQ ID NOs: 11, 12, and 13, and LCDR1, LCDR2, and LCDR3 each include SEQ ID NOs: 14, 15, and 16; b) HCDR1, HCDR2, and HCDR3 each include SEQ ID NOs: 17, 18, and 19, and LCDR1, LCDR2, and LCDR3 each include SEQ ID NOs: 20, 21, and 22; or c) HCDR1, HCDR2, and HCDR3 each include SEQ ID NOs. 23, 24, and 25, and LCDR1, LCDR2, and LCDR3 each include SEQ ID NOs. 26, 27, and 28, The antibody according to claim 1.

5. a) A heavy chain variable domain containing a sequence having at least 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; and b) A light chain variable domain containing a sequence having at least 80% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 The antibody according to any one of claims 1 to 4, comprising:

6. a) A heavy chain variable domain containing a sequence having at least 90% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; and b) A light chain variable domain containing a sequence having at least 90% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 The antibody according to claim 5, comprising:

7. a) A heavy chain variable domain containing a sequence having at least 95% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; and b) A light chain variable domain containing a sequence having at least 95% sequence identity with SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 The antibody according to claim 5, comprising:

8. a) A heavy chain variable domain containing Sequence ID No. 1 and a light chain variable domain containing Sequence ID No. 2; b) A heavy chain variable domain containing SEQ ID NO: 3 and a light chain variable domain containing SEQ ID NO: 4; or c) Heavy chain variable domain containing Sequence ID No. 5 and light chain variable domain containing Sequence ID No. 6 The antibody according to claim 5, comprising:

9. The antibody according to any one of claims 1 to 8, wherein the heavy chain variable domain and the light chain variable domain are linked by a linker.

10. The antibody according to claim 9, wherein the linker comprises a sequence having at least 80% identity with SEQ ID NO:

10.

11. The antibody according to claim 9, wherein the linker comprises SEQ ID NO:

10.

12. The antibody according to any one of claims 1 to 11, wherein the antibody is a single-chain variable region fragment (scFv).

13. An antibody that binds to the target (ToH1) of HGMB1, and is a nanobody having at least 80% identity with SEQ ID NO: 29 or SEQ ID NO:

30.

14. The antibody according to claim 13, having at least 90% identity with SEQ ID NO: 29 or SEQ ID NO:

30.

15. The antibody according to claim 13, having at least 95% identity with SEQ ID NO: 29 or SEQ ID NO:

30.

16. An antibody according to any one of claims 1 to 15, for use in a method for treating, preventing or diagnosing microbial diseases and / or chronic inflammatory diseases in a subject.

17. The antibody according to claim 16, wherein the microbial disease is a microbial infection.

18. The antibody according to claim 17, wherein the microbial infection is a bacterial infection.

19. The antibody according to claim 18, wherein the chronic inflammatory disease is inflammatory bowel disease.

20. A method for treating or preventing microbial diseases and / or chronic inflammatory diseases in a subject, comprising the step of providing an antibody that binds to a target of HGMB1 (ToH1) in the subject.

21. The method according to claim 20, wherein the antibody comprises the antibody described in any one of claims 1 to 15.

22. The method according to claim 20 or claim 21, wherein the microbial disease includes a microbial infection.

23. The method according to claim 22, wherein the microbial infection is a bacterial infection.

24. The method according to claim 22, wherein the chronic inflammatory disease is inflammatory bowel disease.

25. A method for diagnosing a microbial disease and / or chronic inflammatory disease in a subject, comprising the steps of: determining the level of HGMB1 target (ToH1) in a sample obtained from the subject; and determining that the subject has a microbial disease if the level of ToH1 in the sample is equal to or higher than a threshold.

26. The method according to claim 25, wherein the step of determining the level of ToH1 in the sample includes contacting the sample obtained from a subject with an antibody that binds to the target (ToH1) of HGMB1, and detecting the antibody in the sample.

27. The method according to claim 26, wherein the antibody comprises the antibody described in any one of claims 1 to 15.

28. The method according to claim 26 or claim 27, wherein the microbial disease includes a microbial infection.

29. The method according to claim 28, wherein the microbial infection is a bacterial infection.

30. The method according to claim 28, wherein the chronic inflammatory disease is inflammatory bowel disease.

31. A method for generating an antibody that binds to the target of HGMB1 (ToH1), a) A step of sequentially immunizing a host with two or more unique peptides, wherein each of the two or more unique peptides has a [S / T]xExPx[I / V] motif, and each x is a variable amino acid, and b) A step of isolating antibodies produced in response to immunization. Methods that include...

32. The method according to claim 31, wherein two or more unique peptides include three unique peptides.

33. The method according to claim 32, wherein two or more unique peptides comprise four unique peptides.

34. The method according to claim 33, wherein the four unique peptides include SxExPxI, SxExPxV, TxExPxI, and TxExPxV.

35. The method according to claim 34, wherein the four unique peptides include SAENPKI (SEQ ID NO: 42), SPEKPTV (SEQ ID NO: 36), TAEDPRI (SEQ ID NO: 41), and TSETPRV (SEQ ID NO: 33), or the four unique peptides include TSETPRV (SEQ ID NO: 33), SSERPMI (SEQ ID NO: 34), SPEKPTV (SEQ ID NO: 36), and TRELPQI (SEQ ID NO: 37).

36. An antibody produced by the method described in any one of claims 31 to 35.

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