Treatment methods for fibrosis-related disorders associated with IBD

Abstract

The present invention relates to a transglutaminase type 2 (TG2) inhibitor that blocks at least the transamidase activity of an enzyme for use in the treatment of subjects with fibrosis-related disorders associated with inflammatory bowel disease (IBD) or in the prevention of the occurrence of such disorders.

Description

Technology Field

[0001] The present invention relates to a transglutaminase type 2 (TG2) inhibitor that blocks at least the transamidase activity of an enzyme for use in the treatment of subjects with fibrotic-related disorders associated with inflammatory bowel disease (IBD) or in the prevention of the occurrence of such disorders. Background Technology

[0002] Tissue transglutaminase, or transglutaminase type 2 (TG2), is an enzyme that forms crosslinks between proteins via epsilon(gamma-glutamyl) lysine dipeptide bonds. In addition to this direct stabilization of the ECM, TG2 also appears to indirectly promote ECM crosslinking through the activation of the fibrotic cytokine transforming growth factor β (TGF-β) (Nunes et al., 1997; Huang et al ., 2010). Elevated TG2 expression induces abnormal protein cross-linking, which is associated with various pathologies including various types of tissue scarring and fibrosis, the formation of neurofibrillary tangles in several brain disorders, and resistance to chemotherapy in some cancers. Various TG2 inhibitors, such as small molecules, silencing RNA, or antibodies (e.g., Siegel) for the possible treatment of TG2-mediated disorders et al. , 2007, Wang et al. , 2020, WO2006100679, WO2012146901 or WO2013175229) is disclosed.

[0003] Increased levels of TG2 are associated with progressive chronic interstitial lung disease (Olsen), including cases in patients with IPF (WO2023089042) or scleroderma (WO2023089037). et al ., 2011, Olsen et al ., 2020, Philp et al It is associated with several groups of disorders / diseases such as ., 2018).

[0004] Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a chronic disease characterized by recurring relapses and remissions, affecting more than 3 million people in Europe and the United States. IBD-related intestinal fibrosis is a significant clinical issue in patients with CD and UC. More than half of CD patients develop intestinal obstruction due to fibrosis, leading to symptoms that impair function (Cosnes et al. , 2005; Rieder et al ., 2017). Since non-surgical approaches to stricturing CD have limited success, surgical intervention becomes necessary in up to 80% of CD patients. Fibrosis, specifically intestinal fibrosis, is considered an inevitable and irreversible complication of chronic intestinal inflammation. Fibrosis is also found in UC, where it is essentially confined to specific tissues (mucosa and submucosa), affecting, for example, colonic motility.

[0005] Intestinal fibrosis is defined by the excessive accumulation of the extracellular matrix (ECM), which includes increased levels of fibronectin and collagen, ultimately leading to organ dysfunction. Mesenchymal cells (which differentiate primarily into fibroblasts, myofibroblasts, and smooth muscle cells in the intestine / colon) have been identified as the primary effector cells mediating fibrosis. In a normal response to injury or inflammation, these cells act as "repair units," rapidly increasing in population while simultaneously producing profibrotic factors and ECM. However, in a state of chronic inflammation, excessive ECM production leads to irreversible fibrosis.

[0006] There remains a need to identify more effective therapies for use in treating subjects with fibrosis-related disorders associated with inflammatory bowel diseases (including Crohn's disease or ulcerative colitis) or for preventing the occurrence of such disorders.

[0007] Summary of the Invention

[0008] The object of the present invention is to provide a specific transglutaminase 2 (TG2) inhibitor for use in the treatment of fibrosis-related disorders associated with inflammatory bowel disease (IBD) in patients or in the prevention of the occurrence of such disorders.

[0009] In a second aspect, the present invention provides a method for treating a subject with a fibrosis-related disorder associated with inflammatory bowel disease (IBD) or preventing the occurrence of such disorder in the subject, comprising the step of administering a therapeutically effective amount of a transglutaminase 2 (TG2) inhibitor to the subject.

[0010] In a third aspect, the present invention relates to the use of a TG2 inhibitor for the manufacture of a medicine for the treatment of a subject with a fibrosis-related disorder associated with inflammatory bowel disease (IBD) or for the prevention of the occurrence of such disorder. Brief explanation of the drawing

[0011] Fig. 1: Transglutaminase (TG) activity measured using scan images was significantly increased in UCi / CDs compared to NL, and correlated with the fibrosis score but not with the inflammation score. A) Quantification of the integrated density of TG activity was performed in tissue sections from different IBD patients (UCi, CDns, and CDs patients, respectively; NL used as a control) (P values ​​compared to NL). B) As shown in two different layers of intestinal tissues (muscularis propria and muscularis mucosa), TG2 activity in the entire IBD layer correlated with the fibrosis score ("Fibrosis score" column) but not with the inflammation score ("Geboes (inflammation) score" column). Fig. 2:TG2 inhibition using zampilimab confirmed that TG2 is the source of transamidation activity and contributes 80 to 100% of total TG activity. A) Quantification of the integral density of TG2 activity was performed using scan images from myomega-mucosal and muscularis propria tissue sections from different IBD patients (UCi, CDns, and CDs patients; NL used as a control) (scan images not shown). B) Based on images from myomega-mucosal and muscularis propria tissue sections from different IBD patients (UCi, CDns, and CDs patients; NL used as a control), TG2 activity contributes 80 to 100% of total TG activity, as determined by calculating the percentage of TG activity lost after pre-treatment with zampilimab (scan images not shown). (P-value for the treatment group versus the untreated group). Fig. 3: TG2 mRNA and TG2 antigen are increased in UCi and CD stenotic sites: A) Quantification of integrated density for TG2 antigen was performed in tissue sections from different IBD patients (UCi, CDns, and CDs patients; NL used as a control; P-values ​​compared to NL). B) Pathologist scoring of raw images using an RNA-Z-scope revealed that TG2 expression in samples derived from UCi, CDns, and CDs patients was increased by 25% to 100% (in terms of mRNA) compared to NL patients. Scoring: NL = baseline / 0; 0 = no difference; 1 = 1 to 25%; 2 = 26 to 50%; 3 = 51 to 75%; 4 = 76 to 100%. C) Quantitative RT-PCR also showed that TG2 expression was elevated (in terms of mRNA) in patients with UCi and CDs. Fig. 4:Ten rapid-frozen patient tissues each from different IBD patients (UCi, CDns, and CDs patients; NL used as a control) 14 C-putrescine analysis ( 14 Analysis was performed using the C-Putrescine Assay. This figure shows that TG activity was increased in CDs compared to NL (using t-tests and non-parametric tests). Fig. 5: In human intestinal myofibroblasts (HIMFs) derived from NL tissue, TG2 is the source of in vitro transglutaminase activity. Using specific TG2 inhibitors (the anti-TG2 antibody zampilimab and the small molecule Z-Don), it was confirmed that 82–97% of transglutaminase in NL HIMFs is TG2, as determined by calculating the percentage of TG activity lost after pre-treatment with TG2-specific inhibitors. Each dot represents a primary cell line derived from an individual patient. Fig. 6: Mature extracellular matrix (ECM) levels in fibroblasts derived from control (NL) and patients of different types of IBD. This shows baseline production of FN (A) and collagen (B) among NL, UC, CDns, and CDs, and trend changes among them. Each dot represents the primary cell line derived from an individual patient. Fig. 7:Extracellular matrix (ECM) levels were reduced in fibroblasts from NL, UCi, CDns, and CDs patients treated with TG2 inhibitors (anti-TG2 antibody zampilimab and small molecule TG2 inhibitors Z-Don and Boc-Don). A) Scan images showing ECM deposition under different conditions after immunofluorescence staining with antibodies specific to ECM components in tissues derived from NL subjects (top 5 images) and CDs patients (bottom 5 images). BE) ECM levels in fibroblasts from normal and different types of IBD patients, comparing mature ECM levels in fibroblasts from normal and different types of IBD patients in the presence of specific TG2 inhibitors (anti-TG2 antibody zampilimab and small molecule TG2 inhibitors Z-Don and Boc-Don). This graph shows the production of fibronectin (FN) and collagen (Col I & III) among tissues derived from NL (B), UC (C), CDns (D), and CDs (E) patients, and the changes in trends among them. Each point represents a primary cell line derived from an individual patient. Fig. 8: A) Inflammation scores in WT mice and TG2 KO mice in the presence and absence of DSS in various tissues in the mouse DSS chronic model of Crohn's disease. B) Inflammation scores in WT mice and TG2 KO mice in the presence and absence of TNBS in the mouse TNBS pre-sensitization chronic model of Crohn's disease. Each dot represents an individual mouse. Fig. 9:A) PSR staining in tent and muscle regions of WT mice and TG2 KO mice in the presence or absence of DSS. B) Collagen 1 (left graph) and fibronectin (right graph) in WT mice and TG2 KO mice in the presence or absence of DSS (Day 34). C) Thickening of the intestinal barrier in WT mice and TG2 KO mice in the presence or absence of DSS (Day 34); quantitative barrier measurement. Fig. 10: A) PSR staining in tent and muscle regions of WT mice and TG2 KO mice in the presence or absence of TNBS (Day 32). B) Collagen 1 (left graph) and fibronectin (right graph) in WT mice and TG2 KO mice. C) Thickening of the intestinal barrier in WT mice and TG2 KO mice in the presence or absence of TNBS (Day 34); quantitative barrier measurements. Specific details for implementing the invention

[0012] definition

[0013] The entire document is intended to be read as a unified disclosure, and it should be understood that all combinations of features described herein are taken into account, even if such combinations of features are not found together in the same sentence, paragraph, or section of this document. With respect to aspects of the invention described or claimed as “a” or “an,” these terms should be understood to mean “one or more” unless a more limited meaning is clearly required by the context. The term “or” should be understood to include individually or together, unless otherwise clearly required by the context. Where an aspect of the invention is described as “comprising” a feature, the embodiment is also considered to include “consisting of” or “consisting essentially of” said feature.

[0014] - The terms "tissue transglutaminase," "transglutaminase type 2," or "TG2" refer to the enzyme that is the product of the gene TGM2 and forms a cross-link between proteins through epsilon(gamma-glutamyl) lysine dipeptide bonds. TG2 typically refers to a protein having the amino acid sequence presented in UniProt entry P21980 (with or without N-terminal methionine as presented in sequence number 41), namely human TG2. The term "TG2" may also refer to (a) a derivative having one or more amino acid substitutions, modifications, deletions, or insertions to the amino acid sequence of SEQ No. 41 that retains the activity of TG2, or (b) a protein that is a variant thereof, such variants typically retain at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identity with respect to SEQ No. 41 (or even about 96%, 97%, 98%, or 99% identity with respect to SEQ No. 41). The protein TG2 is encoded by the gene Tgm2.

[0015] - The term "TG2 inhibitor" refers to a molecule capable of inhibiting at least one of the TG2 activities, such as the TG2 crosslinking of lysine and glutamine through N-ε(γ-glutamyl)lysine isopeptide bonds. Such a molecule may be a small molecule, silencing RNA, or antibody without limitation.

[0016] Small molecules can be selected from three main classes (as non-limiting examples): 1) competitive amine inhibitors that compete with natural amine substrates, 2) reversible inhibitors, and 3) irreversible inhibitors (Siegel et Khosla, 2007).

[0017] - The terms "inhibition" and synonyms refer to the blocking or neutralization of one or more activities typically associated with a target, such as an enzyme. For example, in the context of the present invention, the term "inhibition" includes the blocking of the enzymatic function of TG2 associated with the transamidation (including cross-linking) and deamidation of proteins. TG2 is also known as GTPase (Im et al. , 1997).

[0018] - As used herein, the term "anti-TG2 antibody" is intended to refer to an antibody molecule that binds to TG2 and blocks its transamidase activity to prevent cross-linking. Examples of such antibodies are described in WO2013175229. Without limitation, anti-TG2 antibodies that may be used according to the present invention include, for example, the light chain variable region defined in SEQ ID NO: 24 and the heavy chain variable region defined in SEQ ID NO: 37.

[0019] - As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies produced by recombinant techniques known in the art. "Antibody" includes antibodies of any species; any isotype of human antibodies, such as IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, and IgD; and antibodies produced as dimers of the basic structure, such as IgGA1 and IgGA2, or as pentamers, such as IgM, and modified variants thereof; non-human primate antibodies, such as antibodies derived from chimpanzees, baboons, rhesus, or cynomolgus monkeys; rodent antibodies, such as antibodies derived from mice or rats; rabbit, goat, or horse antibodies; camelid antibodies (e.g., antibodies derived from camels or llamas, e.g., Nanobodies TM...and derivatives thereof; antibodies of bird species, e.g., chicken antibodies; or antibodies of fish species, e.g., shark antibodies. The term "antibody" also refers to a "chimeric" antibody, wherein at least one first portion of a heavy chain and / or light chain antibody sequence is derived from a first species, and the second portion of said heavy chain and / or light chain antibody sequence is derived from a second species. The chimeric antibodies of interest herein include "primatized" antibodies comprising a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old-World monkey, e.g., baboon, rhesus monkey, or cynomolgus monkey) and a human constant domain sequence. A "humanized" antibody is a chimeric antibody containing a sequence derived from a non-human antibody. In most cases, the humanized antibody is a human antibody (recipient antibody), wherein residues derived from the hypervariable region of the said recipient are replaced with residues derived from the hypervariable region [or complementarity determining region (CDR)] of a non-human species (donor antibody), such as mouse, rat, rabbit, chicken, or non-human primate, having the desired specificity, affinity, and activity. In most cases, residues of the human (recipient) antibody outside the CDR; that is, within the framework region (FR), are further replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or the donor antibody. Such modifications are performed to further improve antibody properties. Humanization reduces the immunogenicity of non-human antibodies in humans, thereby facilitating the application of antibodies for the treatment of human diseases. Humanized antibodies and various techniques for producing them are well known in the art.The term "antibody" above also refers to human antibodies, which can be generated as an alternative to humanization. For example, it is possible to produce genetically modified animals (e.g., mice) capable of producing the entire repertoire of human antibodies without the production of endogenous murine antibodies during immunization. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies, such as phage display or ribosome display technologies, wherein a recombinant DNA library is used that is at least partially artificially generated or generated from the donor’s immunoglobulin variable (V) domain gene repertoire. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies can also be generated from isolated human B cells, which are ex vivo immunized with the antigen of interest and then fused to produce hybridomas capable of screening for optimal human antibodies. The term "antibody" above refers to both glycosylated antibodies and aglycosylated antibodies. Additionally, as used herein, the term "antibody" refers not only to full-length antibodies but also to antibody fragments, more specifically, their antigen-binding fragments. An antibody fragment comprises at least one heavy or light chain immunoglobulin domain known in the art and binds to one or more antigen(s). Examples of antibody fragments according to the present invention include Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv, and Bis-scFv fragments. The fragments also include diabodies, tribodies, triabadies, tetrabodies, minibodies, single-domain antibodies (dAbs) such as sdAb, VL, VH, VHH, or camelid antibodies (e.g., antibodies derived from camels or llamas, e.g., Nanobodies). TMIt may be a ) and VNAR fragment. The antigen-binding fragment according to the present invention may also comprise a Fab connected to one or two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binds to a therapeutic target, and one scFv or dsscFv binds to albumin, for example, to increase the half-life). Examples of such antibody fragments include FabdsscFv (also referred to as BYbe®) or Fab-(dsscFv)2 (also referred to as TrYbe®; see, for example, WO2015 / 197772). The antibody molecules defined above include their antigen-binding fragments and are known in the art.

[0020] - The term "epitope" refers to a region of an antigen to which an antibody binds. Epitopes can be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, consisting of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface agents of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0021] - The terms "treating" or "treatment" of a disease state include: (i) suppression of the disease state, i.e., prevention of the disease state or the occurrence of its clinical symptoms, or (ii) alleviation of the disease state, i.e., inducing temporary or permanent regression of the disease state or its clinical symptoms.

[0022] - The terms "preventing" or "prevention" regarding a disease state include preventing the occurrence of clinical symptoms of a disease state in subjects who may be exposed to or vulnerable to the disease state but have not yet experienced or manifested symptoms of the disease state.

[0023] Detailed description of the invention

[0024] The present invention is based on the inventors' discovery that TG2 expression (e.g., increased TG2 antigen) and its activity were both significantly increased in the tissues of UC and CD patients, specifically in Crohn's disease strictures, and that TG2 activity correlated with the fibrosis score. Additionally, they discovered that TG2 inhibitors can inhibit TG2 activity in situ. The inventors were also able to demonstrate that TG2 inhibitors (e.g., small molecule inhibitors and anti-TG2 antibodies) can attenuate the accumulation of mature fibronectin and collagen mediated by human intestinal myofibroblasts (HIMFs) in in vitro analysis.

[0025] The main object of the present invention is a transglutaminase 2 (TG2) inhibitor for use in the treatment of fibrosis-related disorders associated with inflammatory bowel disease (IBD) in patients or in the prevention of the occurrence of such disorders.

[0026] The present invention also provides a method for treating a subject with a fibrosis-related disorder associated with inflammatory bowel disease (IBD) or preventing the occurrence of such disorder in the subject, comprising the step of administering a therapeutically effective amount of a transglutaminase 2 (TG2) inhibitor to the subject.

[0027] In addition, the use of transglutaminase 2 (TG2) inhibitors is described for the treatment of subjects with fibrosis-related disorders associated with inflammatory bowel disease (IBD) or for the manufacture of medicines for the prevention of such disorders.

[0028] In the context of the present invention as a whole, fibrosis-related disorders associated with IBD are selected from a group consisting of a) strictures including strictures in the intestine, ileum, ileocecal valve, upper gastrointestinal tract, colon, rectum, or anus, b) fibrosis of the intestine, ileum, ileocecal valve, upper gastrointestinal tract, colon, rectum, or anus, and c) bowel obstruction.

[0029] In the context of the present invention as a whole, inflammatory bowel disease (IBD) is selected from the group consisting of Crohn's disease (CD), e.g., non-stricturing CD (CDns) or stricturing CD (stricturing CD: CDs), and ulcerative colitis (UC), e.g., inflamed UC (inflamed UC: UCi).

[0030] In the context of the present invention as a whole, fibrosis-related disorders associated with IBD are characterized by an increase in a marker in a sample of a subject, wherein the marker is, for example, TG2 activity, the level of epsilon(gamma-glutamyl) lysine crosslinking which is a product of TG2, the level of TG2 expression (e.g., an increase in mRNA encoding TG2 or TG2 antigen), TG2 export, or any combination thereof, and the sample of the subject is a cell or tissue associated with said disease (e.g., intestinal cell or colon tissue). The increase in said marker may be determined by any means in the cell / tissue associated with said disease. The increase in a marker in a sample of a subject is typically determined by comparing the level of said marker in the sample of the subject with the level of the same marker in normal cells of the same tissue type (i.e., baseline level; for example, baseline TG2 activity, baseline expression level (mRNA level and / or protein level) and / or baseline level of TG2 export). Samples of a subject in which at least one marker level is 10% or more, 15% or more, 20% or more, 25% or more, or even 30% or more compared to the baseline level for said marker will be considered to indicate an increase in said marker. For example, an increase in TG2 expression (alternatively referred to as TG2 overexpression) can be determined by determining the amount of TG2 mRNA in the patient's intestinal cells. Thus, cells / tissues affected by fibrosis-related disorders associated with IBD may be characterized by an increased amount of TG2 mRNA (indicating overexpression) in the subject's intestinal cells compared, for example, to normal cells of the same tissue type. The expression of TG2 mRNA may increase by any amount compared to the baseline level, e.g., 10% or more, 15% or more, 20% or more, 25% or more, or even 30% or more.The amount of mRNA can be measured using any known method, such as quantitative reverse transcription polymerase chain reaction (quantitative RT-PCR, also called qRT-PCR), real-time qRT-PCR, quantigene assay, northern blotting or microarrays, RNA sequencing, and various types of in situ hybridization (e.g., RNAscope). Alternatively, overexpression can be determined by determining the amount of TG2 antigen in the patient's intestinal cells. Thus, cells affected by fibrosis-related disorders associated with IBD may be characterized by an increased amount (indicating overexpression) of TG2 protein (or TG2 antigen) in the subject's intestinal cells, for example, compared to normal cells of the same tissue type. The expression of TG2 protein may increase by any amount compared to baseline levels, for example, more than 10%, more than 15%, more than 20%, more than 25%, or even more than 30%. The amount of protein can be measured using any known method, such as immunohistochemistry, western blotting, mass spectrometry, or fluorescence-activated cell sorting (FACS), including the use of the anti-TG2 antibody of the present invention. Thresholds for determining expression may vary depending on the technique used and can be validated against immunohistochemistry scores. Alternatively, cells / tissues affected by fibrosis-related disorders associated with IBD may be characterized by increased TG2 activity in the subject's intestinal cells compared to normal cells of the same tissue type. TG2 activity may increase by any amount compared to baseline levels, e.g., more than 10%, more than 15%, more than 20%, more than 25%, or even more than 30%.TG2 activity can be measured using any known method, such as cryo biopsy (TG ISA).

[0031] In one embodiment of the present invention as a whole, the TG2 inhibitor is preferably an anti-TG2 antibody that binds to an epitope within the core region of TG2 and inhibits at least one of the TG2 activities, wherein the at least one TG2 activity inhibited is a TG2 crosslinking of lysine and glutamine through an N-ε(γ-glutamyl) lysine isopeptide bond. In a non-limiting example, when evaluated, for example by a peptide mapping method, the preferred antibody binds to a region or part thereof comprising or consisting of amino acids 304 to 326 of TG2 (e.g., SEQ ID NO: 41). The antibody may comprise or consist of an intact antibody. Alternatively, it may comprise an antigen-binding fragment, e.g. (but not limited thereto) an Fv fragment (e.g., a single-stranded Fv fragment or a disulfide-bound Fv fragment); a Fab fragment; and Fab-like fragments (e.g., Fab' fragment or F(ab)2 fragment), single-domain antibodies (or any other fragment defined herein or known to those skilled in the art) may be included or composed thereof. Preferably, the anti-TG2 antibody used in whole according to the present invention (see also Table A) is:

[0032] a) Includes 6 CDRs selected from a group consisting of:

[0033] (i) KASQDINSYLT (LCDR1; Sequence No.: 1); LVNRLVD (LCDR2; Sequence No.: 2); LQYDDFPYT (LCDR3; Sequence No.: 3); THAMS (HCDR1; Sequence No.: 4); TISSGGRSTYYPDSVKG (HCDR2; Sequence No.: 5); and LISTY (HCDR3; Sequence No.: 6);

[0034] (ii) KASQDINSYLT (LCDR1; Sequence No. 1); LTNRLMD (LCDR2; Sequence No. 7); LQYVDFPYT (LCDR3; Sequence No. 8); SSAMS (HCDR1; Sequence No. 9); TISSGGRSTYYPDSVKG (HCDR2; Sequence No. 5); and LISPY (HCDR3; Sequence No. 10); or

[0035] (iii) KASQDINSYLT (LCDR1; Sequence No.: 1); RTNRLFD (LCDR2; Sequence No.: 11); LQYDDFPYT (LCDR3; Sequence No.: 3); SSAMS (HCDR1; Sequence No.: 9); TISVGGGKTYYPDSVKG (HCDR2; Sequence No.: 42); and LISLY (HCDR3; Sequence No.: 12);

[0036] b) comprising a light chain variable domain having a sequence defined in any one of SEQ ID NO: 13 to SEQ ID NO: 27, and a heavy chain variable domain having a sequence defined in any one of SEQ ID NO: 28 to SEQ ID NO: 40, or

[0037] c) comprising a light chain variable domain having at least 80% identity or similarity, preferably at least 90% identity or similarity, or preferably at least 95% (or even about 96%, 97%, 98%, or 99%) identity or similarity with respect to a sequence defined in any one of SEQ NOs: 13 to 27, and a heavy chain variable domain having at least 80% identity or similarity, preferably at least 90% identity or similarity, or preferably at least 95% (or even about 96%, 97%, 98%, or 99%) identity or similarity with respect to a sequence defined in any one of SEQ NOs: 28 to 40, or

[0038] d) Compete for binding to TG2 with the antibody defined in a), b), or c) above.

[0039] As a non-limiting example, the anti-TG2 antibody comprises the following six CDRs: KASQDINSYLT (LCDR1; Sequence No. 1); LVNRLVD (LCDR2; Sequence No. 2); LQYDDFPYT (LCDR3; Sequence No. 3); THAMS (HCDR1; Sequence No. 4); TISSGGRSTYYPDSVKG (HCDR2; Sequence No. 5); and LISTY (HCDR3; Sequence No. 6). These six exemplary CDRs may each be contained in a light chain variable domain having the sequence defined in Sequence No. 24 and a heavy chain variable domain having the sequence defined in Sequence No. 37. As an additional non-limiting example, the anti-TG2 antibody comprises the following six CDRs: KASQDINSYLT (LCDR1; Sequence No. 1); LTNRLMD (LCDR2; Sequence No. 7); LQYVDFPYT (LCDR3; sequence number: 8); SSAMS (HCDR1; sequence number: 9); TISSGGRSTYYPDSVKG (HCDR2; sequence number: 5); and LISPY (HCDR3; sequence number: 10). These six exemplary CDRs may each be contained in a light chain variable domain having the sequence defined in sequence number: 19 or 25, and a heavy chain variable domain having the sequence defined in sequence number: 32 or 38.

[0040]

[0041] By using conventional methods known in the art, a person skilled in the art can easily determine whether an antibody binds to the same epitope as another antibody or competes with another antibody for binding thereto. For example, to determine whether a test antibody binds to the same epitope as the reference antibody of the present invention, the reference antibody is allowed to bind to a protein or peptide under saturation conditions. Next, the ability of the test antibody to bind to the protein or peptide is evaluated. If the test antibody can bind to the protein or peptide after saturation binding with the reference antibody, it can be concluded that the test antibody binds to an epitope different from that of the reference antibody. On the other hand, if the test antibody cannot bind to the protein or peptide after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope to which the reference antibody of the present invention is bound. To determine whether the antibody competes for binding with the reference antibody, the aforementioned binding method is performed in two orientations. In the first orientation, the reference antibody is allowed to bind to the protein / peptide under saturation conditions, and then the binding of the test antibody to the protein / peptide molecule is evaluated. In the second orientation, the test antibody is allowed to bind to the protein / peptide under saturation conditions, and then the binding of the reference antibody to the protein / peptide is evaluated. In both orientations, if only the first (saturated) antibody is able to bind to the protein / peptide, it can be concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As understood by those skilled in the art, the antibody competing for binding to the reference antibody does not necessarily have to bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0042] When each of the two antibodies competitively inhibits (blocks) the binding of the other antibody to an antigen, these two antibodies bind to the same or overlapping epitopes. That is, as measured in a competitive binding assay, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, 75%, 90%, or even 99%. Alternatively, if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, the two antibodies have overlapping epitopes.

[0043] Then, additional conventional experiments (e.g., peptide mutation and binding assays) may be performed to determine whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody or whether steric blockage (or another phenomenon) is the cause of the observed lack of binding. These kinds of experiments may be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody-binding assay available in the art.

[0044] In another embodiment of the present invention as a whole, the TG2 inhibitor is a small molecule inhibitor that inhibits at least one biological activity of TG2. Such inhibitors are well known to those skilled in the art and may be selected from three major classes (as a non-limiting example): 1) competitive amine inhibitors, 2) reversible inhibitors, and 3) irreversible inhibitors. A non-limiting list of such inhibitors includes the following inhibitors: PX-12, ERW1041, CK805, Z Don, Boc-Don, NC9, VA4, ZED1227, or any inhibitor described in Siegel and Khosla, 2007, WO033784, WO14012858, WO14057266, WO17179018, WO18122419, WO22213198, or WO23135425.

[0045] Any subject may be treated according to the present invention. The subject is preferably a human. However, the subject may be another mammal, such as a non-human primate, horse, cattle, sheep, pig, dog, cat, rabbit, rat, mouse, guinea pig, or hamster. Alternatively, the term patient may be used interchangeably with the subject.

[0046] Any TG2 inhibitor according to the present invention may be incorporated into a pharmaceutical composition suitable for administration to a subject in any manner (but not limited thereto), such as topically, intranasally, deskin, intravenously, subcutaneously, or intramuscularly. Typically, said pharmaceutical composition comprises said TG2 inhibitor and one or more pharmaceutically acceptable adjuvant(s) and / or carrier(s). Thus, the present invention also describes a pharmaceutical composition for use in the treatment of a subject with a fibrosis-related disorder associated with inflammatory bowel disease (IBD) or for the prevention of the occurrence of such disorder, said pharmaceutical composition comprising a TG2 inhibitor and one or more pharmaceutically acceptable adjuvant(s) and / or carrier(s). A pharmaceutical composition according to the present invention may be part of a kit with instructions for use, including instructions and optionally a device for intravenous, subcutaneous, or intramuscular administration to an individual requiring it.

[0047] The "pharmaceuticalally acceptable carrier" as used herein comprises any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and absorption retardants, etc., that are physiologically suitable and suitable for administration to a subject for the methods and uses described herein. Examples of pharmaceutically acceptable carriers include one or more of water, saline solution, phosphate-buffered saline solution, dextrose, glycerol, ethanol, and analogs, as well as combinations thereof. Depending on the route of administration or the type of formulation (e.g., liquid, lyophilized, or spray-dried formulation), isotonic agents, e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be incorporated into the composition. The pharmaceutically acceptable carrier may additionally include small amounts of auxiliary substances that enhance the shelf life or efficacy of the inhibitor, such as wetting agents or emulsifiers, preservatives, or buffers.

[0048] The pharmaceutical composition according to the present invention may be in various forms. These include, for example, liquid solutions (e.g., injectable solutions and infusionable solutions), dispersions or suspensions, powders, and liposomes. The preferred form depends on the intended mode of administration and the mode of therapeutic application. A typical preferred composition is in the form of an injectable solution or an infusionable solution, such as a composition similar to those used for human passive immunization using other therapeutic molecules.

[0049] The appropriate dosage of the TG2 inhibitor according to the present invention can be determined by a skilled medical professional. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be modified to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a specific patient, composition, and mode of administration without toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the route of administration, time of administration, elimination rate of the TG2 inhibitor, duration of treatment, other drugs, compounds, and / or substances concomitantly used with the specific TG2 inhibitor, age, sex, weight, pathological condition, general health status, and medical history of the patient being treated.

[0050] The appropriate dose may be, for example, in the range of about 0.01 pg / kg body weight to about 1000 mg / kg body weight per kg of the patient's body weight, typically about 0.1 pg / kg body weight to about 100 mg / kg body weight. The dosage regimen may be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose may be administered, or multiple divided doses may be administered over time. The dosage unit form used herein refers to a physically separated unit suitable as a unit dose for the subject to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect, along with the necessary pharmaceutical carrier. Administration may be performed as a single dose or multiple doses. Multiple doses may be administered at the same or different locations via the same or different routes.

[0051] In the context of the present invention as a whole, the TG2 inhibitor may be co-administered with one or more other therapeutic agents. Co-administration of two or more agents may be achieved in a number of different ways. All of these may be administered together as a single composition, or as separate compositions as part of a combination therapy. For example, one composition may be administered before or individually, subsequently or sequentially, or together or simultaneously with the administration of another composition.

[0052] Major abbreviations used throughout this description:

[0053] TG: Transglutaminase; TG2: Transglutaminase 2; NL: Non-lesion tissue (tissues from various patients without IBD; by extension, patients from whom such NL is derived are referred to as NL patients); UCi: Inflammatory ulcerative colitis; CDs: Stricter Crohn's disease (CD); CDns: Non-stricter Crohn's disease; FN: Fibronectin; COL: Collagen; KO: Knock-out; WT: Wild type; PSR: PicroSirius red; DSS: Dextran sodium sulfate; TNBS: Trinitrobenzenesulfonic acid solution; HIMF: Human intestinal myofibroblasts; H&E: Hematoxylin and eosin; KO: Knock-out; IF: Immunofluorescence; ID: Integral density.

[0054] Examples

[0055] substance

[0056] Anti-TG2 antibody: The anti-TG2 mAb used in the following examples included a light chain variable region defined at sequence number 24 and a heavy chain variable region defined at sequence number 37. This is known as zampilimab (alternative name UCB7858, derived from antibody DC1).

[0057] Small molecule inhibitors: 1) Z-Don, an irreversible cell-soluble TG2-specific inhibitor, and 2) Boc-Don, an irreversible cell-insoluble TG2-specific inhibitor.

[0058] method

[0059] Patient Sample: Patient-derived human tissue samples with CDs (n=10), CDns (n=20), and UC (n=10), as well as control tissues (NL; n=14), were collected according to industry standard practices. Briefly, whole-thickness specimens of freshly resected intestines from patients with inflammatory bowel disease were obtained by standard methods. Tissue blocks from different tissue experimental groups were secured for further processing.

[0060] Primary human organ myofibroblasts Isolation and Culture: Human intestinal myofibroblasts (HIMF) were obtained as explants of surgically resected intestinal mucosa and isolated and cultured according to standard methods. Briefly, HIMF were grown to subconfluence in DMEM (Dulbecco's minimal essential medium) supplemented with 10% fetal bovine serum (FBS) and antibiotics, and medium was supplied twice a week. Subcultured at confluence, they were established as long-term cultures.

[0061] person Cittu TG2 activity and Extracellular Detection of TG2 antigen: To detect in situ TG activity and extracellular TG2 antigen levels, unfixed, washed cryostat sections from fresh frozen tissue were embedded in OCT mounting media and cut into 8-10 μm thick sections for use in both analyses.

[0062] For the TG activity analysis, the slides were washed three times with PBS. The sections were blocked with 5% BSA and 5 μg / ml streptavidin for 35 minutes at room temperature. Parallel sections obtained from the same sample were blocked in a similar manner, and 30 μg / ml of UCB7858 antibody was added to block TG2 activity. The sections were washed three times with PBS, and a reaction mix containing 1 mM DTT, 5 mM CaCl2, and 100 μm biotin cadaverine in 50 mM Tris buffer (pH 7.4) containing proteinase inhibitors was added. The third section was used as a negative control, and biotin cadaverine was not used in this section.

[0063] For TG2 antigen detection, sections were washed with PBS at room temperature, and 30 μg / ml of CUB7402 antibody (Abcam) was added to blocking buffer supplied with a proteinase inhibitor and incubated at 37°C for 1 hour. After washing with PBS, sections were fixed in cold acetone and incubated with goat anti-mouse Alexa 594 secondary antibody and streptavidin Alexa 488 at 37°C for 30 minutes. Sections were washed with PBS containing 0.1% Tween 20 at room temperature and mounted using Mowiol mounting medium containing Dapi.

[0064] All stained slides were scanned and images were acquired using Aperio Image Scope software (Leica Biosystems). The stained images were quantified using ImageJ software (Bethesda). Starting with the disease-free control slide, the regions of the muscularis mucosa (MM) and muscularis propria (MP) were marked, and thresholds were set. All other diseased slides were quantified using the same set thresholds, integrated densities were measured using ImageJ, and graphs were plotted using GraphPad Prism software (Version 9.3.1, Boston MA).

[0065] Relationship between TG2 activity and the degree of fibrosis in tissues: As evaluated by experienced IBD pathologists in a blind state for diagnosis and TG2 measurement, using Masson trichrome-stained sections for fibrosis and H&E-stained sections for inflammation scoring (Geboes score), the degree of fibrosis and inflammation, as well as the in situ TG2 activity and amount, were correlated. The inflammation score (Geboes) using H&E-stained sections is evaluated based on architectural changes, ranging from no abnormalities (score 0) to severe diffuse or multifocal abnormalities (score 3). In addition, infiltration of neutrophils, eosinophils, and epithelial neutrophils in the lamina propria is evaluated from no increase (score 0) to significant increase (score 3), crypt destruction is evaluated from no (score 0) to >50% destruction (score 3), and erosion or ulceration is evaluated from no (score 0) to ulcer or granulation tissue (score 3). The final Geboes score ranges from 0 to 22 based on the sum of the seven subscores mentioned above. Fibrosis scores using trichrome-stained sections are limited from submucosal ECM deposition <25% of the total submucosal layer (score 0) to massive transmural fibrosis and loss of normal layer (score 3).

[0066] 14 C Putresin By contamination analysis transglutaminase Measurement of activity: Ten rapid-frozen patient tissues, each from NL, UCi, CDns, and CDs patients, were provided from the Clinic. In this analysis, active TG in homogenized tissue [1,4- in the presence of calcium 14[C]-putrescine was incorporated into N,N-dimethylcasein to form radioactive trichloroacetic acid (TCA)-insoluble cross-linked proteins, which were measured by scintillation counting. Briefly, the tissue was homogenized on ice in STE buffer (0.32 M sucrose, 5 mM Tris, 2 mM EDTA) supplemented with a protease inhibitor (Sigma Aldrich), and Pierce TM Protein content was measured using a BCA protein analysis kit. 75 μL of reaction mix containing 14C putrescine + N'N-dimethylcasein was placed in an analysis tube and preheated for 20 minutes on a dry block heater set to 37°C. At T=0, 75 μL of tissue homogenate was added, mixed up and down by pipetting, and incubation was continued on the 37°C dry block heater. At set time points (0, 5, 10, 20, 30, 60 min), a 10 μL aliquot was withdrawn, the reaction was stopped, and 14To precipitate C. putrescine-casein, it was applied to a filter mat pre-soaked in 10% (w / v) ice-cold TCA. Simultaneously, 15 μL of tissue homogenate + 15 μL of control reaction mix (containing EDTA to inhibit enzyme activity) were added to a preheated tube on a heating block set to 37°C at T=0 and mixed; the control mix was sampled at a single time point at the end of the analysis. After the final addition to the filter mat, the filter mat was left to stand for 5 minutes. The filter mat was washed once for 10 minutes with 10% (w / v) ice-cold TCA, washed three times for 5 minutes each with 5% (w / v) ice-cold TCA, washed once for 5 minutes with 1:1 ice-cold acetone:ethanol, and finally washed once for 5 minutes with ice-cold 100% acetone. The filter mat was air-dried, cut into individual squares representing the location of each sample on the filter mat, and placed in one scintillation vial per sample. 12 mL of scintillant was added, and radioactivity was measured using a 1450 microbeta scintillation counter. The incorporation rate in the steady state was corrected for the protein concentration in each sample and reported as cpm / min / mg protein.

[0067] TG2 in Vitro Cell activity analysis ( with fibronectin Biotin and Cadaverine Contamination): Plates were coated with fibronectin, and HIMF 8000 cells were seeded using different conditions including the following (see Example 2): 1. Control: 50 μM biotin cadaverine and 0.1% Tween 20 (positive control), 2. Untreated: 50 μM biotin cadaverine alone, 3. In-house isotype control: 50 μM biotin cadaverine and 150 μg / ml in-house isotype, 4. Zamfilimab: 50 μM biotin cadaverine 1000 nm (150 μg / ml zamfilimab), 5. Z-Don: 50 μM biotin cadaverine in medium containing 200 μM Z-Don, and secondary control: medium alone (no biotin cadaverine), 6. Boc-Don: in-house containing 500 μM Boc-Don. 50 μM biotin cadaverine was added to the prepared medium. Cells were attached under the above conditions at 37°C for 24 hours. After 24 hours, the reaction was stopped with two washes (PBS + 5 mM EDTA), followed by lysis of the cells in 0.25 M NH4OH + 50 mM Tris at room temperature for 10 minutes and washing with PBS. After blocking the cells with BSA, HRP-streptavidin conjugate was added, and biotin cadaverine incorporation was detected by FN. Trimethylbenzidine (TMB) substrate was added to the wells and incubated at room temperature (RT) for 5 minutes. The reaction was stopped with 2 M sulfuric acid (H2SO4), diluted in PBS, and the absorbance was read at 450 nm using a plate reader (Spectra max, 340PC).

[0068] RNA extraction from colon tissue: Up to 25 mg of freshly frozen natural intestinal tissue was finely chopped, and RNA was extracted. RNA extraction was performed using the RNeasy Mini kit (Qiagen) according to the manufacturer's standard protocol. RNA concentration was determined using an ND-1000 UV / Vis spectrometer (NanoDrop, Thermo Scientific).

[0069] Quantitative reverse transcriptase polymerase Chain reaction procedure: Total RNA was isolated as described above, and reverse transcription and quantitative PCR (quantitative RT-PCR) were performed according to the manufacturer's instructions (Applied Biosystems). Products for all primer pairs were verified by sequencing, and relative differences were calculated using the comparative threshold cycle method (ddCt) by normalizing to the CT value of 18S (reference gene). Quantitative RT-PCR was performed on cDNA (synthesized using the iScript cDNA Synthesis Kit, Biorad) using iQ Sybr Green Supermix (Biorad) and gene-specific primers. The fold change in treated samples relative to untreated samples was calculated using the Pfaffl method.

[0070] human organs to myofibroblasts Korea Extracellular Temperament Depth Analysis: The deposition of the extracellular matrix (ECM) by HIMF was analyzed using standard methods. Briefly, cells were plated in 96-well plates in DMEM supplemented with 10% FBS and antibiotics (as described above) and grown for 5 days to induce ECM. Cells were removed using 0.25 M ammonium hydroxide in 50 mM Tris pH 7.4, and the deposited ECM was fixed by exposure to 100% methanol at -20°C. The fixed ECM was stained with Alexa Fluor 488-conjugated anti-fibronectin (Ebioscience, 1:500 dilution), anti-collagen type 1 and type III antibodies (Millipore Sigma, 1:100 dilution), and anti-rabbit Alexa Fluor 594 (Ebioscience, 1:500 dilution). Fluorescence intensity was obtained by scanning the plates containing the ECM using a Thermofisher Cytation 5 scanner. In the experiment, the fluorescence intensity of at least three replicate wells was used to determine the average ECM level.

[0071] Dextran sodium- Sulfate (DSS)-induced colitis and trinitrobenzenesulfonic acid Solution (TNBS)-induced fibrosis: BL6 mice (Jackson Laboratories) were used. DSS-induced colitis and TNBS-induced fibrosis were induced according to standard procedures (e.g., Lawrance et al ., 2003 and Zhao et al. , see 2020).

[0072] DSS and TNBS Evaluation indicators for the colitis experiment: For both DSS and TNBS models, body weight, stool consistency, and the presence of occult blood or visual blood in the rectum were recorded every other day. At the end of the experiment, animals were euthanized by cervical dislocation following CO2 asphyxiation. The entire colon was excised, washed, weighed, and measured from the ileocecal junction to the anus. Tissues were obtained from the descending colon, and paraffin-embedded sections were stained with H&E, Masson Trichrome, or Sirius red for histological examination. Slides were scored by an experienced pathologist blinded to the experimental group according to standard procedures. Images were acquired using an Olympus microscope and ImagePro software. Sirius red images were quantified and analyzed using ImageJ software (NIH & LOCI).

[0073] Statistical Analysis: Data were analyzed using Student's t-test or Analysis of Variance (ANOVA) for independent groups. Values ​​were expressed as mean ± mean standard error (SEM), and statistical significance was set at p≤0.05 or p<0.05. All analyses were performed using GraphPad Prism (version 9.3.1; GraphPad Software Inc.).

[0074] Examples 1 - The Role of TG2 in Stenosis Associated with Crohn's Disease and Its Expression / Activation

[0075] The purpose of this study was to determine 1) the role of TG2 in stenosis associated with IBD, such as Crohn's disease, as well as 2) TG2 expression and activity.

[0076] TG2 is IBD It is the major TG active in related tissues (Figs. 1 and 2): To evaluate the potential involvement of transglutaminase in strictures in IBD, the incorporation of cadaverine, a biotinylated TG substrate, was measured in various tissue sections, more specifically, in unfixed, washed frozen sections taken from fresh human frozen tissues from different patients, including NL, UCi, CDns, and CDs. Using Image J software, the integrated density (ID) of biotin cadaverine incorporation (BCI) in these sections was quantified. As shown in Figure 1A, the total integrated density (ID) for the BCI data indicated increased TG activity in tissues derived from UCi and CDs patients compared to tissues derived from NL patients.

[0077] As shown in Figure 1B, regardless of patient type, TG activity (indicated by integral intensity) was correlated with the histopathological tissue fibrosis score (left fibrosis score diagram: muscularis mucosa: R = 0.31 and p = 0.026; muscularis propria: R = 0.44, p = 0.00087), but was not correlated with the histopathological inflammation score (right Geboes inflammation score diagram: muscularis mucosa: R = 0.12, p = 0.39; muscularis propria: R = 0.24, p = 0.086).

[0078] In addition, to identify the major TG species involved in this activity, the effect of UCB7858, a TG2-specific inhibitor, on parallel sections was also measured. As shown in Figure 2b, it is very clear that TG activity in tissue samples from IBD patients (indicated as UCI, CDns, and CDs) and controls is primarily associated with TG2. 97–99% of TG activity was attributed to TG2.

[0079] As shown in the top graph of Fig. 2a, TG2 activity was increased in mucosal propria tissues derived from UCi and CDs patients compared to tissues derived from NL patients. TG2 activity was statistically significantly reduced in NL, UCi, and CDs tissues upon treatment with an anti-TG2 antibody (here, UCB7858). Additionally, 1) an increasing trend in TG2 activity in mucosal propria tissues derived from CDns patients compared to tissues derived from NL patients, and 2) a decreasing trend in TG2 activity upon treatment with an anti-TG2 antibody in tissues derived from CDns patients were observed. When focusing on mucosal propria (MP) tissues, a higher expression trend was observed, particularly in samples derived from CDs patients (Fig. 2a, bottom graph), and a decreasing trend in TG2 activity was also observed upon treatment with an anti-TG2 antibody regardless of the tissue (UCi, CDns, or CDs).

[0080] TG2 expression (Fig. 3): The presence of TG2 antigens was evaluated by staining identical types of tissue (i.e., unfixed, washed frozen tissue sections from NL, UCi, CDns, and CDs patients) (i.e., TG2 expression was evaluated at the protein level). Images were processed as described in detail in the Methods section. As shown in Figure 3A, a total quantitative analysis of ID from tissue sections of NL, UCi, CDns, and CDs patients revealed that TG2 levels were increased in CDs compared to NL, and also showed an increasing trend in UCi samples (compared to NL). TG2 mRNA (TGM2) expression was visualized using sections from different tissue samples from NL, UCi, CDns, and CDs patients (RNAscope-Z probe approach). Fluorescence microscopy images showed that TG2 mRNA expression was detected in three distinct tissue sections of the mucosa (i.e., muscularis mucosa, submucosal layer, and muscularis propria regions; photographs not shown). Pathologists blindly scoring mRNA signals in three distinct tissue sections for each patient in the UCi, CDns, and CDs groups compared to similar tissue sections derived from NL patients showed that TG2 mRNA expression increased by 25% to 100% in CDs, with this increase being particularly pronounced in the muscularis propria region (Fig. 3B). Additionally, TG2 mRNA expression increased by up to 25% in UCi, with this increase being particularly pronounced in the muscularis propria region (Fig. 3B). In the case of TG2 mRNA expression in CDns, it increased by up to 50%, particularly in the muscularis mucosa and submucosal regions (Fig. 3B). mRNA was extracted using full-thickness tissue samples taken from NL, UC, CDns, and CDs patients, respectively, and then quantified using quantitative RT-PCR.The results show that TG2 has a significant 7 to 9-fold change in mRNA expression in UCi and CDs, and about a 3-fold change in CDns compared to NL (Fig. 3C).

[0081] TG2 activity (Fig. 4): From frozen tissues derived from NL, UCi, CDns, and CDs patients 14 C-putrescine analysis was performed. As shown in Figure 4, when the results were analyzed using a non-parametric t-test, a significant increase in TG activity was observed in tissues derived from CDs patients compared to tissues derived from NL patients, and a tendency for increasing TG activity was observed in tissues derived from UCi and CDns patients compared to NL patients (this trend was more pronounced in CDns patients).

[0082] Examples Conclusion of 1

[0083] Surprisingly, it was confirmed that active TG2 is the dominant transglutaminase in the human gut, and that its activity was increased in intestinal tissues derived from IBD patients, particularly those with UCi and CDs. This activity correlated with fibrosis rather than inflammation. In these tissues, anti-TG2 antibodies were able to significantly inhibit (in situ) TG2 activity. Furthermore, the data showed that TG2 expression, TG2 levels, and TG2 activity were upregulated in tissues derived from CDs patients, as indicated by immunohistochemistry and quantitative RT-PCR.

[0084] Examples 2 - TG2 In vitro inhibitor Inhibitory activity

[0085] Based on the results of Example 1, it was still necessary to prove whether the TG2 inhibitor could inhibit the in vitro activity of TG2.

[0086] TG2 inhibitors TG2 in Vitro It can inhibit cell activity (Fig. 5): TG2 activity was evaluated in HIMF derived from NL samples using specific TG2 inhibitors including Z-Don, Boc-Don, and zampilimab, where "Ig isotype" is an additional control. As shown in Figure 5, TG2 activity can be significantly reduced by using TG2 inhibitors, and optimal results were observed when using zampilimab. Collectively, 82% (using small molecule inhibitors) to 97% (using zampilimab) of TG activity derived from NL HIMF was inhibited, as determined by calculating the percentage of TG activity lost after pre-treatment with the aforementioned TG-specific inhibitors.

[0087] TG2 inhibitors can inhibit ECM deposition (Figs. 6 and 7): Mature extracellular matrix (ECM) levels (expressed as levels of fibronectin and collagen) were studied in various types of samples. Figure 6 shows baseline production of FN in tissues derived specifically from NL, UC, CDns, and CDs patients, and illustrates the trend changes among them. In particular, samples derived from CDs and UCi patients showed a tendency for increasing fibronectin production (Figure 6A). Next, the effect of TG2 inhibition (specific TG2 inhibitors including Z-Don, Boc-Don, and zampilimab; "Ig isotypes" are additional positive controls) on the deposition of extracellular matrix (ECM) within intestinal myofibroblasts was determined. For this purpose, fibronectin (FN) and collagen I / III levels were used as standard measures. As shown in Figures 7b through 7e, respectively, FN and COLI / III levels in HIMF isolated from NL (Figure 7b), UCi (Figure 7c), CDns (Figure 7d), and CDs (Figure 7e) patients were significantly reduced (mostly by more than 50%) in the presence of TG2 inhibitors, and overall similar effects were observed regardless of the type of inhibition (i.e., small molecules such as Z-Don and Boc-Don, or anti-TG2 antibodies such as zampilimab). Zampilimab consistently caused a significant reduction in fibronectin production in all patient groups. The maximum reduction was observed in tissue samples derived from CDs patients treated with zampilimab, and a slightly smaller reduction was observed with small molecule TG2 inhibitors (FN and collagen deposition decreased by approximately 90% and 60%, respectively, with zampilimab, and by approximately 80% and 50%, respectively, with small molecule inhibitors). Figure 7a is an exemplary scan image of different treatments and highlights the effect of TG2 inhibition on ECM deposition. HIMF cell fusion was not affected by the presence of the inhibitor (scan image not shown).

[0088] Examples Conclusion of 2:

[0089] It has been demonstrated here that TG2 inhibitors can inhibit TG2 activity in patient-derived intestinal myofibroblasts, and as a result, significantly reduce the levels of mature ECM deposited in myofibroblasts derived from NL and CDns patients, as well as UCi and CDs patients. Since ECM accumulation is an indicator of fibrosis in tissues, it has been demonstrated here that TG2 inhibitors can be reliably used to prevent and / or reduce fibrosis in IBD patients, including to prevent and / or treat stenosis in UC, CDns, or CDs patients (with optimal results in CDs patients).

[0090] Examples 3 - Effects of TG2 gene knockout in animal models

[0091] Chronic DSS Colitis Model: Using a mouse DSS model of chronic colitis in the presence or absence of TGM2 KO, it was shown that TG2 knockdown (KO) did not affect clinical scores, body weight loss (data not shown), and histopathological inflammation scores (Fig. 8A). Wild-type (WT) and KO DSS chronic colitis mice showed increased inflammation scores in the proximal colon, transverse colon, and rectum compared to WT and KO non-disease model mice (Fig. 8A). PSR staining (i.e., interstitial collagen) revealed increased collagen accumulation in WT mice when comparing "no DSS" and "DSS" in the tent and muscle regions (Fig. 9a). An increase in interstitial collagen was not observed in TG2 knockout mice in response to DSS compared to "no DSS." WT DSS mice showed significantly increased collagen accumulation in the tent region compared to KO DSS mice (Fig. 9a). Collagen 1 and fibronectin protein expression were evaluated by IF staining in DSS mice in the presence or absence of TG2 KO (scans not shown). As shown in Fig. 9b, collagen 1 and fibronectin levels were reduced in tissues derived from TG2 KO DSS mice compared to tissues derived from WT DSS mice (P≤0.05). This reduction was more pronounced in the tent region. The barrier was significantly thicker in WT DSS mice compared to KO DSS mice (Fig. 9c; scans not shown).

[0092] TNBS Fibrosis Model: In the chronic TNBS mouse model, no differences were observed in clinical scores, body weight loss (data not shown), and histopathological inflammation scores between the WT TNBS group and the TG2 KO TNBS group (Fig. 8B). WT and KO TNBS mice exhibited higher inflammation scores compared to WT and KO non-TNBS mice (Fig. 8B). PSR staining showed a nominal increasing tendency of interstitial collagen in WT mice when comparing "non-TNBS" and "TNBS," particularly in the tent region (Fig. 10a). When comparing "non-TNBS" and "TNBS," KO mice showed no changes in interstitial collagen (Fig. 10a). Significantly reduced intensity in chronic TNBS colitis was observed in TG2 knockout mice compared to WT mice (Fig. 10a). As highlighted in Fig. 10b, collagen 1 and fibronectin ECM proteins were reduced in TG2 KO mice, particularly in the TNBS model, compared to WT mice, and barrier thickness was also reduced (Fig. 10c; scan images not shown).

[0093] Examples Conclusion of 3:

[0094] Mature ECM accumulation (consistent with fibrotic remodeling) was reduced in the absence of TG2 (i.e., in TG2 KO mice) and was found to have no effect on inflammation in both the DSS and TNBS prolonged colitis models used to induce early fibrotic remodeling. The absence of TG2 was shown to reduce the degree of fibrosis without having any effect on reducing inflammation. These data presented herein confirm the data of Example 2, that is, TG2 inhibitors may be a viable therapeutic agent for preventing the further accumulation of existing ECM deposits exceeding normal physiological levels in IBD patients, including preventing the progression of already existing ECM accumulation in patients with UC, CDs, or CDns.

[0095]

Claims

Claim 1 Transglutaminase 2 (TG2) inhibitors for use in the treatment of fibrotic-related disorders associated with inflammatory bowel disease (IBD) in patients or for the prevention of the occurrence of such disorders. Claim 2 A TG2 inhibitor according to claim 1, wherein the fibrosis-related disorder associated with IBD is selected from the group consisting of a) strictures including strictures in the intestine, ileum, ileocecal valve, upper gastrointestinal tract, colon, rectum or anus, b) fibrosis of the intestine, ileum, ileocecal valve, upper gastrointestinal tract, colon, rectum or anus, and c) bowel obstruction. Claim 3 A TG2 inhibitor according to any one of the prior claims, wherein the IBD is selected from the group consisting of Crohn's disease (CD) and ulcerative colitis (UC). Claim 4 A TG2 inhibitor according to claim 3, wherein a. the Crohn's disease is selected from the group consisting of stricturing Crohn's disease and non-stricturing Crohn's disease, or b. the ulcerative colitis is inflamed ulcerative colitis. Claim 5 In any one of the prior claims, the fibrosis-related disorder associated with Crohn's disease is characterized by an increase in a marker in a sample of the subject, said marker being selected from the group of TG2 activity, the level of epsilon(gamma-glutamyl) lysine crosslinking which is a product of TG2, mRNA encoding TG2, TG2 antigen, enhanced export of TG2, or any combination thereof. Claim 6 A TG2 inhibitor according to any one of the prior claims, wherein the TG2 inhibitor is an anti-TG2 antibody that binds to an epitope within the core region of TG2 and inhibits at least one of the TG2 activities, and the at least one inhibited TG2 activity is a TG2 cross-linking of lysine and glutamine by N-ε(γ-glutamyl) lysine isopeptide bond. Claim 7 In any one of the prior claims, the anti-TG2 antibody or its antigen-binding fragment is: a. comprising or composed of an intact antibody, or b. comprising or composed of an antigen-binding fragment, a TG2 inhibitor. Claim 8 In any one of the prior claims, the TG2 inhibitor comprising six CDRs selected from the group consisting of the anti-TG2 antibody: (i) KASQDINSYLT (LCDR1; SEQ No. 1); LVNRLVD (LCDR2; SEQ No. 2); LQYDDFPYT (LCDR3; SEQ No. 3); THAMS (HCDR1; SEQ No. 4); TISSGGRSTYYPDSVKG (HCDR2; SEQ No. 5); and LISTY (HCDR3; SEQ No. 6); or (ii) KASQDINSYLT (LCDR1; SEQ No. 1); LTNRLMD (LCDR2; SEQ No. 7); LQYVDFPYT (LCDR3; SEQ No. 8); SSAMS (HCDR1; SEQ No. 9); TISSGGRSTYYPDSVKG (HCDR2; SEQ No. 5); and LISPY (HCDR3; sequence number: 10); or (iii) KASQDINSYLT (LCDR1; sequence number: 1); RTNRLFD (LCDR2; sequence number: 11); LQYDDFPYT (LCDR3; sequence number: 3); SSAMS (HCDR1; sequence number: 9); TISVGGGKTYYPDSVKG (HCDR2; sequence number: 42); and LISLY (HCDR3; sequence number: 12). Claim 9 A TG2 inhibitor according to any one of the prior claims, wherein the anti-TG2 antibody comprises: a) a light chain variable domain having a sequence defined in any one of SEQ ID NO: 13 to SEQ ID NO: 27, and a heavy chain variable domain having a sequence defined in any one of SEQ ID NO: 28 to SEQ ID NO: 40; or b) a light chain variable domain having at least 80% identity or similarity, preferably at least 90% identity or similarity, or preferably at least 95% identity or similarity, with respect to a sequence defined in any one of SEQ ID NO: 13 to SEQ ID NO: 27, and a heavy chain variable domain having at least 80% identity or similarity, preferably at least 90% identity or similarity, or preferably at least 95% identity or similarity with respect to a sequence defined in any one of SEQ ID NO: 28 to SEQ ID NO:

40. Claim 10 A TG2 inhibitor according to any one of claims 1 to 7, wherein the antibody competes for binding to TG2 with the antibody defined in any one of claims 8 and 9. Claim 11 A TG2 inhibitor according to any one of claims 1 to 5, wherein the TG2 inhibitor is a small molecule. Claim 12 The TG2 inhibitor of claim 11, wherein the low molecule is selected from the group consisting of 1) competitive amine inhibitors, 2) reversible inhibitors, and 3) irreversible inhibitors. Claim 13 A TG2 inhibitor according to any one of the prior claims, wherein the TG2 inhibitor is incorporated into a pharmaceutical composition comprising one or more pharmaceutically acceptable adjuvant(s) and / or carrier(s). Claim 14 A pharmaceutical composition for use in the treatment of a subject having a fibrosis-related disorder associated with inflammatory bowel disease (IBD) or in the prevention of the occurrence of such disorder, wherein the pharmaceutical composition comprises a TG2 inhibitor and one or more pharmaceutically acceptable adjuvant(s) and / or carrier(s). Claim 15 A method for treating a subject with a fibrosis-related disorder associated with inflammatory bowel disease (IBD) or preventing the occurrence of such disorder in the subject, comprising the step of administering a therapeutically effective amount of a transglutaminase 2 (TG2) inhibitor to the subject. Claim 16 Use of transglutaminase 2 (TG2) inhibitors for the treatment of subjects with fibrosis-related disorders associated with inflammatory bowel disease (IBD) or for the manufacture of medicines for the prevention of the occurrence of such disorders.

Images