Methods for treating fibrosis-related conditions associated with ibd

Abstract

The present invention relates to a transglutaminase type 2 (TG2) inhibitor which blocks at least the transamidase activity of the enzyme for use in the treatment of a subject suffering from a fibrosis-related condition associated with inflammatory bowel disease (IBD) or for preventing the development of such a condition.

Description

Technical Field

[0001] This invention relates to a type 2 transglutaminase (TG2) inhibitor that at least blocks the transaminastic activity of the enzyme, for use in treating subjects with fibrosis-related conditions associated with inflammatory bowel disease (IBD) or for preventing the development of such conditions. Background Technology

[0002] Tissue transglutaminase, or transglutaminase type 2 (TG2), is an enzyme that cross-links proteins via ε-(γ-glutamyl)-lysine dipeptide bonds. In addition to this direct stabilizing effect on the ECM, TG2 appears to indirectly promote extracellular matrix (ECM) cross-linking by activating the fibrotic cytokine transforming growth factor β (TGF-β) (Nunes et al., 1997; Huang et al., 2010). Elevated TG2 expression leads to aberrant protein cross-linking, which is associated with several pathologies, including various types of tissue scarring and fibrosis, the formation of neurofibrillary tangles in several brain disorders, and resistance to chemotherapy in certain cancers. Several TG2 inhibitors, such as small molecules, silent RNAs, or antibodies (e.g., Siegel et al., 2007; Wang et al., 2020; WO2006100679, WO2012146901, or WO2013175229), have been disclosed for the treatment of possible TG2-mediated conditions.

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

[0004] Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a chronic, relapsing, and relapsing disease affecting more than 3 million people in Europe and the United States. IBD-associated intestinal fibrosis is a significant clinical problem in patients with both CD and UC. More than half of CD patients develop fibrosis-induced bowel obstruction, accompanied by debilitating symptoms (Cosnes et al., 2005; Rieder et al., 2017). Non-surgical approaches have limited success rates in strictured CD, leading to surgical intervention in up to 80% of CD patients. Fibrosis, particularly intestinal fibrosis, appears to be an inevitable and irreversible complication of chronic bowel inflammation. Fibrosis is also seen in UC, primarily confined to specific tissues (mucosa and submucosa), such as affecting colonic motility.

[0005] Intestinal fibrosis is defined as the excessive accumulation of extracellular matrix (ECM), including elevated levels of fibronectin and collagen, ultimately leading to organ dysfunction. Mesenchymal cells (primarily differentiating into fibroblasts, myofibroblasts, and smooth muscle cells in the gut / colon) have been identified as the main effector cells mediating fibrosis. In a healthy response to injury or inflammation, they act as “repair units,” with their population rapidly increasing while producing pro-fibrotic factors and ECM. However, in a chronic inflammatory environment, excessive ECM production leads to irreversible fibrosis.

[0006] It remains necessary to identify other effective therapies for treating subjects with fibrosis-related conditions associated with inflammatory bowel disease (including Crohn's disease or ulcerative colitis) or for preventing the development of such conditions. Summary of the Invention

[0007] One object of the present invention is to provide a specific transglutaminase 2 (TG2) inhibitor for the treatment of patients with fibrosis-related conditions associated with inflammatory bowel disease (IBD) or for the prevention of the development of such conditions.

[0008] In a second aspect, the present invention provides a method for treating a subject suffering from fibrosis-related conditions associated with inflammatory bowel disease (IBD) or for preventing the development of such conditions in a subject, comprising administering to the subject a therapeutically effective amount of a transglutaminase 2 (TG2) inhibitor.

[0009] In a third aspect, the present invention relates to the use of TG2 inhibitors in the preparation of medicaments for treating subjects with fibrosis-related conditions associated with inflammatory bowel disease (IBD) or for preventing the development of such conditions.

[0010] definition

[0011] The entire document is intended to be read as a unified public disclosure, and it should be understood that all combinations of features described herein are considered, even if such combinations do not appear together in the same sentence, paragraph, or section of this document. Regarding the use of “a” or “an” to describe or claim aspects of the invention, it should be understood that these terms mean “one or more” or “one or more types” unless the context explicitly requires a more limited meaning. The term “or” should be understood to cover alternative or concurrent items unless the context explicitly requires otherwise. If an aspect of the invention is described as “comprising” a feature, embodiments “consisting of” or “substantially consisting of” that feature are also considered.

[0012] - The terms “tissue transglutaminase,” “type 2 transglutaminase,” or “TG2” refer to an enzyme, specifically the product of the TGM2 gene, which forms cross-links between proteins via ε(γ-glutamyl)-lysine dipeptide bonds. TG2 refers to a protein that typically has the amino acid sequence shown in UniProt entry P21980 (with or without an N-terminal methionine, as shown in SEQ ID NO: 41), namely human TG2. The term “TG2” may also refer to a protein that: (a) has one or more amino acid substitutions, modifications, deletions, or insertions relative to the amino acid sequence of SEQ ID NO: 41, retaining the activity of TG2; or (b) a variant thereof that typically retains at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identity with SEQ ID NO: 41 (or even about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41). The protein TG2 is encoded by the gene Tgm2.

[0013] - The term "TG2 inhibitor" refers to a molecule capable of inhibiting at least one TG2 activity (e.g., TG2 crosslinks lysine and glutamine via N-ε(γ-glutamyl)lysine isopeptide bonds). Such molecules can be, but are not limited to, small molecules, silent RNA, or antibodies.

[0014] Small molecules can be selected from three main categories (non-limiting examples): 1) competitive amine inhibitors that compete with natural amine substrates, 2) reversible inhibitors, and 3) irreversible inhibitors (Siegel and Khosla, 2007).

[0015] - The term "inhibition," etc., refers to blocking or neutralizing one or more activities typically associated with a target (e.g., an enzyme). For example, in the context of this invention, the term "inhibition" includes blocking the enzymatic function of TG2 associated with transamidation (including cross-linking) and protein deamidation. TG2 is also known as a GTPase (Im et al., 1997).

[0016] - As used herein, the term "anti-TG2 antibody" is intended to refer to an antibody molecule that binds to TG2 and blocks its transaminases to prevent cross-linking. Examples of such antibodies are described in WO2013175229. Without limitation, anti-TG2 antibodies that may be used according to the invention comprise, for example, a light chain variable region as defined in SEQ ID NO: 24 and a heavy chain variable region as defined in SEQ ID NO: 37.

[0017] - As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies generated by recombinant techniques as known in the art. "Antibody" includes antibodies of any species; for example, any isotype of human antibodies, including IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, IgD, and antibodies generated as dimers of this basic structure, including IgGA1, IgGA2, or pentamers such as IgM and their modified variants; non-human primate antibodies, such as those from chimpanzees, baboons, rhesus monkeys, or cynomolgus monkeys; rodent antibodies, such as those from mice or rats; antibodies from rabbits, goats, or horses; camel antibodies (e.g., antibodies from camels or llamas, such as Nanobodies™) and their derivatives; antibodies from bird species, such as chicken antibodies; or antibodies from fish species, such as shark antibodies. The term "antibody" also refers to a "chimeric" antibody, in which the first portion of at least one heavy chain and / or light chain antibody sequence originates from a first species, and the second portion of the heavy chain and / or light chain antibody sequence originates from a second species. Chimeric antibodies of interest herein include "primate-derived" antibodies, which contain a variable domain antigen-binding sequence derived from a non-human primate (e.g., an Old World monkey, such as a baboon, rhesus monkey, or cynomolgus monkey) and a human constant region sequence. "Humanized" antibodies are chimeric antibodies containing sequences derived from non-human antibodies. In most cases, humanized antibodies are human antibodies (receptor antibodies) in which residues from the hypervariable region of the receptor are replaced by residues from a hypervariable region [or complementarity-determining region (CDR)] of a non-human species (donor antibody) (e.g., a mouse, rat, rabbit, chicken, or non-human primate) possessing the desired specificity, affinity, and activity. In most cases, residues outside the CDR of the human (receptor) antibody; i.e., residues in the frame region (FR), are also replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in receptor or donor antibodies. These modifications are made to further optimize antibody properties. Humanization reduces the immunogenicity of non-human antibodies in the human body, thereby facilitating the use of antibodies in the treatment of human diseases. Humanized antibodies and several different techniques for generating them are well known in the art. The term "antibody" also refers to human antibodies, which can be generated as an alternative to humanization. For example, transgenic animals (e.g., mice) can be produced that, upon immunization, generate a full set of human antibodies without producing endogenous mouse antibodies. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display techniques, such as phage display or ribosome display, in which recombinant DNA libraries are used, which are at least partially artificially generated or donor-derived libraries of immunoglobulin variable (V) domain genes. Phage display and ribosome display techniques for generating human antibodies are well known in the art.Human antibodies can also be produced by isolated human B cells, which are immunized in vitro with a target antigen, then fused to generate a hybridoma, which can then be screened to obtain the optimal human antibody. The term "antibody" refers to both glycosylated and non-glycosylated antibodies. Furthermore, as used herein, the term "antibody" refers not only to full-length antibodies but also to antibody fragments, more specifically, to their antigen-binding fragments. Antibody fragments contain at least one heavy or light chain immunoglobulin domain known in the art and bind one or more antigens. Examples of antibody fragments described in this 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 fragment can also be a diabody, tribody, triabody, tetrabody, microbody, single-domain antibody (dAb) (such as sdAb), VL, VH, VHH, or camelid antibody (e.g., antibodies derived from camels or llamas, such as Nanobody™) and VNAR fragment. The antigen-binding fragment of the present invention may also comprise a Fab linked to one or two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binding to a therapeutic target, and one scFv or dsscFv increasing its half-life by binding to, for example, albumin). Examples of such antibody fragments are FabdsscFv (also known as BYbe®) or Fab-(dsscFv)2 (also known as TrYbe®, see, for example, WO2015 / 197772). Antibody molecules as defined above, including their antigen-binding fragments, are known in the art.

[0018] - The term "epitope" refers to the region where an antigen is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and contain those residues that directly contribute to the interaction affinity. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In some embodiments, epitopes may include determinant clusters of molecularly chemically active surface groups, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features.

[0019] - The term “treatment” of a disease state includes: (i) suppressing a disease state, i.e. preventing the development of a disease state or its clinical symptoms, or (ii) alleviating a disease state, i.e. causing a temporary or permanent resolution of a disease state or its clinical symptoms.

[0020] - The term "prevention" of disease state includes preventing the clinical symptoms of disease state from developing in subjects who may be exposed to or susceptible to disease state but have not yet experienced or exhibited symptoms of disease state. Detailed Implementation

[0021] This invention is based on the inventors' discovery that TG2 expression (e.g., increased TG2 antigen) and its activity are significantly increased in the tissues of patients with UC and CD, particularly in Crohn's disease stenosis, where TG2 activity is correlated with fibrosis scores. It was also found that TG2 inhibitors can inhibit TG2 activity in situ. The inventors were also able to demonstrate that TG2 inhibitors (such as small molecule inhibitors and anti-TG2 antibodies) can attenuate the accumulation of mature fibronectin and collagen mediated by human intestinal myofibroblasts (HIMF) in vitro.

[0022] The primary objective of this invention is the use of transglutaminase 2 (TG2) inhibitors for the treatment of patients with fibrosis-related symptoms associated with inflammatory bowel disease (IBD) or for the prevention of the development of such symptoms.

[0023] The present invention also provides a method for treating a subject with fibrosis-related conditions associated with inflammatory bowel disease (IBD) or for preventing the development of such conditions in a subject, comprising administering to the subject a therapeutically effective amount of a transglutaminase 2 (TG2) inhibitor.

[0024] The present invention also describes the use of transglutaminase 2 (TG2) inhibitors in the preparation of medicaments for treating subjects with fibrosis-related conditions associated with inflammatory bowel disease (IBD) or for preventing the development of such conditions.

[0025] In the context of the invention, fibrosis-related conditions associated with IBD are selected from: a) stenosis, including stenosis of 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) intestinal obstruction.

[0026] In the context of the invention, inflammatory bowel disease (IBD) is selected from: Crohn's disease (CD), such as non-strictive CD (CDns) or strictive CD (CDs), and ulcerative colitis (UC), such as inflammatory UC (UCi).

[0027] In the context of this invention, fibrosis-related symptoms associated with IBD are characterized by an increase in biomarkers in a subject sample, wherein the biomarker is, for example, any one of the following: TG2 activity, level of TG2 product ε (γ-glutamyl) lysine crosslinking, TG2 expression level (such as an increase in mRNA encoding TG2 or TG2 antigen), TG2 output, or any combination thereof, and wherein the subject sample is a cell or tissue associated with the disease (e.g., intestinal cells or colon tissue). The increase in said biomarker can be determined in the disease-associated cells / tissue in any way. An increase in a biomarker in a subject sample is typically determined by comparing the level of said biomarker in the subject sample with the level of the same biomarker (i.e., baseline level; e.g., baseline TG2 activity, baseline expression level (mRNA level and / or protein level), and / or baseline TG2 output level) in normal cells of the same tissue type. An increase in the level of at least one biomarker in a subject sample compared to the baseline level of said biomarker by 10%, 15%, 20%, 25%, or even 30% or more will be considered to represent an increase in said biomarker. For example, an increase in TG2 expression (also known as TG2 overexpression) can be determined by measuring the amount of TG2 mRNA in a patient's intestinal cells. Therefore, cells / tissues affected by IBD-related fibrosis can be characterized, for example, by the amount of increased TG2 mRNA in a subject's intestinal cells compared to normal cells of the same tissue type (representing overexpression). TG2 mRNA expression can increase by any amount, such as an increase of 10%, 15%, 20%, 25%, or even 30% compared to baseline levels. The amount of mRNA can be measured using any known method, such as quantitative reverse transcription polymerase chain reaction (quantitative RT-PCR, also known as qRT-PCR), real-time qRT-PCR, quantumgene assays, by RNA blotting, or using microarrays, RNA sequencing, and various types of in situ hybridization (e.g., RNAscope). Alternatively, overexpression can be determined by measuring the amount of TG2 antigen in a patient's intestinal cells. Therefore, cells affected by IBD-related fibrosis can be characterized, for example, by an increased amount of TG2 protein (or TG2 antigen) in the subject's intestinal cells compared to normal cells of the same tissue type (representing overexpression). TG2 protein expression can be increased by any amount, such as an increase of 10%, 15%, 20%, 25%, or even 30% compared to baseline levels. 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 using the anti-TG2 antibody of the present invention.The threshold for determining expression may vary depending on the technique used and can be validated against immunohistochemical scoring. Alternatively, cells / tissues affected by IBD-related fibrosis can 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, such as an increase of 10%, 15%, 20%, 25%, or even 30% compared to baseline levels. TG2 activity can be measured using any known method, such as cryobiopsy (TGISA).

[0028] In one embodiment of the invention, the TG2 inhibitor is an anti-TG2 antibody that preferably binds to an epitope within the core region of TG2 and inhibits at least one TG2 activity, wherein the inhibited at least one TG2 activity is TG2 causing lysine to crosslink with glutamine via an N-ε(γ-glutamyl)lysine isopeptide bond. In a non-limiting example, when evaluated, for example, by peptide mapping analysis, the preferred antibody binding is: a region comprising amino acids 304 to 326 of TG2 (e.g., SEQ ID NO: 41), or a region consisting of amino acids 304 to 326 of TG2 (e.g., SEQ ID NO: 41), or a portion thereof. The antibody may comprise or consist of a complete antibody. Alternatively, it may comprise or consist of an antigen-binding fragment, such as (but not limited to): an Fv fragment (e.g., a single-chain Fv fragment or a disulfide-linked Fv fragment); a Fab fragment; and a Fab-like fragment (e.g., a Fab' fragment or an F(ab)2 fragment), a single-domain antibody (or any other fragment as defined herein or known to those skilled in the art). Preferably, the anti-TG2 antibody used throughout the invention (see also Table A):

[0029] a) Includes six CDRs selected from the following:

[0030] (i) KASQDINSYLT (LCDR1; SEQ ID NO. 1); LVNRLVD (LCDR2; SEQ ID NO. 2); LQYDDFPYT (LCDR3; SEQ ID NO. 3); THAMS (HCDR1; SEQ ID NO. 4); TISSGGRSTYYPDSVKG (HCDR2; SEQ ID NO. 5); NO. 6);

[0031] (ii) KASQDINSYLT (LCDR1; SEQ ID NO. 1); LTNRLMD (LCDR2; SEQ ID NO. 7); LQYVDFPYT (LCDR3; SEQ ID NO. 8); SSAMS (HCDR1; SEQ ID NO. 9); TISSGGRSTYYPDSVKG (HCDR2; SEQ ID NO. 5); and LISPY (HCDR3; SEQ ID NO. 5); NO. 10); or

[0032] and LISLY (HCDR3; SEQ ID NO. 12);

[0033] b) Contains a light chain variable domain having a sequence as defined in any one of SEQ ID NO: 13 to SEQ ID NO: 27 and a heavy chain variable domain having a sequence as defined in any one of SEQ ID NO: 28 to SEQ ID NO: 40.

[0034] c) Containing 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 any of the sequences defined as such as 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% (or even about 96%, 97%, 98%, or 99%) identity or similarity with any of the sequences defined as such as SEQ ID NO: 28 to SEQ ID NO: 40, or

[0035] d) Compete with antibodies as defined in a), b) or c) above for binding to TG2.

[0036] As a non-limiting example, the anti-TG2 antibody comprises the following six CDRs: KASQDINSYLT (LCDR1; SEQ ID NO: 1); LVNRLVD (LCDR2; SEQ ID NO: 2); LQYDDFPYT (LCDR3; SEQ ID NO: 3); THAMS (HCDR1; SEQ ID NO: 4); TISSGGRSTYYPDSVKG (HCDR2; SEQ ID NO: 5); and LISTY (HCDR3; SEQ ID NO: 6). These six exemplary CDRs may be contained in a light chain variable domain having a sequence as defined in SEQ ID NO: 24 and a heavy chain variable domain having a sequence as defined in SEQ ID NO: 37, respectively. As another non-limiting example, the anti-TG2 antibody comprises the following six CDRs: KASQDINSYLT (LCDR1; SEQ ID NO. 1); LTNRLMD (LCDR2; SEQ ID NO. 7); LQYVDFPYT (LCDR3; SEQ ID NO. 8); SSAMS (HCDR1; SEQ ID NO. 9); TISSGGRSTYYPDSVKG (HCDR2; SEQ ID NO. 5); and LISPY (HCDR3; SEQ ID NO. 10). These six exemplary CDRs may be contained in a light chain variable domain having a sequence as defined in SEQ ID NO: 19 or 25 and a heavy chain variable domain having a sequence as defined in SEQ ID NO: 32 or 38, respectively.

[0037]

[0038]

[0039]

[0040] By using conventional methods known in the art, it is readily possible to determine whether an antibody binds to the same epitope or competes with another antibody for binding. 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 subjected to saturation conditions to bind a protein or peptide. Next, the ability of the test antibody to bind the protein or peptide is evaluated. If the test antibody is able to bind the protein or peptide after saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind the protein or peptide after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the reference antibody of the present invention. To determine whether an antibody competes with a reference antibody for binding, the above binding method is performed in two directions. In the first direction, the reference antibody is subjected to saturation conditions to bind a protein / peptide, and then the binding of the test antibody to the protein / peptide molecule is evaluated. In the second direction, the test antibody is subjected to saturation conditions to bind a protein / peptide, and then the binding of the reference antibody to the protein / peptide is evaluated. If, in both directions, only the first (saturated) antibody is able to bind the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As those skilled in the art will understand, an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0041] If two antibodies competitively inhibit (block) the binding of the other to an antigen, they bind to the same or overlapping epitopes. That is, as measured in a competitive binding assay, an excess of one antibody of 1, 5, 10, 20, or 100 times inhibits the binding of the other antibody by at least 50%, 75%, 90%, or even 99%, respectively. Alternatively, if substantially 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 reduce or eliminate the binding of the other antibody, the two antibodies have overlapping epitopes.

[0042] Additional routine experiments (such as peptide mutation and binding assays) can then be performed to confirm whether the observed loss of test antibody binding is actually due to binding to the same epitope as the reference antibody, or whether the observed loss of binding is caused by steric hindrance (or other phenomena). Such experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0043] In another embodiment of the invention, 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 can be selected from three main categories (non-limiting examples): 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 the following literature: Siegel and Khosla, 2007, WO033784, WO14012858, WO14057266, WO17179018, WO18122419, WO22213198, or WO23135425.

[0044] Any subject may be treated according to the present invention. The subject is preferably a human. However, the subject may be other mammals, such as non-human primates, horses, cattle, sheep, pigs, dogs, cats, rabbits, rats, mice, guinea pigs, or hamsters. Alternatively, the term "patient" may be used indiscriminately in place of "subject."

[0045] Any TG2 inhibitor described in this invention may be incorporated into a pharmaceutical composition suitable for administration to a subject in any manner (e.g., (but not limited to) topical, intranasal, intradermal, intravenous, subcutaneous, or intramuscular). Typically, the pharmaceutical composition comprises a TG2 inhibitor and one or more pharmaceutically acceptable adjuvants and / or carriers. Therefore, a pharmaceutical composition is also described herein for the treatment of a subject suffering from fibrosis-related conditions associated with inflammatory bowel disease (IBD) or for the prevention of the development of such conditions, wherein the pharmaceutical composition comprises a TG2 inhibitor and one or more pharmaceutically acceptable adjuvants and / or carriers. The pharmaceutical composition according to the invention may be part of a kit comprising instructions for use (including instructions for use) and optionally a device for intravenous, subcutaneous, or intramuscular administration to an individual in need.

[0046] As used herein, a “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents 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, phosphate-buffered saline, glucose, glycerol, ethanol, and combinations thereof. Depending on the route of administration or type of formulation (e.g., liquid, lyophilized, or spray-dried formulation), isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride, may be added to the composition. Pharmaceutically acceptable carriers may also contain small amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, which may enhance the shelf life or efficacy of the inhibitor.

[0047] The pharmaceutical compositions of this invention can be in various forms. These forms include, for example, liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, powders, and liposomes. Preferred forms depend on the intended method of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusionable solutions, such as compositions used together with other therapeutic molecules for passive immunization in humans.

[0048] The appropriate dosage of the TG2 inhibitor described in this invention can be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition of this invention may vary to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a specific patient, composition, and route of administration without toxicity to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including route of administration, time of administration, excretion rate of the TG2 inhibitor, duration of treatment, other drugs, compounds, and / or materials used in combination with the specific TG2 inhibitor, and the patient's age, sex, weight, condition, general health status, and medical history.

[0049] Appropriate dosages can be, for example, in the range of about 0.01 pg / kg body weight to about 1000 mg / kg body weight, typically about 0.1 pg / kg body weight to about 100 mg / kg body weight, all based on the weight of the patient to be treated. Dosing regimens can be adjusted to provide the best expected response (e.g., therapeutic response). For example, a single dose can be administered, or several fractionated doses can be administered over time. As used herein, unit dosage forms refer to physically separable units suitable for use as a single dose in the subject to be treated; each unit contains a calculated predetermined amount of the active compound to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. Administration can be performed in single or multiple doses. Multiple doses can be administered via the same or different routes to the same or different sites.

[0050] In the context of this invention, TG2 inhibitors can be administered co-administered with one or more other therapeutic agents. The combined administration of two or more agents can be achieved in a variety of different ways. The two agents can be administered together in a single composition or as part of a combination therapy in separate compositions. For example, one agent can be administered before or separately from another agent, after or sequentially, or simultaneously. Attached Figure Description

[0051] Figure 1Transglutaminase (TG) activity, measured using scan images, was significantly increased in UCi / CDs relative to NL and correlated with fibrosis scores but not with inflammation scores. A) TG activity was quantified by integral density (P-value compared to NL) from tissue sections from different IPD patients (UCi, CDns, and CDs patients, respectively; NL used as a control). B) TG2 activity in full-thickness IBD tissue correlated with fibrosis scores (“Fibrosis Score” column) but not with inflammation scores (“Geboes (Inflammation) Score” column), as shown in two different layers of intestinal tissue (muscularis propria and muscularis mucosae).

[0052] Figure 2 The inhibition of TG2 by zobilimab confirmed that TG2 is the source of transaminasing activity and accounts for 80% to 100% of total TG activity. A) Integrated density quantification of TG2 activity was performed using scanned images of muscularis mucosae and muscularis propria tissue sections from different IBD patients (UCi, CDns, and CDs patients; NL as a control) (scanned images not shown). B) TG2 activity accounted for 80% to 100% of total TG activity, as determined by calculating the percentage of TG activity lost after pretreatment with zobilimab based on images (scanned images not shown) of muscularis mucosae and muscularis propria tissue sections from different IBD patients (UCi, CDns, and CDs patients; NL as a control). (P-value between treated and untreated groups).

[0053] Figure 3 Increased TG2 mRNA and TG2 antigen in UCi and CD stenosis: A) TG2 antigen was quantified by integrated density from tissue sections from different IBD patients (UCi, CDns, and CDs patients; NL as a control; P-value compared to NL). B) Using RNA-Z-scope, pathologist scoring of raw images showed that TG2 expression (from the mRNA perspective) was increased by 25% to 100% in samples from UCi, CDns, and CDs patients 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 elevated TG2 expression (from the mRNA perspective) in UCi and CDs patients.

[0054] Figure 4 Ten frozen patient tissues from each of the different IBD patients (UCi, CDns, and CDs patients; NL used as a control) were used. 14 The analysis was performed using the C-putrescine assay. The figure shows an increase in TG activity in CDs compared to NL (using a t-test, a nonparametric test).

[0055] Figure 5 TG2 is the source of transglutaminase activity in human enteric myofibroblasts (HIMF) derived from NL tissue. 82%–97% of transglutaminase in NL HIMF was identified as TG2 using specific TG2 inhibitors (the anti-TG2 antibody zobilimab and the small molecule Z-Don), as determined by calculating the percentage of TG activity lost after pretreatment with a TG2-specific inhibitor. Each point represents a single patient-derived primary cell line.

[0056] Figure 6 : Mature extracellular matrix (ECM) levels between control (NL) and fibroblasts derived from different types of IBD patients. It shows baseline production of fibronectin (FN) (A) and collagen (B) among NL, UC, CDns, and CDs, and illustrates trends of change between them. Each point represents a single patient-derived primary cell line.

[0057] Figure 7 Extracellular matrix (ECM) levels were reduced in fibroblasts from NL, UCi, CDns, and CDs patients treated with TG2 inhibitors (the anti-TG2 antibody zobilimab and the small molecule TG2 inhibitors Z-Don and Boc-Don). A) Scanning images show ECM deposition under different conditions, with immunofluorescence staining using antibodies specifically targeting ECM components in tissues from NL subjects (top 5 images) and CDs patients (bottom 5 images). B) ECM levels between normal and different types of IBD patients, comparing mature ECM levels between normal and different types of IBD patients in the presence of specific TG2 inhibitors (the anti-TG2 antibody zobilimab and the small molecule TG2 inhibitors Z-Don and Boc-Don). Graphs show the production of fibronectin (FN) and collagen (collagen I and III) between tissues from NL (B), UC patients (C), CDns patients (D), and CDs patients (E), and illustrate trends in their variation. Each point represents a single patient-derived primary cell line.

[0058] Figure 8 A) Inflammation scores in different tissues of WT and TG2 KO mice with and without DSS in a mouse chronic model of Crohn's disease with DSS. B) Inflammation scores of WT and TG2 KO mice with and without TNBS in a mouse chronic model of Crohn's disease with TNBS pre-sensitization. Each point represents a single mouse.

[0059] Figure 9A) PSR staining of the tent and muscle regions of WT and TG2 KO mice with and without DSS. B) Collagen 1 (left panel) and fibronectin (right panel) in WT and TG2 KO mice with and without DSS (day 34). C) Intestinal wall thickening in WT and TG2 KO mice with and without DSS (day 34); quantitative measurement of wall thickness.

[0060] Figure 10 A) PSR staining of the tent and muscle regions of WT and TG2 KO mice with and without TNBS (day 32). B) Collagen 1 (left panel) and fibronectin (right panel) in WT and TG2 KO mice. C) Intestinal wall thickening in WT and TG2 KO mice with and without TNBS (day 34); quantitative measurement of wall thickness.

[0061] The P-value in the attached figure:

[0062]

[0063] Key abbreviations used in this specification:

[0064] TG: Transglutaminase; TG2: Transglutaminase 2; NL: Non-lesion tissue (from various patients who do not have IBD; by extension, patients from whom this type of NL tissue is extracted are called NL patients); UCI: Inflammatory ulcerative colitis; CDs: Stenotic Crohn's disease (CD); CDns: Non-stenotic CD; FN: Fibronectin; COL: Collagen; KO: Knockout; WT: Wild type; PSR: Sirius red; DSS: Dextran sulfate sodium; TNBS: Trinitrobenzenesulfonic acid solution; HIMF: Human intestinal myofibroblasts; H&E: Hematoxylin and eosin; KO: Knockout; IF: Immunofluorescence; ID: Integrated density.

[0065] Example

[0066] Material

[0067] Anti-TG2 antibody: The anti-TG2 mAb used in the following examples comprises a light chain variable region as defined in SEQ ID NO: 24 and a heavy chain variable region as defined in SEQ ID NO: 37. This antibody is called zobilimab (also known as UCB7858, derived from antibody DC1).

[0068] Small molecule inhibitors: 1) Z-Don, which is an irreversible, cell-soluble, TG2-specific inhibitor; 2) Boc-Don, which is an irreversible, cell-insoluble, TG2-specific inhibitor.

[0069] method

[0070] Patient sample: Following industry standard practices, human tissue samples were collected from patients with CDs (n=10), CDns (n=20), and UC (n=10), as well as control tissues (NL; n=14). In short, full-thickness fresh resected bowel samples from patients with inflammatory bowel disease were obtained using standard methods. Tissue blocks from different tissue experimental groups were obtained for further processing.

[0071] Isolation and culture of primary human intestinal fibroblasts: Human intestinal myofibroblasts (HIMF) were obtained as explants from surgically resected intestinal mucosa and isolated and cultured according to standard methods. Briefly, HIMF were cultured to subconfluence in Dulbecco minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics, and established as long-term cultures, fed twice a week, and passaged at confluence.

[0072] In situ detection of TG2 activity and extracellular TG2 antigen: To detect in situ TG activity and extracellular TG2 antigen levels, unfixed, washed frozen sections from fresh frozen tissue were embedded in OCT mounting medium and cut to a thickness of 8-10 µM for both assays.

[0073] For the TG activity assay, slides were washed three times in PBS. Sections were blocked for 35 minutes at room temperature with 5% BSA and 5 µg / ml streptavidin. Parallel sections from the same sample were blocked in a similar manner, with the addition of 30 µg / ml UCB7858 antibody to block TG2 activity. Sections were washed three times with PBS and then added to a reaction mixture containing 1 mM DTT, 5 mM CaCl2, and 100 µM biotinylated cadaverine (in 50 mM Tris buffer (pH 7.4) containing a protease inhibitor). A third section served as a negative control, without biotinylated cadaverine.

[0074] For TG2 antigen detection, slides were washed with PBS at room temperature and incubated at 37°C for 1 hour with 30 µg / ml CUB7402 antibody (Abcam) in blocking buffer supplemented with protease inhibitors. After washing with PBS, slides were fixed in cold acetone and incubated at 37°C for 30 minutes with goat anti-mouse Alexa 594 secondary antibody and streptavidin Alexa 488. Slides were washed with PBS containing 0.1% Tween 20 at room temperature and mounted using Mowiol mounting medium containing Dapi.

[0075] All stained slides were scanned and acquired using Aperio Image Scope software (Leica Biosystems). The stained images were quantified using ImageJ software (Bethesda). Starting with non-lesion control slides, regions on the muscularis mucosae (MM) and muscularis propria (MP) were marked and thresholds were set. All other lesion slides were quantified using the same set thresholds, and integrated density was measured using ImageJ. Graphs were plotted using GraphPad Prism software (version 9.3.1, BostonMA).

[0076] Relationship between TG2 activity and amount and the degree of tissue fibrosis: The degree of fibrosis was assessed using Masson trichrome stained slides and the inflammation score (Geboes score) was assessed using H&E stained slides, which correlated in situ TG2 activity and quantity with the degree of fibrosis and inflammation. The inflammation score (Geboes) using H&E stained slides was based on structure (structural changes), ranging from no abnormalities (score 0) to severe diffuse or multifocal abnormalities (score 3). It also included infiltration of lamina propria neutrophils, eosinophils, and epithelial neutrophils, ranging from no increase (score 0) to a significant increase (score 3); crypt destruction, ranging from none (score 0) to >50% destruction (score 3); and erosion or ulceration, ranging from none (score 0) to ulceration or granulation tissue (score 3). The final Geboes score was based on the sum of all seven sub-scores, ranging from 0 to 22. The fibrosis score using trichrome stained sections is limited to submucosal ECM deposition, ranging from <25% of the total submucosal layer (score 0) to extensive transmural fibrosis with loss of normal layers (score 3).

[0077] pass 14 Measurement of transglutaminase activity using the C putrescine incorporation assay: Ten rapidly frozen patient tissue samples each from NL, UCi, CDns, and CDs patients were provided by the clinic. In this assay, active TG in the homogenized tissue was [1,4-] in the presence of calcium. 14 C]-putrescine is incorporated into N,N-dimethylcasein to form a radioactive trichloroacetic acid (TCA)-insoluble cross-linked protein, which is measured by scintillation counting. In short, tissue is homogenized on ice in STE buffer (0.32 M sucrose, 5 mM Tris, 2 mM EDTA) with a protease inhibitor (Sigma Aldrich) added, and protein content is measured using the Pierce™ BCA Protein Assay Kit. [The remaining text appears to be incomplete and requires further context.] 14A 75 µL reaction mixture of C-putrescine and N'N dimethyl casein was preheated in a test tube in a dry bath at 37 °C for 20 min. At T=0, 75 µL of tissue homogenate was added, mixed by up-and-down mixing, and incubated further in a dry bath at 37 °C. At set time points (0, 5, 10, 20, 30, 60 min), 10 µL aliquots were taken and spotted onto a filter mat pre-impregnated with 10% (w / v) ice-cold TCA to terminate the reaction and allow precipitation. 14 C. Putrescine-casein. In parallel experiments, at T=0, 15 µL of tissue homogenate + 15 µL of control reaction mixture (containing EDTA to inhibit enzyme activity) was mixed in a pre-warmed test tube on a heating block set to 37°C; the control mixture was sampled only at a single time point at the endpoint of the assay. The filter membrane was allowed to stand for 5 minutes after its final addition. The filter membrane was washed with 1 × 10% (w / v) ice-cold TCA for 10 minutes, 3 × 5% (w / v) ice-cold TCA for 5 minutes each, 1 × 1:1 ice-cold acetone:ethanol for 5 minutes, and finally 1 × ice-cold 100% acetone for 5 minutes. The filter membrane was air-dried, cut into individual squares representing each sample location on the filter membrane, and placed in scintillation bottles, one bottle per sample. 12 mL of scintillation solution was added, and radioactivity was measured in a 1450 miniature β-scintillation counter. The steady-state incorporation rate was corrected for the protein concentration of each sample and reported as cpm / min / mg protein.

[0078] TG2 in vitro cell viability assay (biotin-cadherin incorporation into fibronectin): Fibronectin was used to coat plates, and 8000 HIMF cells were seeded under different conditions (see Example 2). These conditions included: 1. Control: 50 µM biotinylated cadaverine containing 0.1% Tween 20 (positive control); 2. Untreated: 50 µM biotinylated cadaverine only; 3. Internal isotype control: 50 µM biotinylated cadaverine containing 150 µg / ml internal isotype antibody; 4. Zobilimab: 50 µM biotinylated cadaverine, 1000 nm (150 µg / ml) zobilimab; 5. Z-Don: 50 µM biotinylated cadaverine in medium containing 200 µM Z-Don; and a secondary control: medium only (without biotinylated cadaverine); 6. Boc-Don: 50 µM biotinylated cadaverine in internal medium containing 500 µM Boc-Don. Cells were cultured adherently at 37°C under these conditions for 24 hours. After 24 hours, the reaction was terminated with two washes (PBS + 5 mM EDTA), and the cells were then lysed with 0.25 M NH4OH + 50 mM Tris at room temperature for 10 minutes, followed by washing with PBS. Cells were blocked with BSA, and then HRP-streptavidin conjugate was added to detect biotinylate incorporation into the FN. Trimethylbenzidine (TMB) substrate was added to the wells and incubated at room temperature (RT) for 5 minutes. The reaction was terminated with 2 M sulfuric acid (H2SO4), diluted in PBS, and the absorbance was read at 450 nm using a ELISA reader (Spectra max, 340PC).

[0079] RNA was extracted from colon tissue. Prior to RNA extraction, up to 25 mg of fresh, frozen, natural intestinal tissue was minced. RNA extraction was performed using the RNeasy mini kit (Qiagen) according to the supplier's standard protocol. RNA concentration was determined using an ND-1000 UV / Vis spectrophotometer (NanoDrop, Thermo Scientific).

[0080] Quantitative reverse transcription polymerase chain reaction procedure: Total RNA was isolated as described above and reverse transcribed and quantitative PCR (quantitative RT-PCR) was performed according to the manufacturer's instructions (Applied Biosystems). Products from all primer pairs were validated by sequencing, and relative differences were calculated using the comparison threshold cycling method (ddCt) by normalizing to CT values ​​against the 18S (reference gene). Quantitative RT-PCR was performed using iQ Sybr Green Supermix (Biorad) and gene-specific primer pairs with cDNA (synthesized using Biorad's iScript cDNA Synthesis Kit). The fold change of treated samples relative to untreated samples was calculated using the Pfaffl method.

[0081] Assay of extracellular deposition in human intestinal myofibroblasts: Extracellular matrix (ECM) deposition in HIMF was determined using standard methods. Briefly, cells were seeded into 96-well plates in DMEM supplemented with 10% FBS and antibiotics (as described above) and allowed to grow and produce ECM for 5 days. 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 AlexaFluor488-conjugated anti-fibronectin (Ebioscience, 1:500 dilution), anti-type I and type III collagen antibodies (Millipore Sigma, 1:100 dilution), and anti-rabbit Alexa Fluor 594 (Ebioscience, 1:500 dilution). Fluorescence intensity was obtained by scanning the plates with ECM using a Thermofisher Cytation 5 scanner. The mean ECM level in the experiment was determined using fluorescence intensity from at least three replicate wells.

[0082] Dextran sulfate sodium (DSS)-induced colitis and trinitrobenzenesulfonic acid solution (TNBS)-induced fibrosis: BL6 mice (from Jackson Laboratories) were used. DSS-induced colitis and TNBS-induced fibrosis were induced according to standard procedures (see, for example, Lawrance et al., 2003 and Zhao et al., 2020).

[0083] Endpoints of the DSS and TNBS colitis studies: For the DSS and TNBS models, body weight, fecal consistency, and presence of occult blood or gross rectal blood were recorded every other day. At the end of the experiment, animals were euthanized by CO2 asphyxiation followed by cervical dislocation. The entire colon was removed, cleaned, weighed, and measured from the ileocecal junction to the anus. Tissue was harvested from the descending colon, and paraffin-embedded sections were histologically examined, stained with H&E, Masson trichrome, or Sirius red. Slides were graded according to standard practice by an experienced pathologist who blinded the experimental groups. Images were acquired using an Olympus microscope and ImagePro software. Sirius red images were quantified and analyzed using ImageJ software (NIH & LOCI).

[0084] Statistical analysis: Data from independent groups were analyzed using the Student's t-test or analysis of variance (ANOVA). Values ​​are expressed as 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.).

[0085] Example 1 - The role and expression / activity of TG2 in Crohn's disease-associated stenosis.

[0086] The aim of this study was to determine the role of TG2, and the expression and activity of TG2, in stenosis associated with IBD (such as Crohn's disease).

[0087] TG2 is the main active TG in IBD-related tissues ( Figure 1 and Figure 2 ) To assess the potential involvement of transglutaminase in IBD stenosis, the incorporation of the biotinylated TG substrate cadaverine was measured in various tissue sections, more specifically in unfixed, washed frozen sections of fresh frozen human tissue from different patients (including NL, UCi, CDns, and CDs patients). The integrated density (ID) of biotinylated cadaverine incorporation (BCI) in these sections was quantified using ImageJ software. Figure 1 As shown in Figure A, the global integral density (ID) of the BCI data shows increased TG activity in tissues from UCI and CDs patients compared to tissues from NL patients.

[0088] like Figure 1 As shown in B, regardless of patient type, TG activity (expressed as integral intensity) was correlated with the histopathological fibrosis score (left fibrosis score map: muscularis mucosae: R = 0.31, p = 0.026; muscularis propria: R = 0.44, p = 0.00087), but not with the histopathological inflammation score (right Geboes inflammation score map: muscularis mucosae: R = 0.12, p = 0.39; muscularis propria: R = 0.24, p = 0.086).

[0089] In addition, to identify the main TG species involved in this activity, the effect of the TG2-specific inhibitor UCB7858 on parallel sections was measured. Figure 2 As shown in B, it is clear that TG activity in tissue samples from IBD patients (as indicated by UCi, CDns, and CDs) and controls is primarily associated with TG2. 97-99% of TG activity is attributed to TG2.

[0090] like Figure 2 As shown in the top chart, TG2 activity was increased in muscularis propria tissues from UCi and CDs patients compared to tissues from NL patients. In NL, UCi, and CDs tissues, treatment with an anti-TG2 antibody (UCB7858 in this case) resulted in a statistically significant decrease in TG2 activity. Another trend was observed: 1) increased TG2 activity in muscularis propria tissues from CDns patients compared to tissues from NL patients; and 2) decreased TG2 activity in tissues from CDns patients after treatment with an anti-TG2 antibody. A trend towards higher expression was observed when focusing on muscularis propria (MP) tissues, particularly for samples from CDs patients. Figure 2 (A, bottom chart), and regardless of the tissue (UCi, CDns, or CDs), TG2 activity tended to decrease after treatment with anti-TG2 antibody.

[0091] TG2 expression ( Figure 3 ): Tissue samples of the same type (i.e., unfixed, washed frozen tissue sections from patients with NL, UCi, CDns, and CDs) were stained to assess the presence of the TG2 antigen (i.e., to assess TG2 expression at the protein level). Image processing is detailed in the Methods section. Figure 3 As shown in Figure A, global quantitative visualization of TG2 levels in tissue sections from NL, UCI, CDns, and CDs patients was performed. Compared to NL, TG2 levels were increased in CDs, and there was also a trend of increase in UCI samples (compared to NL). TG2 mRNA (TGM2) expression was visualized using tissue sections from different tissue samples from NL, UCI, CDns, and CDs patients (RNAscope-Z probe method). Fluorescence microscopy images showed TG2 mRNA expression in three different tissue sections of the mucosa (i.e., muscularis mucosae, submucosa, and muscularis propria regions; images not shown). Pathologists scored the mRNA signal in these three different tissue sections from each patient in the UCI, CDns, and CDs groups under blinded conditions and compared them with similar tissue sections from NL patients. The results showed that TG2 mRNA expression was increased by 25% to 100% in CDs, with this increase being particularly significant in the muscularis propria region. Figure 3 B). TG2 mRNA expression in UCI also increased by up to 25%, with this increase being particularly pronounced in the myolamina propria region. Figure 3 B). Regarding TG2 mRNA expression in CDns patients, it was increased by up to 50%, particularly in the muscularis mucosae and submucosal regions. Figure 3 B). Full-thickness tissue samples from NL, UC, CDns, and CDs patients were used to extract mRNA, which was then quantified using quantitative RT-PCR. Results showed that TG2 mRNA expression was significantly different (7-9 fold change) in UCi and CDs patients compared to NL, and approximately 3 fold change in CDns patients. Figure 3 C).

[0092] TG2 activity ( Figure 4 ) Frozen tissues from patients with NL, UCi, CDns, and CDs were analyzed. 14 C-putrescine determination. For example... Figure 4As shown, when the results were analyzed using a nonparametric t-test, a significant increase in TG activity was observed in tissues from CDs patients compared to tissues from NL patients, and there was a trend of increasing TG activity in tissues from UCi and CDns patients compared to NL patients (the trend was more pronounced in CDns patients).

[0093] Conclusion of Example 1

[0094] Surprisingly, this study revealed that active TG2 is the major transglutaminase in the human gut, and its activity is increased in intestinal tissues from IBD patients, particularly those with UCI and CDs. This activity is associated with fibrosis but not inflammation. Anti-TG2 antibodies significantly inhibited TG2 activity (in situ) in these tissues. Data also showed that, as demonstrated by immunostaining and quantitative RT-PCR, TG2 expression, TG2 levels, and TG2 activity were all upregulated in tissues from CDs patients.

[0095] Example 2 - In vitro inhibitory activity of TG2 inhibitors

[0096] Based on the results of Example 1, it is still necessary to prove whether the TG2 inhibitor can inhibit the in vitro activity of TG2.

[0097] TG2 inhibitors can inhibit the in vitro cell activity of TG2 ( Figure 5 ) TG2 activity in HIMF from NL samples was evaluated using TG2-specific inhibitors (including Z-Don, Boc-Don, and zobilimab), with "Ig isotype" as an additional control. Figure 5 As shown, the use of TG2 inhibitors significantly reduced TG2 activity, with the best effect observed with zobilimab. Overall, as determined by calculating the percentage of TG activity lost after pretreatment with the aforementioned TG-specific inhibitors, 82% (using small molecule inhibitors) to 97% (using zobilimab) of TG activity derived from NL HIMF was inhibited.

[0098] TG2 inhibitors can inhibit ECM deposition ( Figure 6 and Figure 7 ) The levels of mature extracellular matrix (ECM) (expressed as fibronectin and collagen levels) have been investigated across various sample types. Figure 6 The baseline production of fibronectin (FN) in tissues from patients with NL, UC, CDns, and CDs was specifically presented, along with trends of change among them. In particular, samples from CDs and UCI patients showed an increasing trend in fibronectin production. Figure 6A). Then, the effect of TG2 inhibition (TG2-specific inhibitors including Z-Don, Boc-Don, and zobilimab; with “Ig isotype” as an additional positive control) on extracellular matrix (ECM) deposition in enteric myofibroblasts was determined. For this purpose, fibronectin (FN) and collagen I / III levels were used according to standard practice. See below. Figure 7 As shown in B to 7E, from NL ( Figure 7 B), UCI ( Figure 7 C) CDns ( Figure 7 D) and CDs Figure 7 E) In HIMF isolated from patients, the levels of FN (fibronectin) and COLI / III (collagen I / III) were significantly reduced (in most cases, by more than 50%) in the presence of TG2 inhibitors, with similar overall effects regardless of the type of inhibition (i.e., using small molecules, as shown with Z-Don and Boc-Don, or using anti-TG2 antibodies, as shown with zobilimab). Zobilimab consistently and significantly reduced fibronectin production in all patient groups. Notably, the reduction was highest in tissue samples from CD patients treated with zobilimab, while the reduction was slightly lower with small molecule TG2 inhibitors (approximately 90% and 60% reductions in FN and collagen deposition, respectively, for zobilimab; and approximately 80% and 50% reductions for small molecule inhibitors, respectively). Figure 7 A shows exemplary scan images of different treatments, highlighting the effect of TG2 inhibition on ECM deposition. The presence of the inhibitor did not affect HIMF cell confluence (scan images not shown).

[0099] Conclusion of Example 2 :

[0100] It has been shown that TG2 inhibitors can suppress TG2 activity in myofibrils derived from patients, thereby significantly reducing the level of mature ECM deposits in myofibrils from UCI and CDs patients (especially these two groups), but also from NL and CDns patients. Since ECM accumulation is a hallmark of fibrosis in tissues, TG2 inhibitors have now been shown to be reliably used to prevent and / or reduce fibrosis in IBD patients, including preventing and / or treating stenosis in UC, CDns, or CDs patients (with the best results in CDs patients).

[0101] Example 3 - Effects of TG2 gene knockout in animal models

[0102] Chronic DSS colitis model Using a mouse DSS model of chronic colitis, with or without TGM2 knockdown (KO), it was shown that TG2 knockdown (KO) did not affect clinical scores, weight loss (data not shown), or histopathological inflammation scores. Figure 8 A). Compared with wild-type (WT) and KO non-disease model mice, WT and KO DSS chronic colitis mice showed increased inflammation scores in the proximal colon, transverse colon, and rectum. Figure 8 A). PSR staining (i.e., interstitial collagen) showed that, when comparing "without DSS" and "with DSS", WT mice had increased collagen accumulation in both the tent and muscle regions. Figure 9 A). In TG2 knockout mice, no increase in interstitial collagen was observed in response to DSS compared to mice without DSS. In the tent region, WT DSS mice showed a significant increase in collagen accumulation compared to KO DSS mice. Figure 9 A). Protein expression of collagen 1 and fibronectin in DSS mice that underwent or did not undergo TG2 KO was assessed by IF staining (scanned images not shown). Figure 9 As shown in Figure B, compared with tissues from WT DSS mice, the levels of collagen 1 and fibronectin were decreased in tissues from TG2 KO DSS mice (P ≤ 0.05). This decrease was more pronounced in the tent region. The intestinal wall of WT DSS mice was significantly thicker than that of KO DSS mice. Figure 9 C; scanned image not shown).

[0103] TNBS fibrosis model In the chronic TNBS mouse model, no differences were observed between the WT TNBS group and the TG2 KO TNBS group in terms of clinical score, weight loss (data not shown), and histopathological inflammation score. Figure 8 B). Compared with WT and KO TNBS-free mice, WT and KO TNBS-free mice showed higher inflammation scores ( Figure 8 B). PSR staining showed a nominally increasing trend in intermediate collagen in WT mice compared to "without TNBS" and "with TNBS", particularly in the tent region (B). Figure 10 A). When comparing "without TNBS" and "with TNBS", KO mice did not show any changes in interstitial collagen. Figure 10 A). In chronic TNBS colitis, the intensity was significantly reduced in TG2 knockout mice compared to WT mice. Figure 10 A). For example Figure 10 As highlighted in Figure B, compared with WT mice, TG2 KO mice showed decreased levels of collagen 1 and fibronectin ECM proteins, particularly in the TNBS model, and also decreased intestinal wall thickness. Figure 10 C; scanned image not shown).

[0104] Conclusion of Example 3:

[0105] It has been shown that the absence of TG2 (i.e., TG2 KO mice) reduces mature ECM accumulation (consistent with fibrotic remodeling) and does not affect inflammation in DSS and TNBS extended colitis models used to induce early fibrotic remodeling. The lack of TG2 has been shown to reduce the degree of fibrosis without affecting inflammation reduction. The data presented in this article confirm the results of Example 2, that TG2 inhibitors may be a viable therapeutic agent for preventing further accumulation of existing ECM deposits beyond normal physiological levels in IBD patients, including preventing the progression of pre-existing ECM accumulation in patients with UC, CDs, or CDns.

[0106] References

[0107] 1) Nunes et al., 1997, J. Cell Biol.;136(5):1151-63

[0108] 2) Huang et al., 2010, Nephrol. Dial. Transplant; 25: 3897–3910

[0109] 3) Siegel et al., 2007, Pharmacol. Ther., 115(2): 232–245

[0110] 4) Wang et al., 2020, 3 Biotech., 10:287

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[0116] 10) Cosnes et al., 2005, Gut, 54:237-241

[0117] 11) Rieder et al., 2017, Gastroenterology, 152:340-350(e346).

[0118] 12) Im et al., 1997, Cell Signal., 9(7):477-82

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[0120] 14) Zhao et al., 2020, Mucosal Immunol., 13:665-678

[0121] 15) Lawrance et al., 2003, Gastroenterology, 125:1750-1761 (2003)

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Claims

1. A transglutaminase 2 (TG2) inhibitor for the treatment of patients with fibrosis-related conditions associated with inflammatory bowel disease (IBD) or for the prevention of the development of such conditions.

2. The TG2 inhibitor for use according to claim 1, wherein the fibrosis-related condition associated with IBD is selected from: a) strictures, including strictures of 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) intestinal obstruction.

3. A TG2 inhibitor for use according to any one of the preceding claims, wherein IBD is selected from Crohn's disease (CD) and ulcerative colitis (UC).

4. The TG2 inhibitor for use according to claim 3, wherein: a. The Crohn's disease is selected from narrow Crohn's disease and non-narrow Crohn's disease, or b. The ulcerative colitis mentioned is inflammatory ulcerative colitis.

5. A TG2 inhibitor for use according to any one of the preceding claims, wherein the fibrosis-related symptoms associated with Crohn's disease are characterized by an increase in a biomarker in the subject's sample, said biomarker being selected from TG2 activity, the level of ε (γ-glutamyl) lysine crosslinking of the TG2 product, mRNA encoding TG2, TG2 antigen, enhanced TG2 output, or any combination thereof.

6. A TG2 inhibitor for use according to any one of the preceding claims, wherein the TG2 inhibitor is an anti-TG2 antibody that binds to an epitope in the core region of TG2 and inhibits at least one TG2 activity, and wherein the inhibited at least one TG2 activity is TG2 causing lysine and glutamine to crosslink via an N-ε (γ-glutamyl)lysine isopeptide bond.

7. A TG2 inhibitor for use according to any one of the preceding claims, wherein the anti-TG2 antibody or its antigen-binding fragment is: a. Contains or is composed of complete antibodies, or b. Contains or consists of antigen-binding fragments.

8. A TG2 inhibitor for use according to any one of the preceding claims, wherein the anti-TG2 antibody comprises six CDRs selected from the following: (i) KASQDINSYLT (LCDR1; SEQ ID NO. 1); LVNRLVD (LCDR2; SEQ ID NO. 2); LQYDDFPYT (LCDR3; SEQ ID NO. 3); THAMS (HCDR1; SEQ ID NO. 4); TISSGGRSTYYPDSVKG (HCDR2; SEQ ID NO. 5); NO. 6); or (ii) KASQDINSYLT (LCDR1; SEQ ID NO. 1); LTNRLMD (LCDR2; SEQ ID NO. 7); LQYVDFPYT (LCDR3; SEQ ID NO. 8); SSAMS (HCDR1; SEQ ID NO. 9); TISSGGRSTYYPDSVKG (HCDR2; SEQ ID NO. 5); and LISPY (HCDR3; SEQ ID NO. 5); NO. 10); or and LISLY (HCDR3; SEQ ID NO. 12).

9. A TG2 inhibitor for use according to any one of the preceding claims, wherein the anti-TG2 antibody comprises: a) A light chain variable domain having a sequence defined as any one of SEQ ID NO: 13 to SEQ ID NO: 27 and a heavy chain variable domain having a sequence defined as 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 any of the sequences defined as such as 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 any of the sequences defined as such as SEQ ID NO: 28 to SEQ ID NO:

40.

10. A TG2 inhibitor for use according to any one of claims 1 to 7, wherein the antibody competes with an antibody as defined in any one of claims 8 and 9 for binding to TG2.

11. The TG2 inhibitor for use according to any one of claims 1 to 5, wherein the TG2 inhibitor is a small molecule.

12. The TG2 inhibitor for use according to claim 11, wherein the small molecule is selected from: 1) competitive amine inhibitors, 2) reversible inhibitors, and 3) irreversible inhibitors.

13. A TG2 inhibitor for use according to any one of the preceding claims, wherein the TG2 inhibitor is incorporated into a pharmaceutical composition comprising one or more pharmaceutically acceptable adjuvants and / or carriers.

14. A pharmaceutical composition for treating a subject with a fibrosis-related condition associated with inflammatory bowel disease (IBD) or for preventing the development of such a condition, wherein the pharmaceutical composition comprises a TG2 inhibitor and one or more pharmaceutically acceptable adjuvants and / or carriers.

15. A method for treating a subject with fibrosis-related conditions associated with inflammatory bowel disease (IBD) or for preventing the development of such conditions in a subject, comprising administering to the subject a therapeutically effective amount of a transglutaminase 2 (TG2) inhibitor.

16. Use of a transglutaminase 2 (TG2) inhibitor in the preparation of a medicament for the treatment of a subject with fibrosis-related symptoms associated with inflammatory bowel disease (IBD) or for the prevention of the development of such symptoms.

Images