Methods for Treating Scleroderma Disease

Abstract

The present invention relates to anti-transglutaminase type 2 (TG2) antibodies for use in the treatment of scleroderma diseases, such as localized or systemic scleroderma.

Description

[Technical field]

[0001] Field of the invention: The present invention relates to anti-transglutaminase type 2 (TG2) antibodies which inhibit the transamidase activity of the enzyme for use in the treatment of scleroderma diseases, such as localized or systemic scleroderma. [Background technology]

[0002] Background of the invention: Tissue transglutaminase or transglutaminase type 2 (TG2) is an enzyme that forms crosslinks between proteins via epsilon (gamma-glutamyl) lysine dipeptide bonds. High expression of TG2 leads to abnormal protein crosslinks and is associated with several pathologies, such as various types of tissue scarring, formation of neurofibrillary tangles in some brain diseases, and resistance to chemotherapy in some cancers. Various TG2 inhibitors, such as small molecules, silencing RNAs, or antibodies (e.g., Non-Patent Document 1, Non-Patent Document 2, Patent Document 1, Patent Document 2, or Patent Document 3), have been disclosed for the potential treatment of TG2-mediated disorders.

[0003] Scleroderma (also called systemic sclerosis) is an immune-mediated rheumatic disease characterized by fibrosis and vascular damage, mainly in the skin and viscera (Non-Patent Documents 3 and 4). Two categories of scleroderma have been identified so far: systemic sclerosis (SSc) and localized scleroderma (LoS) (Non-Patent Document 3). SSc is characterized by skin sclerosis and visceral involvement, usually limited to the skin and / or underlying tissues. LoS is a chronic connective tissue disease with different clinical manifestations depending on the subtype of LoS.

[0004] Currently, immunosuppressive therapy plays a central role in the treatment of SSc, and cyclophosphamide remains the first-line drug for the treatment of SSc interstitial lung disease (ILD), successfully stabilizing respiratory function. However, it has negligible side effects and the efficacy is not sustained after treatment cessation (Non-Patent Document 5). Thus, there remains a need to identify additional effective therapies for use in the treatment and prevention of scleroderma diseases, such as localized or systemic scleroderma. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2006 / 100679 [Patent Document 2] International Publication No. 2012 / 146901 [Patent Document 3] International Publication No. 2013 / 175229 [Non-patent literature]

[0006] [Non-Patent Document 1] Siegel 2007 [Non-Patent Document 2] Wang 2020 [Non-Patent Document 3] Careta&Romiti 2015 [Non-Patent Document 4] Denton&Khanna 2017 [Non-Patent Document 5] Barsotti 2019 Summary of the Invention

[0007] Summary of the invention: The object of the present invention is to provide an anti-transglutaminase type 2 (TG2) antibody for use in the treatment of scleroderma disease or for use in the prevention of the onset of scleroderma disease, preferably localized scleroderma, systemic scleroderma, or systemic scleroderma with interstitial lung disease.

[0008] In a second aspect, the present invention provides a method for treating or preventing the onset of scleroderma disease, the method comprising administering a therapeutically effective amount of an anti-TG2 antibody.

[0009] In a third aspect, the present invention relates to the use of an anti-transglutaminase type 2 (TG2) antibody for the manufacture of a medicament for the treatment of scleroderma disease or for the prevention of the onset of scleroderma disease.

[0010] Definition: The entire specification is intended to be linked as a unified disclosure, and it is to be understood that all combinations of features described herein are contemplated, even if the combinations are not described together in the same sentence or paragraph or section of this specification. With respect to aspects of the invention that may be described or claimed using "a" or "an," these terms should be understood to mean "one or more," unless the context clearly requires a more limited meaning. The term "or" should be understood to encompass alternative or together items, unless the context clearly requires otherwise. When aspects of the invention are described as "comprising" a certain feature, the embodiment is also considered to "consist" of or "consisting essentially of" that feature.

[0011] - The term "tissue transglutaminase", "transglutaminase type 2" or "TG2" refers to an enzyme that forms crosslinks between proteins via epsilon (gamma-glutamyl) lysine dipeptide bonds. TG2 typically refers to the protein having the amino acid sequence set forth in UniProt entry P21980 (SEQ ID NO:41), i.e., human TG2. The term "TG2" may also refer to (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 that retains the activity of TG2, or (b) a protein that is a variant thereof, e.g., a variant typically retains 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).

[0012] - The term "anti-TG2 antibody" as used herein intends an antibody molecule that binds to TG2 and inhibits its transamidase activity, preventing cross-linking. Examples of such antibodies are described in WO 2013 / 175229. Without being limited thereto, an anti-TG2 antibody that can be used according to the present invention includes, for example, a light chain variable region defined in SEQ ID NO. 24 and a heavy chain variable region defined in SEQ ID NO. 37.

[0013] - The term "antibody" as used herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies and recombinant antibodies produced by recombinant techniques as known in the art. "Antibody" includes antibodies of any species, in particular mammalian antibodies, such as human antibodies of any isotype, such as IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, IgD and antibodies produced as dimers of these basic structures, including pentamers such as IgGA1, IgGA2 or IgM, as well as modified variants thereof; non-human primate antibodies, such as antibodies from chimpanzees, baboons, rhesus monkeys or cynomolgus monkeys; rodent antibodies, such as antibodies from mice or rats; rabbit or goat or horse antibodies; camelid antibodies (e.g. antibodies from camels or llamas such as Nanobodies®) and derivatives thereof; antibodies of avian species, such as chicken antibodies; or antibodies of fish species, such as shark antibodies. The term "antibody" also refers to a "chimeric" antibody 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 said heavy and / or light chain antibody sequence is from a second species. Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences from a non-human primate (e.g., an Old World monkey such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences. A "humanized" antibody is a chimeric antibody that contains sequences from a non-human antibody. In most cases, a humanized antibody is a human antibody (recipient antibody) in which residues from the recipient's hypervariable regions are replaced by residues from the hypervariable regions [or complementarity determining regions (CDRs)] of a non-human species (donor antibody) such as mouse, rat, rabbit, chicken, non-human primate, etc., to provide the desired specificity, affinity, and activity. In most cases, residues of the human (recipient) antibody outside the CDRs, i.e., in the framework regions (FR), are further 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 the properties of the antibody. Humanization reduces the immunogenicity of non-human antibodies in humans, facilitating the application of antibodies in the treatment of human diseases. Humanized antibodies and techniques for their production 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 generate transgenic animals (e.g. mice) that are capable of producing a full repertoire of human antibodies upon immunization in the absence of endogenous mouse antibody production. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies, such as phage display and ribosome display technology, using recombinant DNA libraries that are at least partially artificially generated or generated from a donor immunoglobulin variable (V) domain gene repertoire. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies can also be generated from isolated human B cells ex vivo immunized with an antigen of interest, which can then be fused to generate hybridomas and screened for optimal human antibodies. The term "antibody" refers to both glycosylated and aglycosylated antibodies. Furthermore, as used herein, the term "antibody" refers not only to full-length antibodies, but also to antibody fragments, more particularly antigen-binding fragments thereof. Antibody fragments contain at least one heavy or light chain immunoglobulin domain and bind to one or more antigens, as known in the art. Examples of antibody fragments according to the present invention include Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv and Bis-scFv fragments. The fragments may also be single domain antibodies (dAbs) such as diabodies, tribodies, triabodies, tetrabodies, minibodies, sdAbs, VL, VH, VHH or camelid antibodies (e.g. from camel or llama such as Nanobodies®) and VNAR fragments.An antigen-binding fragment according to the invention may also comprise a Fab linked to one or two scFvs or dsscFvs, each scFv or dsscFv binding the same or different targets (e.g. one scFv or dsscFv that binds a therapeutic target and one scFv or dsscFv that increases half-life, e.g. by binding albumin). Examples of such antibody fragments include FabdsscFv (also called BYbe®) or Fab-(dsscFv)2 (also called TrYbe®, see e.g. WO 2015 / 197772). The antibody molecules as defined above, including antigen-binding fragments thereof, are known in the art.

[0014] - The term "epitope" refers to the region of an antigen to which an antibody binds. Epitopes can be structurally or functionally defined. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and, in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0015] - The term "treating" or "treatment" of a disease state includes (i) inhibiting the disease state, i.e., preventing the onset of the disease state or its clinical symptoms, or (ii) alleviating the disease state, i.e., causing the temporary or permanent regression of the disease state or its clinical symptoms.

[0016] - The terms "preventing" or "prevention" of a disease state include preventing the development of clinical symptoms of the disease state in a subject who may be exposed to or susceptible to the disease state, but who has not yet experienced or exhibited symptoms of the disease state.

[0017] Detailed description of the invention: The present invention is based on the inventors' discovery that the total amount of TG2 protein is increased in skin and lung fibroblasts of SSc subjects (in particular, the expression level and activity of TG2 is increased in SSc fibroblasts), and that it is at higher levels in SSc subjects with lung lesions. In contrast, circulating levels of TG2 in serum and plasma are low and detectable only in a subset of SSc subjects and controls. The inventors were then able to surprisingly demonstrate that anti-TG2 antibodies are able to inhibit the deposition of extracellular matrix (ECM) in a subset of SSc fibroblasts, not only under standard 2D culture conditions, but also under 3D culture conditions in full-thickness skin.

[0018] The main object of the present invention is an anti-transglutaminase type 2 (TG2) antibody for use in the treatment of scleroderma disease or for use in preventing the onset of scleroderma disease. Preferably, the scleroderma disease is localized scleroderma or systemic scleroderma. The scleroderma disease may also be systemic scleroderma with interstitial lung disease. For example, an anti-TG2 antibody for use according to the present invention may comprise the following sequence: (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); TISVGGGKTYYPDSVKG (HCDR2; SEQ ID NO. 9); and LISLY (HCDR3; SEQ ID NO. 12), or (iv) It competes with any one of the antibodies (i) to (iii) above.

[0019] The present invention also provides a method for treating or preventing the onset of scleroderma disease, comprising administering a therapeutically effective amount of an anti-TG2 antibody. Preferably, the scleroderma disease is localized scleroderma or systemic scleroderma. The scleroderma disease may also be systemic scleroderma with interstitial lung disease. For example, an anti-TG2 for use in the present invention may comprise the following sequence: (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); TISVGGGKTYYPDSVKG (HCDR2; SEQ ID NO. 9); and LISLY (HCDR3; SEQ ID NO. 12), or (iv) It competes with any one of the antibodies (i) to (iii) above.

[0020] Also described is the use of an anti-transglutaminase type 2 (TG2) antibody for the manufacture of a medicament in the treatment of scleroderma disease or for the prevention of the onset of scleroderma disease. Preferably, the scleroderma disease is localized scleroderma or systemic scleroderma. The scleroderma disease may also be systemic scleroderma with interstitial lung disease. For example, an anti-TG2 for use according to the invention may comprise the following sequence: (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); TISVGGGKTYYPDSVKG (HCDR2; SEQ ID NO. 9); and LISLY (HCDR3; SEQ ID NO. 12), or (iv) It competes with any one of the antibodies (i) to (iii) above.

[0021] In the context of the present invention as a whole, scleroderma disease is characterized by an increase in a marker in a subject's sample, the marker being, for example, one of TG2 expression or TG2 activity, and the subject's sample being a cell or tissue (e.g., fibroblasts or keratinocytes) associated with the disease. Increased TG2 expression (also referred to as overexpression of TG2) can be determined by any means, for example, in fibroblasts or keratinocytes. An increase in a marker in a subject's sample is typically determined by comparing 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). A subject's sample in which the level of at least one marker is equal to or greater than 10%, equal to or greater than 15%, equal to or greater than 20%, equal to or greater than 25%, or even equal to or greater than 30% compared to the basal level of that marker is considered to exhibit an increase in that marker. For example, an increase in TG2 expression (alternatively referred to as TG2 overexpression) can be determined by determining the amount of TG2 mRNA in the dermal fibroblasts or keratinocytes of the subject. Thus, scleroderma disease cells / tissues may be characterized by, for example, an increase in the amount of TG2 mRNA in the dermal fibroblasts or keratinocytes of the subject compared to normal dermal fibroblasts or keratinocytes. It is characterized by an increased amount of mRNA (indicating overexpression). The expression of TG2 mRNA can be increased by any amount, such as 10% or more, 15% or more, 20% or more, 25% or more, or even 30% or more, compared to basal levels.The amount of mRNA can be measured using known methods, such as quantitative reverse transcription polymerase chain reaction (qRT-PCR), real-time qRT-PCR, quantigene assay (Affymetrix / Thermo Fisher), RNA sequencing by Northern blotting or using microarrays, various in situ hybridizations (e.g., RNAscope). Alternatively, overexpression can be determined by measuring the amount of TG2 antigen in some cells of the subject, such as dermal fibroblasts, keratinocytes, lung cells, etc. Thus, scleroderma cells can be characterized by an increased amount (indicating overexpression) of TG2 protein (or TG2 antigen) in the dermal fibroblasts or keratinocytes of the subject, for example, compared to normal dermal fibroblasts or keratinocytes. The expression of TG2 protein can be increased by any amount, such as 10% or more, 15% or more, 20% or more, 25% or more, or even 30% or more, compared to basal levels. The amount of protein can be measured using known methods such as immunohistochemistry, Western blotting, mass spectrometry or fluorescence activated cell sorting (FACS), including the use of the anti-TG2 antibody of the present invention. The threshold for determining expression may vary depending on the technique used and may be verified against the immunohistochemistry score. Alternatively, scleroderma cells are characterized by elevated TG2 activity in the subject's dermal fibroblasts or keratinocytes compared to normal cells of the same tissue type. TG2 activity can be increased by any amount, such as 10% or more, 15% or more, 20% or more, 25% or more, or even 30% or more, compared to basal levels. TG2 activity can be measured using known methods, such as via biopsy.

[0022] The anti-TG2 antibody for use, the method for treatment, or the use of an anti-TG2 antibody according to the invention, e.g., for the treatment or prevention of scleroderma disease in a subject, comprises: Thus, the method can include the steps of: (a) measuring TG2 expression or activity in a sample from a subject (e.g., fibroblasts (such as dermal fibroblasts) or keratinocytes); (b) comparing the measurement results obtained from (a) with corresponding measurement results in normal cells / tissues (e.g., fibroblasts (such as dermal fibroblasts) or keratinocytes); and (c) administering an anti-TG2 antibody to the subject if increased expression (i.e., overexpression of TG2) or increased activity is observed, thereby treating or preventing scleroderma disease. The TG2 expression level measured in step (a) may be either mRNA or protein level, and the increase may be any increase in expression as described above. It is not necessary to obtain a corresponding measurement in normal cells / tissues (e.g., fibroblasts (e.g., dermal fibroblasts) or keratinocytes) each time a comparison is made. The corresponding measurement may be obtained any time before the comparison is made, and may be the average TG2 expression level or TG2 activity in the normal cells / tissues (e.g., fibroblasts (e.g., dermal fibroblasts) or keratinocytes).

[0023] In the context of the present invention as a whole, an anti-TG2 antibody binds to an epitope within the core region of human transglutaminase type 2 (TG2) and inhibits human TG2 activity, wherein said core region consists of amino acids 143-473 of human TG2 and the human TG2 activity inhibited is TG2 cross-linking of lysine and glutamine via an Nε (γ-glutamyl) lysine isopeptide bond. More preferably, the antibody binds to a region comprising or consisting of amino acids 304-326 of human TG2 or a part of this region. The antibody comprises or consists of an intact 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 a F(ab)2 fragment). Preferably, the anti-TG2 antibody used in accordance with the present invention is a) comprises six 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); TISVGGGKTYYPDSVKG (HCDR2; SEQ ID NO. 9); and LISLY (HCDR3; SEQ ID NO. 12), b) 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; c) 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 a sequence defined in any one of SEQ ID NOs: 13 to 27, and a heavy chain variable domain having at least 80% identity or similarity, preferably at least 90% identity or similarity or preferably at least 95% identity or similarity, to a sequence defined in any one of SEQ ID NOs: 28 to 40, d) Competing the binding of an epitope comprising or consisting of amino acids 304 to 326 of human TG2 (SEQ ID NO. 41) or a part of this region with an antibody defined in a), b) or c) above.

[0024] [Table A-1]

[0025] [Table A-2]

[0026] [Table A-3]

[0027] Whether an antibody binds to the same epitope as another antibody or competes for binding with another antibody can be easily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody of the present invention, the reference antibody is bound to a protein or peptide under saturating conditions. The ability of the test antibody to bind to the protein or peptide is then evaluated. If the test antibody can bind to the protein or peptide after saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind to the protein or peptide after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the present invention. To determine whether an antibody competes for binding with a reference antibody, the binding methodology described above is performed in two orientations. In the first orientation, the reference antibody is allowed to bind to the protein / peptide under saturating conditions and then binding of the test antibody to the protein / peptide molecule is assessed. In the second orientation, the test antibody is allowed to bind to the protein / peptide under saturating conditions and then binding of the reference antibody to the protein / peptide is assessed. In either orientation, if only the first antibody (saturating antibody) is able to bind to the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As will be appreciated by those skilled in the art, an antibody that competes with a reference antibody for binding will not necessarily bind to the same epitope as the reference antibody, but may sterically inhibit binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0028] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the binding of the other antibody to the antigen, i.e., a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other antibody by at least 50%, 75%, 90% or even 99% when measured in a competitive binding assay. Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen reduce or eliminate binding of one antibody and reduce or eliminate binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations reduce or eliminate binding of one antibody and reduce or eliminate binding of the other antibody. Additional routine experiments (e.g. peptide mutations and binding analysis) can then be performed to verify whether an observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody or whether steric hindrance (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or other quantitative or qualitative antibody binding assays available in the art.

[0029] Any subject can be treated according to the present invention. Preferably, the subject is a human. However, the subject can also be other mammals, such as non-human primates, horses, cows, sheep, pigs, dogs, cats, rabbits, rats, mice, guinea pigs, or hamsters.

[0030] In the context of the entire present invention, the scleroderma disease is preferably localized scleroderma or systemic scleroderma. Localized scleroderma includes morphea, linear scleroderma, eosinophilic fasciitis or toxin-induced syndrome. Systemic scleroderma includes limited scleroderma, diffuse scleroderma and overlap syndrome. The scleroderma disease may be systemic scleroderma with interstitial lung disease. The scleroderma disease may occur in various tissues and organs, such as skin, lung, liver, gastrointestinal, pancreas, heart and / or kidney.

[0031] Any of the anti-TG2 antibodies of the present invention can be incorporated into a pharmaceutical composition suitable for administration to a subject in any manner, including, but not limited to, topical, intranasal, intradermal, intravenous, subcutaneous, or intramuscular. Typically, the pharmaceutical composition comprises an anti-TG2 antibody and one or more pharma- ceutically acceptable adjuvants and / or carriers. Thus, also described herein is a pharmaceutical composition for use in the treatment of scleroderma diseases, such as localized scleroderma or systemic scleroderma, comprising an anti-TG2 antibody and one or more pharma- ceutical acceptable adjuvants and / or carriers. The pharmaceutical composition may also be used to treat systemic scleroderma associated with interstitial lung disease. The pharmaceutical composition of the present invention may be part of a kit with instructions for use, including instructions for use and, optionally, devices for intravenous, subcutaneous or intramuscular administration to an individual in need thereof.

[0032] As used herein, a "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible and suitable for administration to a subject for the methods and uses described herein. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. Depending on the route of administration or the type of formulation (such as liquid, freeze-dried, or spray-dried formulation), isotonicity agents, e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, may be included in the composition. Pharmaceutically acceptable carriers may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.

[0033] The pharmaceutical compositions of the present invention may be in a variety of forms. These include, for example, liquid solutions (e.g., injection or infusion solutions), dispersions or suspensions, powders, and liposomes. The preferred form will depend on the intended mode of administration or therapeutic application. Exemplary 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 antibodies.

[0034] The appropriate dosage of the anti-TG2 antibody of the present invention can be determined by a skilled physician. The actual dosage of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition and mode of administration and is not toxic to the subject. The selected dosage will depend on various pharmacokinetic factors, such as the route of administration, the time of administration, the rate of excretion of the antibody, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular antibody, the age, sex, weight, condition, general health and medical history of the subject being treated.

[0035] Suitable doses can be, for example, in the range of about 0.01 pg / kg to about 1000 mg / kg body weight of the subject to be treated, typically about 0.1 pg / kg to about 100 mg / kg body weight. The dosage regimen can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single dose can be administered, or several doses can be administered over time. As used herein, a dosage unit form refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical arena. Administration can be a single dose or multiple doses. Multiple doses can be administered by the same or different routes and to the same or different sites.

[0036] In the context of the present invention as a whole, the anti-TG2 antibody can be co-administered with one or more other therapeutic agents. The co-administration of two or more agents can be accomplished in a variety of ways. They can be administered together in a single composition, or in separate compositions as part of a combination therapy. For example, one can be administered before the other, separately, or after the other, sequentially, simultaneously with the other, or simultaneously. [Brief description of the drawings]

[0037] [Figure 1] TG2 detection in serum of SSc subjects and healthy control samples using the TG2 detection MSD. Data are presented as individual data points and mean ± SEM.

[0038] [Diagram 2] TG2 detection in plasma of SSc subjects and healthy control samples using the TG2 detection MSD. Data are shown as box and whisker plots, with boxes representing 10th–90th percentiles and individual values ​​outside (**=p<0.01).

[0039] [Diagram 3]A) TG2 immunofluorescence staining in paraffin-embedded skin sections from SSc subjects and matched healthy controls. Dots represent individual subjects, horizontal lines represent mean ± standard deviation. B) TG2 immunofluorescence staining in paraffin-embedded skin sections from SSc subjects with and without SSc-associated pulmonary fibrosis. Semiquantitative assessment ranging from 0 (no staining), ≥1 (moderate staining), to 2 (strong staining). Dots represent individual subjects, horizontal lines represent mean ± standard deviation.

[0040] [Figure 4] TG2 immunofluorescence staining of frozen sections from healthy and SSc controls. Semiquantitative assessment ranging from 0 (no staining), ≥1 (moderate staining), to 2 (strong staining). Dots represent individual subjects, horizontal lines represent the mean ± standard deviation.

[0041] [Diagram 5] Comparison of TG2 expression patterns across the scleroderma disease spectrum and within specific dermal compartments using IA12. Expression and distribution of staining was assessed and scored between 0 (no staining) and 3 (maximal staining) in increments of 0.5. Data represent the mean staining score ± SD of five biopsy samples. *=p,0.05. **p<0.01, ***p<0.001.

[0042] [Figure 6] TG2 antigen in cultured human dermal or lung fibroblasts from SSc controls and matched healthy volunteers (NF). Data represent mean ± SEM densitometry data from Western blots. *= p<0.05 (t-test).

[0043] [Figure 7] TG2 activity in cultured human dermal fibroblasts from SSc patients and matched healthy controls (NF) (left). In normal cells, TG2 activity is reduced upon application of the anti-TG2 antibody BB7 (right). Dots represent individual patients and horizontal lines indicate the mean.

[0044] [Figure 8]TG2 activity in cultured human dermal fibroblasts from SSc subjects and matched healthy controls from different sites. Comparison between localized and diffuse cutaneous SSc is also shown. Dots represent individual subjects, horizontal lines indicate mean values. **=p<0.01, ***=p<0.001 (ANOVA).

[0045] [Figure 9] TG2 activity in cultured human dermal fibroblasts from SSc subjects with or without pulmonary fibrosis. Dots represent individual subjects, horizontal lines indicate mean values. *=p<0.05, **=p<0.01, ***=p<0.001 (ANOVA).

[0046] [Figure 10] TG2 expression in cultured human dermal fibroblasts isolated from SSc subjects and healthy individuals. Western blot (A) and subsequent quantification (B) of 5 lines from SSc subjects and 5 lines from each control. Dots represent individual subjects, horizontal lines represent mean ± standard deviation. **=p<0.01 (t-test).

[0047] [Figure 11] Inhibitory activity of BB7 on TG2 activity in human dermal fibroblasts. Dose titration of anti-TG2 antibody BB7 in human dermal fibroblasts with isotype antibody 922 as a control. Dots represent individual subjects, bars represent mean values, error bars represent standard deviation.

[0048] [Figure 12] Inhibitory activity of BB7 on ECM deposition in SSc fibroblasts. CX5-based quantification of fibronectin (A) and collagen I / III (B) deposition in TGFβ1-stimulated SSc dermal fibroblasts incubated with anti-TG2 antibody BB7 or isotype antibody 922. Dots represent individual subjects, bars represent mean, error bars represent standard deviation. *=p<0.05 (ANOVA).

[0049] [Figure 13]Inhibitory activity of BB7 on collagen deposition in responsive SSc fibroblast cell lines. CX5-based quantification of collagen (types I and III) deposition in SSc dermal fibroblasts incubated with anti-TG2 antibody BB7 or control isotype antibody 922. Dots represent replicate cultures of cells from one responsive subject, bars represent mean values, error bars represent standard deviation. *=p<0.05, **=p<0.01) (ANOVA).

[0050] [Figure 14] Inhibitory activity of BB7 on ECM deposition in reactive human dermal fibroblast cell "lines." The anti-TG2 antibody BB7 was dose titrated with isotype antibody 922 as a control, followed by quantification of fibronectin (A) and type I collagen (B) deposition using CX5. Bars represent mean values, error bars represent standard deviation of technical replicates. *=p<0.05 (ANOVA).

[0051] [Figure 15]Inhibitory activity of BB7 on mRNA markers of fibroblast activation and TGFβ1-Smad signaling in human dermal fibroblasts. (A) Effect of anti-TG2 antibody BB7 and non-targeting control antibody (both at 1000 nM concentration) on mRNA levels of α-smooth muscle actin (αSMA, ACTA2) and Col1a1 measured by real-time qPCR. (B) mRNA levels of representative Smad target genes Ctgf and soluble collagen in supernatants measured by real-time qPCR and SirCol assay, respectively. (C) Quantification of mean fluorescence intensity of immunofluorescence staining for pSMAD3. (D) Expression of type I collagen, α-smooth muscle actin (a-MA) and CTGF / CCN2 was analyzed by Western blotting and normalized to the expression of GAPDH. Densitometry analysis of Western blots (RHS panel). Bars indicate mean ± SEM. Statistical significance was tested by T-test, *p<0.05. In figures A), B) and C), dots represent individual subjects, bars represent mean values ​​and error bars represent standard deviations of technical replicates.

[0052] [Figure 16] Inhibitory activity of BB7 against ECM components in SSc and normal fibroblasts. Images are greyscale single channel immunofluorescence images for individual ECM components or composite images. Graphs show the abundance of ECM with and without BB7 treatment, showing that BB7 reduces ECM in SSc fibroblasts but not in normal fibroblasts.

[0053] [Figure 17]Inhibitory activity of BB7 on ECM deposition in a full thickness skin model. The full thickness skin model consists of human dermal fibroblasts in a 3D ECM on top of a fully polarized dermis composed of differentiating human keratinocytes. (A) Representative trichrome stained tissue sections and histological quantification of dermal thickness. (B) Macroscopic quantification of gel thickness. (C) Quantification of myofibroblast number. (D) Representative Western blots of collagen I and housekeeping protein β-actin and (E) normalized quantification of expression. Dots represent individual subjects, bars represent mean values, error bars represent standard deviation of technical replicates. *=p<0.05, **=p<0.01 (ANOVA).

[0054] [Figure 18] TG2 deletion protects mice from bleomycin-induced histological changes in the skin. Skin thickness in WT and TG2KO mice injected intradermally with bleomycin. Dots represent individual animals and are the mean of triplicate measurements of skin thickness in Masson's trichrome or PSR stained sections. Individual p values ​​are indicated.

[0055] [Figure 19] (A) Expression of TG2 (IA12 antibody) in primary fibroblasts from healthy controls (NF) (n=6) and scleroderma fibroblasts (n=6) was analyzed by Western blot and normalized to GAPDH expression. (B) TG2 expression in primary fibroblasts from healthy controls (NF) (n=3) was measured after treating fibroblasts with TGFβ1 (4mg / ml) for 24 hours.

[0056] [Figure 20]TG2 inhibition suppresses the expression of TGFβ1-induced fibrotic protein markers in control and SSc dermal fibroblasts. Dermal fibroblasts isolated from healthy controls (NF; n=3) or scleroderma fibroblasts (SSc: n=1) were cultured with TGFβ1 (4ng / ml) alone or with recombinant TGFβ1, and then treated in the presence of control IgG, antibody BB7, a combination of pan-TGFβ1 inhibitor antibodies, or a small molecule inhibitor of ALK5 / TGFβ1RI. Expression of collagen type I and α-smooth muscle actin (α-SMA) was analyzed by Western blot.

[0057] [Figure 21] Figure 2. Activity of BB7 in inhibiting TGFβ1 expression in primary SSc dermal fibroblasts compared to control IgG and no treatment. Data represent mean luminescence ± SEM.

[0058] [Figure 22] Skin of TG2 knockout mice is thinner and less cross-linked under polarized light. The total percentage of green, yellow, orange and red collagen fibers was measured under polarized light after Picrosirius Red staining in wild-type and TG2-deficient mice. The lower red bar, followed by the orange bar, yellow bar and upper green bar indicate generally thick, highly cross-linked to thin collagen fibers, respectively. N=6 mice per group.

[0059] [Figure 23] TG2 deletion protects mice from bleomycin-induced changes in collagen in the skin. Dermal collagen content using SirCol assay from 4 mm dermal biopsies after bleomycin-induced skin injury in WT and TG2KO mice. Dots represent individual subjects. Measurements are presented as fold change relative to saline-treated controls (WT mice). Data are presented as fold change in collagen levels. p=0.0001**** (ANOVA).

[0060] [Figure 24]Assessment of dermal fibroblast migration using a scratch wound assay. Primary dermal fibroblasts were cultured to confluence and the monolayer was scratched to induce a single injury in the monolayer. The left panel shows the time point 0 hours post-wounding. The middle and right panels show scratch repair after 48 hours for WT and TG2-deficient fibroblast populations, respectively. The top and bottom panels are in the absence and presence of TGFβ1, respectively. Panels are in the presence of 0.5% BSA.

[0061] [Diagram 25] Biomechanical forces using 3D collagen gel contraction assay. WT and TG2KO fibroblast populations derived from excised skin cultures were placed in 3D collagen gels. The level of contraction was assessed after 48 hours in the absence or presence of TGFβ1. Contraction was determined by quantification of gel weight after contraction. *=p<0.05, ***=p<0.0001 (ANOVA).

[0062] Working Example: material: Anti-TG2 antibody: - One of the anti-TG2 mAbs used in the examples was BB7, which contained a light chain variable region defined in SEQ ID NO. 19 and a heavy chain variable region defined in SEQ ID NO. 32. - Another anti-TG2 mAb used in the examples contained a light chain variable region as defined in SEQ ID NO.25 and a heavy chain variable region as defined in SEQ ID NO.38. This is a rabbitised version of the original BB7 and is herein named rbBB7 or mAb1 in the following examples. As both have similar IC50 against human TG2 (data not shown), they are used interchangeably in the following examples, depending on availability. Zampilimab (also known as UCB7858, derived from antibody DC1) is an anti-TG2 antibody with a variable light chain according to SEQ ID NO.24 and a variable heavy chain according to SEQ ID NO.37, a humanized antibody that specifically binds to human TG2. rbBB7 was developed to mimic its effect in animal models such as rabbits, mAb1 (rbBB7). Zampilimab / DC1 and rbBB7 / BB7 have been shown to behave similarly. They bind to the same epitope in the TG2 core (aa 313-325 of SEQ ID NO.41), have nearly identical IC50 (0.25 vs. 0.3 nM) and Kd (<50 vs. <60 pm) against human TG2, and inhibit ECM accumulation comparably in in vitro cell-based evaluations. The only notable difference is the inferior IC50 of Zampilimab against rabbit TG2 (103 vs. 8 nM). Thus, the findings from the following examples using BB7 / rbBB7 are fully applicable to Zampilimab and other anti-TG2 antibodies as described herein.

[0063] Anti-TG2 antibody IA12: The anti-TG2 mAb used in the following examples contained a light chain variable region defined in SEQ ID NO:26 and a heavy chain variable region defined in SEQ ID NO:39. In the following examples, it is designated mAb2. This antibody binds to an epitope distinct from that of BB7 / Zampilimab.

[0064] Antibody 922: This is a control antibody that binds to Clostridium Difficile toxin.

[0065] method: Human samples: Obtained from Friederich-Alexander-University Erlangen-Nuremberg and Royal Free Hospital London.

[0066] Assessment of TG2 in serum, plasma and urine samples: The MSD® assay using the LGC protocol was used. Plated was measured on an MSD SI600.

[0067] Tissue sample preparation: Formalin-fixed, paraffin-embedded skin samples were serially sectioned and stained to correlate the extent of SSc / fibrosis with TG2. Staining was performed to assess SSc / fibrosis as measured by collagen content, tissue transglutaminase type 2 protein expression and enzyme activity.

[0068] TG2 antigen and activity immunohistochemistry: TG2 antigen and isopeptidase activity (ISA) were detected by immunostaining of frozen slices of lung tissue (which were swollen in 30% sucrose at the time of collection and then snap frozen) according to standard protocols.

[0069] TG2 Expression: Histological examinations were scored semi-quantitatively by experienced scientists, and Western blots were determined by densitometry.

[0070] Western Blot: Western blots were performed according to standard protocols. Membranes were incubated overnight with antibodies against TG2 (1:100 dilution), β-actin (1:5000 dilution), α-SMA (71 ng / mL), collagen type I / Col-1 (0.4 μg / mL), or GAPDH (0.2 μg / mL). The membranes were then incubated with secondary antibodies for 1 h at room temperature. Blots were revealed using enhanced chemiluminescence (ECL). For growth factor treatment, cells were incubated with TGFβ (4 ng / ml) and incubated for an additional 24 h before being lysed for Western blot analysis.

[0071] TG2 MSD: TG2 in patient plasma was measured by MSD assay according to standard protocols.

[0072] Extracellular TG activity assessment: Cells were cultured in 96-well plates for 7 days. The medium was replaced with medium containing 1 mM calcium and 100 uM biotin-cadaverine. This was incubated at 37 degrees for 1 hour and then washed twice with 10 mM EDTA. Cells were then lysed with 100 μL of 0.25 mol / L ammonium hydroxide in 50 mmol / L TRIS for 5 minutes. Lysed cells were washed with PBS and blocked with 5% BSA for 30 minutes. Blocking buffer was removed and Streptavidin-HRP was added for 1 hour at RT. Wells were then washed three times with PBS. 100 μL of TMB solution was added and color was developed for 5-10 minutes. 50 μL of stop solution was added and absorbance was measured at 450 nm.

[0073] Full thickness skin equivalent (3D skin model): Fibroblasts were suspended in collagen neutralizing solution (containing DMEM, fetal calf serum, HEPES and chondroitin sulfate) and mixed with rat collagen type I. This solution was dispensed into transwells. After 45 min of incubation at 37°C to polymerize the collagen, DMEM-F12 (further containing heat-inactivated fetal calf serum, penicillin / streptomycin, L-glutamine and amphotericin B) was added into each transwell on top of the collagen matrix. The next day, keratinocytes were added. Prior to addition, keratinocytes were carefully detached using Accutase (Thermo Fisher Scientific), centrifuged and diluted with 0.5.10 mL of ethanol. 6Keratinocytes were resuspended in E2 medium (EpiLife basal medium supplemented with Human Keratinocyte Growth Supplement, penicillin / streptomycin, and CaCl2). The medium in the Transwells was also replaced with E2 medium. The following day, the medium was changed to E3 (E2 medium supplemented with 2-phospho-L-ascorbic acid trisodium salt and keratinocyte growth factor). The medium was removed from the top of the Transwells, exposing the keratinocyte layer to air. From day 3 onwards, E3 medium was replaced every other day. In a subset of samples, TGFβ (10 ng / mL) and antibodies (BB7 or control antibody, both at 1000 nM) were added at each medium replacement.

[0074] TGFβ1 evaluation: Primary cultures of cells from four dcSSC patients were treated with 1000 nM BB7 or control IgG for 48 hours, after which the medium was removed and levels of active TGFβ1 were measured overnight using a mink lung cell bioassay (following standard protocols).

[0075] Scratch Wound Assessment: Wound closure assays were performed by wounding fibroblast monolayers from WT or TG2KO mice and creating a cell-free line on the confluent cell monolayer with a sterile plastic pipette tip. Cell migration into the clearing space was then monitored for 48 h and photographed according to standard protocols. Cells were treated with 0.5% BSA (negative control, no FBS) or 10% FBS (positive control), alone or with 0.1% 2ng / ml TGFβ1.

[0076] 3D collagen matrix contraction: Twenty-four-well tissue culture plates were precoated with sterile 2% bovine serum albumin in PBS (2 ml / well) to prevent gel binding to the plastic. Gel contraction assays were performed using 10% fetal bovine serum (FBS), 1.0 mg / ml bovine type I collagen, and 8 × 10 fibroblasts / pericytes from WT or TG2KO mice. 4 The gels were gelled in 2 ml of DMEM containing 10% fetal calf serum (FCS) or 2 ng / ml TGFβ1 for 3 h at 37°C, and then released from the dish. Gel contraction was quantified by the loss of gel weight and gel diameter over 48 h. Gels were then treated with DMEM containing 10% fetal calf serum (FCS) or 2 ng / ml TGFβ1 and maintained for 48 h to allow mechanical tension to develop. To initiate contraction, gels were gently released from the dish using a sterile pipette tip, and contraction was monitored over 48 h.

[0077] Statistical analysis: Statistical significance was calculated by one way ANOVA or unpaired student two-tailed t-test using Microsoft Excel or GraphPad Prism V8.43. A p value of <0.05 was considered significant.

[0078] Example 1 - Expression of TG2 in Scleroderma Patients: The aim of this study was to investigate whether the presence of collagen and expression of TG2 correlate with scleroderma and its severity. The presence of TG2 was evaluated in serum samples of 200 SSc subjects and 26 healthy volunteers. TG2 was detected in 29 of the 200 SSc subjects and in only 2 of the 26 healthy volunteers (Figure 1). TG2-positive and TG2-negative subjects did not differ with regard to disease subtype, antibody profile, disease duration, disease activity, organ involvement, or treatment with potential disease-modifying antirheumatic drugs. In addition to the serum samples analyzed above, we also assessed the presence of TG2 in plasma samples from 76 SSc subjects and 20 healthy volunteers, in which TG2 was clearly increased in the plasma of SSc patients compared to controls (Figure 2). They then analyzed urine samples from 30 SSc subjects and 20 healthy volunteers to detect the presence of TG2: the protein was not detected in any of the samples. As no clear clinical subtypes were identified from serum, plasma and urine samples, for inclusion, subjects were categorized according to recognized clinical phenotypes: Progressive / active disease, Subjects with stable or regressing disease, Subjects with established disease (i.e., >5 years), Subjects with newly diagnosed disease (<18 months), - With major fibrotic visceral lesions.

[0079] Antibody IA12 was used for staining. Sections were scored in a blinded manner by an experienced researcher, with 0 being absent, 1 being moderate, and 2 being strong. Skin sections from 56 SSc subjects and 13 controls were stained and observed for semiquantitative evaluation (Figure 3A). The results highlighted increased staining in SSc compared to controls. Diffuse cutaneous SSc (dcSSc) showed more intense staining compared to localized cutaneous SSc (lcSSc). Furthermore, SSc subjects with pulmonary fibrosis showed more intense staining than SSc subjects without pulmonary fibrosis (Figure 3B). Elevated TG2 staining profile in inflammatory infiltrates is prominent in non-lesional and lesional tissues of DcSSc (Figure 3A). Other clinical features such as disease duration, inflammatory subtype, and other organ involvement were not associated with changes in TG2 expression.

[0080] TG2 needs to be extracellular to be active and targetable with antibodies. This can be assessed using immunofluorescence for TG2 (antigen) and TG2 ISA (activity). Staining of skin cryosections from 18 healthy subjects showed either no TG2 staining or TG2 staining restricted to the epidermal layer of the skin, except for weak to moderate staining in two subjects (Figure 4). In contrast, staining of 14 cryosections from dcSSc and ILD-rich SSc subjects showed TG2 staining not only in the epidermis but also in the dermis, especially the papillary dermis (data not shown).

[0081] Figure 5 aims to compare the expression patterns of TG2 in the scleroderma disease spectrum. An elevated profile of TG2 expression in dermal fibroblasts was observed in the dermis of DcSSc lesions, established SSc, and two types of scleroderma (Morphea), while expression levels were unchanged in LcSSc and nonlesional DcSSc (Figure 5C). TG2 vascular and perivascular staining levels did not change across the scleroderma disease spectrum, except for a slight increase in established SSc. A decrease in TG2 vascular staining levels was observed in LcSSc samples (Figure 5D). An elevated TG2 staining profile in the inflammatory infiltrate is prominent in DcSSc nonlesional and lesional tissues, and in established SSc. However, TG2 staining in the inflammatory infiltrate of LcSSc was unchanged (Figure 5B). TG2 expression levels in keratinocytes within the epidermal layer are uniformly distributed in healthy controls (HC) and scleroderma tissues. A slight decrease in the level of staining was observed in nonlesional samples of DcSSc and LcSSc (Fig. 5E).TG2 antigen was measured by Western blotting in dermal and lung fibroblasts from SSc patients and healthy volunteers.

[0082] Conclusions of Example 1: These results indicate that TG2 is highly expressed in SSc lesions and is widely distributed in the skin, where it is associated with many cell types, including epidermal cells, fibrotic dermal cells, fibroblast-like cells, and inflammatory cells, and is also closely associated with the microvasculature.

[0083] Example 2 - Intervention study with primary human cells: First, TG2 antigen was assessed in dermal and lung fibroblasts from dcSSc patients and in dermal fibroblasts from lcSSc patients and shown to be elevated in patient cells compared to cells from healthy controls (Figure 6). TG2 activity was also assessed and shown to be elevated in fibroblasts from SSc patients. TG2 activity in normal fibroblasts was shown to be reduced by BB7 antibody treatment (Figure 7).

[0084] Next, TG2 activity was assessed in cultured human dermal fibroblasts from a second site. Elevated TG2 activity was detected in SSc fibroblasts compared to fibroblasts from healthy individuals (Figure 8). Furthermore, diffuse cutaneous SSc subjects showed higher levels than focal cutaneous SSc controls.

[0085] Fibroblasts from subjects with lung lesions tended to have higher TG2 levels, although numbers were too small to allow for accurate statistical assessment (Figure 9). Total levels of TG2 in dermal fibroblasts from SSc subjects and healthy volunteers were quantified by Western blot (Figures 10, 19A). Total levels of TG2 were increased in SSc fibroblasts compared to matched healthy fibroblasts. Pretreatment with TGFβ1 for 24 h increased TG2 in dermal fibroblasts from healthy volunteers, to levels similar to those seen in some SSc dermal cells (Figure 19B).

[0086] Furthermore, the inhibitory activity of the inhibitory TG2 antibody BB7 was verified in six dermal fibroblast cell lines with high TG2 activity. A dose response of the TG2 inhibitory antibody was performed at the following antibody concentrations: 1000, 750, 500, 250, 100, 50, 25, 10, 5 nM to evaluate the effect of the antibody on TG2 activity in the six fibroblast cell lines. As shown in Figure 11, a dose-dependent decrease in TG2 activity was observed with BB7, but not with the control antibody 922. Comparison of the mean matrix deposition (collagen I / III and fibronectin) in fibroblast cell lines incubated with TGFβ and the anti-TG2 antibody BB7 at concentrations of 250 and 1000 nM showed no consistent statistical difference from cells incubated with the same concentration of the control antibody in the five different lines (Figure 12). It is noteworthy that some fibroblast cell lines incubated with the control IgG showed a mild inhibitory effect on fibronectin and collagen I / III deposition, especially at a dose of 1000 nM.

[0087] Analysis of individual fibroblast "lines" demonstrated that TG2 inhibition strongly reduced deposition of fibronectin and collagen I / III (Figure 14). No differences were observed for collagens 2, 4, 5, and 6 between the same concentrations of anti-TG2 and control antibodies (data not shown).

[0088] The most anti-TG2 responsive cell lines were used for dose titration studies, changes in phosphorylated SMAD 2 / 3, and measurements of cellular production of SMA, collagen 1, and CTGF (Figures 14-15). No difference in pSMAD2 / 3 levels was detected in fibroblast cell lines incubated with anti-TG2 antibody compared to control antibody (Figure 15C). TG2 inhibition did not reduce basal levels of pSMAD2 / 3, nor did it reduce the accumulation of pSMAD2 / 3 induced by TGFβ1 (Figure 15C). Furthermore, no reduction in CTGF mRNA was detected in fibroblasts incubated with anti-TG2 antibody compared to control antibody (Figure 15B). However, a mild downregulation of CTGF mRNA was observed with the control antibody. Similar to the results for CTGF mRNA, the levels of COL1A1 mRNA and ACTA2 mRNA were also not different between fibroblasts incubated with anti-TG2 antibody compared to control antibody (Figure 15A). As for CTGF, the control antibody mildly reduced COL1A1 and ACTA2 mRNA.

[0089] Although no strong effect was observed at the mRNA level, the results highlighted in Figure 15D show that treatment with BB7 dramatically and significantly reduced Col-1, α-SMA, and CTGF protein expression by scleroderma fibroblasts. We found that the protein expression levels of the three markers in scleroderma-derived fibroblasts were reduced to 70-90% of that in the absence of BB7. Treatment with BB7 also reduced Col-1 and α-SMA expression when dermal fibroblasts were cultured with TGFβ1 (Figure 20). This reduction was similar to that observed with a pan TGFβ1 blocking antibody and a small molecule inhibitor of ALK5. This reduction was evident in both fibroblasts from healthy controls (NF) and scleroderma fibroblasts. Compared to healthy control fibroblasts, scleroderma fibroblasts showed significant basal levels of Col-1 and α-SMA even without the addition of TGFβ1 (Figure 20B). Treatment with the IgG control had no effect on control and scleroderma fibroblasts.

[0090] These surprising findings indicated that inhibiting TG2 activity altered TGFβ1-driven SMAD signaling. Confirming this, we measured the levels of active TGFβ1 in the culture medium of cells exposed to BB7 using a mink lung bioassay and showed a >80% decrease (Figure 21).

[0091] Individual ECM components were stained by immunofluorescence and quantified by automated image analysis. Fibronectin, collagens 1 and 2 (analyzed together), and collagen IV were all decreased upon application of BB7 to SSc fibroblasts but not to healthy fibroblasts (FIG. 16).

[0092] The experiments were reproduced in a skin composite model. For such modeling, primary cultures from identified clinical phenotypes were used, in which TG2 appears to play a key role. In this so-called full-thickness skin model, fibroblasts grow normally in a three-dimensional collagen matrix. The dermis-like part is overlaid by epidermal keratinocytes, which are induced to differentiate and polarize, and within one week a fully polarized epidermis is formed, separated from the dermis by a functional basement membrane. Not only SSc fibroblasts but also fibroblasts from healthy individuals can be used. A major advantage of this model, besides the opportunity to study the crosstalk between the two main cell populations in the skin, is that the fibroblasts are not themselves preactivated by the hard plastic surface of conventional culture dishes.

[0093] Given the mode of action of TG2 as a cross-linking enzyme, we hypothesized that this mode of action might be more relevant in a 3D culture environment that more closely resembles the physiological environment of fibroblasts in the skin than in standard 2D culture of fibroblasts on a stiff plastic surface. Four different fibroblast “lines” were tested. The fibroblast “lines” were preliminarily tested in 2D, and only those that showed at least a mild response to TG2 inhibition were selected for further validation in a full-thickness skin model. These four “lines” include the two fibroblast “lines” mentioned above that showed a mild to moderate response, as well as two “lines” identified by screening another six SSc “lines” (four of the eleven lines screened). In the presence of anti-TG2 antibody (1000 nM), a significant antifibrotic effect was observed, with a statistically significant reduction in TGFβ1-induced gel thickening, dermal thickening, myofibroblast numbers, and collagen I deposition compared to untreated skin equivalents and compared to skin equivalents incubated with 1000 nM of control antibody (FIG. 17). A mild effect of the control antibody was also observed in this experimental setting.

[0094] Conclusions of Example 2: Targeting TG2 inhibition was found to be promising for ECM deposition in a subset of SSc fibroblast "lines" in traditional 2D culture systems and in a full-thickness 3D skin model. The effect was more pronounced in the full-thickness skin model, suggesting that standard cell culture approaches may not be optimal for evaluating the therapeutic potential of TG2 inhibition. These antifibrotic effects appear to be independent of TGFβ1 / SMAD signaling.

[0095] Example 3 - Protection of a skin fibrosis model by TG2 knockout: To investigate the effect of TG2 deletion on the development of dermal fibrosis, the extent of bleomycin-induced skin remodeling was examined in global TG2 KO mice. To this end, wild-type (WT) or transglutaminase 2 knockout (TG2KO) mice were injected intradermally with 50 μL of saline (control) or 2 U / mL bleomycin (Bleo) in a 1 cm × 1 cm skin patch on the back every other day for 28 days. After 28 days, animals were withdrawn and skin was harvested for histology, collagen assays, and TG2 cross-linking quantification.

[0096] Skin sections were stained with Masson's Trichrome and Picrosirius Red to visualize the ECM and collagen, respectively. Dermal thickness was then measured and averaged across the cross section in triplicate. Picrosirius Red stained sections were placed under polarized light to visualize collagen thickness.

[0097] WT animals--those with normal levels of TG2--had a significant increase in skin thickness (represented by collagen staining) in response to bleomycin injury (Figure 18). In contrast, the fibrotic response in TG2 null mice was clearly attenuated, and no significant differences or changes in dermal collagen content were observed between TG2 null mice treated with saline or bleomycin. The findings of skin thickness were confirmed by measuring dermal collagen content in response to bleomycin injury (Figure 23). WT mice injected with bleomycin had a significantly higher increase in dermal collagen compared to saline-injected WT and TG2KO mice, and bleomycin-injected WT mice.

[0098] When viewed under polarized light, thick collagen fibers appear red, then gradually thin, orange, yellow, and the thinnest collagen fibers appear green. The distribution of collagen fiber thickness was calculated (Figure 22). WT skin was predominantly red and orange, which means that there was a high proportion of thick collagen fibers. TG2KO mouse skin was predominantly green and yellow, which means that there was a high proportion of thin collagen fibers. This observation was confirmed by scanning electron microscopy, where the thick "bright" collagen fibers most commonly seen in WT skin were completely absent in TG2KO mice (data not shown). Treatment of TG2KO mice with bleomycin did not result in any changes in the dermis as seen in bleomycin-treated WT mice.

[0099] The effects of TG2 deletion were also examined functionally using a scratch assay to examine fibroblast migration and response to TGFβ1 (Figure 24). After injury to the fibroblast monolayer, WT fibroblasts migrated into the wound gap and began to repair the scratch wound starting at 6 h, and after 48 h a significant number of cells were observed to be present at the wound site. During the same 48 h period, only a few TG2KO fibroblasts penetrated into the wound site. Addition of TGFβ1 did not affect the migration of TG2KO fibroblasts. When TG2KO cells were cultured in vitro, they showed reduced migration ability compared to WT cells.

[0100] The contractile ability of dermal WT and TG2KO fibroblast populations was examined using a 3D type I collagen gel contraction assay (Figure 25). TGFβ1 stimulates fibroblasts to contract relaxed collagen gels, a process that depends on the traction forces of migrating cells and their differentiation into contractile fibroblasts (myofibroblasts). The WT group contracted significantly more when incubated with carrier alone than the TG2KO group (P<0.0001). Gel contraction of TG2KO cells incubated in the presence of TGFβ1 was significantly greater than that of TG2KO cells incubated with carrier alone (P<0.0001). This indicates that knockout of the TG2 gene significantly affected gel contraction, confirming that cell migration and differentiation were impaired, and that the addition of exogenous TGFβ1 restored the fibroblasts' migratory function and contraction-promoting activity.

[0101] Loss of TG2 expression by fibroblasts resulted in several impairments important for scar formation and fibrosis, as shown by the effects on cell migration and the ability of TG2-deficient cells to remodel type I collagen 3D matrices. The significantly reduced migration of these fibroblasts after scratch wounding and their inability to effectively contract collagen gels suggest alterations in cell adhesion, attachment and motility, and impaired migration of fibroblasts into activated contractile myofibroblasts. These surprising findings deepen our understanding of the role of TG2 in fibrosis and scleroderma.

[0102] Overall conclusion: These studies provide good evidence for the association between TG2 expression and skin fibrosis and also provide some insight into the potential molecular mechanisms involved. These findings suggest that TG2 inhibition, such as with anti-TG2 antibodies as shown herein, may be a promising treatment for connective tissue fibrosis. These data add to the evidence supporting the association between TG2 and fibrotic pathology and provide further insight into the potential role of TG2 in promoting tissue fibrosis in scleroderma.

[0103] References: Siegel&Khosla(2007),Pharmacol.Ther.,115(2):232-245 Wang et al. (2020)3 Biotech.,10:287 International Publication No. 2006 / 100679 International Publication No. 2012 / 146901 International Publication No. 2013 / 175229 Careta&Romiti(2015),An.Bras.Dermatol.,90(1):62-73. Denton&Khanna(2017),Lancet,390:1685-99 Barsotti et al.(2019),Clin.Exp.Rheumatol.,37(Suppl.119):S3-S14 International Publication No. 2013 / 175229 International Publication No. 2015 / 197772

Claims

1. An anti-transglutaminase type 2 (TG2) antibody for use in treating a subject with scleroderma disease or in preventing the onset of scleroderma disease.

2. The anti-TG2 antibody for use according to claim 1, wherein the scleroderma disease is localized scleroderma, systemic sclerosis, or systemic scleroderma with interstitial lung disease.

3. 2. The anti-TG2 antibody for use according to claim 1, wherein the scleroderma disease is characterized by an increase in a marker in a sample from the subject, the marker being, for example, one of TG2 expression or TG2 activity.

4. 2. The anti-TG2 antibody for use according to claim 1, wherein the antibody binds to an epitope within the core region of human transglutaminase type 2 (TG2) and inhibits human TG2 activity, the core region consisting of amino acids 143 to 473 of human TG2, and the human TG2 activity inhibited is TG2 cross-linking of lysine and glutamine via an Nε(γ-glutamyl)lysine isopeptide bond.

5. The antibody or antigen-binding fragment thereof, a. comprises or consists of a whole antibody, or b. The anti-TG2 antibody for use according to claim 1, comprising or consisting of an antigen-binding fragment selected from the group consisting of an Fv fragment (e.g., a single-chain Fv fragment or a disulfide-linked Fv fragment); an Fab fragment; and an Fab-like fragment (e.g., an Fab' fragment or an F(ab)2 fragment).

6. 2. The anti-TG2 antibody for use according to claim 1, wherein the antibody comprises the following sequence: (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); TISVGGGKTYYPDSVKG (HCDR2; SEQ ID NO: 9); and LISLY (HCDR3; SEQ ID NO: 12).

7. An anti-TG2 antibody for use according to claim 1, comprising: a) a light chain variable domain having the sequence defined in any one of SEQ ID NO: 13 to SEQ ID NO: 27 and a heavy chain variable domain having the sequence defined in any one of SEQ ID NO: 28 to SEQ ID NO: 40; 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 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 defined in any one of SEQ ID NO: 28 to SEQ ID NO:

40.

8. An anti-TG2 antibody for use according to claim 1, wherein the antibody binds to TG2 in competition with antibody X and / or antibody Y; said antibody X comprising the sequence: (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); TISVGGGKTYYPDSVKG (HCDR2; SEQ ID NO: 9); and LISLY (HCDR3; SEQ ID NO: 12), The antibody Y comprises an anti-TG2 antibody: a) a light chain variable domain having the sequence defined in any one of SEQ ID NO: 13 to SEQ ID NO: 27 and a heavy chain variable domain having the sequence defined in any one of SEQ ID NO: 28 to SEQ ID NO: 40; 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 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 defined in any one of SEQ ID NO: 28 to SEQ ID NO:

40.

9. A method for treating a subject having scleroderma disease or for preventing the onset of scleroderma disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-TG2 antibody.

10. 1. Use of an anti-transglutaminase type 2 (TG2) antibody for the manufacture of a medicament in the treatment of a subject with scleroderma disease or for the prevention of the onset of scleroderma disease in a subject.

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