Method for the treatment of fibrotic-related disorder associated to ibd

Abstract

The present invention relates to a transglutaminase type 2 (TG2) inhibitor that blocks at least the transamidase activity of the enzyme for use in the treatment of a subject having a fibrotic-related disorder associated with inflammatory bowel disease (IBD) or in the prevention of the development of such disorder.

Description

[0001] METHOD FOR THE TREATMENT OF FIBROTIC-RELATED DISORDER ASSOCIATED TO IBD

[0002] Field of invention

[0003] The present invention relates to a transglutaminase type 2 (TG2) inhibitor that blocks at least the transamidase activity of the enzyme for use in the treatment of a subject having a fibrotic-related disorder associated with inflammatory bowel disease (IBD) or in the prevention of the development of such disorder.

[0004] Background of the invention

[0005] Tissue transglutaminase or transglutaminase type 2 (TG2) is an enzyme which forms crosslinks between proteins via epsilon(gamma-glutamyl) lysine di-peptide bonds. In addition to this direct stabilization of the ECM, TG2 also appears to indirectly promote ECM crosslinking via activation of the fibrotic cytokine transforming growth factor 3 (TGF-0) (Nunes et al., 1997; Huang et al., 2010). Elevated expression of TG2 leads to aberrant protein cross-linking which has been 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 some cancers. Various TG2 inhibitors, such as small molecules, silencing RNA or antibodies (e.g. Siegel et al., 2007, Wang et al., 2020, WG2006100679, W02012146901 or WO2013175229), have been disclosed for the possible treatment of TG2-mediated disorders.

[0006] Increased levels of TG2 have been associated with a few groups of disorders / diseases, such as progressive chronic interstitial lung diseases (Olsen et al., 2011 , Olsen et al., 2020, Philp et al., 2018), including in IPF patients (W02023089042) or yet in scleroderma (W02023089037).

[0007] Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic relapsing and remitting type of disease, affecting more than 3 million people in Europe and in the USA. IBD-associated intestinal fibrosis is a significant clinical problem in CD and UC patients. More than half of CD patients develop fibrosis-induced intestinal obstruction, with debilitating symptoms (Cosnes et al., 2005; Rieder et al., 2017). Non-surgical approaches have limited success in stricturing CD, which makes it necessary to have surgical interventions in up to 80% of CD patients. It appears that fibrosis, and in particular intestinal fibrosis, is an inevitable and irreversible complication of chronic gut inflammation. Fibrosis is also found in UC, where it is essentially restricted to specific tissues (the mucosa and submucosa), impacting colonic motility for instance.

[0008] Intestinal fibrosis is defined as excessive accumulation of extracellular matrix (ECM), including increased levels of fibronectin and collagen, ultimately leading to organ dysfunction. Mesenchymal cells (differentiating mainly into fibroblasts, myofibroblasts and smooth muscle cells in the intestine / colon) have been found as the main effector cells mediating fibrosis. In a healthy response to injury or inflammation, they act as “repair units” whose populations increase rapidly while producing profibrotic factors and ECM. However, in a chronic inflammatory setting, excess ECM production leads to irreversible fibrosis.

[0009] There remains a need to identify further effective therapies for use in the treatment of a subject having a fibrotic-related disorder associated with inflammatory bowel disease (including Crohn’s disease or ulcerative colitis ) or in the prevention of the development of such disorder.

[0010] Summary of the invention

[0011] It is an object of the present invention to provide a specific transglutaminase 2 (TG2) inhibitor for use in the treatment of a fibrotic-related disorder associated with inflammatory bowel disease (I BD) in a patient or in the prevention of the development of such disorder.

[0012] In a second aspect, the invention provides a method for treating a subject having a fibrotic-related disorder associated with inflammatory bowel disease (IBD) or for the prevention of the development of such disorder in a subject comprising administering a therapeutically effective amount of a transglutaminase 2 (TG2) inhibitor to said subject.

[0013] In a third aspect, the invention relates to the use of an TG2 inhibitor for the manufacturing of a medicament for the treatment of a subject having a fibrotic-related disorder associated with inflammatory bowel disease (IBD) or for the prevention of the development of such disorder.

[0014] Definitions

[0015] 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 contemplated, even if the combination of features is not found together in the same sentence, or paragraph, or section of this document. With respect to aspects of the invention described or claimed with "a" or "an", it should be understood that these terms mean "one or more" unless context unambiguously requires a more restricted meaning. The term "or" should be understood to encompass items in the alternative or together, unless context unambiguously requires otherwise. If aspects of the invention are described as "comprising" a feature, embodiments also are contemplated "consisting of' or "consisting essentially of" the feature.

[0016] - The term “Tissue transglutaminase”, “Transglutaminase type 2” or “TG2” refers to an enzyme, product of the gene TGM2, which forms crosslinks between proteins via epsilon(gamma-glutamyl) lysine di-peptide bonds. TG2 refers to a protein that typically has the amino acid sequence as set out in the UniProt entry P21980 (with or without the N-terminal methionine as set out in SEQ ID NO: 41), i.e. human TG2. The term “TG2” may also refer to protein which is (a) a derivative having one or more amino acid substitutions, modifications, deletions or insertions relative to the amino acid sequence of SEQ ID NO: 41 which retains the activity of TG2, or (b) a variant thereof, such variants typically retain at least about 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94% or 95% identity to SEQ ID NO: 41 (or even about 96%, 97%, 98% or 99% identity to SEQ ID NO: 41). The protein TG2 is encoded by the gene Tgm2. - The term “TG2 inhibitor” refers to a molecule that is able to inhibit at least one of the TG2 activities, such as TG2 cross-linking of lysine and glutamine with N-e(y-glutamyl)lysine isopeptide bonds. Such molecule can be, without any limitation, a small molecule, a silencing RNA or an antibody.

[0017] Small molecules can be selected among (non-limiting examples) three main classes: 1) competitive amine inhibitors that compete with natural amine substrates, 2) reversible inhibitors and 3) irreversible inhibitors (Siegel et Khosla, 2007).

[0018] - The term “inhibition” and the like refers to the blocking or neutralisation of one or more activities usually associated with a target, such as an enzyme. For instance, in the context of the present invention, the term “inhibition” includes the blocking of TG2's enzymatic functions related to transamidation (including crosslinking) and deamidation of proteins. TG2 is also known as a GTPase (Im ef al., 1997).

[0019] - The term "anti-TG2 antibody", as used herein, is intended to be an antibody molecule which binds TG2 and block its transamidase activity to prevent crosslinking. Examples of such antibodies are described in WO2013175229. Without any limitation, an anti-TG2 antibody that can be used according to the present invention comprises for instance 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.

[0020] - The term "antibody" as used herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies and recombinant antibodies that are generated by recombinant technologies as known in the art. "Antibody" include antibodies of any species; such as human antibodies of any isotype, including lgG1 , lgG2a, lgG2b, lgG3, lgG4, IgE, IgD and antibodies that are produced as dimers of this basic structure including IgGAI , lgGA2, or pentamers such as IgM and modified variants thereof; non-human primate antibodies, e.g. from chimpanzee, baboon, rhesus or cynomolgus monkey; rodent antibodies, e.g. from mouse, or rat; rabbit, goat or horse antibodies; camelid antibodies (e.g. from camels or llamas such as Nanobodies™) and derivatives thereof; antibodies of bird species such as chicken antibodies; or antibodies of fish species such as shark antibodies. The term "antibody" also refers to "chimeric" antibodies in which a first portion of at least one heavy and / or light chain antibody sequence is from a first species and a second portion of the heavy and / or light chain antibody sequence is from a second species. Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old-World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences. "Humanized" antibodies are chimeric antibodies that contain a sequence derived from non-human antibodies. For the most part, humanized antibodies are human antibodies (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a 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 instances residues of the human (recipient) antibody outside of the CDR; i.e. in the framework region (FR), are additionally replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody properties. Humanization reduces the immunogenicity of non-human antibodies in humans, thus facilitating the application of antibodies to the treatment of human disease. Humanized antibodies and several different technologies to generate 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, it is possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of production of endogenous murine antibodies. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies such as phage display or ribosome display technology, wherein recombinant DNA libraries are used that are either generated at least in part artificially or from immunoglobulin variable (V) domain gene repertoires of donors. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies may also be generated from isolated human B cells that are ex vivo immunized with an antigen of interest and subsequently fused to generate hybridomas which can then be screened for the optimal human antibody. The term “antibody” refers to both glycosylated and aglycosylated antibodies. Furthermore, the term "antibody" as used herein not only refers to full-length antibodies, but also refers to antibody fragments, more particularly to antigen-binding fragments thereof. A fragment of an antibody comprises at least one heavy or light chain immunoglobulin domain as known in the art and binds to one or more antigen(s). Examples of antibody fragments according to the invention include a Fab, modified Fab, Fab’, modified Fab’, F(ab’)2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv and Bis-scFv fragment. Said fragment can also be a diabody, tribody, triabody, tetrabody, minibody, single domain antibody (dAb) such as sdAb, VL, VH, VHH orcamelid antibody (e.g. from camels or llamas such as a Nanobody™) and VNAR fragment. An antigenbinding fragment according to the invention can also comprise a Fab linked to one or two scFvs or dsscFvs, each scFv or dsscFv binding the same or a different target (e.g., one scFv or dsscFv binding a therapeutic target and one scFv or dsscFv that increases half-life by binding, for instance, albumin). Exemplary of such antibody fragments are FabdsscFv (also referred to as BYbe®) or Fab-(dsscFv)2 (also referred to as TrYbe®, see e.g. WO2015 / 197772). Antibody molecules as defined above, including antigen-binding fragments thereof, are known in the art.

[0021] - The term “epitope” refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings 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. - The term “treating” or “treatment” of a disease state includes: (i) inhibiting the disease state, i.e. arresting the development of the disease state or its clinical symptoms, or (ii) relieving the disease state, i.e. causing temporary or permanent regression of the disease state or its clinical symptoms.

[0022] - The term “preventing” or “prevention” of a disease state includes causing the clinical symptoms of the disease state not to develop in a subject that may be exposed to or predisposed to the disease state but does not yet experience or display symptoms of the disease state.

[0023] Detailed description of the invention

[0024] The invention is based on the finding from the inventors that TG2 expression (such as increase of TG2 antigen) and its activity were both significantly increased in tissues from UC and CD patients, including in particular in Crohn’s strictures, with TG2 activity being correlated with fibrosis score. It was also a finding that TG2 inhibitors were able to 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 accumulation of mature fibronectin and collagen mediated by human Intestinal Myofibroblasts (HIMFs) in in vitro assays.

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

[0026] The invention also provides a method for treating a subject having a fibrotic-related disorder associated with inflammatory bowel disease (IBD) or for the prevention of the development of such disorder in a subject comprising administering a therapeutically effective amount of a transglutaminase 2 (TG2) inhibitor to said subject.

[0027] Also described is the use of a transglutaminase 2 (TG2) inhibitor for the manufacturing of a medicament for the treatment of a subject having a fibrotic-related disorder associated with inflammatory bowel disease (IBD) or for the prevention of the development of such disorder.

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

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

[0030] In the context of the present invention as a whole, the fibrotic-related disorder associated to IBD is characterised by an increase of a marker in a subject’s sample, wherein the marker is for instance any one of TG2 activity, level of TG2’s product epsilon (gamma-glutamyl) lysine crosslink, level of TG2 expression (such as increase of mRNA encoding TG2 or of TG2 antigen), export of TG2 or any combination thereof, and wherein the subject’s sample is a cell or a tissue associated with said disease (e.g. a intestine cell or a colon tissue). Said increase of the marker may be determined by any means in cells / tissues associated with said disease. An increase of a marker in one subject’s sample is typically determined by comparison of the level of said marker in the subject’s sample to the level of the same marker in normal cells of the same tissue type (i.e. basal level; e.g. basal TG2 activity, basal expression level (mRNA level and / or protein level) and / or basal level of TG2 export). A subject’s sample having a level of at least one marker equal or higher than 10%, equal or higher than 15%, equal or higher than 20%, equal or higher than 25% or even equal or higher than 30% compared to the basal level for said marker will be considered as presenting an increase of said marker. For example, an increase of TG2 expression (alternatively called TG2 overexpression) can be determined via determination of the amount of TG2 mRNA in intestine cells of a patient. Cells / tissues affected by a fibrotic-related disorder associated with IBD may thus be characterised for instance by an increased amount (representing overexpression) of TG2 mRNA in intestine cells of a subject, compared with normal cells from the same tissue type. The expression of TG2 mRNA may be increased by any amount, such as equal or higher than 10%, equal or higher than 15%, equal or higher than 20%, equal or higher than 25% or even equal or higher than 30% compared to the basal level. The amount of mRNA can be measured using any known methods such as quantitative reverse transcription polymerase chain reaction (quantitative RT-PCR also called qRT-PCR), real time qRT-PCR, quantigene assay, by northern blotting or using microarrays, RNA sequencing and various types of in situ hybridisation (e.g. RNAscope). Alternatively, overexpression can be determined via determination of the amount of TG2 antigen in intestine cells of a patient. Cells affected by a fibrotic-related disorder associated with IBD may thus be characterised for instance by an increased amount (representing overexpression) of TG2 protein (or TG2 antigen) in intestine cells of a subject, such as compared with normal cells of the same tissue type. The expression of TG2 protein may be increased by any amount, such as equal or higher than 10%, equal or higher than 15%, equal or higher than 20%, equal or higher than 25% or even equal or higher than 30% compared to the basal level. The amount of protein can be measured using any known methods such as immunohistochemistry, western blotting, mass spectrometry or fluorescence-activated cell sorting (FACS), including by use of an anti-TG2 antibody of the invention. The thresholds for determining expression may vary depending on the techniques that are used and may be validated against immunohistochemistry scores. Alternatively, the cells / tissues affected by a fibrotic-related disorder associated with IBD may be characterised by an increase of the TG2 activity in intestine cells of a subject, compared with normal cells of the same tissue type. TG2 activity may be increased by any amount, such as equal or higher than 10%, equal or higher than 15%, equal or higher than 20%, equal or higher than 25% or even equal or higher than 30% compared to the basal level. TG2 activity can be measured using any known methods such as via cryo biopsy (TG ISA).

[0031] In one embodiment of the invention as a whole, 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 of the TG2 activities, and wherein the at least one TG2 activity that is inhibited is the TG2 cross-linking of lysine and glutamine with N-e(y-glutamyl)lysine isopeptide bonds. In a non-limiting example, when assessed for instance by a peptide mapping method, the preferred antibody binds to region comprising or consisting of amino acids 304 to 326 of TG2 (e.g. of SEQ ID NO.41) or part of this region. Said antibody can comprise or consist of an intact antibody. Alternatively, it can comprise or consist of an antigen-binding fragment such as (but not limited to): an Fv fragment (for example a single chain Fv fragment or a disulphide-bonded Fv fragment); a Fab fragment; and a Fab-like fragment (for example a Fab' fragment or an F(ab)2 fragment), single domain antibody (or any other fragments as herein defined or known by the skilled person). Preferably, the anti-TG2 antibody to be used according to the invention as a whole (see also Table A): a) comprises 6 CDRs selected from the group consisting of:

[0032] (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); and LISTY (HCDR3; SEQ ID NO. 6);

[0033] (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. 10); or

[0034] (iii) KASQDINSYLT (LCDR1 ; SEQ ID NO. 1); RTNRLFD (LCDR2; SEQ ID NO. 11 ); LQYDDFPYT (LCDR3; SEQ ID NO. 3); SSAMS (HCDR1 ; SEQ ID NO. 9); TISVGGGKTYYPDSVKG (HCDR2; SEQ ID NO. 42; and LISLY (HCDR3; SEQ ID NO. 12); b) comprises a light chain variable domain having the sequence as defined in any one of SEQ ID NO: 13 to SEQ ID No. 27 and a heavy chain variable domain having the sequence as defined in any one of SEQ ID NO: 28 to SEQ ID No. 40, c) comprises 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 to the sequence as 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% (or even about 96%, 97%, 98% or 99%) identity or similarity to the sequence as defined in any one of SEQ ID NO: 28 to SEQ ID No. 40, or d) competes for binding to TG2 with an antibody as defined in a), b) or c) above.

[0035] As a non-limiting example, the anti-TG2 antibody comprises the six following 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 6 exemplary CDRs can be comprised respectively in a light chain variable domain having the sequence as defined SEQ ID NO: 24 and a heavy chain variable domain having the sequence as defined in SEQ ID NO: 37. As a further non-limiting example, the anti-TG2 antibody comprises the six following 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 6 exemplary CDRs can be comprised respectively in a light chain variable domain having the sequence as defined SEQ ID NO: 19 or 25 and a heavy chain variable domain having the sequence as defined in SEQ ID NO: 32 or 38.

[0036] Table A - Amino acid sequences

[0037] One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, another antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference antibody of the invention, the reference antibody is allowed to bind to a protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the protein or peptide is assessed. If the test antibody is able to bind to the protein or peptide following 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 is not able to bind to protein or peptide following saturation binding with the reference antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the invention. To determine if an antibody competes for binding with a reference antibody, the above-described binding methodology is performed in two orientations. In a first orientation, the reference antibody is allowed to bind to a protein / peptide under saturating conditions followed by assessment of binding of the test antibody to the protein / peptide molecule. In a second orientation, the test antibody is allowed to bind to the protein / peptide under saturating conditions followed by assessment of binding of the reference antibody to the protein / peptide. If, in both orientations, only the first (saturating) antibody is capable of binding to the protein / peptide, then it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As will be appreciated by the skilled person, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

[0038] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50%, 75%, 90% or even 99% as measured in a competitive binding assay. Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0039] Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.

[0040] In another embodiment of the invention as a whole, the TG2 inhibitor is a small molecule inhibitor inhibiting at least one biological activity of TG2. Such inhibitors are well known to the skilled person in the art and could be selected among (non-limiting examples) three main classes: 1) competitive amine inhibitors 2) reversible inhibitors and 3) irreversible inhibitors. A non-limiting list of such inhibitors comprises the following inhibitors: PX-12, ERW1041 , CK805, Z Don, Boc-Don, NC9, VA4, ZED1227, or yet any of those described in Siegel and Khosla, 2007, WO033784, WO14012858, WO14057266, WO17179018, WO18122419, WO22213198 or WO23135425.

[0041] Any subject may be treated in accordance with the invention. The subject is preferably human. However, the subject may be another mammalian animal, such as a non-human primate, a horse, a cow, a sheep, a pig, a dog, a cat, a rabbit, a rat, a mouse, a guinea pig ora hamster. Alternatively, the term patient can be used indifferently instead of subject.

[0042] Any TG2 inhibitor according to the invention may be incorporated into pharmaceutical compositions suitable for administration to a subject in any way, such as (but not limited to) topically, intra nasally, intradermally, intravenously, subcutaneously or intramuscularly. Typically, the pharmaceutical composition comprises the TG2 inhibitor and one or more pharmaceutically acceptable adjuvant(s) and / or carrier(s). Therefore, herein described is also a pharmaceutical composition for use in the treatment of a subject having a fibrotic-related disorder associated with inflammatory bowel disease (IBD) or in the prevention of the development of such disorder, wherein said pharmaceutical composition comprises a TG2 inhibitor and one or more pharmaceutically acceptable adjuvant(s) and / or carrier(s). The pharmaceutical composition according to the invention can be part of a kit with instructions for use, including instructions and optionally a device for intravenous, subcutaneous or intramuscular administration to the individual in need thereof.

[0043] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible and are 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, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Depending on the route of administration or the type of formulation (such as liquid, freeze-dried or spray-dried formulation), isotonic agents can be incorporated, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the inhibitor.

[0044] The pharmaceutical compositions according to the present invention may be in a variety of forms. These include, for example, liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, powders and liposomes. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other therapeutic molecules.

[0045] A suitable dosage of a TG2 inhibitor according to the present invention may be determined by a skilled medical practitioner. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the route of administration, the time of administration, the rate of excretion of the TG2 inhibitor, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular TG2 inhibitor, the age, sex, weight, condition, general health and prior medical history of the patient being treated.

[0046] A suitable dose may be, for example, in the range of from about 0.01 pg / kg to about 1000 mg / kg body weight, typically from about 0.1 pg / kg to about 100 mg / kg body weight of the patient to be treated. Dosage regimens may be adjusted to provide the optimum desired response (e.g. a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered overtime. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical earner. Administration may be in single or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different locations. In the context of the invention as a whole the TG2 inhibitor may be co-administered with one or other more other therapeutic agents. Combined administration of two or more agents may be achieved in a number of different ways. Both may be administered together in a single composition, or they may be administered in separate compositions as part of a combined therapy. For example, the one may be administered before or separately, after or sequential, or concurrently or simultaneously with the other. Description of the Figures:

[0047] Figure 1 : Transglutaminase (TG) activity as measured using scanned images significantly increases in UCi / CDs compared to NL and is correlated with Fibrosis score but not with inflammation score. A) Quantitation of integrated density for TG activity was performed in tissue sections from different IPD patients (respectively UCi, CDns and CDs patients; using NL as control) (P values compared to NL). B) TG2 activity in IBD, full thickness is correlated with fibrosis score (“Fibrosis score” column) but not with inflammation score (“Geboes (inflammation) score” column), as shown in two different layers of gut tissues (muscularis propria and muscularis mucosa).

[0048] Figure 2: TG2 inhibition with zampilimab confirms TG2 is the source of transamidation activity and contributes to 80 to 100% of total TG activity. A) Quantitation of integrated density for TG2 activity was performed using scanned images in muscularis mucosa and muscularis propria tissue sections from different IBD patients (UCi, CDns and CDs patients; using NL as control) (scanned images not shown). B) TG2 activity contributes to 80 to 100% of total TG activity as determined by calculating percentage of TG activity lost following pre-treatment with zampilimab, based on images in muscularis mucosa and muscularis propria tissue sections from different IBD patients (UCi, CDns and CDs patients; using NL as control) (scanned images not shown). (P values for treated vs untreated groups).

[0049] Figure 3: TG2 mRNA and TG2 antigen are increased in UCi and CD strictures: A) Quantitation of integrated density for TG2 antigen was performed in tissue sections from different IBD patients (UCi, CDns and CDs patients; using NL as control; P values compared to NL). B) Using RNA-Z- scope, pathologist scoring of raw images showed 25% to 100% increase in TG2 expression (from an mRNA viewpoint) 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 an mRNA viewpoint) in UCi and CDs patients.

[0050] Figure 4: Ten snap frozen patient tissues each from different IBD patients (UCi, CDns and CDs patients; using NL as control) were analysed using14C-Putrescine Assay. This Figure shows the increase in TG activity in CDs compared to NL (using t-test, non-parametric test).

[0051] Figure 5: TG2 is the source of Transglutaminase activity in vitro in Human intestinal myofibroblast (HIMF) from NL tissues . By using specific TG2 inhibitors (anti-TG2 antibody zampilimab and small molecule Z-Don) it was confirmed that 82-97% of transglutaminase in NL HIMF were TG2 as determined by calculating percentage of TG activity lost following pre-treatment with TG2 specific inhibitor. Each dot represents a separate patient derived primary cell line.

[0052] Figure 6: Mature Extracellular Matrix (ECM) levels across control (NL) and different types of IBD patient derived fibroblasts. It shows the baseline production of FN (A) and Collagen (B) between NL, UC, CDns, CDs and shows trend changes between them. Each dot represents a separate patient derived primary cell line. Figure 7: Extracellular Matrix (ECM) levels are reduced in NL, UCi, CDns and CDs patient fibroblasts treated with TG2 inhibitors (anti-TG2 antibody zampilimab and small molecule TG2 inhibitors Z-Don and Boc-Don). A) scanned images showing ECM deposition under different conditions, further to immunofluorescence staining with antibodies specific to ECM components in tissues from NL subjects (top 5 images) and CDs patients (bottom 5 images). B-E) ECM levels across normal and different types of IBD patient fibroblasts, comparing mature ECM levels across normal and different types of IBD patient fibroblasts, in the presence of specific TG2 inhibitors (anti-TG2 antibody zampilimab and small molecule TG2 inhibitors Z-Don and Boc-Don). The graph shows the production of Fibronectin (FN) and collagen (Col l&lll) between tissues from NL (in B), UC (in C), CDns (in D), CDs (in E) patients and shows trend changes between them. Each dot represents a separate patient derived primary cell line.

[0053] Figure 8: A) Inflammation score in WT and TG2 KO Mice in the presence and absence of DSS in various tissues in a mouse DSS chronic Model of Crohn’s disease. B) Inflammation Score in WT and TG2 KO Mice in the presence and absence of TNBS in a mouse TNBS pre-sensitization chronic Model of Crohn’s disease. Each dot represents a separate mouse.

[0054] Figure 9: A) PSR staining in the tent and muscle areas in WT and TG2 KO mice, in presence or absence of DSS. B) Collagen 1 (left hand graphs) and Fibronectin (right hand graphs) in WT and TG2 KO mice, in presence or absence of DSS (day 34). C) Thickening of intestinal wall in WT and TG2 KO mice, in presence or absence of DSS (day 34); Quantitative wall measurement.

[0055] Figure 10: A) PSR staining in the tent and muscle areas in WT and TG2 KO mice, in presence or absence of TNBS (day 32). B) Collagen 1 (left hand graphs) and Fibronectin (right hand graphs) in WT and TG2 KO mouse. C) Thickening of intestinal wall in WT and TG2 KO mice, in presence or absence of TNBS (day 34); Quantitative wall measurement

[0056] P values in the Figures:

[0057] Main abbreviations used throughout this description:

[0058] TG: Transglutaminase ; TG2: Transglutaminase 2; NL: non-lesional tissues (from various patients who do not have IBD; by extension patients from which such NL patients are extracted are called NL patients); UCi: inflamed ulcerative colitis; CDs: Crohn’s disease (CD) strictures; CDns: CD non- strictured; 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: knockout; IF: immunofluorescence; ID: integrated density. Examples

[0059] Material

[0060] Anti-TG2 antibody: the anti-TG2 mAb that was used in the following examples comprised 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. It is known as Zampilimab (alternative names UCB7858, derived from the antibody DC1).

[0061] Small molecule inhibitors: 1) Z-Don which is an irreversible cell soluble TG2 specific inhibitor, and 2) Boc-Don which is an irreversible cell in-soluble TG2 specific inhibitor.

[0062] Methods

[0063] Patient Samples: Human tissue samples from patients having CDs (n=10), CDns (n=20), UC (n=10) and control tissues (NL; n=14) were collected according to standard practice in the industry. Briefly, full thickness freshly resected intestinal specimens from Inflammatory bowel disease patients were obtained by standard methods. Tissue blocks from different experimental groups of tissue were procured for further processing.

[0064] Isolation and culture of primary human intestinal myofibroblasts: Human intestinal myofibroblast (HI MF) were obtained as explants of surgically resected intestinal mucosa, isolated and cultured according to standard methods. Briefly, HIMF were grown to subconfluence in Dulbecco’s minimal essential medium supplemented (DMEM) with 10% fetal bovine serum (FBS) and antibiotics and established as long-term cultures fed twice weekly and subcultured at confluence.

[0065] Detection of TG2 activity and extracellular TG2 antigen in situ: To detect in situ TG activity and extracellular TG2 antigen levels, unfixed, washed cryostat sections from fresh frozen tissues were embedded in OCT mounting media and cut at 8-1 OpM thickness to be used for both assays.

[0066] For the TG activity assay, slides were washed three times in PBS. Sections were blocked with 5% BSA and 5pg / ml streptavidin at room temperature for 35 minutes. Parallel sections were blocked from the same sample in a similar manner, with the addition of 30 pg / ml UCB7858 antibody to block TG2 activity. Sections were washed three times with PBS and the reaction mix containing 1 mM DTT, 5mM CaCh and 100pM biotin cadaverine in 50mM Tris buffer (pH7.4) containing proteinase inhibitors was added. A third section was used as negative control, for which no Biotin cadaverine was used.

[0067] For the TG2 antigen detection, sections were washed with PBS at room temperature and incubated with CUB7402 antibody (Abeam) at 30pg / ml in blocking buffer supplied with proteinase inhibitor 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 media with Dapi. All stained slides were scanned and acquired using Aperio Image Scope software (Leica Biosystems). Stained images were quantified using Imaged software (Bethesda). Starting with a non-diseased control slide, areas on muscularis mucosa (MM) and muscularis propria (MP) were marked and threshold was set. All other diseased slides were quantified using the same set threshold and integrated density was measured using Imaged and graphs plotted using GraphPad Prism software (Version 9.3.1 , Boston MA).

[0068] Relationship of TG2 activity and amount to degree of fibrosis in the tissue’. In situ TG2 activity and amount were correlated to the degree of fibrosis and inflammation using Masson’s trichrome stained sections for fibrosis and H&E stained sections for inflammation scoring (Geboes score) as evaluated by a trained IBD pathologist blinded to the diagnosis and TG2 measurements. Inflammation scores (Geboes) using H&E stained sections are based on structural (architectural changes) from no abnormality (score of 0) to severe diffuse or multifocal abnormalities (score of 3). It also includes infiltration of lamina propria neutrophils, eosinophils and epithelial neutrophils from no increase (score of 0) to marked increase (score of 3), includes crypt destruction from none (score of 0) to >50% destruction (score of 3), erosion or ulceration from none (score of 0) to ulcer or granulation tissue (score of 3). The final Geboes score is based on the sum of all above 7 subscores and ranges from 0 to 22. Fibrosis score using trichrome stained sections are limited to submucosal ECM deposition from <25% of total submucosa (score of 0) to massive transmural fibrosis, effacement of normal layers (score of 3).

[0069] Measuring Transglutaminase Activity by14C putrescine Incorporation Assay: Ten snap frozen patient tissues each from NL, UCi, CDns and CDs patients were provided by a Clinic. In this assay, active TG in the homogenised tissue incorporates [1 ,4-14C]-putrescine into N,N-dimethylcasein in the presence of calcium, forming a radioactive, trichloroacetic acid (TCA)insoluble, cross-linked protein which is measured by scintillation counting. Briefly, tissues were homogenized on ice in STE buffer (0.32M sucrose, 5mM Tris, 2mM EDTA) with Protease inhibitors added (Sigma Aldrich) and protein content was measured using Pierce™ BCA Protein Assay Kit. 75 pL reaction mix containing 14C putrescine + N’N dimethyl casein was pre-warmed in assay tubes for 20 minutes on a dry block heater set to 37°C. 75 pL tissue homogenate was added at T=0, mixed by pipetting up and down, and incubation continued on the dry block heater at 37°C. At set timepoints (0, 5, 10, 20, 30, 60 minutes), 10 pL aliquots were removed and spotted onto a filtermat pre-soaked in 10% (w / v) ice-cold TCA to stop the reaction and precipitate the14C putrescine-casein. In parallel 15pL tissue homogenate + 15pL control reaction mix (containing EDTA to inhibit enzyme activity) were mixed at T=0 in pre-warmed tubes on the heating block set to 37°C; the control mix was sampled at a single timepoint only at assay endpoint. Filtermats were left for 5 minutes after the final additions to the filtermat. Filtermats were washed 1 x 10% (w / v) ice-cold TCA for 10 minutes, 3 x 5% (w / v) ice-cold TCA for 5 minutes each, 1 x 1 :1 ice-cold acetone:ethanol for 5 minutes, and finally 1 x ice-cold 100% acetone for 5 minutes. The filtermats were air dried, cut into individual squares representing each sample position on the filtermat and placed in scintillation vials, 1 per sample. 12mL scintillant was added and radioactivity was measured in a 1450 microbeta scintillation counter. The rate of incorporation at steady state was corrected for protein concentration in each sample and reported as cpm / minute / mg protein.

[0070] TG2 in vitro cell activity assay (Biotin cadaverine incorporation into fibronectin): Plates were coated with fibronectin and HIMF 8000 cells were seeded using different conditions (see example 2) including: 1. Control: 50pM biotin cadaverine with 0.1 % Tween 20 (positive control), 2. Untreated: 50 pM biotin cadaverine only, 3. In-house isotype control : 50 pM biotin cadaverine with 150 pg / ml in-house isotype, 4. Zampilimab: 50 pM biotin cadaverine 1000 nm (150 pg / ml Zampilimab), 5. Z- Don: 50 pM biotin cadaverine in media with 200 pM Z-Don and secondary control: media alone (without biotin cadaverine), 6. Boc-Don: 50 pM biotin cadaverine in in-house media with 500 pM Boc-Don. Cells were adhered for 24h in the above conditions at 37°C. After 24h, reactions were stopped with two washes (PBS + 5 mM EDTA) and then the cells were lysed with 0.25M NH4OH + 50mM Tris for 10 minutes at room temperature and washed with PBS. Cells were blocked with BSA and then HRP-streptavadin conjugate was added to detect biotin cadaverine incorporation into the FN. Tri methyl benzidine (TMB) substrate was added to the wells and incubated for 5 mins at room temperature (RT). The reaction was stopped with 2M Sulfuric acid (H2SO4), diluted in PBS and absorbance was read at 450nm on a plate reader (Spectra max, 340PC).

[0071] RNA extraction from colon tissue’. Up to 25 mg of freshly frozen native intestinal tissues were minced prior to RNA extraction. RNA extraction was performed with RNeasy Mini Kit (Qiagen), applying the supplier’s standard protocol. RNA concentrations were determined using the ND-1000 UV / Vis Spectrophotometer (NanoDrop, Thermo Scientific).

[0072] Quantitative reverse transcriptase polymerase chain reaction procedure’. Total RNA was isolated as described above, and reverse transcription and quantitative PCR (quantitative RT-PCR) performed according to manufacturer's instructions (Applied Biosystems). The products for all primer pairs were verified by sequencing and relative differences were calculated using the comparative threshold cycle method (ddCt) by normalizing to CT values of 18S (reference gene). Quantitative RT-PCR was performed on cDNA (synthesized with iScript cDNA Synthesis Kit, Biorad) with iQ Sybr Green Supermix (Biorad) and gene specific primers. The Pfaffl method was used to calculate fold changes in treated versus untreated sample.

[0073] Extracellular deposition assay for human intestinal myofibroblasts: Deposition of extracellular matrix (ECM) by HIMF was assayed using a standard method. Briefly, cells were plated into 96- well plates in DMEM supplemented with 10% FBS and antibiotics (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. Fixed ECM was stained with Alexa Fluor488-conjugated anti-fibronectin (Ebioscience, 1 :500 dilution), Anti-Collagen Type 1 and Type III antibody (Millipore Sigma, 1 :100 dilution) and antirabbit Alexa Fluor 594 (Ebioscience, 1 :500 dilution). Fluorescence intensities were obtained by scanning the plates with ECM using a Thermofisher Cytation5 scanner. Fluorescence intensities of at least three replicate wells were used to determine the mean ECM levels in an experiment.

[0074] Dextran sodium-sulfate (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 processes (see for instance Lawrance et al., 2003 and Zhao et al., 2020).

[0075] Endpoints for DSS and TNBS colitis experiments: For both DSS and TNBS models, body weights, stool consistency and occult blood or the presence of gross blood per rectum 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, weighted and measured from the ileocaecal junction to the anus. Tissues were obtained from the descending colon and histology was performed on paraffin-embedded sections stained with H&E, Masson trichrome or Sirius red. Slides were scored by an experienced pathologist blinded to the experimental groups as per standard practice. Images were acquired using an Olympus microscope and ImagePro software. Sirius red images were quantified and analysed using Imaged software (NIH & LOCI).

[0076] Statistical analysis: Data were analysed using analysis of Student’s t test or variance (ANOVA) for independent groups. Values were expressed as mean ± standard error of mean (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.).

[0077] Example 1 -TG2 role and its expression / activity in strictures related to Crohn’s disease

[0078] The objective of this study was to determine 1) the role of TG2 as well as 2) TG2 expression and activity, in strictures related to IBD, such as in Crohn’s disease.

[0079] TG2 is the main TG active in IBP related tissues (Figures 1&2): T 0 assess the possible involvement of transglutaminases in strictures in IBD, the incorporation of biotinylated TG substrate cadaverine was measured in various tissues sections, more particularly in unfixed, washed cryostat sections taken from fresh human frozen tissues from different patients including NL, UCi, CDns and CDs patients. Using image J software, integrated density (ID) of biotin cadaverine incorporation (BCI) was quantified in these sections. As shown in Figure 1 A, global integrated density (ID) for BCI data demonstrated increased TG activity in tissues from UCi and CDs patients compared to tissues from NL patients.

[0080] As shown in Figure 1 B, whatever the type of patients, TG activity (represented by integrated intensity) was correlated with the histopathologic tissue fibrosis score (left Fibrosis score Figures: muscularis mucosa: R = 0.31 and p = 0.026; muscularis propria: R = 0.44, p = 0.00087) but not with histopathologic inflammation score (right Geboes inflammation score Figures: muscularis mucosa: R = 0.12, p = 0.39; muscularis propria: R = 0.24, p = 0.086).

[0081] In addition, to identify the main TG species involved in this activity, the effect of TG2 specific inhibitor UCB7858 on parallel sections was also measured. As shown in Figure 2B, it is quite clear that the TG activity in tissue samples from IBD patients (as shown with UCi, CDns, CDs) and controls is mainly linked to TG2.97-99% of the TG activity was attributed to TG2.

[0082] As shown in Figure 2A, top graph, there was an increase in TG2 activity in muscularis mucosa tissues from UCi and CDs patients compared to tissues from NL patients. The TG2 activity was statistically significantly reduced upon treatment with an anti-TG2 antibody (here UCB7858) in NL, UCi and CDs tissues. There was also a trend for 1) an increase in TG2 activity in muscularis mucosa tissues from CDns patients compared to tissues from NL patients and 2) reduction in TG2 activity upon treatment with an anti-TG2 antibody in tissues from CDns patients. When focusing on mucosa propria (MP) tissues, a trend to higher expression was observed, in particular for samples from CDs patients (Figure 2A, bottom graph), also with a trend to reduction in TG2 activity upon treatment with an anti-TG2 antibody, whatever the tissues (UCi, CDns or CDs).

[0083] TG2 expression (Figure 3): Same kind of tissues (i.e. unfixed, washed cryostat tissue sections from NL, UCi, CDns and CDs patients) were stained in order to assess presence of TG2 antigen (i.e. assess TG2 expression at the protein level). Images were processed as detailed in the Methods section. As shown in Figure 3A, global quantitation of ID from tissue sections of NL, UCi, CDns, and CDs patients showed an increased TG2 amount in CDs compared to NL, with a trend to increase for UCi samples (compared to NL). Sections from different tissue samples from NL, UCi, CDns and CDs patients were used to visualize TG2 mRNA (TGM2) expression (RNAscope- Z probe approach). Fluorescence microscope images showed that TG2 mRNA expression was detected in three separate tissues sections in mucosa (i.e. Mucosa Muscularis, Sub Mucosa and Muscularis Propria areas; pictures not shown). Blinded pathologist scoring of mRNA signal in these 3 separate tissue sections for each patient from the UCi, CDns, CDs groups compared to similar tissues sections from NL patients showed 25% to 100% increase in TG2 mRNA expression in CDs, the increase was particularly pronounced in the Muscularis Propria area (Figure 3B). There is also an increase in TG2 mRNA expression in UCi of up to 25%, the increase being particularly significant in the Muscularis Propria area (Figure 3B). When it comes to TG2 mRNA expression in CDns, there is up to 50% increase, in particular in the Mucosa Muscularis and Sub-Mucosa areas (Figure 3B). Full thickness tissue samples each from NL, UC, CDns and CDs patients were used for extracting mRNA, then quantified using quantitative RT-PCR. The results show that TG2 had a significant 7-to-9-fold change in mRNA expression in UCi and CDs and around 3-fold change in CDns, compared to NL (Figure 3C).

[0084] TG2 activity (Figure 4): A14C-putrescine assay was performed on frozen tissues from NL, UCi, CDns and CDs patients. As shown in Figure 4, a significant increase in TG activity in tissues from CDs patients compared to tissues from NL patients was observed when the results were analysed using non-parametric t-test, and there is a trend for increased TG activity in tissues from UCi and CDns patients (trend more pronounced for CDns patients) compared to NL patients. Conclusion of example 1

[0085] It has been surprisingly shown that active TG2 is the predominant transglutaminase in the human intestine and that its activity is increased in intestinal tissues from IBD patients, in particular from UCi and CDs patients. This activity was correlated with fibrosis but not with inflammation. In those tissues, an anti-TG2 antibody was able to inhibit drastically TG2 activity (in situ). The data also show that TG2 expression, TG2 amount and TG2 activity is upregulated in tissues from CDs patients, as shown by immunostaining and quantitative RT-PCR.

[0086] Example 2 - In vitro inhibiting activities of TG2 inhibitors

[0087] Based on the results of Example 1 , it remained to be shown whether TG2 inhibitors could inhibit in vitro activities of TG2.

[0088] TG2 inhibitors can inhibit TG2 in vitro cell activity (Figure 5): TG2 activity was assessed on HI MF from NL samples, using specific inhibitors of TG2 including Z-Don, Boc-Don and Zampilimab, with “Ig Isotype” being an additional control. As shown in Figure 5, TG2 activity could be drastically reduced by using TG2 inhibitors, the best results having been observed with Zampilimab. All in all, 82% (with a small molecule inhibitor) to 97% (with zampilimab) of NL HIMF derived TG activity had been inhibited, as determined by calculating the percentage of TG activity lost following pretreatment with the above-mentioned TG specific inhibitors.

[0089] TG2 inhibitors can inhibit ECM deposition (Figures 6&7): Mature Extracellular Matrix (ECM) levels (represented by the levels of fibronectin and collagen) have been studied across various types of samples. Figure 6 represents in particular the baseline production of FN in tissues from NL, UC, CDns and CDs patients, and shows the trend for changes between them. Particularly the samples from CDs and UCi patients demonstrate an increased trend of fibronectin production (Figure 6A). Then, the effect of TG2 inhibition (specific inhibitors of TG2 including Z-Don, Boc-Don and Zampilimab; with “Ig Isotype” being an additional positive control) on the deposition of extracellular matrix (ECM) in intestinal myofibroblasts was determined. Fibronectin (FN) and collagen l / lll levels were used for this purpose, as standard practice. As shown respectively in Figure 7B to 7E, 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 (most of the time more than 50% reduction) in the presence of TG2 inhibitors, with overall similar effects whatever the type of inhibition (i.e. with a small molecule, such as shown with Z-Don and Boc-Don, orwith an anti-TG2 antibody, such as shown with zampilimab). Zampilimab consistently caused a significant reduction in the fibronectin production across all patient groups. It is noted that the highest reduction was reached in tissues samples from CDs patients treated with Zampilimab and at a slightly lesser extend small molecules TG2 inhibitors (with respectively about 90% and 60% reduction for FN and collagen deposition for zampilimab and respectively about 80% and 50% reduction for FN and collagen deposition for small molecule inhibitors). Figure 7A are exemplary scanned imagines for the different treatments, highlighting the effect of TG2 inhibition on the deposition of ECM. HIMF cell confluency was not affected by the presence of the inhibitors (scanned images not shown).

[0090] Conclusions of example 2:

[0091] It has now been shown that TG2 inhibitors are able to inhibit TG2 activity in patient derived intestinal myofibroblasts, leading to a heavily reduction in mature ECM deposited ECM levels myofibroblasts from UCi and CDs patients more especially but also from NL and CDns patients. As ECM accumulation is a mark of fibrosis in tissues, it has therefore now been shown that TG2 inhibitors could credibly be used to prevent and / or reduce fibrosis in IBD patients, including to prevent and / or treat strictures in UC, CDns or yet CDs patients (best results among the CDs patients).

[0092] Example 3 - Effect of TG2 gene knockout in animal models

[0093] Chronic DSS colitis model’. Using mouse DSS model of chronic colitis model, with or without TGM2 KO, it was shown that TG2 knockdown (KO) affected neither the clinical score, weight loss (data not shown) nor the histopathologic inflammation score (Figure 8A). The wildtype (WT) and KO DSS chronic colitis mice showed an increased inflammation score in the proximal colon, transverse colon and rectum compared to the WT and KO non-disease model mice (Figure 8A). PSR staining (i.e. interstitial collagen) showed an increased collagen accumulation in WT mice when comparing “No DSS” and “DSS” in the tent area and the muscle area (Figure 9A). The increase in interstitial collagen did not occur in the TG2 knock out mice in response to DSS compared to No DSS. The WT DSS mice showed a significant increase in collagen accumulation compared to the KO DSS mice in the tent area (Figure 9A). Collagen 1 and fibronectin protein expressions were assessed by IF staining in DSS mice, with or without TG2 KO (scanned pictures not shown). As shown on Figure 9B, collagen 1 and fibronectin levels were decreased in the tissues from TG2 KO DSS mice compared with those from the WT DSS mice (P < 0.05). The decrease is more pronounced in the tent area. The gut wall was significantly thicker in the WT DSS mice compared to the KO DSS mice (Figure 9C; scanned pictures not shown).

[0094] TNBS fibrosis models: In the chronic TNBS mice model, no difference in the clinical score, weight loss (data not shown) and histopathologic inflammation score was noted (Figure 8B) between WT TNBS and TG2 KO TNBS groups. The WT and KO TNBS mice displayed higher inflammation scores compared to the WT and KO non-TNBS mice (Figure 8B). PSR staining showed a nominally increased trend in interstitial collagen in WT mice when comparing “No TNBS” and “TNBS”, particularly in the tent area (Figure 10A). KO mice when comparing “No TNBS” and “TNBS” did not show any changes in the interstitial collages (Figure 10A). A significantly decreased intensity was found in the TG2 knock out mice compared to WT mice in chronic TNBS colitis (Figure 10A). As highlighted in Figure 10B, collagen 1 and fibronectin ECM proteins were decreased in the TG2 KO mice compared with the WT mice, particularly in the TNBS models, as was the decrease in the gut wall thickness (Figure 10C; scanned pictures not shown).

[0095] Conclusions of example 3: It has been shown that mature ECM accumulation (consistent with fibrotic remodelling) was reduced in absence of TG2 (i.e. TG2 KO mice) and that it did not affect inflammation in both the DSS and TNBS extended colitis models used to induce early fibrotic remodelling. It has been demonstrated that the absence of TG2 reduced the degree of fibrosis, without any effect on reduction of inflammation. This data presented herein confirms those of example 2, i.e. the TG2 inhibitors could be viable therapeutics to prevent further accumulation of existing ECM deposits above normal physiological levels in IBD patients, including preventing progression of already existing ECM accumulation in UC, CDs or yet CDns patients.

[0096] REFERENCES

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Claims

CLAIMS1. A transglutaminase 2 (TG2) inhibitor for use in the treatment of a fibrotic-related disorder associated with inflammatory bowel disease (IBD) in a patient or in the prevention of the development of such disorder.

2. The TG2 inhibitor for use according to claim 1 , wherein the fibrotic-related disorder associated with IBD is selected from the group consisting of a) strictures, including strictures in the intestines, ileum, the ileocecal valve, the upper gastrointestinal tract, the colon, the rectum, or the anus, b) fibrosis of the intestines, ileum, the ileocecal valve, the upper gastrointestinal tract, the colon, the rectum, or the anus, and c) bowel obstruction.

3. The TG2 inhibitor for use according to any one of the preceding claims, wherein IBD is selected from the group consisting of 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 the group consisting of stricturing Crohn’s disease and non-stricturing Crohn’s disease, or b. the ulcerative colitis is inflamed ulcerative colitis.

5. The TG2 inhibitor for use according to any one of the preceding claims, wherein the fibrotic- related disorder associated with Crohn’s disease is characterised by an increase of a marker in a subject’s sample, said marker being selected from the group of TG2 activity, level of TG2’s product epsilon (gamma-glutamyl) lysine crosslink, mRNA encoding TG2, TG2 antigen, enhanced export of TG2 or any combination thereof.

6. The TG2 inhibitor for use according to any one of the preceding claims, wherein said 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 wherein the at least one TG2 activity that is inhibited is the TG2 cross-linking of lysine and glutamine with N-e(y-glutamyl)lysine isopeptide bonds.

7. The TG2 inhibitor for use according to any one of the preceding claims, wherein the anti-TG2 antibody or antigen-binding fragment thereof: a. comprises or consists of an intact antibody, or b. comprises or consists of an antigen-binding fragment.

8. The TG2 inhibitor for use according to any one of the preceding claims, wherein said anti-TG2 antibody comprises 6 CDRs selected from the group consisting of:(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); and LISTY (HCDR3; SEQ ID 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. 10); or(iii) KASQDINSYLT (LCDR1 ; SEQ ID NO. 1); RTNRLFD (LCDR2; SEQ ID NO. 11 ); LQYDDFPYT (LCDR3; SEQ ID NO. 3); SSAMS (HCDR1 ; SEQ ID NO. 9); TISVGGGKTYYPDSVKG (HCDR2; SEQ ID NO. 42); and LISLY (HCDR3; SEQ ID NO. 12).

9. The 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 the sequence as defined in any one of SEQ ID NO: 13 to SEQ ID No. 27 and a heavy chain variable domain having the sequence as 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 to the sequence as 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 to the sequence as defined in any one of SEQ ID NO: 28 to SEQ ID No. 40.

10. The TG2 inhibitorfor use according to any one of claims 1 to 7, wherein said antibody competes for binding to TG2 with an antibody as defined in any one of claims 8 and 9.11 . 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 the group consisting of: 1) competitive amine inhibitors, 2) reversible inhibitors, and 3) irreversible inhibitors.

13. The TG2 inhibitor for use according to any one of the preceding claims, wherein said TG2 inhibitor is incorporated in a pharmaceutical composition comprising one or more pharmaceutically acceptable adjuvant(s) and / or carrier(s).

14. A pharmaceutical composition for use in the treatment of a subject having a fibrotic-related disorder associated with inflammatory bowel disease (IBD) or in the prevention of the development of such disorder, wherein said pharmaceutical composition comprises a TG2 inhibitor and one or more pharmaceutically acceptable adjuvant(s) and / or carrier(s).

15. A method for treating a subject having a fibrotic-related disorder associated with inflammatory bowel disease (IBD) or for the prevention of the development of such disorder in a subject comprising administering a therapeutically effective amount of a transglutaminase 2 (TG2) inhibitor to said subject.

16. Use of a transglutaminase 2 (TG2) inhibitor for the manufacturing of a medicament for the treatment of a subject having a fibrotic-related disorder associated with inflammatory bowel disease (IBD) or for the prevention of the development of such disorder.

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