Pharmaceutical composition for ameliorating acute lung injury and acute worsening of pulmonary fibrosis

Abstract

Provided are a colicin-fighting monoclonal antibody molecule or a derivative thereof, and a pharmaceutical composition containing the same, for ameliorating acute lung injury and acute worsening of pulmonary fibrosis.　The pharmaceutical composition of the present invention contains a monoclonal antibody molecule that fights against colicin, which is a peptide that stores a variety of staphylococcus strains including staphylococcus nepalensis, or a derivative of the monoclonal antibody molecule.

Description

Pharmaceutical composition for ameliorating acute lung injury and acute exacerbation of pulmonary fibrosis

[0001] This patent application claims priority and the benefit of Japanese Patent Application No. 2021-046301 (filed March 19, 2021) and Japanese Patent Application No. 2021-173381 (filed October 22, 2021) under the Paris Convention, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a pharmaceutical composition for ameliorating acute exacerbation of pulmonary fibrosis and a method therefor, and more particularly to a pharmaceutical composition for ameliorating acute exacerbation of pulmonary fibrosis, which contains an inhibitor of a peptide derived from pulmonary microbiota, and a method therefor.

[0003] Idiopathic pulmonary fibrosis (IPF) is a chronic, incurable disease of unknown etiology. Increased apoptosis of alveolar epithelial cells and subsequent abnormal lung tissue repair are central to IPF progression. Two antifibrotic drugs currently used to treat IPF, pirfenidone and nintedanib, only provide a survival benefit of approximately 2–3 years after diagnosis (Non-Patent Document 1). These drugs only slightly reduce the loss of lung function and do not improve patient survival or quality of life (Non-Patent Documents 2–4). Recent epidemiological studies have shown that IPF affects more than 5 million people worldwide, and the number of patients is increasing worldwide (Non-Patent Document 5). Alveolar epithelial cell damage and abnormal tissue repair, resulting from increased secretion of pulmonary fibrotic factors such as transforming growth factor-β1, excessive recruitment of myofibroblasts in the lung, and deposition of extracellular matrix, play a central role in the pathogenesis of IPF (Non-Patent Document 1). This progression ultimately leads to lung tissue scarring, structural destruction of the lung, and respiratory failure (Non-Patent Document 5). The natural history of IPF is unpredictable and variable (Non-Patent Document 6), and can be characterized by slow progression, rapid progression, or sudden clinical deterioration called acute exacerbations (AEs) (Non-Patent Documents 7, 8). AEs are the most common cause of death in IPF, occurring in approximately 46% of cases (Non-Patent Documents 9, 10). AEs predict high mortality during the disease (up to 50%) and short survival after the acute episode; once diagnosed, patients survive only 3–4 months (Non-Patent Documents 8, 9). In cases requiring mechanical ventilation, mortality increases by up to 90% (Non-Patent Document 9). Infections and diagnostic or surgical procedures may precipitate AEs, although the exact mechanisms are unknown (Non-Patent Document 8). Currently, there are no effective treatments for AEs (Non-Patent Document 3).

[0004] Increasing evidence suggests that the pulmonary microbiome plays a role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). In patients with IPF, dysbiotic or abundant pulmonary microbiomes are associated with persistent altered expression of genes involved in host defense, impaired immune responses, lung epithelial cell damage, abnormal fibroblast reactivity, severe pulmonary function abnormalities, worsening chest radiographic findings, disease progression, and reduced survival (Non-Patent Documents 11-16). Alterations in the pulmonary microbiome and bacterial burden are further exacerbated in IPF with acute exacerbations (Non-Patent Documents 17, 18). However, the mechanisms associated with pulmonary microbiome involvement in pulmonary fibrosis remain unclear. We recently identified proapoptotic peptides, termed colicins, in the pulmonary microbiome, which may explain their pathogenic role in pulmonary fibrosis (Non-Patent Document 19). Colicins are peptides conserved in diverse staphylococci and various pathogenic bacterial strains (Non-Patent Document 19).

[0005] The amino acid sequence of colicin corresponds to a bacterial transglycosylase fragment (Non-Patent Document 19). Intrapulmonary administration of colicin or colicin-carrying bacteria to mice with established pulmonary fibrosis induces apoptosis in alveolar epithelial cells and acute exacerbations (AE) (Non-Patent Document 19). Furthermore, IPF patients with slowly progressing disease have increased pulmonary colicin levels compared with healthy controls, and patients with AE have significantly higher pulmonary colicin levels than stable patients (Non-Patent Document 19).

[0006] Therefore, the present inventors have already hypothesized that treatment with antibodies against colicin can directly inhibit AE (AE-IPF) of pulmonary fibrosis by blocking the apoptotic activity of colicin, and after various experiments, have produced antibodies against colicin and demonstrated that they can be used for Western blotting of colicin in lung tissue (Patent Document 1).

[0007] WO2021 / 126957

[0008] 1 King, T. E., Jr., Pardo, A. & Selman, M. Idiopathic pulmonary fibrosis. Lancet 378, 1949-1961, doi: 10.1016 / S0140-6736(l 1)60052-4 (2011).2 Richeldi, L. et al. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis. N EnglJ Med370, 2071-2082, doi: 10.1056 / NEJMoal402584 (2014).King, T. E., Jr., Noble, P. W. & Bradford, W. Z. Treatments for idiopathic pulmonary fibrosis. N Engl J Med 371, 783-784, doi: 10.1056 / NEJMcl407776 (2014).King, T. E., Jr. et al. A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis. N Engl J Med 370, 2083-2092, doi: 10.1056 / NEJMoal402582 (2014).5 Lederer, D. J. & Martinez, F. J. Idiopathic Pulmonary Fibrosis. N Engl J Med 378, 1811-1823, doi: 10.1056 / NEJMral705751 (2018).6 Sgalla, G., Biffi, A. & Richeldi, L. Idiopathic pulmonary fibrosis: Diagnosis, epidemiologyand natural history. Respirology21, 427-437, doi: 10.1111 / resp.l2683 (2016).Kreuter, M. et al.Acute exacerbation of idiopathic pulmonary fibrosis: international survey and call for harmonisation. Eur Respir J 55, doi: 10.1183 / 13993003.01760-2019(2020).Collard, H. R. et al. Acute Exacerbation of Idiopathic Pulmonary Fibrosis. An International Working Group Report. Am J Respir Crit Care Med 194, 265-275, doi: 10.1164 / rccm.201604-0801CI (2016).Natsuizaka, M. et al. Epidemiologic survey of Japanese patients with idiopathic pulmonary fibrosis and investigation of ethnic differences. Am J Respir Crit Care Med 190, 773-779, doi: 10.1164 / rccm.201403-05660C (2014).Ley, B., Collard, H. R. & King, T. E., Jr. Clinical course and prediction of survival in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 183, 431-440, doi: 10.1164 / rccm.201006-0894CI (2011).

[0009] Dickson, R. P. et al. Radiographic Honeycombing and Altered Lung Microbiota in Patients with Idiopathic Pulmonary Fibrosis. Am J Respir Crit Care Med200, 1544-1547, doi: 10.1164 / rccm.201903-0680LE (2019).Han, M. K. et al. Lung microbiome and disease progression in idiopathic pulmonary fibrosis: an analysis of the COMET study. Lancet Respir Med 2, 548-556, doi: 10.1016 / S2213-2600(14)70069-4 (2014).Huang, Y. et al. Microbes Are Associated with Host Innate Immune Response in Idiopathic Pulmonary Fibrosis. Am J Respir Crit Care Med 196, 208-219, doi: 10.1164 / rccm.201607-15250C (2017).Molyneaux, P. L. et al. Host-Microbial Interactions in Idiopathic Pulmonary Fibrosis. Am J Respir Crit Care Med 195, 1640-1650, doi:10.1164 / rccm.201607-14080C (2017).Takahashi, Y. et al. Impaired diversity of the lung microbiome predicts progression of idiopathic pulmonary fibrosis. Respir Resl9, 34, doi:10.1186 / sl2931-018-0736-9 (2018).Wang, J. et al.Lung microbiome and host immune tone in subjects with idiopathic pulmonary fibrosis with inhaled interferon-gamma. ERJ Open Res 3, doi: 10.1183 / 23120541.00008-2017 (2017). Molyneaux, PL et al. Changes in the respiratory microbiome during acute exacerbations of idiopathic pulmonary fibrosis. Respir Res 18, 29, doi: 10.1186 / sl2931-017-0511-3 (2017). Weng, D. et al. The Role of Infection in Acute Exacerbation of Idiopathic Pulmonary Fibrosis. Mediators Inflamm 2019, 5160694, doi: 10.1155 / 2019 / 5160694 (2019).D'Alessandro-Gabazza, CN etal. A Staphylococcus pro-apoptotic peptide induces acute exacerbation of pulmonary fibrosis. Nat Commun 11, 1539, doi: 10.1038 / s41467-020-15344-3 (2020).

[0010] Further studies by the present inventors demonstrated the presence and apoptotic activity of colicin-like peptides from known pathogenic bacteria other than Staphylococcus nepalensis strain CNDG, thus indicating that this toxic peptide is more widely distributed. Furthermore, by generating and characterizing several monoclonal antibodies, we identified monoclonal antibody A21, which has the ability to neutralize the toxic activity of colicin and related peptides.

[0011] Utilizing the above findings, the efficacy of a neutralizing monoclonal antibody was tested in two different pulmonary fibrosis models, and it was demonstrated that this antibody significantly inhibits acute exacerbation in a human transforming growth factor-β1 transgenic mouse model and acute lung injury in a bleomycin model, thereby improving pulmonary fibrosis, thereby completing the present invention.

[0012] That is, the present invention includes the following aspects. <Monoclonal antibody molecule> [1] An isolated monoclonal antibody molecule or derivative thereof that specifically binds to colicin. [2] The monoclonal antibody molecule or derivative thereof according to [1], wherein the colicin is IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1). [3] The monoclonal antibody molecule or derivative thereof according to [1] or [2], wherein the epitope is PESSGNP (SEQ ID NO: 68) or NPAGY (SEQ ID NO: 69).

[0013] <Monoclonal antibody molecules specified by CDRs: Series A> [4] (IA) (i) an H-chain variable region comprising the H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 37, the H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 38, and the H-chain variable region CDR3 amino acid sequence of SEQ ID NO: 39, and (ii) an L-chain variable region comprising the L-chain variable region CDR1 amino acid sequence of SEQ ID NO: 41, the L-chain variable region CDR2 amino acid sequence of SEQ ID NO: 42, and the L-chain variable region CDR3 amino acid sequence of SEQ ID NO: 43; (IIA) (i) an H-chain variable region comprising the H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 70, the H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 49, and the H-chain variable region CDR3 amino acid sequence of SEQ ID NO: 50, and (ii) an L-chain variable region comprising the L-chain variable region CDR1 amino acid sequence of SEQ ID NO: 52, the L-chain variable region CDR2 amino acid sequence of SEQ ID NO: 53, and the L-chain variable region CDR3 amino acid sequence of SEQ ID NO: 54; or (IVA) The monoclonal antibody molecule or derivative thereof according to [1] or [2], comprising: (i) an H chain variable region comprising the H chain variable region CDR1 amino acid sequence of SEQ ID NO: 72, the H chain variable region CDR2 amino acid sequence of SEQ ID NO: 73, and the H chain variable region CDR3 amino acid sequence of SEQ ID NO: 74; and (ii) an L chain variable region comprising the L chain variable region CDR1 amino acid sequence of SEQ ID NO: 76, the L chain variable region CDR2 amino acid sequence of SEQ ID NO: 77, and the L chain variable region CDR3 amino acid sequence of SEQ ID NO: 78.

[0014] <Monoclonal antibody molecules identified by heavy chain variable region and variants: Series B> [5] (IB) an heavy chain variable region comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 36, or an amino acid sequence at least 80% identical thereto, or an heavy chain variable region comprising at least one framework (FW) region comprising an amino acid sequence in the heavy chain variable region amino acid sequence of SEQ ID NO: 36, which has one or several amino acid residue substitutions, deletions, insertions, or additions compared to the heavy chain variable region amino acid sequence of SEQ ID NO: 36; (IIB) an heavy chain variable region comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 48, or an amino acid sequence at least 80% identical thereto, or an heavy chain variable region comprising at least one framework (FW) region comprising an amino acid sequence in the heavy chain variable region amino acid sequence of SEQ ID NO: 48, which has one or several amino acid residue substitutions, deletions, insertions, or additions compared to the heavy chain variable region amino acid sequence of SEQ ID NO: 48; or (IVB) A monoclonal antibody molecule or derivative thereof according to any one of [1] to [3], having an H chain variable region comprising the H chain variable region amino acid sequence of SEQ ID NO: 71, or an amino acid sequence at least 80% identical thereto, or an H chain variable region comprising at least one framework (FW) region comprising an amino acid sequence in the H chain variable region amino acid sequence of SEQ ID NO: 71 that has one or several amino acid residue substitutions, deletions, insertions or additions compared to the H chain variable region amino acid sequence of SEQ ID NO: 71.

[0015] <Monoclonal antibody molecules characterized by light chain variable region and variants: Series C> [6] (IC) An light chain variable region comprising the light chain variable region amino acid sequence of SEQ ID NO: 40, or an amino acid sequence at least 80% identical thereto, or an light chain variable region comprising at least one framework region comprising an amino acid sequence in the light chain variable region amino acid sequence of SEQ ID NO: 40, which has one or several amino acid residue substitutions, deletions, insertions, or additions compared to the light chain variable region amino acid sequence of SEQ ID NO: 40; (IIC) An light chain variable region comprising the light chain variable region amino acid sequence of SEQ ID NO: 51, or an amino acid sequence at least 80% identical thereto, or an light chain variable region comprising at least one framework region comprising an amino acid sequence in the light chain variable region amino acid sequence of SEQ ID NO: 51, which has one or several amino acid residue substitutions, deletions, insertions, or additions compared to the light chain variable region amino acid sequence of SEQ ID NO: 51; (IIIC) (IVC) an L-chain variable region comprising the L-chain variable region amino acid sequence of SEQ ID NO: 75 or an amino acid sequence at least 80% identical thereto, or an L-chain variable region comprising at least one framework region comprising an amino acid sequence which has one or several amino acid substitutions, deletions, insertions, or additions compared to the L-chain variable region amino acid sequence of SEQ ID NO: 44; or (IVC) an L-chain variable region comprising the L-chain variable region amino acid sequence of SEQ ID NO: 75 or an amino acid sequence which is at least 80% identical thereto, or an L-chain variable region comprising at least one framework region comprising an amino acid sequence which has one or several amino acid substitutions, deletions, insertions, or additions compared to the L-chain variable region amino acid sequence of SEQ ID NO: 75. [7] The monoclonal antibody molecule or derivative thereof according to any of [1] to [3], comprising any H-chain variable region according to [5] and any L-chain variable region according to [6].

[0016] <Identifying the combination of H-chain and L-chain variable regions of a monoclonal antibody molecule> [8] The monoclonal antibody molecule or derivative thereof described in [7], which has the H-chain variable region (IB) described in [5] and the L-chain variable region (IC) described in [6], the H-chain variable region (IIB) described in [5] and the L-chain variable region (IIC) described in [6], or the H-chain variable region (IVB) described in [5] and the L-chain variable region (IVC) described in [6]. <Monoclonal antibody molecule identified by heavy and light chain variable regions> [9] The monoclonal antibody molecule or derivative thereof according to [8], wherein the heavy chain variable region of (IB) according to [5] has the heavy chain variable region amino acid sequence of SEQ ID NO: 36, and the light chain variable region of (IC) according to [6] has the light chain variable region amino acid sequence of SEQ ID NO: 40; or the heavy chain variable region of (IIB) according to [5] has the heavy chain variable region amino acid sequence of SEQ ID NO: 48, and the light chain variable region of (IIC) according to [6] has the light chain variable region amino acid sequence of SEQ ID NO: 51; or the heavy chain variable region of (IVB) according to [5] has the heavy chain variable region amino acid sequence of SEQ ID NO: 71, and the light chain variable region of (IVC) according to [6] has the light chain variable region amino acid sequence of SEQ ID NO: 75.

[0017] <Monoclonal antibody molecule variant 1>

[10] The H-chain variable region of (IB) according to [5] is an H-chain variable region comprising an amino acid sequence at least 80% identical to the H-chain variable region amino acid sequence of SEQ ID NO: 36, and the L-chain variable region of (IC) according to [6] is an L-chain variable region comprising an amino acid sequence at least 80% identical to the L-chain variable region amino acid sequence of SEQ ID NO: 40; or The H-chain variable region of (IIB) according to [5] is an H-chain variable region comprising an amino acid sequence at least 80% identical to the H-chain variable region amino acid sequence of SEQ ID NO: 48, and the L-chain variable region of (IIC) according to [6] is an L-chain variable region comprising an amino acid sequence at least 80% identical to the L-chain variable region amino acid sequence of SEQ ID NO: 51; or The monoclonal antibody molecule or derivative thereof according to [8], wherein the H chain variable region of (IVB) according to [5] is an H chain variable region comprising an amino acid sequence that is at least 80% identical to the H chain variable region amino acid sequence of SEQ ID NO: 71, and the L chain variable region of (IVC) according to [6] is an H chain variable region comprising an amino acid sequence that is at least 80% identical to the H chain variable region amino acid sequence of SEQ ID NO: 75.

[0018] <Monoclonal antibody molecule variant 2>

[11] The heavy chain variable region of (IB) according to [5] is an heavy chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the heavy chain variable region amino acid sequence of SEQ ID NO: 36, and the light chain variable region of (IC) according to [6] is an light chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the light chain variable region amino acid sequence of SEQ ID NO: 40; or The H-chain variable region of (IIB) according to [5] is an H-chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions, or additions compared to the H-chain variable region amino acid sequence of SEQ ID NO: 48, and the L-chain variable region of (IIC) according to [6] is an L-chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions, or additions compared to the L-chain variable region amino acid sequence of SEQ ID NO: 51; or The monoclonal antibody molecule or derivative thereof according to [8], wherein the H-chain variable region of (IVB) according to [5] is an H-chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the H-chain variable region amino acid sequence of SEQ ID NO: 71, and the L-chain variable region of (IVC) according to [6] is an L-chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the L-chain variable region amino acid sequence of SEQ ID NO: 75.

[12] Fab, F(ab') ２

[13] The antibody molecule or derivative thereof according to any one of [1] to

[12] , which neutralizes and / or inhibits colicin-induced apoptosis.

[0019] <Pharmaceutical Compositions>

[14] A pharmaceutical composition for treating or preventing acute lung injury or fibrosis, comprising an effective amount of the monoclonal antibody molecule or derivative thereof according to any one of [1] to

[13] .

[15] The pharmaceutical composition according to

[14] , wherein the acute lung injury is life-threatening pneumonia that causes acute respiratory distress syndrome (ARDS), resulting in a decline in pulmonary function and requiring respiratory management.

[16] The pharmaceutical composition according to

[14] , wherein the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and mammary fibrosis.

[0020] <Vaccine>

[17] A vaccine composition for preventing acute lung injury or fibrosis, comprising as an active ingredient a colicin mutant having one or several mutations in the sequence of colicin having the amino acid sequence: IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1), wherein the mutant does not exhibit apoptotic activity.

[0021] <Biomarkers>

[18] Use of colicins as biomarkers for determining the progression of acute lung injury or fibrosis.

[0022] The present inventors further investigated the effects of anti-colicin monoclonal antibodies in a common model of acute lung injury and obtained results regarding the potential impact of colicin on such diseases. The present invention is believed to further clarify the role of colicin in the pathogenesis and progression of pulmonary fibrosis and possibly other acute lung injury-related lung diseases, and to provide new approaches, i.e., new medicines, therapeutic methods, etc., for treating these incurable diseases.

[0023] Figure 1 shows the homology alignment of the putative transglycosylase containing colicin peptides from Staphylococcus nepalensis strain CNDG (CNDG) with three strains of Staphylococcus haemolyticus isolated from the same pulmonary fibrosis tissue of a mouse. The colicin peptides are indicated by bars in the fourth column. Outside the colicin peptide sequence, conserved amino acids are shaded in black, and similar amino acids are shaded in gray. Staphylococcus haemolyticus is Staphylococcus haemolyticus, and 1bp_00353, 12bp_00350, and 7bp_00350 are S. haemolyticus strain 1 protein number 353, S. haemolyticus strain 12 protein number 350, and S. haemolyticus strain 7 protein number 350, respectively. The protein of S. nepalensis strain CNDG is encoded by position 351 of the genome's open reading frame (ORF), counting from the gene encoding DnaA (ORF00001). Amino acids with similar properties are grouped as LIMV, AG, YWF, DEQN, KRH, and ST. Alignment was performed using multiple sequence comparison with Log-Expectation (MUSCLE (https: / / www.ebi.ac.uk / Tools / msa / muscle / )).

[0024] Figure 2 shows that culture supernatants from three strains of Staphylococcus haemolyticus induce apoptosis in alveolar epithelial cells. Figure 2a shows flow cytometry results for A549 alveolar epithelial cells cultured for 48 hours in the presence of culture supernatants from Staphylococcus haemolyticus strains 1, 7, and 12 (1 / 10 dilution). Fluorescein-labeled annexin V and propidium iodide staining were used as indicators. A549 cells treated with culture supernatant from Staphylococcus nepalensis strain CNDG served as a positive control, and saline-treated cells served as a negative control. Figure 2b shows the percentage of apoptotic cells determined and quantified by flow cytometry. N = 4 per group. Data are presented as mean ± SD. Statistical analysis was performed using Student's t-test. *** p < 0.0001. Figure 2c: A549 cells were cultured for 48 hours in the presence of S. haemolyticus culture supernatant and evaluated by transmission electron microscopy. Figure 2d: Western blotting results for caspase-3 cleavage in A549 cells cultured in the presence of the three S. haemolyticus strains 1, 7, and 12 and the culture supernatant of S. nepalensis. Figure 2e: A graph showing the results of caspase-3 cleavage in A549 cells cultured in the presence of the three S. haemolyticus strains 1, 7, and 12 and the culture supernatant of S. nepalensis. Data are presented as mean ± SD. Statistical analysis was performed using an unpaired t-test. **p < 0.001, ***p < 0.0001.

[0025] Figure 3 shows that synthetic colicin-like peptides of transglycosylases from several pathogens induce apoptosis in alveolar epithelial cells. Figure 3a, b: Flow cytometry analysis of A549 alveolar epithelial cells cultured in the presence of 10 μM colicin-like peptides to assess apoptosis. A549 cells treated with the synthetic colicin sequence or scrambled peptide served as positive and negative controls, respectively. Figure 3b, c: Graphs showing the percentage of apoptotic cells determined and quantified by flow cytometry. N = 4 per group. Data are presented as mean ± SD. Statistical analysis was performed using Student's t-test. *p < 0.05; ***p < 0.0001.

[0026] FIG. 4 shows an alignment of colicin-containing putative transglycosylases from various bacterial pathogens. The organism names are Wconf (Weissella confusa, GeneBank Protein Accession Number WP_112464134.1), CNDG-00351 (S. nepalensis strain CNDG Protein 00351), L. monocyt-2 (Listeria monocytogenes, GeneBank Protein Accession Number HAB0417320.1), Myabs-2 (Mycobacteroides abscessus, GeneBank Protein Accession Number SKR69498.1), Myabs-1 (Mycobacteroides abscessus, GeneBank Protein Accession Number SKT99287.1), Shem_00350 (Staphylococcus hemolyticus 12b Protein 00350), and L. monocyt-1 (L. monocytogenes - GeneBank Protein Accession Number ECO1693478.1). Colicin peptides are shown in bold, and conserved amino acids are labeled colicin (fifth row). Outside the colicin peptide sequence, conserved amino acids are shaded in black, and similar amino acids are shaded in gray. Amino acids with similar properties are grouped as LIMV, AG, YWF, DEQN, KRH, and ST. S, Staphylococcus aureus. Alignment was performed using multiple alignment comparison with Log-Expectation (MUSCLE (https: / / www.ebi.ac.uk / Tools / msa / muscle / )).

[0027] Figure 5 shows the apoptotic pathways induced by S. haemolyticus culture supernatant. Figure 5a: Apoptosis occurs via the extrinsic and intrinsic pathways. The extrinsic pathway is initiated by ligand binding to membrane-bound death receptors, resulting in activation of intracellular signaling and cleavage of procaspase-8 to caspase-8. The intrinsic pathway is regulated by mitochondria. In the presence of stimuli, perturbation of the mitochondrial membrane increases its permeability, triggering the release of suppressor of apoptosis repeat-containing (BIRC) proteins and proapoptotic factors, baculoviral inhibitor of apoptosis repeat-containing (BIRC) proteins, which contribute to the formation of the apoptosome, which cleaves procaspase-9 to caspase-9. Both caspase-8 and caspase-9 can activate caspase-3, an effector of apoptosis. Figure 5b: A549 alveolar epithelial cells were cultured for 24 hours in the presence of culture supernatant from S. haemolyticus strain 12 (1 / 10 dilution), and the percentage of cells positive for cleaved caspase-8, cleaved caspase-9, and cleaved caspase-3 was analyzed by flow cytometry. The bottom graph shows the percentage of positive cells relative to the control. N = 6 per group. Data are presented as mean ± SD. Statistical analysis was performed using Student's t-test. **p <0.01; ***p <0.0001.

[0028] Figure 6 is a graph showing that colicin alters mRNA expression of pro- and anti-apoptotic factors. N = 6 in each group. Data are presented as mean ± SD. Statistical analysis was performed using an unpaired t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.

[0029] Figure 7 shows the degradation of colicin-containing transglycosylase by the culture supernatant of Staphylococcus nepalensis strain CNDG and the preparation of a peptidase fraction. Figure 7a shows the amino acid sequences of the transglycosylase and colicin moieties of Staphylococcus nepalensis strain CNDG. Figure 7b shows the procedure for concentrating Staphylococcus nepalensis culture supernatant into specific fractions. Figure 7c shows the data and procedure for obtaining fractions with high digestive activity. Figure 7d shows the data and procedure for obtaining fractions with high digestive activity against recombinant transglycosylase 351 from fractions with high absorbance at OD (280 nm).

[0030] Figure 8 shows that bacterial secretory products induce apoptosis in lung epithelial cells by degrading transglycosylase. Figure 8a: Photographs showing sodium dodecyl sulfate-polyacrylamide gels after 2 hours of incubation of recombinant transglycosylase in the presence of various amounts of peptidase fractions prepared from Staphylococcus nepalensis culture supernatant. Figure 8b: Photographs showing sodium dodecyl sulfate-polyacrylamide gels after incubation of recombinant transglycosylase in the presence of a fixed amount of peptidase fraction for various time intervals. Figure 8c: Flow cytometry analysis of A549 alveolar epithelial cells after treatment with the indicated reaction mixtures. The dots in the upper panel (excluding the third quadrant) represent apoptotic cells, and the hump on the right side of the x-axis in the lower panel (c) represents apoptotic cells. Figure 8d: Graph showing the percentage of apoptotic cells relative to the control. N = 4 per group. Data are mean ± SD. Statistical analysis by one-way ANOVA with Tukey's test. *** p < 0.0001.

[0031] 9 is a photograph of a sodium dodecyl sulfate-polyacrylamide gel showing that culture supernatant of Staphylococcus haemolyticus degrades recombinant transglycosylase. Staphylococcus nepalensis supernatant served as a control.

[0032] Figure 10 shows that a putative bacterial serine protease cleaves transglycosylase. Figure 10a, b: Photographs and graphs showing the effect of the serine protease inhibitor Pefabloc SC (a, b) on transglycosylase degradation. Figure 10c, d: Photographs and graphs showing the effect of diisopropylfluorophosphate (DFP) on transglycosylase degradation. Figure 10e, f: Photographs and graphs showing the effect of the chelator ethylenediaminetetraacetic acid (EDTA) on transglycosylase degradation. Figure 10g, h: Photographs and graphs showing the effect of the cysteine ​​protease inhibitor E-64 on transglycosylase degradation.

[0033] Figure 11 shows that different classes of anti-colicin monoclonal antibodies bind to colicin peptides and sequences on transglycosylase 351. Figure 11a: The isotype of each monoclonal antibody is shown. Figure 11b: Western blotting results showing the binding of each monoclonal antibody to transglycosylase 351. Figure 11c: A graph evaluating the binding of each clone of anti-colicin monoclonal antibody. Data are presented as mean ± SD. The small band below the 26 kDa molecular weight marker is a degradation product of transglycosylase 351, which is known to be degraded during recombinant protein purification.

[0034] Figure 12 shows that rat anti-colicin monoclonal antibodies bind to the colicin consensus motifs PESSGNP or NPAGY. Figure 12a, b: Staining results of peptide microarray copies verifying overall peptide microarray integrity and assay quality. Figure 12c, d: Scan intensity of rat monoclonal (mAtb) antibody 9A. Figure 12e, f: Scan intensity of rat monoclonal (mAtb) antibody 21A. Figure 12g, h: Scan intensity of rat monoclonal (mAtb) antibody 2A. Figure 12i, j: Scan intensity of rat monoclonal (mAtb) antibody 4A.

[0035] Figure 13 shows the sequences of the variable framework and complementarity determining regions of anti-colicin monoclonal antibody clones. Lowercase letters indicate framework regions (FR1, FR2, FR3, FR4), and bold capital letters indicate CDR regions (CDR1, CDR2, CDR3) of the antibody variable regions.

[0036] Figure 14 shows graphs demonstrating that anti-colicin monoclonal antibody clone 21A potently neutralizes colicin-induced apoptosis in human alveolar epithelial cells. Figure 14a shows flow cytometry analysis results demonstrating the inhibitory effect of anti-colicin monoclonal antibodies on the proapoptotic activity of colicin on cultured alveolar epithelial cells. In the upper panel of Figure 14a, the dots in the third quadrant of the panel represent apoptotic cells. In the lower panel of Figure 14a, the hump on the right side of the x-axis represents apoptotic cells. Figure 14b shows the percentage of apoptotic cells relative to the control, as determined and quantified by flow cytometry. N = 3 per group. Data are presented as mean ± SD. Statistical analysis was performed using Student's t-test. *** p < 0.0001.

[0037] Figure 15 shows that anti-colicin monoclonal antibody clone 21A neutralizes the apoptotic activity of culture supernatant from Staphylococcus haemolyticus on human alveolar epithelial cells. Figure 15a: Flow cytometry analysis showing the inhibitory effect of anti-colicin monoclonal antibody clone 21A on the pro-apoptotic activity of Staphylococcus haemolyticus colicin. In the upper panel of Figure 15a, the dots in the third quadrant of the panel represent apoptotic cells. In the lower panel of Figure 15a, the hump on the right side of the x-axis represents apoptotic cells. Figure 15b: Graph showing the percentage of apoptotic cells relative to the control, as determined and quantified by flow cytometry. N = 4 per group. Data are presented as mean ± SD. Statistical analysis was performed using one-way analysis of variance with Tukey's test. *** p < 0.0001.

[0038] Figure 16 shows that anti-colicin neutralizing monoclonal antibodies inhibit apoptosis of lung cells induced by transglycosylase degradation products. Figure 16a: Flow cytometry analysis showing the inhibitory effect of anti-colicin neutralizing monoclonal antibodies on apoptosis of lung cells induced by transglycosylase degradation products. In the upper panel of Figure 16a, the dots in the third quadrant of the panel represent apoptotic cells. In the lower panel of Figure 16a, the bumps on the right side of the x-axis represent apoptotic cells. Figure 16b: Graph showing the percentage of apoptotic cells compared to the control, as determined by flow cytometry. N = 4 per group. Data are presented as mean ± SD. Statistical analysis was performed using one-way analysis of variance with Tukey's test. *** p < 0.0001. Figure 16c: The reaction mixture was electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and stained with silver.

[0039] FIG. 17 is a graph showing the mean half-life of neutralizing anti-colicin antibodies in plasma.

[0040] Figure 18 shows the concordance scores for pulmonary fibrosis between TGFβ1 TG mice administered anti-colicin antibody and TGFβ1 TG mice administered isotype antibody before intratracheal instillation of colicin. Figure 18a: Results of computed tomography (CT) performed before the start of treatment with anti-colicin monoclonal antibody (mAtb) or isotype antibody (Atb). N = 6 for the TGFβ1-TG / fibrosis(+) / anti-colicin antibody / colicin and TGFβ1-TG / fibrosis(+) / isotype antibody / colicin groups, and n = 5 for the TGFβ1-T / fibrosis(-) / colicin group. Figure 18b: Graph showing the mean scores scored by eight experts to grade pulmonary fibrosis on CT. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey's test. TGFβ1: transforming growth factor β1; TG: transgenic.

[0041] Figure 19 shows that monoclonal anti-colicin antibodies prevent the deterioration of lung CT findings in TGFβ1 TG mice. Figure 19a shows the schedule for obtaining CT findings in TGFβ1 TG mice with pulmonary fibrosis. The grade of pulmonary fibrosis was scored by blinded experts. Figures 19b and 19c show graphs of CT findings and scores before and after intratracheal instillation of colicin in the TGFβ1-TG / fibrosis(-) / colicin group. Statistical analysis was performed by paired Student's t-test. ns, not significant. Figures 19d and 19e show graphs of CT findings and scores before and after intratracheal instillation of colicin in the TGFβ1-TG / fibrosis(+) / colicin / isotype group. Bars indicate mean scores. Statistical analysis was performed by paired t-test. *p<0.05. TGFβ1: transforming growth factor β1; TG: transgenic. Figure 19f, g: Graphs showing CT findings and scores before and after intratracheal instillation of colicin in the TGFβ1-TG / fibrosis(+) / colicin / anti-colicin group. Bars indicate mean scores. Statistical analysis was performed by paired t-test. **p<0.01.

[0042] Figure 20 shows that monoclonal anti-colicin antibodies suppress acute exacerbations (AEs) of pulmonary fibrosis in TGFβ1-TG mice. Figure 20a: Giemsa staining of differential cell counts. The scale bar indicates 50 μm. Figure 20b: Graphs showing the total number of cells and neutrophils in BALF. Data are means ± SD. Statistical analysis was performed by one-way analysis of variance with the Neuman-Keuls test. * p <0.05; ** p <0.01; *** p <0.0001. Figure 20c: Graphs showing the levels of surfactant protein D (SP-D), MUC5B protein, matrix metalloproteinase-1 (MMP-1), decorin, and MUC-1 measured by commercially available enzyme immunoassay kits. Hydroxyproline levels were measured using a commercially available colorimetric kit according to the manufacturer's instructions. Data are means ± SD. Statistical analysis was performed by one-way analysis of variance with the Neuman-Keuls test. *p<0.05; **p<0.01; ***p<0.0001. Figure 20d, e: Photographs and graphs showing collagen deposition in lung tissues of each group by Masson staining. The scale bar indicates 200 μm. Data are mean ± SD. Statistical analysis was performed by one-way analysis of variance with the Neuman-Keuls test. *p<0.05; ***p<0.0001. Figure 20f: Graph showing the correlation between the fibrotic area and the total number of inflammatory cells described in Figure 20b. Statistical analysis was performed using Spearman's correlation coefficient.

[0043] Figure 21 shows the experimental schedule for induction of acute exacerbations in mice with bleomycin-induced pulmonary fibrosis and the CT criteria for pulmonary fibrosis. Figure 21a shows the experimental schedule for induction of acute exacerbations in mice with bleomycin-induced pulmonary fibrosis. Figure 21b shows the CT criteria for pulmonary fibrosis. The following CT scoring system was used to determine the grade of pulmonary fibrosis: Score 1: normal findings; Score 2: slightly increased lung density; Score 3: patchy ground-glass opacity and septal thickening; Score 4: moderate ground-glass opacity and septal thickening; Score 5: subpleural thickening, diffuse ground-glass opacity, and reticular septal thickening; Score 6: subpleural thickening, diffuse ground-glass opacity, reticular septal thickening, and traction bronchiectasis; Score 7: subpleural thickening, severe ground-glass opacity, reticular septal thickening, and diffuse traction bronchiectasis.

[0044] Figure 22 shows the worsening of lung CT findings in mice with bleomycin-induced pulmonary fibrosis after intratracheal instillation of colicin. Figure 22a, b, c: Graphs showing lung CT findings and scores in mice with bleomycin-induced pulmonary fibrosis after intratracheal instillation of colicin. Number of mice in the WT / BLM / scrambled peptide group: n = 5, WT / BLM / colicin group: n = 6. Statistical analysis by Student's paired t-test. *p < 0.05; ns, not significant. Figure 22d, e, f: Graphs showing lung CT findings and scores in mice with saline-administered pulmonary fibrosis after intratracheal instillation of colicin. Number of mice: n = 3 in the WT / SAL / scrambled peptide and WT / SAL / colicin groups. Statistical analysis by paired t-test. ns, not significant.

[0045] Figure 23 shows the acute exacerbation of pulmonary fibrosis in mice with bleomycin-induced pulmonary fibrosis after intratracheal instillation of colicin. Figure 23a shows representative photomicrographs of BALF cell staining in each treatment group in mice with BLM-induced pulmonary fibrosis administered with colicin. Figure 23b shows a graph showing the number of stained BALF cells in each treatment group in mice with BLM-induced pulmonary fibrosis administered with colicin. The scale bar indicates 20 μm. Data are mean ± SD statistical analysis by t-test. *p <0.05; *p <0.01. Number of mice in the WT / SAL / scrambled peptide and WT / SAL / colicin groups: n = 3, and in the WT / BLM / scrambled peptide and WT / BLM / colicin groups: n = 5. Figure 23c: Graphs showing serum amyloid P component (SAP), MUC-1, monocyte chemoattractant protein-1 (MCP-1), periostin, collagen I, and osteopontin levels in colicin-treated BLM-induced pulmonary fibrosis mice. Data are mean ± SD statistical analysis by unpaired t-test. *p <0.05; *p <0.01; ***p <0.001. Figure 23d: Western blotting analysis of caspase-3 cleavage in colicin-treated BLM-induced pulmonary fibrosis mice, assessing and quantifying the cleavage. Figure 23e: Graphs showing the ratio of cleaved caspase-3 to β-actin in colicin-treated BLM-induced pulmonary fibrosis mice. Bars represent mean ± SD statistical analysis by unpaired t-test. *p <0.05. Figure 23f: Quantification of collagen deposition. Figure 23g: Quantification of collagen deposition. Data are mean ± SD statistical analysis by unpaired t-test. *p<0.05. Figure 23h: Graph showing the measurement results of lung tissue content of hydroxyproline. Bars represent mean ± SD statistical analysis by unpaired t-test. *p<0.05, **p<0.001.

[0046] Figure 24 shows the improvement of lung CT findings and the reduction of pulmonary inflammatory cell infiltration in mice with bleomycin-induced pulmonary fibrosis treated with anti-colicin monoclonal antibodies. Figure 24a shows the schedule for investigating the pulmonary fibrosis-inhibitory effect of anti-colicin monoclonal antibodies. Figures 24b and 24c show lung CT findings in mice with bleomycin-induced pulmonary fibrosis treated with anti-colicin monoclonal antibodies. Figure 24c shows a graph of the CT scores reflecting the results of Figure 24b. Number of mice in the WT / SAL / isotype and WT / SAL / anti-colicin groups: n = 4, and in the WT / BLM / anti-colicin and WT / BLM / isotype groups: n = 9. Data are mean ± SD statistical analysis by one-way analysis of variance with Tukey's test. *** p < 0.001. Figures 24d and 24e show photographs of cells in BALF from mice with bleomycin-induced pulmonary fibrosis treated with anti-colicin monoclonal antibodies. e) Total and differential cell counts in BALF from mice with bleomycin-induced pulmonary fibrosis treated with anti-colicin monoclonal antibody. Number of mice in the WT / SAL / isotype and WT / SAL / anti-colicin groups: n = 4, and in the WT / BLM / anti-colicin and WT / BLM / isotype groups: n = 9. Scale bars represent 50 μm. Data are mean ± SD statistical analysis by one-way ANOVA with Tukey's test. **p <0.01, ***p <0.0001.

[0047] Figure 25 shows the improvement of acute tissue injury, parenchymal cell apoptosis, and tissue fibrosis in the lungs of pulmonary fibrosis mice treated with anti-colicin monoclonal antibodies. Figure 25a: Graph showing osteopontin, MUC-1, and MUC5B levels in the lungs of pulmonary fibrosis mice treated with anti-colicin monoclonal antibodies. N = 4 for the WT / SAL / isotype and WT / SAL / anti-colicin groups, and n = 9 for the WT / BLM / isotype and WT / BLM / anti-colicin groups. Bars represent mean ± SD statistical analysis by one-way ANOVA with Tukey's test. * p < 0.05, ** p < 0.01, *** p < 0.0001. Figure 25b: Photograph showing DNA fragmentation in the lungs of pulmonary fibrosis mice treated with anti-colicin monoclonal antibodies. Arrows indicate apoptotic cells. Figure 25c: Graph showing the quantification of apoptotic cells in the lungs of pulmonary fibrosis mice treated with anti-colicin monoclonal antibodies. Scale bars represent 20 μm. N = 4 for the WT / SAL / isotype and WT / SAL / anti-colicin groups, and n = 9 for the WT / BLM / isotype and WT / BLM / anti-colicin groups. Bars represent mean ± SD statistical analysis by one-way analysis of variance with Tukey's test. ***p <0.0001. Figure 25d, e: The grade of pulmonary fibrosis in the lungs of pulmonary fibrosis mice treated with anti-colicin monoclonal antibody was scored blindly by seven experts using the Ashcroft score. Scale bars represent 200 μm. N = 4 for the WT / SAL / isotype and WT / SAL / anti-colicin groups, and n = 9 for the WT / BLM / isotype and WT / BLM / anti-colicin groups. Bars represent mean ± SD statistical analysis by one-way analysis of variance with Tukey's test. *p <0.05, ***p <0.0001. Figure 25f, g: (f) Masson's trichrome stained photographs of lung collagen deposition, and (g) a graph quantified using WindRoof imaging software. N = 4 for the WT / SAL / isotype and WT / SAL / anti-colicin groups, and n = 9 for the WT / BLM / isotype and WT / BLM / anti-colicin groups.Bars represent mean ± SD statistical analysis by one-way ANOVA with Tukey's test. *p<0.05, ***p<0.0001. Figure 25h: Graph showing lung tissue content of hydroxyproline measured by colorimetry. N=4 for WT / SAL / isotype and WT / SAL / anticolicin groups, n=9 for WT / BLM / isotype and WT / BLM / anticolicin groups. Bars represent mean ± SD statistical analysis by one-way ANOVA with Tukey's test. ***p<0.0001.

[0048] Figure 26 shows the reduction in lung CT abnormalities and pulmonary inflammatory cells on day 9 in mice with bleomycin-induced pulmonary fibrosis treated with anti-colicin monoclonal antibodies during the acute phase of the disease. Figure 26a shows the timeline for examining the ameliorative effect of anti-colicin monoclonal antibodies treated during the acute phase on BLM-induced lung injury and pulmonary fibrosis. Figures 26b and 26c show: (a) a photograph showing CT lung opacity in BLM-induced lung injury and pulmonary fibrosis treated with anti-colicin monoclonal antibodies during the acute phase, and (c) a graph of CT scores reflecting the results of (b). N = 7 mice treated with anti-colicin monoclonal antibodies (WT / BLM / anti-colicin) or isotype antibodies (WT / BLM / isotype). Data are means ± SD. Statistical analysis was performed by unpaired t-test. * p < 0.05. Figure 26d, e: (d) is a photograph showing blood cells in BLM-induced lung injury and pulmonary fibrosis in anti-colicin monoclonal antibody treated acutely, and (e) is a graph showing the total and differential cell counts. N = 7 mice treated with anti-colicin monoclonal antibody (WT / BLM / anti-colicin) or isotype antibody (WT / BLM / isotype). The scale bar represents 20 μm. Data are mean ± SD statistical analysis by unpaired t-test. ** p < 0.01.

[0049] Figure 27 shows the reduction of lung CT abnormalities and lung inflammatory cells on day 22 in mice with bleomycin-induced pulmonary fibrosis treated with anti-colicin monoclonal antibody during the acute phase of the disease. Figures 27a and 27b: (a) is a photograph showing CT images 21 days (chronic phase) after BLM pump implantation in mice with bleomycin-induced pulmonary fibrosis treated with anti-colicin monoclonal antibody during the acute phase of the disease, and (b) is a graph showing the CT fibrosis score on day 22. N = 7 for both treatment groups. Data are mean ± SD. Statistical analysis was performed by unpaired t-test. ** p < 0.01. Figures 27c and d: (c) is a photograph showing cell differential staining after Giemsa staining in bronchoalveolar lavage fluid (BALF) 22 days after BLM pump implantation in mice with bleomycin-induced pulmonary fibrosis treated with anti-colicin monoclonal antibody during the acute phase of the disease, and (d) is a graph showing the total cell count. N = 7 for both treatment groups. Scale bar indicates 20 μm. Data are mean ± SD. Statistical analysis by unpaired t-test. *** p < 0.0001.

[0050] Figure 28 shows the improvement of lung injury, lung parenchymal apoptosis, and collagen deposition on day 22 in mice with bleomycin-induced pulmonary fibrosis treated with anti-colicin monoclonal antibody during the acute phase of the disease. Figure 28a: Graphs showing the levels of MUC-1, surfactant protein C (SP-C), SP-D, collagen I, periostin, osteopontin, and total TGF-β1 in BALF and plasma collected 22 days after BLM pump infusion. Data are means ± SD. Statistical analysis was performed by unpaired t-test. * p <0.05, ** p <0.01, *** p <0.001. Figures 28b and c: (b) shows the results of Western blotting of cleaved caspase-3 and β-actin, and (c) shows a graph of the ratio of cleaved caspase-3 to β-actin, which is the result of (b). N = 7 for the WT / BLM / isotype group and n = 6 for the WT / BLM / anti-colicin group. Data are means ± SD. Statistical analysis was performed by unpaired t-test. *p<0.05. Figure 28d, e: Seven experts blindly scored the grade of pulmonary fibrosis using the Ashcroft score. Scale bar indicates 200 μm. N = 7 in both the WT / BLM / isotype and WT / BLML / anti-colicin groups. Bars indicate mean ± SD. Statistical analysis was performed by unpaired t-test. *p<0.05. Figure 28f, g: (f) Photographs of Masson's trichrome stained lung collagen deposition, and (g) graphs of quantification using WindRoof Image software. Figure 28h: Graph showing lung hydroxyproline content. N = 7 in both the WT / BLM / isotype and WT / BLML / anti-colicin groups. Bars indicate mean ± SD. Statistical analysis was performed by unpaired t-test. *p<0.05; ***p<0.0001.

[0051] Figure 29 shows that anti-colicin monoclonal antibodies prolong the survival rate of mice with acute exacerbation of pulmonary fibrosis. Figures 29a and 29b: (a) shows the results of computed tomography (CT) in mice with acute exacerbation of pulmonary fibrosis, and (b) is a graph of the CT scores reflecting the results of (b). N = 22 in both the WT / BLM / isotype and WT / BLM / anti-colicin groups, and n = 5 in both the WT / SAL / isotype and WT / SAL / anti-colicin groups. Bars indicate mean ± SD. Statistical analysis was performed by one-way analysis of variance with Tukey's test. * p < 0.05. Figure 29c: A graph showing the results of monitoring survival. Statistical analysis was performed by the log-rank test. p = 0.001.

[0052] Figure 30 shows that anti-colicin monoclonal antibodies attenuate severe lipopolysaccharide-induced acute lung injury. Figure 30a shows the timeline for investigating the ability of anti-colicin monoclonal antibodies to attenuate severe lipopolysaccharide-induced acute lung injury. Figure 30b shows representative photomicrographs of BALF cell staining for each treatment group. Figure 30c shows a graph showing the number of cells in BALF for each treatment group. The scale bar represents 20 μm. N = 4 for the SAL / isotype and SAL / anti-colicin groups, n = 5 for the LPS / isotype group, and n = 8 for the LPS / anti-colicin group. Data are mean ± SD statistical analysis by one-way ANOVA with Neuman-Keuls test. #p = 0.1, ***p < 0.0001. Figure 30d: Graph showing the levels of lactate dehydrogenase A (LDHA), surfactant protein D (SP-D), matrix metalloproteinase-1 (MMP-1), MUC-1, and tumor necrosis factor alpha (TNFα) in mice with severe lipopolysaccharide-induced acute lung injury. N = 4 for the SAL / isotype and SAL / anticolicin groups, n = 5 for the LPS / isotype group, and n = 8 for the LPS / anticolicin group. Data are mean ± SD statistical analysis by Newman-Keuls analysis of variance. #p = 0.1, *p < 0.05, **p < 0.01, ***p < 0.001.

[0053] Figure 31 shows that treatment with anti-colicin monoclonal antibody ameliorates CT findings in mice with severe lipopolysaccharide-induced acute lung injury. Figure 31a shows the results of computed tomography (CT) performed 1 day after intratracheal instillation of lipopolysaccharide (LPS). N = 4 for the WT / SAL / isotype and WT / SAL / anti-colicin groups, n = 5 for the WT / LPS / isotype group, and n = 8 for the WT / LPS / anti-colicin group. Figure 31b shows a graph showing CT opacity from the apex to the basal region of the lung, quantified using WindRoof imaging software. Data are mean ± SD. Statistical analysis was performed by one-way analysis of variance with the Neuman-Keuls test. *p <0.05, ***p <0.001.

[0054] Figure 32 shows that anti-colicin monoclonals attenuate moderate lipopolysaccharide-induced acute lung injury. Figure 32a: Schedule for investigating whether anti-colicin monoclonals attenuate moderate lipopolysaccharide-induced acute lung injury. Figure 32b: Representative photomicrographs of BALF cell staining for each treatment group. Figure 32c: Graph showing the number of stained BALF cells for each treatment group. Scale bar indicates 20 μm. N = 4 for the LPS / isotype group and n = 5 for the LPS / anti-colicin group. Data are mean ± SD statistical analysis by unpaired test. *** p < 0.001. Figure 32d: BALF and plasma levels of surfactant protein D (SP-D), monocyte chemoattractant protein-1 (MCP-1), TNFα, lactate dehydrogenase A (LDHA), MUC-5B, and matrix metalloproteinase-1 (MMP-1) in mice with moderate lipopolysaccharide-induced acute lung injury treated with anti-colicin monoclonal antibody. N = 4 in the LPS / isotype group and n = 5 in the LPS / isotype group. Data are mean ± SD. Statistical analysis by analysis of variance using the Newman-Keuls method. * p < 0.05, ** p < 0.01, *** p < 0.001.

[0055] Figure 33 shows that elevated circulating colicin levels correlate with markers of pulmonary fibrosis. Figure 33a shows the timeline for measuring plasma colicin levels in TGFβ1 transgenic (TG) and wild-type (WT) mice and assessing correlation with fibrosis markers. N = 16 for WT mice and n = 20 for TGFβ1TG mice. Figure 33b shows the Ashcroft score for pulmonary fibrosis and the measurement of plasma colicin and lung hydroxyproline in TGFβ1TG and WT mice. Statistical differences between groups were analyzed by unpaired t-test, and the strength of the relationship was analyzed by Pearson product-moment correlation. * p < 0.05, ** p < 0.01, *** p < 0.001.

[0056] Figure 34 shows an increase in pulmonary fibrosis CT score in TGFβ1TG mice. Figure 34a: Results of pulmonary fibrosis CT score in TGFβ1TG mice. Figure 34b: Graph showing CT opacity from the apex to the basal region of the lung quantified using WindRoof imaging software. N = 16 for WT mice and n = 20 for TGFB1TG mice. WT: wild-type; TGFβ1TG: transforming growth factor-β1; CT: computed tomography. Data are mean ± SD. Statistical analysis was performed by unpaired Student's t-test. *** p < 0.001.

[0057] FIG. 35 is a graph showing the apoptotic activity of colicin peptides with specific amino acid mutations.

[0058] Figure 36 shows a multiple sequence alignment of the conserved sequences of the pro-apoptotic segments of transglycosylases in several species of staphylococci and streptococci. Figure 37 shows a multiple sequence alignment of the conserved sequences of the pro-apoptotic segments of transglycosylases in several species of staphylococci and streptococci. Figure 38 shows a multiple sequence alignment of the conserved sequences of the pro-apoptotic segments of transglycosylases in several species of staphylococci and streptococci. Colicins shown in Figures 36, 37, and 38C include, for example, the amino acid sequences of IVMPESGGNPNAVNPAGYR (SEQ ID NO:58), IIMPESGGNPNIVNPYGYS (SEQ ID NO:59), IVMPESGGNPNAVNPYGYR (SEQ ID NO:60), IVLPESSGNPNAVNPAGYR (SEQ ID NO:61), IVLPESSGNPNAVNELGYR (SEQ ID NO:62), IVMPESGGNPNAVNELGYR (SEQ ID NO:63), IVMPESSGNPNAVNELGYR (SEQ ID NO:64), IVMPESSGNPDAVNELGYR (SEQ ID NO:65), IAQRESGGDLKAVNPSSGA (SEQ ID NO:66), or IAERESGGDLKAVNPSSGA (SEQ ID NO:67), which can be used in one or more aspects of the present invention.

[0059] Figure 39 shows photographs of cleaved caspase-3-positive cells in colonic epithelial cells, small intestinal epithelial cells, skin keratinocytes, retinal epithelial cells, kidney podocytes, and ureteral epithelial cells cultured with colicin, colicin-like peptides, or scrambled peptides, and a graph of the ratio of caspase-3-positive cells to control β-actin-positive cells. Figure 40 shows a graph comparing serum colicin concentrations in healthy subjects, patients with stable idiopathic pulmonary fibrosis (IPF), and patients with acute exacerbations of IPF. Colicin was measured by enzyme immunoassay using rabbit polyclonal anti-transglycosylase and rat monoclonal anti-colicin antibodies. Healthy subjects, n = 8; IPF patients, n = 22; IPF patients with acute exacerbations, n = 22. Bars indicate mean ± SD. Statistical analysis was performed using a t-test. *p <0.05, **p <0.001. Figures 41a and b: Flow cytometry analysis of apoptosis in A549 alveolar epithelial cells cultured with Staphylococcus nepalensis culture supernatant (1:10 dilution) in the presence of anti-colicin antibodies. Bars represent mean ± SD. Statistical analysis was performed using ANOVA and Newman-Keuls test. ****p <0.0001. Figure 41c: Concentration of native colicin in undiluted culture supernatants of Staphylococcus nepalensis and Staphylococcus haemolyticus. Bars represent mean ± SD. Figure 41d: A549 alveolar epithelial cells were cultured in bronchoalveolar lavage fluid (BALF) (1:2 dilution) from healthy subjects (HC; n = 5) and patients with acute exacerbation of idiopathic pulmonary fibrosis (IPF; n = 14) in the presence of anti-colicin neutralizing antibody (clone 21A) (Anticorisin) or control IgG (Control IgG). Flow cytometry analysis revealed apoptosis. Bars represent mean ± SD. Figure 41e: Comparison of the percentage of apoptotic cells induced by BALF from all healthy subjects (n = 5) and IPF patients (n = 14) in Figure 41d between the anti-colicin antibody and control IgG groups. Bars represent mean ± SD.Statistical analysis was performed using the Wilcoxon test. ***p<0.001. ns, not significant. Figure 41f: Graph showing the concentration of natural colicin in undiluted BALF from healthy subjects (n = 5) and IPF patients (n = 14). Bars indicate mean ± SD. **p<0.01. Figure 41g: A549 alveolar epithelial cells were cultured, and synthetic colicin was added to the cell culture medium at a final concentration of 10 μg / ml. The medium was collected after 0, 1, 3, 6, 12, and 24 hours, and the colicin concentration was measured. Results: The in vitro half-life of colicin is shown.

[0060] Monoclonal Antibody Molecules In one aspect, the present invention relates to isolated monoclonal antibody molecules or derivatives thereof that specifically bind to colicins, which in the context of the present invention generally refer to staphylococcal pro-apoptotic peptides that have been found to induce acute exacerbations of pulmonary fibrosis. The colicin may have, for example, one of the amino acid sequences IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1), IVMPESGGNPNAVNPAGYR (SEQ ID NO: 58), IIMPESGGNPNIVNPYGYS (SEQ ID NO: 59), IVMPESGGNPNAVNPYGYR (SEQ ID NO: 60), IVLPESSGNPNAVNPAGYR (SEQ ID NO: 61), IVLPESSGNPNAVNELGYR (SEQ ID NO: 62), IVMPESGGNPNAVNELGYR (SEQ ID NO: 63), IVMPESSGNPNAVNELGYR (SEQ ID NO: 64), IVMPESSGNPDAVNELGYR (SEQ ID NO: 65), IAQRESGGDLKAVNPSSGA (SEQ ID NO: 66), or IAERESGGDLKAVNPSSGA (SEQ ID NO: 67). Preferably, the colicin is a peptide having the amino acid sequence IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1).

[0061] The sequence of SEQ ID NO: 1 may originate from Staphylococcus nepalensis. With reference to Figures 36, 37 and 38, the origin of the sequences of other SEQ ID NOs: 58: 1 wp022791177 (top row 236) 59: 2 wp049409534 wp103371985 (top row 248) 60: 2 wp103328722 wp126565453 (top rows 239, 244) 61: 2 wp069827045 wp099091381 (top row 246) 62: 1 wp061853755 (top row 244) 63: 2 suk04795 wp105995336 (top row 116)・SEQ ID NO: 64: wp112369066 (top row 252) 1 ・SEQ ID NO: 65: wp099090334 (top row 120) 1 ・SEQ ID NO: 66: wp107644182 to wp119486153 (top row 222 to 223) 92 ・SEQ ID NO: 66: wp096808177 to CNDG00159 (top row 229) 2

[0062] The conservation of colicins was revealed by phylogenetic analysis. To clarify the evolutionary relationships of transglycosylases expressed by different bacteria, a phylogenetic tree was constructed based on the amino acid sequences of six transglycosylases identified in the genome of Staphylococcus nepalensis strain CNDG and their homologs, according to a publicly available database (www.ncbi.nlm.nih.gov / pubmed). The topology of the tree indicates that transglycosylase derivatives close to the ancestral sequence are divided into two IsaA clusters (IsaA-1 and IsaA-2), and that proteins termed SceD members likely evolved from IsaA-1-related sequences (SceD-1, SceD-2, SceD-3, and SceD-4). Multiple alignment of IsaA and SceD amino acid sequences generally revealed the conservation of amino acid residues that represent pro-apoptotic colicins, highlighting their functional significance (Figures 36, 37, and 38). The amino acid sequence identity of colicin-homologous transglycosylases from Staphylococcus xylosus, Staphylococcus cohnii, and Staphylococcus nepalensis was 100%. Furthermore, these staphylococci shared over 98% identity with the corresponding colicin regions of transglycosylases from other members of the IsaA-1 and IsaA-2 clusters and 60% identity with the corresponding regions of members of the SceD cluster (Figures 36, 37, and 38). The genomic organization of gene clustering around the transglycosylases (synteny) tended to be conserved in Staphylococcus cohnii and Staphylococcus nepalensis.

[0063] In one embodiment, the present invention relates to a monoclonal antibody molecule or derivative thereof of the present invention, which has an epitope consisting of an amino acid sequence of PESSGNP (SEQ ID NO: 68) or NPAGY (SEQ ID NO: 69). In the present invention, various anti-colicin monoclonal antibodies having binding properties to colicin molecules were prepared, and epitope mapping to identify their binding sites was performed, resulting in the discovery of an epitope consisting of the amino acid sequence of PESSGNP (SEQ ID NO: 68) or NPAGY (SEQ ID NO: 69) ( FIG. 12 ). It is believed that a monoclonal antibody molecule or derivative thereof targeting this epitope has equivalent binding properties to the colicin and equivalent functions, such as apoptosis inhibition and / or neutralizing activity. Therefore, a preferred monoclonal antibody molecule or derivative thereof of the present invention has neutralizing activity against colicin. This includes neutralizing antibodies.

[0064] In one specific embodiment, the present invention provides: (IA) (i) an H-chain variable region comprising the H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 37, the H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 38, and the H-chain variable region CDR3 amino acid sequence of SEQ ID NO: 39, and (ii) an L-chain variable region comprising the L-chain variable region CDR1 amino acid sequence of SEQ ID NO: 41, the L-chain variable region CDR2 amino acid sequence of SEQ ID NO: 42, and the L-chain variable region CDR3 amino acid sequence of SEQ ID NO: 43; (IIA); (i) an H-chain variable region comprising the H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 70, the H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 49, and the H-chain variable region CDR3 amino acid sequence of SEQ ID NO: 50, and (ii) an L-chain variable region comprising the L-chain variable region CDR1 amino acid sequence of SEQ ID NO: 52, the L-chain variable region CDR2 amino acid sequence of SEQ ID NO: 53, and the L-chain variable region CDR3 amino acid sequence of SEQ ID NO: 54; or (IVA) The present invention relates to an A-series monoclonal antibody molecule or a derivative thereof, which specifies the monoclonal antibody of the present invention by its CDRs, comprising: (i) an H-chain variable region comprising the H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 72, the H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 73, and the H-chain variable region CDR3 amino acid sequence of SEQ ID NO: 74; and (ii) an L-chain variable region comprising the L-chain variable region CDR1 amino acid sequence of SEQ ID NO: 76, the L-chain variable region CDR2 amino acid sequence of SEQ ID NO: 77, and the L-chain variable region CDR3 amino acid sequence of SEQ ID NO: 78.

[0065] In the present invention, a "monoclonal antibody molecule" refers to an immunoglobulin molecule having a tetrapeptide chain structure in which two identical heavy chains (H chains) and two identical light chains (L chains) are interconnected by disulfide bonds. Approximately 110 amino acids adjacent to the N-terminus of each antibody H and L chain are highly variable and known as the variable region (Fv region). The remaining amino acid sequences near the C-terminus are relatively stable and known as the constant region. The variable region comprises three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and each heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3, and the three heavy chain CDRs are referred to as HCDR1, HCDR2, and HCDR3. Antibody molecules include non-human antibody molecules, humanized antibody molecules, and human antibody molecules.

[0066] In the present invention, "isolated" refers to a sample that has been separated from its original natural environment. For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide that has been artificially separated from some or all of the samples with which it coexists in the natural system is isolated.

[0067] In the present invention, "derivatives of monoclonal antibody molecules" refer to diabodies and single-chain molecules, as well as "antigen-binding fragments." "Antigen-binding fragments" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, and colicin-binding Fv and scFv fragments that have antigen-binding activity, and may contain one or more CDR regions of the antibody defined in the present invention. An Fv fragment is the minimum antibody fragment that contains the entire antigen-binding site without the heavy chain variable region, light chain variable region, and constant region. Generally, Fv antibodies further contain a polypeptide linker between the heavy chain variable region and light chain variable region domain, allowing them to form the structure necessary for antigen binding. Alternatively, different linkers can be used to link two antibody variable regions to form a polypeptide called a single-chain antibody or single-chain Fv (scFv). As used herein, the "antigen-binding site" of the present invention refers to a discrete, three-dimensional site on the antigen recognized by the antibody or antigen-binding fragment of the present invention. As used herein, "specifically recognizing a colicin" means having the ability to bind to a colicin, which is synonymous with "an antibody that binds to a colicin" or "an antibody against a colicin."

[0068] In another specific embodiment, the present invention provides: (IB) an H-chain variable region comprising the H-chain variable region amino acid sequence of SEQ ID NO: 36, or an amino acid sequence at least 80% identical thereto, or an H-chain variable region comprising at least one framework (FW) region comprising an amino acid sequence in the H-chain variable region amino acid sequence of SEQ ID NO: 36 that has one or several amino acid residue substitutions, deletions, insertions, or additions compared to the H-chain variable region amino acid sequence of SEQ ID NO: 36; (IIB) an H-chain variable region comprising the H-chain variable region amino acid sequence of SEQ ID NO: 48, or an amino acid sequence at least 80% identical thereto, or an H-chain variable region comprising at least one framework (FW) region comprising an amino acid sequence in the H-chain variable region amino acid sequence of SEQ ID NO: 48 that has one or several amino acid residue substitutions, deletions, insertions, or additions compared to the H-chain variable region amino acid sequence of SEQ ID NO: 48; or (IVB) The present invention relates to a B-series monoclonal antibody molecule or derivative thereof, which specifies the monoclonal antibody of the present invention by its H-chain variable region and a mutant type, having an H-chain variable region comprising the H-chain variable region amino acid sequence of SEQ ID NO: 71 or an amino acid sequence at least 80% identical thereto, or an H-chain variable region comprising at least one framework (FW) region comprising an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted, or added compared to the H-chain variable region amino acid sequence of SEQ ID NO: 71. In the present invention, a monoclonal antibody molecule or derivative thereof having a mutation has binding characteristics and functions equivalent to those of the original monoclonal antibody molecule without the mutation.

[0069] In yet another specific embodiment, the present invention provides: (IC) an L chain variable region comprising the L chain variable region amino acid sequence of SEQ ID NO: 40, or an amino acid sequence at least 80% identical thereto, or an L chain variable region comprising at least one framework region comprising an amino acid sequence in the L chain variable region amino acid sequence of SEQ ID NO: 40 which has one or several amino acid residue substitutions, deletions, insertions or additions compared to the L chain variable region amino acid sequence of SEQ ID NO: 40; (IIC) an L chain variable region comprising the L chain variable region amino acid sequence of SEQ ID NO: 51, or an amino acid sequence at least 80% identical thereto, or an L chain variable region comprising at least one framework region comprising an amino acid sequence in the L chain variable region amino acid sequence of SEQ ID NO: 51 which has one or several amino acid residue substitutions, deletions, insertions or additions compared to the L chain variable region amino acid sequence of SEQ ID NO: 51; (IIIC) an L chain variable region comprising the L chain variable region amino acid sequence of SEQ ID NO: 44, or an amino acid sequence at least 80% identical thereto, or an L chain variable region comprising at least one framework region comprising an amino acid sequence in the L chain variable region amino acid sequence of SEQ ID NO: 44 which has one or several amino acid residue substitutions, deletions, insertions or additions compared to the L chain variable region amino acid sequence of SEQ ID NO: 44; or (IVC) The present invention relates to a C-series monoclonal antibody molecule or derivative thereof, which specifies the monoclonal antibody of the present invention by its L-chain variable region and mutant type, having an L-chain variable region comprising the L-chain variable region amino acid sequence of SEQ ID NO: 75 or an amino acid sequence at least 80% identical thereto, or an L-chain variable region comprising at least one framework region comprising an amino acid sequence in which the L-chain variable region amino acid sequence of SEQ ID NO: 75 has one or more amino acid substitutions, deletions, insertions, or additions compared to the L-chain variable region amino acid sequence. In the present invention, a monoclonal antibody molecule or derivative thereof having a mutation has binding characteristics and functions equivalent to those of the original monoclonal antibody molecule without the mutation.

[0070] In another specific embodiment, the present invention relates to a monoclonal antibody molecule or a derivative thereof comprising any one of the heavy chain variable regions of the present invention and any one of the light chain variable regions of the present invention. Preferred combinations of heavy and light chain variable regions are those of the isolated monoclonal antibody clones 4A, 9A, 2A, and 21A, respectively. Specifically, the following combinations are used:

[0071] Anti-colicin monoclonal antibody clone 4A: Monoclonal antibodies (IA), (IB), (IC) related: H chain (4A-HC.7331): SEQ ID NO: 36 evqvlesggglvqpgnslklscatsGFTFSTAWmywyrqfpekrlewvarIKAKSNSYATdytesvkgrftisrddskgsiylrmnnlkeedtaiyycASTDAFYFSHSYwgqgvlvtvss, and L chain (4A-LC.7395): SEQ ID NO: 40 divmtqtpssqavsagekvtmrcrssQSLLYSENKKNYlawyqqkpgrspklliyWASTgesgvpdrfigsgsgtdftltissvqaedlavyycQQYYNFPLTfgsgtkleik

[0072] Anti-colicin monoclonal antibody clone 9A: Monoclonal antibody (IIIC) related: L chain (9A-LC.7402): SEQ ID NO: 44 divmtqspssqavsagekvtmsckssQNLLYSEDKKNYlawyqqkpgqspklliyWAStresgvpdrfigsgsgtdftltvtsvqaedlavyycQQYYNFPRTfgggtklelk

[0073] Anti-colicin monoclonal antibody clone 2A: Monoclonal antibodies (IIA), (IIB), (IIC) related: H chain (2A-HC.7352): SEQ ID NO: 48 evklvesggglvqpgnsltlscvasGFTFTNYGmhwirqapkkglewiamIYYDSSKMsyadtvkgrftisrdnskntlylemnslrsedtamyycAAEGFGTPFPYwgqgtlvtvss, and L chain (2A-LC.7413): SEQ ID NO: 51 divmtqtpssravsagekvtmsckssQSLLYSENEKNYlawyqqrpgqspklliyWAStresgvpdrfigtgsgtdftltissvqaedlavyycQQYYHFPRTfgggtrlelk

[0074] Anti-colicin monoclonal antibody clone 21A: monoclonal antibodies (IVA), (IVB), (IVC) related: H chain (KK1410-2_VH_pep): SEQ ID NO: 71 evklvesggglvqpgnsltlscgasgftftNYGMHwirqapkkglewigMIYYDSSKMSYADTVKGrftisrdnsknilylemnslrsedtamyycaaEGFGTPFPYwgqgtlvtvss, and L chain (KK1410-2_VL_pep): SEQ ID NO: 75 divmtqtpssqavsagekvtmscKSSQSLLYREDTKNYLAwyqqrpgqspklliyWASTRESgvpdrfigsgsgtdftltissvqaedlavyycQQYYHFPRTfgggtrlelk.

[0075] As described above, the monoclonal antibody molecules of the present invention can have any of the heavy chain variable regions and / or any of the light chain variable regions. In (IB), (IIB), (IVB), (IC), (IIC), (IIIC), and (IVC), 80%, preferably 85%, more preferably 90%, and even more preferably 95%. The heavy and light chain variable regions are highly variable because they can be subdivided into regions of hypervariability termed "complementarity-determining regions" (CDRs) interspersed with more conserved regions termed "framework regions" (FR or FW).

[0076] In some embodiments, the light or heavy chain variable framework of a monoclonal antibody molecule against a colicin can comprise a light or heavy chain variable framework that comprises at least 80%, 85%, 87%, 90%, 93%, 95%, 97%, or 99% of the light or heavy chain variable framework residues derived from a human consensus sequence.

[0077] In another specific embodiment, the present invention relates to a mutant monoclonal antibody molecule of the present invention or a derivative thereof, which comprises any of the mutant H-chain variable regions of the present invention and any of the mutant L-chain variable regions of the present invention. Preferred combinations of mutant H-chain and mutant L-chain variable regions are those of the isolated monoclonal antibody clones 4A, 9A, 2A, and 21A, respectively. Specifically, the following combinations are used:

[0078] Anti-colicin monoclonal antibody clone 4A: monoclonal antibodies (IA), (IB), (IC) related: H chain: an H chain variable region comprising an amino acid sequence that is at least 80% identical to the H chain variable region amino acid sequence of SEQ ID NO: 36 evqvlesggglvqpgnslklscatsGFTFSTAWmywyrqfpekrlewvarIKAKSNSYATdytesvkgrftisrddskgsiylrmnnlkeedtaiyycASTDAFYFSHSYwgqgvlvtvss, and L chain: an L chain variable region comprising an amino acid sequence that is at least 80% identical to the L chain variable region amino acid sequence of SEQ ID NO: 40 divmtqtpssqavsagekvtmrcrssQSLLYSENKKNYlawyqqkpgrspklliyWASTgesgvpdrfigsgsgtdftltissvqaedlavyycQQYYNFPLTfgsgtkleik

[0079] Anti-colicin monoclonal antibody clone 2A: monoclonal antibodies (IIA), (IIB), (IIC) related: H chain: an H chain variable region comprising an amino acid sequence that is at least 80% identical to the H chain variable region amino acid sequence of SEQ ID NO: 48 evklvesggglvqpgnsltlscvasGFTFTNYGmhwirqapkkglewiamIYYDSSKMsyadtvkgrftisrdnskntlylemnslrsedtamyycAAEGFGTPFPYwgqgtlvtvss, and L chain: an L chain variable region comprising an amino acid sequence that is at least 80% identical to the L chain variable region amino acid sequence of SEQ ID NO: 51 divmtqtpssravsagekvtmsckssQSLLYSENEKNYlawyqqrpgqspklliyWAStresgvpdrfigtgsgtdftltissvqaedlavyycQQYYHFPRTfgggtrlelk

[0080] Anti-colicin monoclonal antibody clone 21A: monoclonal antibodies (IVA), (IVB), (IVC) related: H chain: an H chain variable region comprising an amino acid sequence that is at least 80% identical to the H chain variable region amino acid sequence of SEQ ID NO: 71 evklvesggglvqpgnsltlscgasgftftNYGMHwirqapkkglewigMIYYDSSKMSYADTVKGrftisrdnsknilylemnslrsedtamyycaaEGFGTPFPYwgqgtlvtvss, and L chain: an H chain variable region comprising an amino acid sequence that is at least 80% identical to the H chain variable region amino acid sequence of SEQ ID NO: 75 divmtqtpssqavsagekvtmscKSSQSLLYREDTKNYLAwyqqrpgqspklliyWASTRESgvpdrfigsgsgtdftltissvqaedlavyycQQYYHFPRTfgggtrlelk.

[0081] In another specific embodiment, the present invention relates to a mutant monoclonal antibody molecule of the present invention or a derivative thereof, which comprises any of the mutant H-chain variable regions of the present invention and any of the mutant L-chain variable regions of the present invention. Preferred combinations of mutant H-chain and mutant L-chain variable regions are those of the isolated monoclonal antibody clones 4A, 9A, 2A, and 21A, respectively. Specifically, the following combinations are used:

[0082] Anti-colicin monoclonal antibody clone 4A: Monoclonal antibodies (IA), (IB), (IC) related: H chain: SEQ ID NO: 36, an H chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions, or additions compared to the H chain variable region amino acid sequence of evqvlesggglvqpgnslklscatsGFTFSTAWmywyrqfpekrlewvarIKAKSNSYATdytesvkgrftisrddskgsiylrmnnlkeedtaiyycASTDAFYFSHSYwgqgvlvtvss; and L chain: SEQ ID NO: 40 an L-chain variable region comprising at least one framework (FW) region containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions, or additions compared to the L-chain variable region amino acid sequence of divmtqtpssqavsagekvtmrcrssQSLLYSENKKNYlawyqqkpgrspklliyWASTgesgvpdrfigsgsgtdftltissvqaedlavyycQQYYNFPLTfgsgtkleik;

[0083] Anti-colicin monoclonal antibody clone 2A: Monoclonal antibodies (IIA), (IIB), (IIC) related: H chain: SEQ ID NO: 48 evklvesggglvqpgnsltlscvasGFTFTNYGmhwirqapkkglewiamIYYDSSKMsyadtvkgrftisrdnskntlylemnslrsedtamyycAAEGFGTPFPYwgqgtlvtvss H chain variable region comprising at least one framework (FW) region containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions, or additions compared to the H chain variable region amino acid sequence, and L chain: SEQ ID NO: 51 an L-chain variable region comprising at least one framework (FW) region containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions, or additions compared to the L-chain variable region amino acid sequence of divmtqtpssravsagekvtmsckssQSLLYSENEKNYlawyqqrpgqspklliyWAStresgvpdrfigtgsgtdftltissvqaedlavyycQQYYHFPRTfgggtrlelk;

[0084] Anti-colicin monoclonal antibody clone 21A: monoclonal antibodies (IVA), (IVB), (IVC) related: H chain: SEQ ID NO: 71 evklvesggglvqpgnsltlscgasgftftNYGMHwirqapkkglewigMIYYDSSKMSYADTVKGrftisrdnsknilylemnslrsedtamyycaaEGFGTPFPYwgqgtlvtvss H chain variable region comprising at least one framework (FW) region containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the H chain variable region amino acid sequence, and L chain: SEQ ID NO: 75 An L-chain variable region comprising at least one framework (FW) region containing an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the L-chain variable region amino acid sequence of divmtqtpssqavsagekvtmscKSSQSLLYREDTKNYLAwyqqrpgqspklliyWASTRESgvpdrfigsgsgtdftltissvqaedlavyycQQYYHFPRTfgggtrlelk.

[0085] The monoclonal antibody molecules of the present invention may be antibodies produced by hybridomas or recombinant antibodies produced by transformed cells transformed with an expression vector containing antibody genes. The monoclonal antibody molecules of the present invention, monoclonal antibody molecules of the present invention having any of the heavy chain variable regions of the present invention and any of the light chain variable regions of the present invention, and mutant monoclonal antibody molecules can be prepared by methods known in the art. For example, nucleic acid molecules encoding the antibody molecules of the present invention are incorporated into vectors that can be introduced into host cells. Because antibody molecules of the present invention necessarily contain at least two polypeptide chains, the nucleic acids encoding those chains can be present in either a single vector or two or more vectors. When two or more vectors are used, the vectors can be introduced together into host cells. The vectors containing nucleic acids encoding such antibody molecules and host cells can then be cultured under conditions that allow the protein to be expressed. The expressed antibody molecules can then be obtained from the medium in which the cells are cultured or from the cells themselves, and can be purified by any of a number of suitable means known in the art.

[0086] In the present study, colicin-induced apoptosis occurs through activation of either the extrinsic or intrinsic pathway (Ref. 25). The extrinsic pathway is mediated by membrane-bound death receptors, which transmit intracellular death signals via their intracellular death domains after ligand binding and cleavage of procaspase-8 into caspase-8 (Figure 5a) (Ref. 25). The intrinsic pathway is regulated by mitochondria. Proapoptotic stimuli induce mitochondrial perturbations, increasing mitochondrial membrane permeability, leading to the release of baculovirus inhibitor of apoptosis repeat-containing (BIRC) proteins and proapoptotic factors, which contribute to apoptosis formation and cleave procaspase-9 into caspase-9 (Refs. 26, 27). The cleaved forms of caspase-8 and caspase-9 activate caspase-3, the executioner of apoptosis (Figure 5a). These apoptotic pathways can be used to assess apoptotic activity using a variety of well-known methods. The antibody molecule or derivative thereof of the present invention is preferably a colicin-neutralizing antibody molecule or derivative thereof, more preferably an antibody molecule or derivative thereof that neutralizes and / or inhibits colicin-induced apoptosis.

[0087] <Pharmaceutical Composition> In one aspect, the present invention relates to a pharmaceutical composition for treating or preventing acute lung injury or fibrosis, which comprises an effective amount of the monoclonal antibody molecule of the present invention or a derivative thereof.

[0088] In this invention, acute lung injury is a fatal pneumonia that leads to acute respiratory distress syndrome (ARDS), resulting in a decline in lung function and the need for respiratory support. Important human lung injuries, including COVID-19 and severe acute respiratory syndrome (SARS), are characterized by acute lung injury with sequelae of pulmonary fibrosis.

[0089] In the present invention, fibrosis includes idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and / or mammary fibrosis. In particular, idiopathic pulmonary fibrosis is an incurable disease of unknown etiology. Increased apoptosis of alveolar epithelial cells and subsequent abnormal lung tissue repair are central to its progression. The present inventors discovered the present invention by identifying a peptide, colicin, that induces apoptosis of alveolar epithelial cells and triggers acute exacerbation of pulmonary fibrosis. The monoclonal antibody molecule or derivative thereof of the present invention may be a neutralizing antibody, e.g., an antibody that blocks or inhibits the negative action of colicin in the lungs or other tissues of patients with fibrosis.

[0090] As used herein, "treatment" means a method or process intended to (1) delay or prevent the onset of a disease or condition; (2) slow or halt the progression, worsening, or deterioration of the symptoms of a disease or condition; (3) bring about the amelioration of the symptoms of a disease or condition; or (4) cure a disease or condition. Treatment may be administered prior to the onset of a disease or condition as a preventative measure, or alternatively, treatment may be administered after the onset of the disease.

[0091] In the present invention, "prevention" means preventing the onset of an inflammatory and / or fibrotic condition.

[0092] In the present invention, the pharmaceutical composition generally means an agent for treating, managing or preventing a disease or pathological condition, or for testing or diagnosing the disease or pathological condition.

[0093] As used herein, "subject" or "patient" refers to an animal, which is the object of treatment, observation, or testing. By way of example only, the subject may be a mammal, including a human. Antibodies against colicin may be provided alone or as pharmaceutical compositions, preferably for humans. To this end, the compounds may be prepared into various pharmaceutical dosage forms according to the preventive or therapeutic purpose. Examples of pharmaceutical dosage forms are oral agents, injections, suppositories, ointments, salves, etc. Such dosage forms are well known to those skilled in the art and may be formulated by conventional methods.

[0094] In the present invention, a "pharmaceutical composition" contains one or more antibodies against a colicin, together with one or more other chemical components, such as stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical composition facilitates administration of the antibodies against a colicin to an organism. Several techniques for administering antibodies against a colicin are known, including, for example, oral administration or parenteral administration, such as topical, intravenous, aerosol, or ophthalmic administration.

[0095] The term "diluent" refers to a compound used to dilute a desired agent (e.g., an antibody against a colicin) prior to delivery. Diluents can also be used as stabilizers, as they can provide a more stable environment. Salts dissolved in buffers that can control or maintain pH are useful as diluents, including phosphate buffered saline.

[0096] An "effective amount" or "therapeutically effective amount" refers to a sufficient amount of an antibody to a colicin of the invention administered to alleviate to some extent one or more of the symptoms of the disease, disorder, or condition being treated, thereby allowing for the reduction and / or alleviation of the signs, symptoms, or causes of the disease. For example, an "effective amount" in therapeutic applications is the amount of an antibody to a colicin required to provide the desired pharmacological effect, therapeutic improvement, or clinically significant reduction in disease symptoms, without undue side effects. An "effective amount" may vary from subject to subject due to differences in metabolism, genetics, combinations of antibodies to a colicin, or differences in the age, weight, general condition, condition being treated, severity of the condition being treated, and the judgment of the prescribing physician.

[0097] "Effective amount" includes "prophylactically effective amount," which means the amount of antibody to a colicin of the invention administered to a patient to alleviate the severity of one or more of the symptoms of the disease, condition, or disorder being treated. In such prophylactic applications, such amounts may vary depending on the subject's health status, weight, etc.

[0098] The amount of antibody against colicin incorporated into each unit dosage form may vary depending on the subject's health condition or the type of preparation. Preferred amounts per unit dosage form are about 1 to 1000 mg for oral administration and about 0.1 to 500 mg for injection. The daily dose varies depending on the patient's condition, weight, age, sex, and other factors, and is usually in the range of about 0.1 to 5000 mg, preferably about 1 to 1000 mg, per day for an adult. The preparation is preferably administered in a single dose or in 2 to 4 divided doses.

[0099] To prepare a solid preparation for oral administration, excipients, and if necessary, binders, disintegrants, lubricants, colorants, correctives, flavors, and the like are added to the anti-PD-1 preparation, and the preparation is formulated into tablets, coated tablets, granules, powders, capsules, and the like by a conventional method. Such additives are well known in the art, and useful examples include excipients such as lactose, sucrose, sodium chloride, starch, calcium carbonate, kaolin, crystalline cellulose, and silicic acid; binders such as water, ethanol propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone; disintegrants such as dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose; lubricants such as purified talc, stearates, borax, and polyethylene glycol; and correctives such as sucrose, orange peel, citric acid, and tartaric acid.

[0100] For the preparation of liquid preparations for oral administration, a corrective agent, a buffer, a stabilizer, a flavoring agent, etc. may be added to the compound, and the mixture may be formulated into an oral liquid preparation, a syrup, an elixir, etc. by conventional methods. Examples of useful corrective agents are as described above. An example of a buffer is sodium citrate, etc. Examples of stabilizers are tragacanth, gum arabic, gelatin, etc. As a non-limiting example, a syrup may be prepared by conventional methods using the ingredients in the proportions shown below.

[0101] Injections are prepared as subcutaneous, intramuscular, or intravenous injections in a conventional manner by adding a pH adjuster, buffer, stabilizer, isotonicity agent, local anesthetic, etc. to an antibody against colicin. Examples of pH adjusters and buffers include sodium citrate, sodium acetate, sodium phosphate, etc. Examples of stabilizers include sodium pyrosulfite, EDTA, thioglycolic acid, etc. Examples of local anesthetics include procalcin hydrochloride, lidocaine hydrochloride, etc. Examples of isotonicity agents include sodium chloride, glucose, etc. As a non-limiting example, an injection can be prepared in a conventional manner using the components in the proportions shown below.

[0102] In another aspect of this aspect, the present invention relates to a method for treating or preventing acute lung injury or fibrosis, which comprises administering a monoclonal antibody molecule or a derivative thereof of the present invention to a subject in need of such treatment, preferably a method comprising administering an effective amount of a monoclonal antibody molecule or a derivative thereof of the present invention to such a subject. Preferably, in this method of the present invention, the acute lung injury is life-threatening pneumonia that causes acute respiratory distress syndrome (ARDS), resulting in a decline in lung function and the need for respiratory support, or the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and mammary fibrosis.

[0103] In another aspect, the present invention relates to the monoclonal antibody molecule of the present invention or a derivative thereof for treating or preventing acute lung injury or fibrosis. Preferably, the acute lung injury is life-threatening pneumonia that leads to acute respiratory distress syndrome (ARDS), resulting in a decline in lung function and the need for respiratory support, or the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and mammary fibrosis. In yet another aspect, the present invention relates to use of the monoclonal antibody molecule of the present invention or a derivative thereof for the manufacture of a medicament for treating or preventing acute lung injury or fibrosis. Preferably, the acute lung injury is life-threatening pneumonia that leads to acute respiratory distress syndrome (ARDS), resulting in a decline in lung function and the need for respiratory support, or the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and mammary fibrosis.

[0104] In another aspect, the present invention relates to a vaccine composition for preventing acute lung injury or fibrosis, which comprises as an active ingredient a colicin mutant having one or more mutations in the sequence of colicin having the amino acid sequence: IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1), wherein the mutant does not exhibit apoptotic activity. Preferred colicin mutants are colicin N14S: IVMPESSGNPNAVSPAGYR (SEQ ID NO: 55) and colicin P15A: IVMPESSGNPNAVNAAGYR (SEQ ID NO: 56).

[0105] In the present invention, a "vaccine composition" refers to a biological preparation containing an antigen that induces a specific immune response (the production of specific antibodies). In the present invention, a vaccine may contain an optionally modified peptide. The colicin mutant contained in the vaccine composition of the present invention is one that attenuates the pro-apoptotic activity of colicin through specific mutations or changes in amino acid positions, while retaining antigenicity. That is, the peptide is one in which amino acids at different positions in the colicin peptide are changed, and the peptide is sufficiently similar to colicin to induce antibodies that neutralize the pro-apoptotic colicin peptide without inducing apoptosis.

[0106] In the present invention, mutations include "amino acid residue substitutions," "amino acid residue deletions," "amino acid residue insertions," and "amino acid residue additions." Examples of "amino acid residue substitutions" include non-conservative amino acid substitutions. Non-conservative amino acid substitutions refer to the replacement of a specific amino acid with an amino acid having a side chain with properties different from those of the original amino acid. Specifically, in non-conservative amino acid substitutions, a specific amino acid is replaced with another amino acid belonging to a different group from the original amino acid. Groups of amino acids with side chains of different properties are known in the art. Examples of such groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), and neutral side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan).

[0107] Amino acids with neutral side chains can be further classified into those with polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine) and those with nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan). Again, amino acids from groups with different properties are selected. Other groups that can be used include, for example, amino acids with aromatic side chains (e.g., phenylalanine, tryptophan, tyrosine) and amino acids with side chains containing hydroxyl groups (alcoholic hydroxyl groups, phenolic hydroxyl groups) (e.g., serine, threonine, tyrosine).

[0108] "Deletion of an amino acid residue" includes, for example, deleting any amino acid residue selected from a partial sequence of a colicin peptide. "Insertion of an amino acid residue" or "addition of an amino acid residue" includes, for example, inserting an amino acid residue into the partial sequence of a colicin peptide, or adding an amino acid residue to the N-terminus or C-terminus of the partial sequence.

[0109] In another aspect of this aspect, the present invention relates to a vaccine therapy for preventing acute lung injury or fibrosis, which comprises administering to a subject in need of such treatment a colicin mutant having one or several mutations in the sequence of colicin having the amino acid sequence IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1), said mutant not exhibiting apoptotic activity, or preferably a therapy comprising administering to such a subject an effective amount of the monoclonal antibody molecule of the present invention or a derivative thereof. Preferred colicin mutants are colicin N14S: IVMPESSGNPNAVSPAGYR (SEQ ID NO: 55) and colicin P15A: IVMPESSGNPNAVNAAGYR (SEQ ID NO: 56).

[0110] In another aspect, the present invention relates to a colicin mutant having one or several mutations in the sequence of colicin having the amino acid sequence IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1), which does not exhibit apoptotic activity, for use in vaccine therapy to prevent acute lung injury or fibrosis. In yet another aspect, the present invention relates to the use of a colicin mutant having one or several mutations in the sequence of colicin having the amino acid sequence IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1), which does not exhibit apoptotic activity, for the manufacture of a medicament for use in vaccine therapy to prevent acute lung injury or fibrosis.

[0111] <Biomarker> In another aspect, the present invention relates to the use of colicin as a biomarker for determining the progression of acute lung injury or fibrosis. The use of colicin as a biomarker also includes the evaluation or diagnosis of a subject suspected of having or developing fibrosis. A biological sample is collected from the subject, and the amount of colicin present in the biological sample is detected. The detected amount of colicin in the biological sample may be compared with one or more predetermined thresholds. The predetermined thresholds may be set, for example, based on the level of colicin normally present that is typical of healthy individuals.

[0112] The biological sample can be, for example, sputum, bronchial secretions, pleural effusion, bronchoalveolar lavage fluid (BALF), and tissue collected from the bronchi or lungs. The biological sample can also be blood. The colicin can have, for example, one of the amino acid sequences: IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1), IVMPESGGNPNAVNPAGYR (SEQ ID NO: 58), IIMPESGGNPNIVNPYGYS (SEQ ID NO: 59), IVMPESGGNPNAVNPYGYR (SEQ ID NO: 60), IVLPESSGNPNAVNPAGYR (SEQ ID NO: 61), IVLPESSGNPNAVNELGYR (SEQ ID NO: 62), IVMPESGGNPNAVNELGYR (SEQ ID NO: 63), IVMPESSGNPNAVNELGYR (SEQ ID NO: 64), IVMPESSGNPDAVNELGYR (SEQ ID NO: 65), IAQRESGGDLKAVNPSSGA (SEQ ID NO: 66), or IAERESGGDLKAVNPSSGA (SEQ ID NO: 67). Preferably, the colicin is a peptide having the amino acid sequence IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1).

[0113] Colicins may be detected by mass spectrometry, Western blotting, or enzyme-linked immunosorbent assay (e.g., ELISA) using, for example, IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1), IVMPESGGNPNAVNPAGYR (SEQ ID NO: 58), IIMPESGGNPNIVNPYGYS (SEQ ID NO: 59), IVMPESGGNPNAVNPYGYR (SEQ ID NO: 60), IVLPESSGNPNAVNPAGYR (SEQ ID NO: 61), IVLPESSGNPNAVNELGYR (SEQ ID NO: 62), IVMPESGGNPNAVNELGYR (SEQ ID NO: 63), IVMPESSGNPNAVNELGYR (SEQ ID NO: 64), IVMPESSGNPDAVNELGYR (SEQ ID NO: 65), IAQRESGGDLKAVNPSSGA (SEQ ID NO: 66), or IAERESGGDLKAVNPSSGA (SEQ ID NO: 67), for example, by binding a labeled antibody to an antibody that binds to a substrate. Kits for carrying out such methods can include such antibodies and one or more reagents for detecting colicin in a biological sample.

[0114] In this embodiment, the present invention provides a method for determining the stage of fibrosis, comprising: (a10) measuring the amount of colicin in a subject (amount of test biomarker); (b10) comparing the amount of test biomarker with a reference amount of colicin (amount of control biomarker); and (c10) determining that the subject has a high stage of acute lung injury or fibrosis when the amount of test biomarker is higher than the amount of control biomarker. The reference amount of colicin may be the amount of colicin in the same subject measured before step (a10), or may be the amount of colicin in myofibroblasts of a healthy subject.

[0115] The present inventors have found that colicin is a fibrosis-specific marker molecule, which is associated with fibrotic diseases, particularly severe fibrotic diseases. The use of the method, biomarker, and colicin of the present invention makes it possible to predict the progression of acute lung injury or fibrosis, and is beneficial for the prevention or treatment of acute lung injury or fibrosis.

[0116] The correspondence between the three-letter and one-letter codes of amino acids used in this specification is as follows: Alanine: Ala: A, Arginine: Arg: R, Asparagine: Asn: N, Aspartic acid: Asp: D, Cysteine: Cys: C, Glutamine: Gln: Q, Glutamic acid: Glu: E, Glycine: Gly: G, Histidine: His: H, Isoleucine: Ile: I, Leucine: Leu: L, Lysine: Lys: K, Methionine: Met: M, Phenylalanine: Phe: F, Proline: Pro: P, Serine: Ser: S, Threonine: Thr: T, Tryptophan: Trp: W, Tyrosine: Tyr: Y, Valine: Val: V

[0117] The present invention will be described in detail below with reference to examples. These examples are merely illustrative, and it should be noted that the present invention can be modified in various ways within the scope of the invention described in the claims, and such modifications are also included within the scope of the present invention.

[0118] Reference Result 1: A Wide Range of Pathogens Possess Proapoptotic Colicin-Like Peptides We previously demonstrated that culture supernatants from a mixture of Staphylococcus nepalensis CNDG strain and Staphylococcus spp. grown in media inoculated with pulmonary fibrosis specimens from mice with end-stage pulmonary fibrosis induce apoptosis in alveolar epithelial cells (Non-Patent Document 19). We also reported that transglycosylase fragments released by Staphylococcus nepalensis, designated colicins, cause apoptosis in pulmonary epithelial cells in established transgenic mice and induce acute exacerbations of chronic pulmonary fibrosis (Non-Patent Document 19). Because the sequences of colicins or their derivatives are highly conserved among many members of the Staphylococcus family (Non-Patent Document 19), we predicted that colicin-like peptides would be released from transglycosidases in Staphylococcus nepalensis as well as other staphylococcal species, and induce apoptosis in lung epithelial cells.

[0119] To test this prediction, a mixture of Staphylococcus species grown in a medium inoculated with lung fibrosis specimens was streaked and individual bacterial colonies were isolated. Twelve resulting colonies were screened, and three colonies (strains 1, 7, and 12) secreted apoptotic factors, whereas the remaining isolates showed no detectable apoptotic activity. These results indicate that the previously discovered culture of strain 6 (Non-Patent Document 19) contained a mixture of pro-apoptotic and non-apoptotic bacteria. Whole-genome sequencing revealed that these three apoptotic bacterial isolates were distinct strains of Staphylococcus haemolyticus. The whole-genome sequences of Staphylococcus haemolyticus strains 1, 7, and 12 have been deposited in the GeneBang database under accession numbers. These genome analyses revealed the presence of conserved colicin-like fragments in strain 1 (transglycosylase_1bp_353), strain 7 (transglycosylase_7bp_350), and strain 12 (transglycosylase_12bp_350) (Fig. 1). The sequences of the colicin-like fragments were identical in the corresponding transglycosylases of each strain (Non-Patent Document 19) (Fig. 1).

[0120] Next, we cultured A549 alveolar epithelial cells in the presence of bacterial culture supernatants from each bacterial strain and assessed apoptosis. Culture supernatants from each strain induced apoptosis in alveolar epithelial cells (Figure 2a, b). Transmission electron microscopy confirmed an increase in apoptotic cells after treatment with culture supernatant from Staphylococcus haemolyticus strain 12 (Figure 2c). A549 alveolar epithelial cells cultured in the presence of culture supernatants from each Staphylococcus haemolyticus strain also showed a significant increase in caspase-3 cleavage (Figure 2d, e). Synthetic peptides of the Staphylococcus haemolyticus collicin-like sequence prepared by the manufacturer (ThermoFisher Scientific) differed at only a single position compared to the previously published sequence (Non-Patent Document 19), and this recapitulated the pro-apoptotic effect of the supernatants from each bacterial strain (strains 1, 7, and 12) on A549 lung epithelial cells (Figure 3a, b, c). Staphylococcus haemolyticus is a commensal bacterium, but it is also well known as an opportunistic, multidrug-resistant pathogen (Ref. 20). This bacterium grows optimally under oxygen and salt (10% NaCl) conditions (Ref. 21). Human infections caused by S. haemolyticus include sepsis, peritonitis, endocarditis, meningitis, and wound, bone, and urinary tract infections. Interestingly, the colicin-like sequence of the transglycosylase homologue from the S. haemolyticus strain isolated here is identical to that observed in transglycosylases from Mycobacteroides abscessus subsp. abscessus (GenBank protein accession number SKR 69498.1) and Listeria monocytogenes (GenBank protein accession number ECO 1693478.1) strains (Figure 4). Listeria monocytogenes is the most virulent foodborne pathogen (Reference 22), and Mycobacteroides abscessus subsp. abscessus (Reference 23) is a multidrug-resistant nontuberculous mycobacterium that commonly causes chronic pulmonary infections.Homologous transglycosylases from other strains of Listeria monocytogenes and Mycobacteroides abscessus subsp. abscessus, as well as a hypothetical protein from the opportunistic pathogen Weissella confusa (24), also contain colicin-like sequences with proapoptotic activity (Fig. 3a, b, c). These observations indicate that a wide range of pathogens possess derivatives of this toxic peptide.

[0121] Reference Results 2: Activation of Intracellular Apoptotic Pathways by Bacterial Culture Supernatant and Colicin. Figure 5a shows the extrinsic and intrinsic pathways of apoptosis. A549 alveolar epithelial cells were cultured for 24 hours in the presence of culture supernatant from S. haemolyticus strain 12 (1 / 10 dilution). The percentages of cells positive for cleaved caspase-8, cleaved caspase-9, and cleaved caspase-3 were determined and quantified by flow cytometry. We found that S. haemolyticus culture supernatant significantly increased the number of alveolar epithelial cells with activated caspase-8 and activated caspase-3 compared with control stimulation (Figure 5b).

[0122] Next, we examined the effect of colicin on the mRNA expression of pro-apoptotic factors such as Bax and anti-apoptotic factors BIRC1, BIRC5, BIRC7, Bcl2, BclXL, cyclin D1, proliferating cell nuclear antigen, and proliferation marker Ki-67. A549 alveolar epithelial cells were cultured in the presence of 50 μM colicin for 24 h, and total mRNA was extracted, cDNA was prepared, and amplified by RT-PCR. We found that colicin significantly reduced the mRNA expression of anti-apoptotic factors, but significantly increased the mRNA expression of pro-apoptotic factors such as Bax and apoptotic protease-activating factor-1 (APAF-1) compared with the scrambled peptide (Figure 6, Table 1). These results indicate that bacterial-derived peptides selectively activate the intrinsic pathway of apoptosis.

[0123]

[0124] [In the table, BIRC, baculovirus inhibitor of apoptosis repeat-containing (BIRC) protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; BclXL, B-cell lymphoma-extra-large; APAF-1, apoptotic protease-activating factor 1; CCND1, cyclin D1; PCNA, proliferating cell nuclear antigen; MPKi-67, proliferation marker Ki-67]

[0125] Reference Result 3: Ability of Bacterial Secreted Factors to Degrade Colicin-Containing Transglycosylase and Induce Apoptosis in Lung Epithelial Cells. We previously reported that the apoptotic peptide colicin can be cleaved from S. nepalensis transglycosylase and induce AE in pulmonary fibrosis (Non-Patent Document 19). The factors involved in this cleavage are unknown. Here, we predicted that colicin-excreting bacteria secrete a protease that cleaves the apoptotic peptide from the respective transglycosylase (Fig. 7a). To verify this prediction, we first partially purified a peptide fraction from the culture supernatant of S. nepalensis CNDG strain. The culture supernatant of S. nepalensis was sequentially concentrated using a >50 kDa filter, and then the flow-through (<50 kDa) fraction was concentrated using a >10 kDa filter (Fig. 7b). The concentrated fractions were loaded onto a Cefracryl S-300 column, and the products were separated by gel electrophoresis. The digestion activity of the collected fractions against the colicin-containing recombinant transglycosylase was then measured (Fig. 7c). Fractions with high digestion activity were pooled, concentrated, and loaded onto a Cefracryl S-300 column for a second time to isolate the highly digestively active fractions (Fig. 7d).

[0126] Next, reaction mixtures containing digestion buffer, recombinant transglycosylase, and various amounts of peptidase fractions (>10 kDa) prepared from Staphylococcus nepalensis culture supernatant were incubated at 37°C for 2 hours, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Reaction mixtures containing digestion buffer, recombinant transglycosylase, and various amounts of peptidase fractions (>10 kDa) prepared from Staphylococcus nepalensis culture supernatant were also incubated at 37°C for various time intervals, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Silver staining of the gels revealed dose- and time-dependent degradation of the recombinant transglycosylase (Figure 8a, b). In a separate experiment, the degradative effect of transglycosylase from Staphylococcus haemolyticus culture supernatant was examined. The reaction mixture containing digestion buffer, recombinant transglycosylase, and culture supernatant of Staphylococcus haemolyticus strain 12 was incubated at 37°C for 14 hours, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining. The results showed that the culture supernatant of Staphylococcus haemolyticus also cleaved the recombinant transglycosylase (Figure 9). Next, A549 alveolar epithelial cells were cultured to subconfluence and treated with the cleavage product prepared by incubating recombinant transglycosylase in bacterial supernatant of Staphylococcus nepalensis for 2 hours. Flow cytometry analysis performed after 48 hours of cell culture revealed significantly increased apoptosis in epithelial cells cultured in the presence of the cleavage product compared to the control (Figure 8c, d). These results indicate that bacterially secreted factors, such as proteases, cleave the transglycosylase, thereby producing colicin peptides that induce apoptosis in lung epithelial cells.

[0127] Reference Result 4: Bacterial secreted factor cleaving transglycosylase: putative serine protease. To determine whether the transglycosylase cleavage factor was a protease, we added various concentrations (0.1, 0.3, 1 mM) of the serine protease inhibitors Pefabloc SC or diisopropylfluorophosphate (DFP), the cysteine ​​protease inhibitor E-64, or the chelating agent ethylenediaminetetraacetic acid (EDTA) to a reaction mixture containing recombinant transglycosylase (2 mg / ml) and a peptidase fraction (>10 kDa) prepared from the culture supernatant of Staphylococcus nepalensis, and incubated for 0.5, 1, and 2 hours at 37°C. After separating the products on a silver-stained protein gel, we found that the serine protease inhibitors Pefabloc SC and DFP inhibited transglycosylase degradation (Fig. 10 a, b, c, d). However, neither E-64 nor EDTA inhibited transglycosylase degradation (Fig. 10 e, f, g, h). These results indicate that a putative serine protease secreted by Staphylococcus nepalensis degrades its colicin-containing transglycosylase into smaller peptides containing colicin.

[0128] Materials and Methods: Reagents used in the reference results and examples herein were obtained or prepared as follows. Reagents: Human lung epithelial cell line A549 and hypersaline medium (ATCC media 1097, 2168) were purchased from the American Type Culture Collection (Manassas, VA, USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from BioWhittaker (Walkersville, MD, USA). L-glutamine, penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA). Synthetic colicin and the corresponding synthetic scrambled peptide: NRVYNGPAASPVSEGMPIN (SEQ ID NO: 1) were prepared and provided by Peptide Institute Incorporation (Osaka, Japan) and ThermoFisher Scientific (Waltham, MA). Bleomycin (BLM) was purchased from Nippon Kayaku (Tokyo, Japan), and Alzet osmotic minipumps (Model 2001) were purchased from Alza Corporation (Palo Alto, CA). Here, the scrambled peptide is a peptide whose amino acid sequence has been broken down to disable its function, specifically, a peptide whose colicin amino acid sequence has been broken down to disable its function.

[0129] Animals: Female wild-type C57BL / 6J mice were purchased from Japan SLC (Hamamatsu, Japan) and used to induce lung injury, pulmonary fibrosis, and acute exacerbations (Ref. 29). WT mice weighing 20–22 g and 8–9 weeks of age were used for BLM-induced pulmonary fibrosis experiments. We also used female and male transforming growth factor beta (TGFβ1) TG mice and their wild-type littermates on a C57BL / 6J background, which develop spontaneously progressive and fatal pulmonary fibrosis (Non-Patent Document 19, Refs. 31 and 61). TG mice and wild-type littermates weighed 20–24 g and were 8–10 weeks of age. Mice were housed in a specific pathogen-free environment at 21°C with a 12-h light / dark cycle in the Mie University Laboratory Animal Facility. Mouse cages were provided with wood-wool nesting material, and mice had free access to water and food. TG mice were genotyped by standard PCR analysis using DNA isolated from mouse tails and specific primer pairs (Non-Patent Document 19).

[0130] Ethics Statement: The Recombinant DNA Experimental Safety Committee (Permit Number: I-614 (Henko 1); Date: 2013 / 15 / 12; Permit Number: I-708, Date: 13 / 02 / 2019) and the Mie University Animal Experimentation Committee approved the experimental protocol (Permit Number: 25-20-Hen 1-Sai 1, Date: 23 / 07 / 2015; Permit Number: 29-23, Date: 15 / 01 / 2019). All experimental procedures were conducted in accordance with the internationally accepted principles of laboratory animal care published by the National Institutes of Health (https: / / olaw.nih.gov / ). The study followed the ARRIVE guidelines for animal research, and variables were measured blinded to treatment groups.

[0131] Preparation of peptidase fractions: 100 ml of serially concentrated Staphylococcus nepalensis culture supernatant was filtered through a >50 kDa filter, and the resulting flow-through fraction was concentrated through a >10 kDa filter as described in Figure 7b. The >10 kDa concentrated sample was then loaded onto a Sephacryl S-300 column, and several fractions (1 ml each) were isolated. The proteolytic activity of each fraction on the colicin-containing recombinant transglycosylase was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining (Figure 7c). Silver staining was performed using the Daiichi 2-D Silver Staining Kit (Daiichi, Tokyo, Japan). The reaction mixture contained 0.5 μL of 1x digestion buffer (20 mM TrisCl, 140 mM NaCl, pH 7.5), 2 mg / ml recombinant transglycosylase, and 6 μL of each fraction. Fractions with high digestion activity (fractions 10 to 13 in Figure 7c) were then pooled and concentrated to 0.5 ml. The concentrated sample was then loaded onto a Sephacryl S-300 column, separated into several fractions, and the absorbance at 280 nm was measured (Figure 7d). The digestion activity of each fraction on recombinant transglycosylase 351 was then evaluated as described above (Figure 7d). The fractions with high proteolytic activity were used in the experiment.

[0132] Degradation assay of recombinant transglycosylase: To a 50 μl reaction mixture, 12.5 μl of recombinant transglycosylase (2 μg / mL), 5 μl of 10× digestion buffer (TrisCl, 1.4% NaCl, pH 7.5), 25 μl of Staphylococcus nepalensis culture supernatant, and 7.5 μl of distilled water were added. The reaction mixture was incubated overnight at 37°C, and then 10 μl of the reaction mixture was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the products were visualized by silver staining. In separate experiments, several amounts of Staphylococcus nepalensis culture supernatant, anti-colicin monoclonal antibody, or isotype antibody were added to the reaction mixture and run on SDS-PAGE for subsequent silver staining.

[0133] Assay for transglycosylase degradation inhibition: Reaction mixtures were prepared containing recombinant transglycosylase (2 μg / mL) in digestion buffer, Staphylococcus nepalensis culture supernatant, and various concentrations (0.1, 0.3, and 1 mM) of serine protease inhibitors (Pefabloc SC or diisopropylfluorophosphate [DFP]), cysteine ​​protease inhibitors (E-64), or chelators (ethylenediaminetetraacetic acid [EDTA]). The mixtures were then incubated for 0.5, 1, and 2 hours before SDS-PAGE and silver staining.

[0134] Cell Culture: Human A549 alveolar epithelial cells were cultured in DMEM supplemented with 10% fetal bovine serum, 0.03% (w / v) L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin at 37°C in a 5% CO2 humidified atmosphere.

[0135] Apoptosis assay: A549 cells (4 x 10 cells / well) were cultured to subconfluency in 12-well plates and serum-starved for 24 h. After 48 h, they were treated with colicin or colicin-like peptides, or a scrambled peptide based on the colicin sequence to assess apoptosis. Apoptosis was assessed by flow cytometry (BD Biosciences FACScan, Oxford, UK) after staining with fluorescein-labeled annexin V and propidium iodide (FITC Annexin V Apoptosis Detection Kit with PI, Bioelgend, San Diego, CA). The percentage of cells positive for cleaved caspase-8, cleaved caspase-9, and cleaved caspase-3 was assessed by flow cytometry using antibodies from Cell Signaling Technology (Beverly, MA, USA).

[0136] Transmission electron microscopy of apoptotic cells. A549 cells (10 × 104 cells / ml) were plated onto collagen-coated 8-well chamber slides (BD Bioscience, San Jose, CA) and grown to semiconfluence. Cells were serum-starved for 6 hours and stimulated with a proapoptotic peptide (5 μM) for 16 hours. Cells were fixed with 2% fresh formaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 hours at room temperature. After washing with 0.1 M cacodylate buffer (pH 7.4), cells were postfixed with 1% OsO4 in the same buffer at 4°C for 2 hours. They were rinsed with distilled water, stained with 1% uranyl acetate in water for 2 hours or overnight at room temperature, dehydrated with ethanol and propylene oxide, and embedded in Epon (Epon 812 resin, Nakarai). After removing the cells from the glass, ultrathin sections (94 nm) were cut, stained with uranyl acetate and Reynolds' lead citrate, and observed under a transmission electron microscope (JEOL, JEM-1010, Tokyo, Japan).

[0137] Assessment of transglycosylase degradation product activity. A549 alveolar epithelial cells (2 × 10 cells / well) were cultured to confluence in 12-well plates. After overnight serum starvation, a reaction mixture containing 5 μM recombinant transglycosylase 351, 1 / 10 volume of Staphylococcus nepalensis supernatant, and 5 μM anti-colicin monoclonal or isotype antibody was added to the cell culture and incubated for 48 hours. Apoptosis was assessed as described above.

[0138] Genomic DNA sequencing and genome annotation. Genome sequencing was performed using a combination of Oxford Nanopore sequencing and Illumina Miseq nanosequencing, as previously reported (19). Briefly, genomic DNA from each bacterial strain (400 ng) was converted into a nanopore library using the Rapid Barcoding library kit SQK-RAD 004. The resulting library was sequenced for 48 hours using a GridION sequencer on a SpotON® 9.4.1 FLOMIN-MIN 106 flow cell. The majority of reads were 6 kb to 30 kb in length, but reads up to 94 kb in length were also obtained. Illumina Miseq sequencing was performed by preparing a shotgun genomic library using the Hyper Library Construction Kit from Kapa Biosystems (Roche). The library was quantified by qPCR and sequenced for 251 cycles on a single MiSeq Nano flow cell. Fastq files were generated and separated with bcl 2 fastq v 2.20 conversion software (Illumina).

[0139] An initial assembly of the Oxford Nanopore data was performed using Canu (62), followed by refinement using Nanopolish (63) and Pilon (using Illumina MiSeq reads) (64), and finally genome reorientation using Circlator (65). Another hybrid genome assembly was performed using SPAdes (66), followed by genome reorientation using Circlator. Another hybrid genome assembly was performed using Unicycler (67). The final hybrid genome assembly was generated using Unicycler with the Canu assembly as the assembly scaffold.

[0140] All assemblies were quality assessed using BUSCO (68) and QUAST (69) and compared to the relevant reference genome using MUMmer (70). Genome assemblies were annotated using the tool Prokka (71). After evaluation, the best overall assembly was determined by combining the best overall BUSCO score with global assembly metrics.

[0141] Example 1: Inhibition of Lung Cell Apoptosis Induced by Transglycosylase Degradation Products by Anti-colicin Neutralizing Monoclonal Antibodies 1-1: Preparation of Monoclonal Antibodies Against the Proapoptotic Peptide IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1) Based on the findings of the Reference Results above, we predicted that anti-colicin neutralizing antibodies would inhibit the proapoptotic activity of transglycosylase degradation products derived from the proteolytic activity of a putative bacterial serine protease. To verify this prediction, we first prepared and characterized a monoclonal antibody against the proapoptotic peptide IVMPESSGNPNAVNPAGYR as an anti-colicin neutralizing monoclonal antibody. The proapoptotic peptide IVMPESSGNPNAVNPAGYR is a colicin-like sequence from Staphylococcus nepalensis shown in Figures 3 and 4 and was prepared by the manufacturer (ThermoFisher Scientific).

[0142] Monoclonal antibodies were prepared at Eurofins Genomics Inc. (Tokyo, Japan). Briefly, the peptide used for immunization (NH2-C+IVMPESSGNPNAVNPAGYR-COOH) was synthesized, purified by high-performance liquid chromatography, and then conjugated to keyhole limpet hemocyanin, a commonly used carrier protein, by the maleimide method (Refs. 72, 73). For experimental animal immunization, two Wistar rats were given a single subcutaneous injection of the immunogen at weeks 0, 2, 4, and 6, followed by a single intravenous injection of the same peptide at week 8 after the first immunization. Blood was collected 5 weeks after the first immunization, and antibody titers were measured by immunoassay. Spleen cells from one rat were used for cell fusion with myeloma cells (P3U1 and Sp2). Cell fusion was performed using the polyethylene glycol method in hypoxanthine-aminopterin-thymidine medium (Ref. 74). Monoclonal antibody-producing hybridomas were cloned by limiting dilution (Reference 74). The resulting hybridomas were stored in liquid nitrogen for future use. Isotyping was performed using the Rapid RAT Monoclonal Antibody Isotyping XpressCard purchased from Antagen Pharmaceuticals, Inc. (Boston, MA, USA).

[0143] Various amounts of recombinant transglycosylase 351 were electrophoresed on polyacrylamide gels, and Western blotting was performed using a 1:1000 dilution of the supernatant of a hybridoma producing each clone of anti-colicin monoclonal antibody. Specifically, tissues or samples were washed twice with ice-cold phosphate-buffered saline and then lysed in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease / phosphatase inhibitors (1 mM orthovanadate, 50 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 5 μg / mL leupeptin, 2 μg / mL aprotinin, 5 mM sodium fluoride). After centrifugation at 17,000 × g for 10 minutes at 4°C, protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Samples containing equal amounts of protein were mixed with Laemmli sample buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting was then performed using nitrocellulose membranes and anti-cleaved caspase-3 or anti-β-actin antibodies (Cell Signaling, Danvers, MA, USA) (61). Band intensities on the blots were quantified using the public domain NIH ImageJ program (wayne@codon.nih.gov; Wayne Rasband, NIH, Research Services Division).

[0144] The recombinant transglycosylase 351 used here was expressed as follows. To prepare recombinant transglycosylase 351, the gene encoding the protein was synthesized using Escherichia coli-optimized codons, amplified with a terminal A, and cloned into the TA cloning vector pGEM-T Easy (Promega, Madison, Wisconsin, USA). The gene was then excised and cloned into a modified pET 28a vector, transformed into E. coli BL 21 DE 3 cells, and expressed and purified as a 6-histidine-tagged (His-tagged) protein (Non-Patent Document 19).

[0145] As described above, hybridoma clones 2A, 3Aa, 4A, 9A, and 21A were established, producing immunoglobulin (Ig) IgG2bκ, IgMκ, IgG2aκ, IgG2aκ, IgG2aκ, and IgG2bκ, respectively (Fig. 11a). The isotype of each monoclonal antibody was determined using the Rapid Monoclonal Antibody Isotyping Kit.

[0146] Western blotting performed with increasing amounts of recombinant transglycosylase revealed that all monoclonal antibody clones recognized and bound to the colicin-containing transglycosylase from S. nepalensis (Fig. 11b). Next, colicin peptides were coated onto microplates, and the binding of each anti-colicin monoclonal antibody clone was assessed using various dilutions of the hybridoma supernatant. The monoclonal antibodies produced by each hybridoma clone differentially bound to the surface-coated colicin at various dilution levels. In particular, clones 9A and 21A showed strong binding activity (Fig. 11c).

[0147] 1-2: Epitope mapping of monoclonal antibodies against the pro-apoptotic peptide IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1) Epitope mapping was performed to identify the binding site of anti-colicin monoclonal antibodies on the colicin molecule. To this end, the binding of each monoclonal antibody to a microarray of small peptides of different lengths from the colicin sequence was evaluated. Hemagglutinin peptides and anti-hemagglutinin antibodies were used as controls to verify the integrity of the peptide microarray and the quality of the assay. Mapping was performed using PEPPerPRINT Incorporation (Heidelberg).

[0148] Specifically, the peptide IVMPESSGNPNAVNPAGYR was first extended with neutral GSGSG linkers at the C- and N-termini to avoid truncated peptides. The extended peptide sequences were translated into 5-, 10-, and 15-amino acid peptides with 4-, 9-, and 14-amino acid peptide-peptide overlaps. The resulting peptide microarray contained 72 different linear peptides in duplicate (144 spots) and an additional hemagglutinin (YPYDVPDYAG, 17 spots) control peptide. Peptide microarray copies were prestained with secondary (goat anti-rat IgG [H+L] DyLight 680) and control (mouse monoclonal anti-HA [12 CA 5] DyLight 800) antibodies in incubation buffer (wash buffer [phosphate-buffered saline, pH 7.4, 0.05% Tween 20, 10% Rockland blocking buffer MB-070) to determine background interactions with the 72 different peptides on the microarray. Other peptide microarray copies were then incubated with rat monoclonal antibodies 9A, 21A, 2A, and 4A at a concentration of 1 μg / ml in incubation buffer, then stained with the secondary and control antibodies and read using a LI-COR Odyssey imaging system at a scan intensity of 7 / 7 (center spot / periphery spot (top and left border spot)). Additional hemagglutinin peptides comprising the peptide microarray were simultaneously stained as internal quality controls to confirm the quality of the assay and the integrity of the peptide microarray (Fig. 12a, b).

[0149] Quantification of spot intensity and peptide annotation was based on 24-bit grayscale tiff files with a scan intensity of 7 / 7, which exhibits a higher dynamic range than 16-bit colorized tiff files. Microarray image analysis was performed using a PepSlide® Analyzer, and the results are described in the Supplementary Microarray Data. A software algorithm decomposed the fluorescence intensity of each spot into raw, foreground, and background signals, and calculated the median mean foreground intensity and the spot-to-spot deviation of spot overlap. Intensity maps were generated based on the median mean foreground intensity, and an intensity color code highlighted interactions in the peptide map, with medium spots representing high spot intensity and white for low spot intensity. The maximum spot-to-spot deviation was 40%. Otherwise, the corresponding intensity value was set to zero. Additionally, the average spot intensity from assays using a rat antibody against the antigen sequence from the N- to C-terminus of the peptide IVMPESSGNPNAVNPAGYR was plotted to visualize the total spot intensity and signal-to-noise ratio. To identify the epitopes of rat antibodies 9A, 21A, 2A, and 4A, the intensity plots were correlated with peptide and intensity maps and visual inspection of the microarray. In this case, it is unknown whether a particular amino acid contributes to antibody binding, but the corresponding amino acid letter is drawn in gray.

[0150] As a result of the epitope mapping described above, only the staining of the hemagglutinin peptide with the control antibody showed a well-established spot pattern without background interactions at any scan intensity level (Figure 12a, b). Subsequent incubation of other peptide microarray copies with rat monoclonal antibodies 9A (c, d), 21A (e, f), 2A (g, h), and 4A (i, j) at a concentration of 1 μg / ml in buffer followed by staining with the secondary and control antibodies was performed using a LI-COR Odyssey Imaging System at a scan intensity of 7 / 7 (center spot / peripheral spot). The results showed moderate and clear antibody responses to epitope-like spot patterns of medium spots containing the consensus motif PESSGNP (sequence number 68) for the 9A, 21A, and 2A monoclonal antibody clones (Fig. 12c, d, e, f, g, h), and the consensus motif NPAGY (sequence number 69) for the 4A monoclonal antibody clone (Fig. 12i, j), with high signal-to-noise ratios (supplementary microarray data).

[0151] 1-3: Sequencing of the variable framework and complementarity-determining regions of anti-colicin monoclonal antibody clones. The sequences of the variable regions of anti-colicin monoclonal antibody clones 4A, 9A, and 2A were determined by syb labs Incorporation (Hopkinton, Massachusetts, USA). cDNA was synthesized using total RNA from each hybridoma, and 5' rapid amplification of cDNA ends was performed. Positive bands obtained by PCR analysis were cloned and sequenced. The complementarity-determining regions (CDRs) of each clone were sequenced. The CDR regions were defined using IMGT / V-QUEST (http: / / www.imgt.org). The sequence of the variable region of anti-colicin monoclonal antibody clone 21A was also determined in a similar manner. CDR identification was performed using the Kabat system. Figure 13 shows the variable framework and complementarity-determining regions of the monoclonal antibodies.

[0152] In detail, they are as follows: Anti-colicin monoclonal antibody clone 4A H chain (4A-HC.7331): Variable region evqvlesggglvqpgnslklscatsGFTFSTAWmywyrqfpekrlewvarIKAKSNSYATdytesvkgrftisrddskgsiylrmnnlkeedtaiyycASTDAFYFSHSYwgqgvlvtvss (SEQ ID NO: 36) H chain variable region CDR1 amino acid sequence: GFTFSTAW (SEQ ID NO: 37) H chain variable region CDR2 amino acid sequence: IKAKSNSYAT (SEQ ID NO: 38) H chain variable region CDR3 amino acid sequence: ASTDAFYFSHSY (SEQ ID NO: 39) H chain (4A-HC.7331): Full length L chain (4A-LC.7395): MELCMMWIFLVAFLKGVQCevqvlesggglvqpgnslklscatsGFTFSTAWmywyrqfpekrlewvarIKAKSNSYATdytesvkgrftisrddskgsiylrmnnlkeedtaiyycASTDAFYFSHSYwgqgvlvtvssAETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVTVPSSTWSSQAVTCNVAH (SEQ ID NO: 79) (wherein the sequence: MELCMMWIFLVAFLKGVQC (SEQ ID NO: 80) is the signal peptide, and the sequence: AETTAAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVTVPSSTWSSQAVTCNVAH (SEQ ID NO: 81) is the constant region) Variable region divmtqtpssqavsagekvtmrcrssQSLLYSENKKNYlawyqqkpgrspklliyWASTgesgvpdrfigsgsgtdftltissvqaedlavyycQQYYNFPLTfgsgtkleik (SEQ ID NO: 40) L chain variable region CDR1 amino acid sequence: QSLLYSENKKNY (SEQ ID NO: 41) L chain variable region CDR2 amino acid sequence: WAST (SEQ ID NO: 42) L chain variable region CDR3 amino acid sequence: QQYYNFPLT (SEQ ID NO: 43) L chain (4A-LC.7395): Full length MESQTQVLMSLLLWVSGTCGdivmtqtpssqavsagekvtmrcrssQSLLYSENKKNYlawyqqkpgrspklliyWASTgesgvpdrfigsgsgtdftltissvqaedlavyycQQYYNFPLTfgsgtkleikRADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVTDQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTS (SEQ ID NO: 82) (wherein the sequence: MESQTQVLMSLLLWVSGTCG (SEQ ID NO: 83) is the signal peptide, The sequence: RADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVTDQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTS (SEQ ID NO: 84) is the constant region).

[0153] Anti-colicin monoclonal antibody clone 9A L chain (9A-LC.7402): Variable region divmtqspssqavsagekvtmsckssQNLLYSEDKKNYlawyqqkpgqspklliyWAStresgvpdrfigsgsgtdftltvtsvqaedlavyycQQYYNFPRTfgggtklelk (SEQ ID NO: 44) L chain variable region CDR1 amino acid sequence: QNLLYSEDKKNY (SEQ ID NO: 45) L chain variable region CDR2 amino acid sequence: WAS (SEQ ID NO: 46) L chain variable region CDR3 amino acid sequence: QQYYNFPRT (SEQ ID NO: 47) L chain (9A-LC.7402): Full length MESQTQVLMSLLLWVSGTCGdivmtqspssqavsagekvtmsckssQNLLYSEDKKNYlawyqqkpgqspklliyWAStresgvpdrfigsgsgtdftltvtsvqaedlavyycQQYYNFPRTfgggtklelkRADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVTDQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTS (SEQ ID NO: 85) (wherein the sequence: MESQTQVLMSLLLWVSGTCG (SEQ ID NO: 86) is the signal peptide, The sequence: RADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVTDQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTS (SEQ ID NO: 87) is the constant region)

[0154] Anti-colicin monoclonal antibody clone 2A H chain (2A-HC.7352): Variable region evklvesggglvqpgnsltlscvasGFTFTNYGmhwirqapkkglewiamIYYDSSKMsyadtvkgrftisrdnskntlylemnslrsedtamyycAAEGFGTPFPYwgqgtlvtvss (SEQ ID NO: 48) H chain variable region CDR1 amino acid sequence: GFTFTNYG (SEQ ID NO: 70) H chain variable region CDR2 amino acid sequence: IYYDSSKM (SEQ ID NO: 49) H chain variable region CDR3 amino acid sequence: AAEGFGTPFPY (SEQ ID NO: 50) H chain (2A-HC.7352): Full length L chain: MDFRLNLVFLVFILKGVWCevklvesggglvqpgnsltlscvasGFTFTNYGmhwirqapkkglewiamIYYDSSKMsyadtvkgrftisrdnskntlylemnslrsedtamyycAAEGFGTPFPYwgqgtlvtvssAQTTAPSVYPLAPGCGDTTSSTVTLGCLVKGYFPEPVTVTWNSGALSSDVHTFPAVLQSGLYTLTSSVTSSTWPSQTVTCNVAH (SEQ ID NO: 88) (wherein the sequence: MDFRLNLVFLVFILKGVWC (SEQ ID NO: 89) is the signal peptide, and the sequence: AQTTAPSVYPLAPGCGDTTSSTVTLGCLVKGYFPEPVTVTWNSGALSSDVHTFPAVLQSGLYTLTSSVTSSTWPSQTVTCNVAH (SEQ ID NO: 90) is the constant region.) (2A-LC.7413): Variable region divmtqtpssravsagekvtmsckssQSLLYSENEKNYlawyqqrpgqspklliyWAStresgvpdrfigtgsgtdftltissvqaedlavyycQQYYHFPRTfgggtrlelk (SEQ ID NO: 51) L chain variable region CDR1 amino acid sequence: QSLLYSENEKNY (SEQ ID NO: 52) L chain variable region CDR2 amino acid sequence: WAS (SEQ ID NO: 53) L chain variable region CDR3 amino acid sequence: QQYYHFPRT (SEQ ID NO: 54) L chain (2A-LC.7413): Full length MESQTQVLMSLLLWVSGSCGdivmtqtpssravsagekvtmsckssQSLLYSENEKNYlawyqqrpgqspklliyWAStresgvpdrfigtgsgtdftltissvqaedlavyycQQYYHFPRTfgggtrlelkRADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVTDQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTS (SEQ ID NO: 91) (wherein the sequence: MESQTQVLMSLLLWVSGSCG (SEQ ID NO: 92) is the signal peptide, The sequence: RADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVTDQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTS (SEQ ID NO: 93) is the constant region).

[0155] Anti-colicin monoclonal antibody clone 21A: H chain (KK1410-2_VH_pep): variable region evklvesggglvqpgnsltlscgasgftftNYGMHwirqapkkglewigMIYYDSSKMSYADTVKGrftisrdnsknilylemnslrsedtamyycaaEGFGTPFPYwgqgtlvtvss (SEQ ID NO: 71) H chain variable region CDR1 amino acid sequence: NYGMH (SEQ ID NO: 72) H chain variable region CDR2 amino acid sequence: MIYYDSSKMSYADTVKG (SEQ ID NO: 73) H chain variable region CDR3 amino acid sequence: EGFGTPFPY (SEQ ID NO: 74) H chain (KK1410-2_VH_pep): full length MDFRLNLVFLVFILKGVWCevklvesggglvqpgnsltlscgasgftftNYGMHwirqapkkglewigMIYYDSSKMSYADTVKGrftisrdnsknilylemnslrsedtamyycaaEGFGTPFPYwgqgtlvtvss (SEQ ID NO: 94) (wherein the sequence: MDFRLNLVFLVFILKGVW (SEQ ID NO: 95) is the signal peptide) L chain (KK1410-2_VL_pep): Variable region divmtqtpssqavsagekvtmscKSSQSLLYREDTKNYLAwyqqrpgqspklliyWASTRESgvpdrfigsgsgtdftltissvqaedlavyycQQYYHFPRTfgggtrlelk (SEQ ID NO: 75) L chain variable region CDR1 amino acid sequence: KSSQSLLYREDTKNYLA (SEQ ID NO: 76) L chain variable region CDR2 amino acid sequence: WASTRES (SEQ ID NO: 77) L chain variable region CDR3 amino acid sequence: QQYYHFPRT (SEQ ID NO: 78) L chain (KK1410-2_VL_pep): Full length MESQTQVLMSLLLWVSGTYGdivmtqtpssqavsagekvtmscKSSQSLLYREDTKNYLAwyqqrpgqspklliyWASTRESgvpdrfigsgsgtdftltissvqaedlavyycQQYYHFPRTfgggtrlelk (SEQ ID NO: 96) (Note that the sequence: MESQTQVLMSLLLWVSGTYG (SEQ ID NO: 97) is the signal peptide.)

[0156] 1-4: Inhibition of colicin proapoptotic activity by anti-colicin monoclonal antibody clones. We evaluated whether monoclonal antibodies (2A, 9A, and 21A) with high binding affinity to colicin could inhibit the proapoptotic activity of colicin on cultured alveolar epithelial cells. Apoptosis inhibition assays were performed on A549 alveolar epithelial cells cultured to subconfluence, serum-starved overnight, and then pretreated with anti-colicin monoclonal antibody clones 2A, 9A, or 21A for an additional 48 hours. The number of apoptotic cells was assessed by flow cytometry as described above. A549 alveolar epithelial cells were pretreated with each anti-colicin monoclonal antibody clone (hybridoma supernatant diluted 1:100) and then cultured in the presence of colicin or scrambled peptide. A549 cells pretreated with rat anti-colicin serum served as a positive control, and untreated cells served as a negative control. Flow cytometry analysis revealed that only the clone 21A monoclonal antibody significantly neutralized colicin-induced apoptosis of alveolar epithelial cells in vitro (FIG. 14a, b).

[0157] We further investigated whether the clone 21A neutralizing monoclonal antibody inhibited the apoptotic activity of both Staphylococcus nepalensis and Staphylococcus haemolyticus. A549 alveolar epithelial cells were pretreated with the hybridoma supernatant of the anti-colicin monoclonal antibody clone A21 (diluted 1:100) and then cultured in the presence of culture supernatant of S. haemolyticus strain 12. A549 cells pretreated with monoclonal antibody A21 or isotype IgG alone served as a negative control, and cells pretreated with S. nepalensis supernatant served as a positive control. The results showed that the clone 21A neutralizing monoclonal antibody significantly inhibited the apoptotic activity of both S. nepalensis and S. haemolyticus bacterial supernatants on A549 alveolar epithelial cells (Fig. 15a, b).

[0158] Therefore, in subsequent experiments, we used the clone 21A monoclonal antibody to evaluate whether anti-colicin monoclonal antibodies could inhibit the proapoptotic activity of the degradation products of Staphylococcus nepalensis lysin-containing transglycosylase. A reaction mixture containing recombinant transglycosylase (2 mg / ml), a peptidase fraction (>10 kDa) from Staphylococcus nepalensis culture supernatant, and 20 μg / ml of anti-colicin monoclonal antibody clone A21 or an isotype antibody was incubated for 2 hours and then added to A549 cell cultures. After 48 hours of incubation, apoptosis was assessed by flow cytometry analysis. Cells treated with colicin and anti-colicin antibody or isotype antibody served as positive controls, and cells treated with anti-colicin antibody or isotype antibody alone served as negative controls. Flow cytometry analysis demonstrated that the anti-colicin monoclonal antibody clone A21 significantly inhibited the apoptotic activity of transglycosylase degradation products on alveolar epithelial cells compared with the isotype antibody control (Figure 16a, b). However, noteworthy results from gel electrophoresis and silver staining revealed that the anti-colicin neutralizing monoclonal antibody clone 21A failed to inhibit the proteolysis of recombinant transglycosylase (Figure 16c). Overall, these observations indicate that bacterial secretion of a putative serine protease triggers transglycosylase proteolysis, which releases colicin and induces apoptosis in lung epithelial cells, and that binding of the anti-colicin neutralizing monoclonal antibody to transglycosylase does not interfere with transglycosylase degradation.

[0159] Example 2: Inhibitory Effect of Anti-colicin Neutralizing Antibodies on Colicin-Induced AEs in Pulmonary Fibrosis We previously demonstrated that colicin induces AEs in pulmonary fibrosis in TGFβ1 TG mice. Based on this finding, we investigated whether anti-colicin neutralizing antibodies could suppress AEs in fibrotic disease. First, we evaluated the changes in circulating levels of anti-colicin monoclonal antibody clone 21A after intraperitoneal administration and calculated its half-life. Five TGFβ1 transgenic mice (TGFβ1 TG mice) with pulmonary fibrosis induced by lung-specific overexpression of the gene encoding full-length human TGFβ1 were intraperitoneally injected with 20 mg / kg of anti-colicin antibody clone A21, and blood was collected at 3, 6, 24, 48, 168, 336, and 504 hours. Plasma was separated after centrifugation, and anti-colicin antibody levels were measured using an enzyme immunoassay as described in Materials and Methods. As a result, the mean plasma half-life of the anti-colicin neutralizing monoclonal antibodies was 3.8±2.5 days (FIG. 17).

[0160] TGFβ1 TG mice with pulmonary fibrosis were then assigned to two groups with corresponding computed tomography (CT) scores (Figure 18a, b). CT examinations were performed as follows: Lung CT was performed using a microCT Latheta LCT-200 purchased from Hitachi Aloka Medical (Tokyo, Japan). Mice were anesthetized with isoflurane inhalation and placed in a prone position for data collection (Non-Patent Document 19, Reference 61). Seven to nine pulmonary radiology experts blinded to the treatment groups scored the CT findings in TGFβ1 TG mice with pulmonary fibrosis based on previously reported and validated criteria: score 1, normal lung findings; score 2, intermediate findings; score 3, mild pulmonary fibrosis; score 4, intermediate findings; score 5, moderate pulmonary fibrosis; score 6, intermediate findings; and score 7, advanced pulmonary fibrosis (Non-Patent Document 19). The same criteria were used to score lung inflammation and fibrosis in a BLM-induced pulmonary fibrosis mouse model (Fig. 21b).

[0161] A group of TGFβ1-TG mice without fibrosis that received intratracheal colicin injection served as a control (Fig. 18a, b). One group with pulmonary fibrosis received anti-colicin monoclonal antibody clone 21A, and the other group with pulmonary fibrosis received an irrelevant isotype antibody intraperitoneally once every 3 days for 2 weeks prior to intratracheal colicin injection to induce AE (Fig. 19a). Specifically, a group of TGFβ1-TG mice with pulmonary fibrosis (n = 6) received intraperitoneal injections of anti-colicin monoclonal antibody (mAtb), and another group with pulmonary fibrosis (n = 6) received five doses of isotype antibody (Atb) 2 days prior to intratracheal colicin injection. A group of TGFβ1-TG mice without fibrosis (n = 5) received intratracheal colicin alone. For comparison, CT was performed before and after intratracheal colicin or saline injection.

[0162] Control TGFβ1 TG mice without fibrosis treated with saline, isotype antibody, or anti-colicin antibody showed no changes (Fig. 18b). TGFβ1 TG mice administered colicin and treated with isotype antibody showed a significant worsening of CT findings (Fig. 19c). On the other hand, mice treated with anti-colicin monoclonal antibody showed a significant improvement in pulmonary fibrosis CT scores (Fig. 19d).

[0163] We next evaluated whether monoclonal anti-colicin antibodies suppressed acute exacerbations (AEs) of pulmonary fibrosis in TGFβ1-TG mice. TGFβ1-TG mice were randomly assigned to two groups matched for pulmonary fibrosis grade and one group without pulmonary fibrosis. The TGFβ1-TG mice with pulmonary fibrosis (n = 6) were intraperitoneally injected with anti-colicin monoclonal antibodies, while the other group with pulmonary fibrosis (n = 6) received five doses of isotype antibodies 2 days prior to instillation. Colicin was administered intratracheally. The TGFβ1-TG mice without fibrosis (n = 5) received only intratracheal colicin. After euthanasia by anesthesia overdose, bronchoalveolar lavage fluid (BALF) was collected from each group. The BALF fluid was centrifuged, and the pellet was used to assess total and differential cell counts between treatment groups.

[0164] Results showed that TGFβ1 TG mice treated with anti-colicin monoclonal antibody had significantly reduced total cell and lymphocyte counts in BALF compared with control and isotype antibody-treated mice (Fig. 20a, b). Markers of lung injury, such as surfactant protein-D (SP-D), MUC5B, matrix metalloproteinase-1 (MMP-1), and decorin, as well as bronchoalveolar lavage fluid (BALF) levels of SP-D, MUC5B, and MUC-1, were significantly reduced in anti-colicin monoclonal antibody-treated mice compared with control and isotype antibody-treated mice (Fig. 20c). Hydroxyproline content and collagen deposition in the lungs were significantly lower in anti-colicin monoclonal antibody-treated mice than in isotype antibody-treated mice (Fig. 20c, d, e). The grade of pulmonary fibrosis significantly correlated with the number of pulmonary inflammatory cells (Fig. 20f). These observations indicate that colicins are potential therapeutic targets for AE in pulmonary fibrosis.

[0165] Example 3: Effect of colicin on AE induction in bleomycin-induced pulmonary fibrosis. Bleomycin (BLM)-induced pulmonary fibrosis in mice or rats is the most characterized and commonly used preclinical model for IPF research (Reference 28). Whether colicin exacerbates BLM-induced pulmonary fibrosis is unknown. To address this issue, we developed an experimental model in wild-type (WT) mice by infusing BLM via an osmotic minipump and administering colicin via intratracheal instillation (Figure 21a). According to the experimental schedule for induction of acute exacerbation in mice with bleomycin-induced pulmonary fibrosis shown in Figure 21a, bleomycin (BLM) or saline (SAL) was infused through an osmotic minipump implanted subcutaneously in the backs of mice. Computed tomography (CT) imaging was performed before and after intratracheal administration of colicin or scrambled peptide before mouse euthanasia. Mice administered SAL via the osmotic minipump and intratracheal colicin or scrambled peptide were used as controls. Mice were euthanized 22 days after BLM or SAL pump implantation.

[0166] After confirming the development of pulmonary fibrosis by chest CT in this way, mice were assigned to two groups with consistent CT scores using an autologous CT score criterion (FIG. 21b).

[0167] Wild-type (WT) mice were administered bleomycin (BLM) via osmotic minipumps on day 0 and intratracheally instilled (75 μl) colicin or scrambled peptide (300 μg / mouse) 20 days after pump implantation. CT was performed as described in Example 2. The CT scores of mice with pulmonary fibrosis were significantly worsened after intratracheal colicin administration (Fig. 22a, b, c). Next, wild-type (WT) mice were administered saline (SAL) via osmotic minipumps on day 0 and intratracheally instilled (75 μl) colicin or scrambled peptide (300 μg / mouse) 20 days after pump implantation. The CT scores were unchanged in mice with pulmonary fibrosis that received intratracheal instillation of scrambled peptide or mice without pulmonary fibrosis that received either intratracheal colicin or scrambled peptide (Fig. 22d, e, f).

[0168] Next, we examined inflammatory cells in mice with BLM-induced pulmonary fibrosis treated with intratracheal colicin. Wild-type (WT) mice were administered bleomycin (BLM) or saline (SAL) via osmotic minipumps on day 0, and colicin or scrambled peptide (300 μg / mouse) was intratracheally instilled (75 μl) on day 20 after pump implantation. Total cell counts in BALF were determined using a NucleoCounter, and differential cell counts were performed after Giemsa staining using WindRoof Image software. Serum amyloid P component (SAP), MUC-1, monocyte chemoattractant protein-1 (MCP-1), periostin, collagen I, and osteopontin levels were measured by enzyme immunoassay according to the manufacturer's instructions. Results showed that mice with BLM-induced pulmonary fibrosis treated with intratracheal colicin had significantly increased total lymphocyte numbers in BALF, increased circulating levels of serum amyloid P component, and increased levels of MUC-1, monocyte chemoattractant protein-1 (MCP-1), periostin, collagen I, and osteopontin compared with control mice treated with intratracheal scrambled peptide (Fig. 23a, b, c).

[0169] Next, caspase-3 cleavage was assessed and quantified by Western blotting. Caspase-3 cleavage was significantly increased in mice administered intratracheally colicin compared with their counterparts administered scrambled peptide (Figure 23d, e). Collagen deposition was assessed by Masson's trichrome staining and quantified using WindRoof imaging software. Lung tissue hydroxyproline content was measured colorimetrically using a commercially available kit according to the manufacturer's instructions. Results showed that collagen deposition and hydroxyproline content in the lungs were also increased in mice administered colicin compared with mice treated with scrambled peptide (Figure 23f, g, h). Mice administered saline via osmotic minipump and treated with colicin or scrambled peptide showed no significant changes. These findings indicated that colicin also exacerbates BLM-induced pulmonary fibrosis.

[0170] Example 4: Inhibitory effect of anti-colicin monoclonal antibodies on BLM-induced pulmonary fibrosis. Because colicin exacerbates BLM-induced pulmonary fibrosis, we predicted that this lethal peptide would play a role in this experimental model. To test this prediction, mice were infused with BLM via osmotic minipumps and treated intraperitoneally with anti-colicin neutralizing monoclonal or isotype antibodies three times a week for three weeks (Figure 24a). Wild-type (WT) mice were administered bleomycin (BLM) via osmotic minipumps and treated intraperitoneally with anti-colicin monoclonal antibodies (WT / BLM / anti-colicin) or isotype antibodies (WT / BLM / isotype) three times a week for three weeks. WT mice were administered saline (SAL) via osmotic minipumps and treated intraperitoneally with anti-colicin monoclonal antibodies (WT / SAL / anti-colicin) or isotype antibodies (WT / SAL / isotype) three times a week for three weeks as controls. Computed tomography (CT) scans were performed 1 day before euthanasia, and nine experts blinded to the treatment groups recorded the CT findings. Mice were euthanized 21 days after implantation of the BLM or saline (SAL) pump. Mice receiving saline (SAL) via osmotic minipumps served as controls to rule out secondary effects of the antibody.

[0171] As a result, mice with BLM-induced pulmonary fibrosis treated with anti-colicin monoclonal antibodies had significantly reduced CT fibrosis scores compared with matched mice treated with isotype antibodies (Fig. 24b, c). Mice treated with anti-colicin monoclonal antibodies or isotype antibodies after saline injection did not show any changes in CT.

[0172] Next, the total cell counts in the BALF were determined using a NucleoCounter, and differential cell counts were performed after Giemsa staining using WindRoof Image software. Evaluation of inflammatory cells in the BALF showed that BLM-induced pulmonary fibrosis mice treated with anti-colicin monoclonal antibodies had significantly reduced numbers of all cell types, lymphocytes, and neutrophils compared with mice treated with isotype antibodies ( Figure 24 d, e).

[0173] In addition, we examined acute tissue damage, parenchymal cell apoptosis, and tissue fibrosis in the lungs of pulmonary fibrosis mice treated with anti-colicin monoclonal antibody. Wild-type (WT) mice received bleomycin (BLM) via osmotic minipumps and were treated intraperitoneally with anti-colicin monoclonal antibody (WT / BLM / anti-colicin) or isotype antibody (WT / BLM / isotype) three times a week for three weeks. WT mice received saline (SAL) via osmotic minipumps and were treated intraperitoneally with anti-colicin monoclonal antibody (WT / SAL / anti-colicin) or isotype antibody (WT / SAL / isotype) three times a week for three weeks as controls. Osteopontin, MUC-1, and MUC5B levels were measured by enzyme immunoassay using commercially available kits. DNA fragmentation was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Pulmonary fibrosis grade was scored by seven blinded experts using the Ashcroft score. Lung collagen deposition was assessed by Masson's trichrome staining and quantified using WindRoof imaging software. Lung tissue hydroxyproline content was measured by colorimetry using a commercially available kit according to the manufacturer's instructions.

[0174] Results showed that mice with BLM-induced pulmonary fibrosis treated with anti-colicin monoclonal antibodies showed lower plasma and BALF levels of osteopontin, MUC-1, and MUC5B, a reduced number of apoptotic lung epithelial cells, and reduced Ashcroft scores, collagen deposition, and hydroxyproline in the lungs compared with mice treated with isotype antibodies (Figures 25a, b, c, d, e, f, g, h). Overall, these findings indicate that colicins also play a role in the development of BLM-induced pulmonary fibrosis.

[0175] Example 5: Effect of anti-colicin monoclonal antibodies administered during the acute phase on improving BLM-induced lung injury and pulmonary fibrosis. Lung injury induced by BLM administered subcutaneously via an osmotic minipump is characterized by acute lung inflammation that peaks on day 11, followed by chronic lung fibrosis (Reference 29). We investigated whether anti-colicin monoclonal antibodies improve pulmonary fibrosis by inhibiting BLM-induced early acute lung injury.

[0176] Bleomycin (BLM) was infused via an osmotic minipump implanted subcutaneously in the backs of mice. On days 2, 4, 6, 9, and 11 after injection, mice were treated intraperitoneally with an isotype antibody (WT / BLM / isotype) or an anti-colicin monoclonal antibody (WT / BLM / anti-colicin) at a dose of 20 mg / kg. Mice were not treated during the chronic phase from day 12 until euthanasia on day 22. Blood was collected on day 9, and computed tomography (CT) scans were performed on day 9 and before euthanasia. Mice were euthanized on day 22 after BLM pump implantation. CT lung opacity was measured in each group using WindRoof image software. Blood was collected on day 9 after BLM pump implantation, and the total cell count in the blood was determined using a NucleoCounter, and the differential cell count was calculated after Giemsa staining. WindRoof image software was used.

[0177] As a result, CT findings of lung injury and blood neutrophil counts were significantly improved on day 9 in mice treated with anti-colicin monoclonal antibody compared to controls treated with IgG2 isotype antibody (Figure 26b, c, d, e).

[0178] Next, wild-type (WT) mice were administered bleomycin (BLM) via an osmotic minipump and treated with anti-colicin monoclonal antibody (WT / BLM / anti-colicin) or isotype antibody (WT / BLM / isotype) via the intraperitoneal route on days 2, 4, 6, 9, and 11 after bleomycin pump implantation. CT was performed on day 21 after BLM pump implantation (chronic phase). Bronchoalveolar lavage fluid (BALF) was also collected 22 days after BLM pump implantation. Total cell counts in BALF were determined using a NucleoCounter, and differential cell counts were performed after Giemsa staining using WindRoof Image software.

[0179] Results showed that mice treated with anti-colicin monoclonal antibodies showed significant improvements in CT fibrosis scores and lymphocyte counts in BALF during the chronic phase of the disease (day 22 after the start of BLM infusion) compared to mice receiving control antibodies (Figure 27a, b, c, d).

[0180] Furthermore, we examined lung injury, pulmonary parenchymal apoptosis, and collagen deposition on day 22 in mice with bleomycin-induced pulmonary fibrosis treated with anti-colicin monoclonal antibody during the acute phase of the disease. Wild-type (WT) mice were administered bleomycin (BLM) via osmotic minipumps and then treated intraperitoneally with anti-colicin monoclonal antibody (WT / BLM / anti-colicin) or isotype antibody (WT / BLM / isotype) on days 2, 4, 6, 9, and 11 after BLM pump implantation. Bronchoalveolar lavage fluid (BALF) and plasma were collected 22 days after BLM pump implantation. Levels of MUC-1, interface protein C (SP-C), SP-D, collagen I, periostin, osteopontin, and total TGF-β1 were measured using commercially available enzyme immunoassay kits according to the manufacturer's instructions (Figure 28a). Western blotting for cleaved caspase-3 and β-actin was also performed (Figure 28b, c). Lung collagen deposition was quantified using Masson's trichrome-stained photographs and WindRoof image software (Fig. 28f, g), and lung hydroxyproline content was measured by a commercial colorimetric assay according to the manufacturer's instructions (Fig. 28h).

[0181] Results showed that plasma levels of MUC-1, SP-C, SP-D, collagen I, periostin, and osteopontin, as well as BALF levels of MUC-1 and total TGF-β1, were significantly lower in mice treated with anti-colicin monoclonal antibodies than in mice treated with isotype antibodies at day 22 (Figure 28a). Mice treated with anti-colicin monoclonal antibodies had significantly reduced Ashcroft scores, collagen deposition, and hydroxyproline content, as well as reduced caspase-3 cleavage in the lungs compared with mice treated with isotype antibodies (Figures 28b, c, d, e, f, and g). These observations indicate that colicin-associated acute lung injury during the acute phase is an important determinant of pulmonary fibrosis during the chronic phase of the disease.

[0182] Example 6: Effect of Anti-colicin Monoclonal Antibody on Survival of AE Mice with Pulmonary Fibrosis. Computed tomography (CT) was performed on human transforming growth factor β1 (TGFβ1) transgenic (TG) mice. Seven experts blindly scored the grade of pulmonary fibrosis, and the average score of the seven experts for each mouse was considered the mouse's CT score. TGFβ1TG mice with pulmonary fibrosis were divided into two groups based on matched CT fibrosis scores and administered bleomycin (BLM) via osmotic minipumps. One of these two groups received an intraperitoneal injection of anti-colicin monoclonal antibody (TGFβ1-TG / BLM / anti-colicin), while the other group received an intraperitoneal injection of an isotype antibody (TGFβ1-TG / BLM / isotype). Treatment with anti-colicin monoclonal antibody or isotype antibody was performed three times a week for three weeks. Control mice without pulmonary fibrosis were treated with anti-colicin monoclonal antibody (TGFβ1-TG / SAL / anti-colicin) or isotype antibody (TGFβ1-TG / SAL / isotype) and infused with saline via osmotic minipumps (Fig. 29a, b). The survival rates of these treated mice are shown in Fig. 29c.

[0183] The results showed that TGFβ1 TG mice with pulmonary fibrosis and BLM-induced AE treated with anti-colicin monoclonal antibodies showed significantly longer survival than mice treated with isotype antibodies (Figure 29c). As expected, there was no change in survival in the control group administered saline via osmotic minipumps.

[0184] Example 7: Suppression of lipopolysaccharide-induced acute lung injury in anti-colicin monoclonal antibodies. Based on the above results, we investigated whether colicin also affects the development of acute lung injury caused by other substances, such as lipopolysaccharide (LPS). Wild-type (WT) mice were treated with isotype antibodies or anti-colicin monoclonal antibodies at a dose of 20 mg / kg intraperitoneally, once daily every other day, for three consecutive treatments. Two days after the last antibody treatment, mice were intratracheally instilled with a high dose (150 μg) of lipopolysaccharide (LPS) or saline (SAL). Two days after the last antibody treatment, mice were euthanized two days after LPS instillation. Mice administered intratracheal saline (SAL) and treated with isotype antibodies (WT / SAL / isotype) or anti-colicin antibodies (WT / SAL / anti-colicin) served as control mice (Figure 30a). Bronchoalveolar lavage fluid (BALF) was collected from mice euthanized 2 days after intratracheal LPS instillation. BALF cells were counted using a NucleoCounter, and the cell fraction was Giemsa stained and counted (Fig. 30b, c). The levels of lactate dehydrogenase A (LDHA), surfactant protein D (SP-D), matrix metalloproteinase-1 (MMP-1), MUC-1, and tumor necrosis factor-α (TNFα) were measured using commercially available immunoassay kits according to the manufacturer's instructions (Fig. 30d).

[0185] We also investigated whether treatment with anti-colicin monoclonal antibodies improved CT findings in mice with severe lipopolysaccharide-induced acute lung injury. Wild-type (WT) mice were treated with isotype antibodies (WT / LPS / isotype) or anti-colicin antibodies (WT / LPS / anti-colicin) at a dose of 20 mg / kg intraperitoneally, once daily every other day, for 1 week, and then euthanized. Mice were intratracheally instilled with a high dose (150 μg) of lipopolysaccharide (LPS) or saline. Mice administered intratracheal saline (SAL) and treated with isotype antibodies (WT / SAL / isotype) or anti-colicin antibodies (WT / SAL / anti-colicin) served as controls. Computed tomography (CT) scans were performed 1 day after intratracheal instillation of LPS (Figure 31a). CT opacity from the apex to the basal region of the lung was quantified using WindRoof imaging software as described in Materials and Methods (Fig. 31b).

[0186] The results showed that mice treated with anti-colicin monoclonal antibodies had significantly reduced lung CT opacity compared with mice pretreated with the isotype control (Fig. 31a, b), a lower number of lung-infiltrating neutrophils, and significantly reduced plasma and BALF levels of lactate dehydrogenase A (LDHA), SP-D, and MMP-1, as well as BALF levels of MUC-1 and tumor necrosis factor-α (TNFα), indicating the involvement of colicin in the acute inflammatory response to LPS (Fig. 30c, d).

[0187] We next investigated whether anti-colicin monoclonal antibodies could attenuate moderate lipopolysaccharide-induced acute lung injury. WT mice were treated intraperitoneally with either isotype or anti-colicin monoclonal antibodies at a dose of 20 mg / kg, once daily on alternate days, for three treatments. Two days after the final antibody treatment, mice were intraperitoneally injected with a low dose (75 μg) of LPS and euthanized two days after LPS injection. BALF was then collected, and BALF cells were counted using a nucleocounter and stained with Giemsa for differential cell counting, as described in Materials and Methods (Figure 32b, c). BALF and plasma levels of surfactant protein D (SP-D), monocyte chemotactic protein-1 (MCP-1), TNFα, lactate dehydrogenase A (LDHA), MUC-5B, and matrix metalloproteinase-1 (MMP-1) were measured using commercially available immunoassay kits according to the manufacturer's instructions (Figure 32d).

[0188] Thus, in another experiment inducing milder acute lung injury using a lower dose of intratracheal LPS, mice pretreated with anti-colicin monoclonal antibody showed significantly reduced numbers of lung-infiltrating neutrophils, plasma and BALF levels of SP-D, monocyte chemoattractant protein-1 (MCP-1), and TNFα, and significantly reduced BALF levels of LDHA and plasma levels of MUC5B and MMP-1 compared with isotype-pretreated control mice (Fig. 32a, b, c, d). Overall, these observations further support the involvement of colicin in the development of acute lung injury.

[0189] Example 8 Plasma levels of colicin as a potential biomarker for pulmonary fibrosis The occurrence and frequency of AEs in IPF patients predicts accelerated pulmonary fibrosis and adverse clinical outcomes (Non-Patent Document 1). We predicted that circulating colicins might serve as biomarkers for pulmonary fibrosis. To test this prediction, we designed an enzyme immunoassay using anti-colicin monoclonal antibodies.

[0190] 8-1: Colicin Assay. An enzyme immunoassay was developed using anti-colicin monoclonal antibody clone 9A as the capture antibody and biotinylated anti-colicin monoclonal antibody as the detection antibody. Briefly, the capture antibody was coated onto a 96-well plate at a final concentration of 2 μg / ml in phosphate-buffered saline overnight at 4°C. After blocking with 1% bovine serum albumin in phosphate-buffered saline and appropriate washing with phosphate, buffered saline in Tween, various concentrations of colicin as a standard, and plasma samples were added to the wells and incubated overnight at 4°C. After washing the wells, horseradish peroxidase-conjugated streptavidin (R&D Systems) was added in phosphate-buffered saline. After appropriate washing and incubation, a substrate solution was added for color development, and the absorbance was read at 450 nm. Values ​​were extrapolated from a standard curve constructed using several concentrations of colicin.

[0191] 8-2: Evaluation of colicin as a potential biomarker for pulmonary fibrosis. Plasma levels of colicin were measured in TGFβ1 transgenic (TG) and wild-type (WT) mice with pulmonary fibrosis induced by lung-specific overexpression of the gene encoding full-length human TGFβ1, and correlations with fibrosis markers were assessed (Figure 33a). Specifically, transforming growth factor β1 (TGFβ1) transgenic (TG) mice were genotyped, computed tomography (CT) was performed, and findings regarding the degree of pulmonary fibrosis were recorded. Mice were euthanized for blood and lung tissue sampling. Ashcroft scores for pulmonary fibrosis, as well as measurements of plasma colicin and lung hydroxyproline, were performed as described in Materials and Methods.

[0192] As expected, the computed tomography (CT) score of pulmonary fibrosis, Ashcroft score of fibrosis, and lung hydroxyproline content were significantly increased in TGFβ1 TG mice compared with WT mice (Fig. 32b; Fig. 34a, b). Plasma colicin concentrations were significantly elevated in TGFβ1 TG mice compared with WT mice, and the plasma colicin concentrations were significantly correlated with the CT fibrosis score, Ashcroft score, and lung hydroxyproline content, supporting the potential application of circulating colicins as biomarkers of disease progression in pulmonary fibrosis (Fig. 31b).

[0193] Example 9 Vaccine Preparation The amino acid sequences of the colicin positions of the colicin-containing transglycosylase of S. nepalensis (i.e., S. nepalensis strain CNDG protein 00351) and several transglycosylases in the same family were examined, as shown in the alignment in Figure 4. It is very clear that certain bacteria have amino acid changes at specific positions. The Weissella confusa colicin peptide showed two amino acid changes: Asp14Ser (or N14S) and Ala16Asp (or A16N). All nomenclature of mutations / changes is based on the S. nepalensis colicin numbering system; i.e., position 1 is isoleucine (I) and position 19 is arginine (R). As shown in Figure 3(3), W. confusa peptides (Shem_00350 or 1b or 7b or 12b) were synthesized (accession number WP_112464134.1), and their apoptotic activity was compared with that of a Slaphylococcus haemolyticus colicin peptide with the same sequence except for two changes shown in the W. confusa peptide (i.e., N14S and A16N—peptide accession number WP_112464134.1). As shown in Figure 3(3), it is very clear that these mutations dramatically reduced the apoptotic activity of the W. confusa peptide (25% of the activity of Shem_00350 or 1b or 7b or 12b). Another example of a mutation that reduces the apoptosis-inducing ability of a colicin peptide is Mycobacterium abscessus colicin peptide 1 (Absc-1 in the Myco alignment or GenBank accession number SKT99287.1). The Myco abc-1 peptide has three amino acid changes and one amino acid deletion compared to the S. haemolyticus corisin peptide, as shown in the alignment in Figure 4 (4). The data on apoptosis induction ability shown in Figure 3 (3) indicate that the amino acid changes and deletion resulted in a much lower induction ability of the Myabs-1 peptide (approximately 30% of the activity of the hemolytic streptococcal peptides - Shem 00350 or 1 b or 7 b or 12 b).The opposite effect was observed for the original colicin, i.e., the CNDG peptide from S. nepalensis (or CNDG-00351 in the alignment) and the S. haemolyticus peptide (Shem_00350 or 1 b or 7 b or 12 b). Here, there was a single amino acid change in the S. nepalensis peptide compared to the S. haemolyticus peptide (i.e., Gly 7 Ser or G 7 S in the S. nepalensis peptide). Unlike previous examples, the S. nepalensis peptide always showed slightly higher apoptosis-inducing activity compared to the S. haemolyticus peptide.

[0194] Based on the above analysis, amino acid mutations were performed in colicins and the following three peptides were synthesized: colicin N14S: IVMPESSGNPNAVSPAGYR (SEQ ID NO: 55) colicin P15A: IVMPESSGNPNAVNAAGYR (SEQ ID NO: 56) colicin A16N: IVMPESSGNPNAVNPNGYR (SEQ ID NO: 57)

[0195] After synthesis, the colicins were compared to their parent (peptide) sequence (IVMPESSGNPNAVNPAGYR) to determine whether their activity was reduced. It was predicted that a single amino acid change would render the colicin peptide inactive. The results are shown in Figure 35. As expected, colicins N14S and P15A were found to have no apoptotic activity, comparable to the nonfunctional scrambled peptide.

[0196] We will continue to make amino acid changes (focusing on the C-terminal half, since antibody binding is likely to occur in the N-terminal half). We are currently investigating single amino acid changes, but based on these results, we believe that a combination of two or three amino acids should be able to completely eliminate the apoptotic properties of the colicin peptide. This dead (or attenuated) peptide can then be tested in mouse models via either intramuscular injection or intranasal (inhalation) administration to determine its ability to protect mouse patients from colicin-released bacteria or colicin peptide challenge.

[0197] Example 10: Additive Activities of Colicins. The Caki-2 cell line derived from proximal renal tubule epithelium, the HaCaT cell line derived from adult skin keratinocytes, the ARPE-19 cell line derived from retinal pigment epithelium, the Caco-2 cell line derived from colon carcinoma, and the HIEC-6 cell line derived from normal human small intestinal epithelial cells were obtained from the American Type Culture Collection (Manassas, VA). RPMI 1640 medium was purchased from Sigma-Aldrich (St. Louis, MO), and fetal bovine serum (FBS) was purchased from BioWhittaker (Walkersville, MD). L-glutamine, penicillin, and streptomycin were obtained from Invitrogen (Carlsbad, CA). Caki-2, Caco-2, and ihPOD cells were cultured in RPMI 1640 medium, HaCaT cells in DMEM medium, ARPE-19 cells in DMEM / F12 medium, and HIEC-6 cells in DMEM medium.

[0198] To examine caspase-3 cleavage by Western blotting, each cell line was cultured to subconfluence. Cells were then washed and cultured overnight in medium without FCS. Cells were stimulated with 10 μg / ml of scrambled peptide, colicin, or colicin-like peptide for 24 hours before Western blotting.

[0199] The results are shown in Figure 39. Caspase-3 activity analysis revealed that caspase-3 cleavage was significantly increased in all cells treated with colicin and colicin-like peptides compared to cells treated with scrambled peptides, indicating activation of the apoptotic pathway. Specifically, Figure 39 demonstrates that colicin induces apoptosis in colonic epithelial cells, small intestinal epithelial cells, skin keratinocytes, retinal epithelial cells, renal podocytes, and ureteral epithelial cells. These results suggest that the anti-colicin monoclonal antibody of the present invention may be effective in treating diseases caused by these cells, such as ulcerative colitis, Crohn's disease, retinopathy, nephritis / renal fibrosis, chronic dermatitis, scleroderma, and liver cirrhosis, in addition to pulmonary fibrosis.

[0200] Example 11: Comparison of Serum Colicin Concentrations An enzyme immunoassay was developed using a rabbit polyclonal anti-transglycosylase polyclonal antibody and a biotinylated anti-colicin 9A monoclonal antibody. Serum colicin levels were compared between healthy subjects, stable idiopathic pulmonary fibrosis (IPF) patients, and IPF patients with acute exacerbations. The results are shown in Figure 40. Figure 40 shows that serum colicin concentrations were significantly higher in stable IPF patients compared to healthy subjects, and were also significantly increased in IPF patients with acute exacerbations compared to stable IPF patients. These results demonstrate the usefulness of colicin measurement as a biomarker for IPF patients.

[0201] Example 12: Natural colicin exhibits stronger apoptotic activity than synthetic colicin. Previous experiments have shown that synthetic colicin has apoptotic activity at a concentration of 10 μg / ml. We investigated the relative activity of natural colicin secreted by bacteria compared to synthetic colicin. First, we examined the apoptotic activity of bacterial culture supernatants. Ten-fold (1 / 10) diluted Staphylococcus nepalensis culture supernatant was added to the medium of A59 alveolar epithelial cells in the presence or absence of anti-colicin monoclonal antibody (clone A21) (20 μg / ml). After culturing A59 cells for 48 hours, apoptosis was examined by flow cytometry.

[0202] The results are shown in Figures 41a, b and c. Figure 41a shows that apoptosis of A549 cells induced by 10-fold diluted bacterial supernatant was significantly and almost completely inhibited by anti-colicin monoclonal antibody.

[0203] The concentrations of native colicins in the undiluted bacterial supernatants were then measured by enzyme immunoassay. The results are shown in Figures 41b and 41c. Figures 41b and 41c show that the mean concentration of native colicins in undiluted culture supernatants of Staphylococcus nepalensis was 1042±174.6 pg / ml, and that the mean concentration of native colicins in undiluted culture supernatants of Staphylococcus haemolyticus was 802.4±57.7 pg / ml.

[0204] These results demonstrate that the native colicin has potent biological activity at approximately 100 pg / ml, since bacterial supernatants were diluted 1:10 in the apoptosis induction assay for A549 cells. While the synthetic colicin exhibited apoptotic activity at 10 μg / ml, as shown in Figures 41a, b, and c, the native colicin exhibited apoptotic activity at approximately 100 pg / ml. Therefore, the native colicin (10 μg / 100 pg = 100,000) has 100,000-fold greater apoptotic activity than the synthetic colicin.

[0205] Example 13: Anti-colicin monoclonal antibody (clone A21) also inhibits the apoptotic activity of native colicin in human body fluids (bronchoalveolar lavage fluid) Similar experiments were performed to investigate the proapoptotic activity of native colicin present in bronchoalveolar lavage fluid (BALF) from IPF patients. BALF collected from IPF patients with acute exacerbations (n ​​= 14) and healthy controls (n = 5) was added to the culture medium of A59 alveolar epithelial cells in the presence of anti-colicin monoclonal antibody (clone A21) or control IgG (20 μg / ml). 48 hours later, apoptosis was examined by flow cytometry.

[0206] Figure 41d shows the results obtained for each IPF patient or each healthy subject. Figure 41e shows the results obtained for all IPF patients or all healthy subjects. Figure 41e shows that the anti-colicin monoclonal antibody of the present invention significantly inhibits the apoptotic activity of BALF from IPF patients. Meanwhile, Figure 41f shows the concentrations of native colicin in BALF from IPF patients (n = 14) (650.9 ± 218.3 pg / ml) and healthy subjects (n = 5) (458.4 ± 60.7 pg / ml). Because BALF was used at a 1:2 dilution in the apoptosis induction assay for A549 cells, native colicin in human body fluids also possesses potent biological activity.

[0207] Figure 41g also shows the half-life of colicin in vitro and in cell culture supernatants. To determine how rapidly colicin is metabolized in a cell culture system, we investigated the half-life of colicin. A549 alveolar epithelial cells were cultured to confluence in DMEM supplemented with 10% fetal bovine serum. Colicin was then inoculated into the cell culture medium at a final concentration of 10 micrograms / ml, and cell supernatants were collected after 0, 1, 3, 6, 12, and 24 hours. The colicin concentration was measured by enzyme immunoassay to examine the half-life of colicin. As shown in Figure 41g, the half-life of (synthetic) colicin in solution was found to be less than 1 hour in a human alveolar cell culture system. These results suggest that although serum (natural) colicin concentrations increase during acute exacerbations in IPF patients, their concentrations rapidly decline due to their short half-life. Therefore, serum colicin measurements should be performed immediately after the onset of an acute exacerbation.

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Claims

1. An isolated monoclonal antibody molecule or derivative thereof which specifically binds to a colicin.

2. The monoclonal antibody molecule or derivative thereof according to claim 1, wherein the colicin is IVMPESSGNPNAVNPAGYR (SEQ ID NO: 1).

3. A monoclonal antibody molecule or a derivative thereof according to claim 1 or 2, which has PESSGNP (SEQ ID NO: 68) or NPAGY (SEQ ID NO: 69) as an epitope.

4. (IA) (i) an H-chain variable region comprising the H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 37, the H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 38, and the H-chain variable region CDR3 amino acid sequence of SEQ ID NO: 39, and (ii) an L-chain variable region comprising the L-chain variable region CDR1 amino acid sequence of SEQ ID NO: 41, the L-chain variable region CDR2 amino acid sequence of SEQ ID NO: 42, and the L-chain variable region CDR3 amino acid sequence of SEQ ID NO: 43; (IIA) (i) an H-chain variable region comprising the H-chain variable region CDR1 amino acid sequence of SEQ ID NO: 70, the H-chain variable region CDR2 amino acid sequence of SEQ ID NO: 49, and the H-chain variable region CDR3 amino acid sequence of SEQ ID NO: 50, and (ii) an L-chain variable region comprising the L-chain variable region CDR1 amino acid sequence of SEQ ID NO: 52, the L-chain variable region CDR2 amino acid sequence of SEQ ID NO: 53, and the L-chain variable region CDR3 amino acid sequence of SEQ ID NO: 54; or (IVA) The monoclonal antibody molecule or derivative thereof according to claim 1 or 2, comprising: (i) an H chain variable region comprising the H chain variable region CDR1 amino acid sequence of SEQ ID NO: 72, the H chain variable region CDR2 amino acid sequence of SEQ ID NO: 73, and the H chain variable region CDR3 amino acid sequence of SEQ ID NO: 74; and (ii) an L chain variable region comprising the L chain variable region CDR1 amino acid sequence of SEQ ID NO: 76, the L chain variable region CDR2 amino acid sequence of SEQ ID NO: 77, and the L chain variable region CDR3 amino acid sequence of SEQ ID NO:

78.

5. (IB) a heavy chain variable region comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 36, or an amino acid sequence at least 80% identical thereto, or a heavy chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the heavy chain variable region amino acid sequence of SEQ ID NO: 36; (IIB) a heavy chain variable region comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 48, or an amino acid sequence at least 80% identical thereto, or a heavy chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the heavy chain variable region amino acid sequence of SEQ ID NO: 48; or (IVB) a heavy chain variable region comprising the heavy chain variable region amino acid sequence of SEQ ID NO: 71, or an amino acid sequence at least 80% identical thereto, or a heavy chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the heavy chain variable region amino acid sequence of SEQ ID NO: 71; A monoclonal antibody molecule or derivative thereof according to any one of claims 1 to 3, having the following structure:

6. (IC) an L chain variable region comprising the L chain variable region amino acid sequence of SEQ ID NO: 40, or an amino acid sequence at least 80% identical thereto, or an L chain variable region comprising at least one framework region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the L chain variable region amino acid sequence of SEQ ID NO: 40; (IIC) an L chain variable region comprising the L chain variable region amino acid sequence of SEQ ID NO: 51, or an amino acid sequence at least 80% identical thereto, or an L chain variable region comprising at least one framework region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the L chain variable region amino acid sequence of SEQ ID NO: 51; (IIIC) an L chain variable region comprising the L chain variable region amino acid sequence of SEQ ID NO: 44, or an amino acid sequence at least 80% identical thereto, or an L chain variable region comprising at least one framework region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the L chain variable region amino acid sequence of SEQ ID NO: 44; or (IVC) A monoclonal antibody molecule or derivative thereof according to any one of claims 1 to 3, comprising: an L chain variable region comprising the L chain variable region amino acid sequence of SEQ ID NO: 75, or an amino acid sequence that is at least 80% identical thereto; or an L chain variable region comprising at least one framework region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the L chain variable region amino acid sequence of SEQ ID NO:

75.

7. A monoclonal antibody molecule or a derivative thereof according to any one of claims 1 to 3, which has any one of the H-chain variable regions according to claim 5 and any one of the L-chain variable regions according to claim 6.

8. The monoclonal antibody molecule or derivative thereof according to claim 7, comprising: an H chain variable region (IB) according to claim 5 and an L chain variable region (IC) according to claim 6; an H chain variable region (IIB) according to claim 5 and an L chain variable region (IIC) according to claim 6; or an H chain variable region (IVB) according to claim 5 and an L chain variable region (IVC) according to claim 6.

9. The monoclonal antibody molecule or derivative thereof according to claim 8, wherein the heavy chain variable region of (IB) according to claim 5 has the heavy chain variable region amino acid sequence of SEQ ID NO: 36, and the light chain variable region of (IC) according to claim 6 has the light chain variable region amino acid sequence of SEQ ID NO: 40; or the heavy chain variable region of (IIB) according to claim 5 has the heavy chain variable region amino acid sequence of SEQ ID NO: 48, and the light chain variable region of (IIC) according to claim 6 has the light chain variable region amino acid sequence of SEQ ID NO: 51; or the heavy chain variable region of (IVB) according to claim 5 has the heavy chain variable region amino acid sequence of SEQ ID NO: 71, and the light chain variable region of (IVC) according to claim 6 has the light chain variable region amino acid sequence of SEQ ID NO:

75.

10. The heavy chain variable region of (IB) according to claim 5 is an heavy chain variable region comprising an amino acid sequence at least 80% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 36, and the light chain variable region of (IC) according to claim 6 is an light chain variable region comprising an amino acid sequence at least 80% identical to the light chain variable region amino acid sequence of SEQ ID NO: 40; or the heavy chain variable region of (IIB) according to claim 5 is an heavy chain variable region comprising an amino acid sequence at least 80% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 48, and the light chain variable region of (IIC) according to claim 6 is an light chain variable region comprising an amino acid sequence at least 80% identical to the light chain variable region amino acid sequence of SEQ ID NO: 51; or The monoclonal antibody molecule or derivative thereof according to claim 8, wherein the H chain variable region of (IVB) according to claim 5 is an H chain variable region comprising an amino acid sequence that is at least 80% identical to the H chain variable region amino acid sequence of SEQ ID NO: 71, and the L chain variable region of (IVC) according to claim 6 is an H chain variable region comprising an amino acid sequence that is at least 80% identical to the H chain variable region amino acid sequence of SEQ ID NO:

75.

11. The heavy chain variable region of (IB) according to claim 5 is an heavy chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the heavy chain variable region amino acid sequence of SEQ ID NO: 36, and the light chain variable region of (IC) according to claim 6 is an light chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the light chain variable region amino acid sequence of SEQ ID NO: 40; or The H chain variable region of (IIB) according to claim 5 is an H chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the H chain variable region amino acid sequence of SEQ ID NO: 48, and the L chain variable region of (IIC) according to claim 6 is an L chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the L chain variable region amino acid sequence of SEQ ID NO: 51; or The monoclonal antibody molecule or derivative thereof according to claim 8, wherein the H chain variable region of (IVB) according to claim 5 is an H chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the H chain variable region amino acid sequence of SEQ ID NO: 71, and the L chain variable region of (IVC) according to claim 6 is an L chain variable region comprising at least one framework (FW) region comprising an amino acid sequence having one or several amino acid residue substitutions, deletions, insertions or additions compared to the L chain variable region amino acid sequence of SEQ ID NO:

75.

12. Fab, F(ab') ２ 12. A derivative of an antibody molecule according to any one of claims 1 to 11, which is an Fv or a single chain Fv fragment (scFv).

13. An antibody molecule or derivative thereof according to any one of claims 1 to 12, which neutralises and / or inhibits colicin-induced apoptosis.

14. A pharmaceutical composition for treating or preventing acute lung injury or fibrosis, comprising an effective amount of a monoclonal antibody molecule or derivative thereof according to any one of claims 1 to 13.

15. The pharmaceutical composition according to claim 14, wherein the acute lung disorder is life-threatening pneumonia that induces acute respiratory distress syndrome (ARDS), reduces pulmonary function, and requires respiratory care.

16. The pharmaceutical composition of claim 14, wherein the fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, renal fibrosis, cystic fibrosis, myelofibrosis, and mammary fibrosis.

17. A vaccine composition for preventing acute lung injury or fibrosis, comprising as an active ingredient a colicin mutant having one or several mutations within the sequence of colicin having the amino acid sequence: IVMPESSGNPNAVNPAGYR (sequence number 1), said mutant not exhibiting apoptotic activity.

18. Use of colicins as biomarkers for determining the progression of acute lung injury or fibrosis.

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