Patent Application
20250026856
The present invention relates to an anti-transglutaminase type 2 (TG2) antibody that blocks transamidase activity of the enzyme for use in the treatment of a scleroderma disease, such as localized or systemic scleroderma.
Tissue transglutaminase or transglutaminase type 2 (TG2) is an enzyme which forms crosslinks between proteins via epsilon(gamma-glutamyl) lysine di-peptide bonds. Elevated expression of TG2 leads to aberrant protein cross-linking which has been associated with several pathologies including various types of tissue scarring, 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 2007, Wang 2020, WO2006100679, WO2012146901 or WO2013175229), have been disclosed for the possible treatment of TG2-mediated disorders.
Scleroderma (also called Systemic sclerosis) is an immune-mediated rheumatic disease, mainly characterised by fibrosis of the skin and internal organs as well as vasculopathy (Careta & Romiti 2015; Denton & Khanna, 2017). Two categories of scleroderma have been identified so far: systemic sclerosis (SSc) and localized scleroderma (LoS) (Careta & Romiti 2015). SSc is characterized by cutaneous sclerosis and visceral involvement typically limited to the skin and/or underlying tissues. LoS is a chronic connective tissue disease having different clinical manifestations depending on the LoS subtypes.
Immunosuppressive treatments have a central role for the treatment of SSc currently, with cyclophosphamide remaining the first choice for treatment of SSc interstitial lung disease (ILD), successful in stabilising respiratory functions. However, there are none negligible side effects and the benefits are not maintained after the cessation of the therapy (Barsotti 2019).
Therefore, there remains a need to identify further effective therapies for use in treatment and prevention of scleroderma diseases, such as localized or systemic scleroderma.
It is an object of the present invention to provide an anti-Transglutaminase type 2 (TG2) antibody for use in the treatment of a scleroderma disease or for use in the prevention of the development of a scleroderma disease. Preferably, the scleroderma disease is localized scleroderma, systemic scleroderma or systemic scleroderma with interstitial lung disease.
In a second aspect, the invention provides a method for treating or for preventing the development of a scleroderma disease comprising administering a therapeutically effective amount of an anti-TG2 antibody.
In a third aspect, the invention relates to the use of an anti-Transglutaminase type 2 (TG2) antibody for the manufacturing of a medicament in the treatment of a scleroderma disease or for the prevention of the development of a scleroderma disease.
The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are 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.
The invention is based on the finding from the inventor that total amount of TG2 protein was increased in the skin and the lung fibroblasts of SSc subjects (in particular, expression levels and the activity of TG2 were increased in SSc fibroblasts), with higher levels in SSc subjects with pulmonary involvement. In contrast, the 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 could attenuate extracellular matrix (ECM) deposition in a subset of SSc fibroblasts not only under standard 2D culture conditions, but also under 3D culture conditions in full thickness skin.
The main object of the present invention is an anti-Transglutaminase type 2 (TG2) antibody for use in the treatment of a scleroderma disease or for use in the prevention of the development of a scleroderma disease. Preferably, the scleroderma disease is localized scleroderma or systemic scleroderma. The scleroderma disease can also be systemic scleroderma with interstitial lung disease. For instance, the anti-TG2 antibody for use according to the invention can comprises the following sequences:
The invention also provides a method for the treatment or for the prevention of the development of a 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 can also be systemic scleroderma with interstitial lung disease. For instance, the anti-TG2 for use according to the invention can comprises the following sequences:
Also described is the use of an anti-Transglutaminase type 2 (TG2) antibody for the manufacturing of a medicament in the treatment of a scleroderma disease or for the preventing the development of a scleroderma disease. Preferably, the scleroderma disease is localized scleroderma or systemic scleroderma. The scleroderma disease can also be systemic scleroderma with interstitial lung disease. For instance, the anti-TG2 for use according to the invention can comprises the following sequences:
In the context of the present invention as a whole, the scleroderma disease is characterised by an increase of a marker in a subject's sample, wherein the marker is for instance any one of TG2 expression or TG2 activity, and wherein the subject's sample is a cell or a tissue associated with said disease (e.g. fibroblasts or keratinocytes). The increase of TG2 expression (alternatively called overexpression of TG2) may be determined by any means in fibroblasts or keratinocytes for instance. 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). 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 consider 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 dermal fibroblasts or keratinocytes of a subject. Scleroderma disease cells/tissues may thus be characterised for instance by an increased amount (representing overexpression) of TG2 mRNA in dermal fibroblasts or keratinocytes of a subject, compared with normal dermal fibroblasts or keratinocytes. 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 (qRT-PCR), real time qRT-PCR, quantigene assay (Affymetrix/Thermo Fisher), 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 some cells of a subject, for instance in dermal fibroblasts, keratinocytes or lung cells. The sclerodermic cells may thus be characterised for instance by an increased amount (representing overexpression) of TG2 protein (or TG2 antigen) in dermal fibroblasts or keratinocytes of a subject, such as compared with normal dermal fibroblasts or keratinocytes. 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 sclerodermic cells may be characterised by an increase of the TG2 activity in dermal fibroblasts or keratinocytes 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 biopsy.
The anti-TG2 antibodies for use, method for treating or use of anti-TG2 according to the invention, i.e. for treating or preventing scleroderma disease in a subject, may thus comprise the steps of (a) measuring TG2 expression or TG2 activity in a sample (e.g. fibroblasts (such as dermal fibroblasts) or keratinocytes) from the subject, (b) comparing the result of the measure obtained from a) to the corresponding measure in a normal cell/tissue (e.g. fibroblasts (such as dermal fibroblasts) or keratinocytes), and c) if an increase of expression (i.e. overexpression of TG2) or an increase of activity is observed, administering to the subject an anti-TG2 antibody, thereby treating or preventing the scleroderma disease. TG2 expression that is measured in step a) can be the mRNA or protein amount, and the increase can be any increase of expression as discussed above. The corresponding measure in a normal cell/tissue (e.g. fibroblasts (such as dermal fibroblasts) or keratinocytes) does not need to be obtained each time a comparison is to be made. Said corresponding measure can be obtained any time before the comparison is to be made and can be the average TG2 expression or TG2 activity in said normal cell/tissue (e.g. fibroblasts (such as dermal fibroblasts) or keratinocytes).
In the context of the invention as a whole, the 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 to 473 of human TG2, and wherein the human TG2 activity that is inhibited is the TG2 cross-linking of lysine and glutamine with Nε(γ-glutamyl)lysine isopeptide bonds. Even preferably, the antibody binds to region comprising or consisting of amino acids 304 to 326 of human TG2 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 an Fab′ fragment or an F(ab)2 fragment). Preferably, the anti-TG2 antibody to be used according to the invention as a whole (see also Table A):
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.
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.
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.
Any subject may be treated in accordance with the invention. The subject is preferably a 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 or a hamster. In the context of the invention as a whole, the scleroderma disease is preferably a localized scleroderma or a systemic scleroderma. Localised scleroderma encompasses Morphea, linear scleroderma, eosinophilic fascilitis or yet toxin-induced syndromes. Systemic scleroderma encompasses limited scleroderma, diffuse scleroderma as well as overlap syndromes. The scleroderma disease can also be systemic scleroderma with interstitial lung disease. Scleroderma diseases can happen in various different tissues or organs, such as skin, lung, liver, gastrointestinal, pancreas, heart and/or kidney.
Any anti-TG2 antibody 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 anti-TG2 antibody 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 scleroderma disease, such as localized scleroderma or systemic scleroderma, wherein said pharmaceutical composition comprises an anti-TG2 antibody and one or more pharmaceutically acceptable adjuvant(s) and/or carrier(s). The pharmaceutical composition can also be for use in the treatment of systemic scleroderma with interstitial lung disease. 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.
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 antibody or antibody portion.
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 antibodies.
A suitable dosage of an anti-TG2 antibody 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 subject, composition, and mode of administration, without being toxic to the subject. 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 antibody, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular antibody, the age, sex, weight, condition, general health and prior medical history of the subject being treated.
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 subject 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 over time. 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 anti-TG2 antibody 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.
As they both have similar IC50 for human TG2 (data not shown), both were used indifferently in the following examples, depending on availability.
Zampilimab (also known as UCB7858; derived from the antibody DC1), an anti-TG2 antibody having a variable light chain according to SEQ ID NO: 24 and a variable heavy chain according to SEQ ID NO: 37 is a humanised antibody binding specifically human. In order to be able to mimic its effects on animal models, such as rabbit, mAb1 (rbBB7) has been developed. Zampilimab/DC1 and rbBB7/BB7 have been shown to behave in a similar way. They bind to the same epitope in the TG2 core (aa 313-325 of SEQ ID NO: 41), have almost 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 assays. The only notable difference is the inferior IC50 of Zampilimab against rabbit TG2 (103 vs 8 nM). Therefore, the findings from the following examples using BB7/rbBB7 are fully applicable to zampilimab and any other of the anti-TG2 antibodies such as the ones herein described.
Anti-TG2 antibody IA12: the anti-TG2 mAb that was used in the following examples comprised a light chain variable region as defined in SEQ ID NO: 26 and a heavy chain variable region as defined in SEQ ID NO: 39. It is named mAb2 in the following examples. This antibody binds to an epitope that is different from the one of BB7/Zampilimab.
The antibody 922: is a control antibody binding a Clostridium Difficile toxin.
Human samples: they were obtained from the Friederich-Alexander-University Erlangen-Nuremberg and the Royal Free Hospital London.
Assessment of TG2 in Serum, plasma and Urine samples: an MSD® assay was used, using the LGC protocol. The plated were read on the MSD S1600.
Preparation of the tissue's samples: Formalin fixed, paraffin embedded skin samples were serial sectioned and stained to correlate extent of SSc/fibrosis to TG2. Staining was made to assess SSc/fibrosis as measured by collagen content and tissue transglutaminase type 2 protein expression, and enzymatic activity.
TG2 Antigen and Activity Immunohistochemistry: TG2 antigen and isopeptidase activity (ISA) was detected by immunostaining on frozen slices of lung tissue (that had been inflated with 30% sucrose at harvest and then snap-frozen), according to standard protocols.
TG2 expression: For the histology, this was scored semi-quantitatively by an experienced scientist, and for the western blot, this was determined by densitometry.
Western blot: Western blots were performed according to standard protocols. The membranes were incubated either with antibodies against TG2 (dilution of 1:100), with antibodies against β-actin (dilution of 1:5000). with antibodies against α-SMA (71 ng/mL), with antibodies against collagen type I/Col-1 (0.4 μg/mL), or with antibodies against GAPDH (0.2 μg/mL) overnight. Membranes were then incubated with the secondary antibodies for 1 hour 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 a further 24 hours before being lysed for Western blot analysis.
TG2 MSD: TG2 in patient plasma was determined by an MSD assay according to standard protocols.
Extracellular TG activity assay: Cells were grown in 96-well plates for 7 days. Culture media was replaced with media containing 1 mM calcium, and 100 uM biotin-cadaverine. This was incubated at 37 degrees for 1 hour before being 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 then washed with PBS and blocked with 5% BSA for 30 minutes. Blocking buffer was removed and Streptavidin-HRP added for 1 hour at RT. Wells were then washed with PBS 3 times. 100 μL TMB solution was added, and colour allowed to develop for 5-10 minutes. 50 μL stop solution was added and the absorbance read at 450 nm.
Full thickness skin equivalent (3D skin model): Fibroblasts were suspended in collagen neutralization solution (comprising DMEM, foetal calf serum, HEPES and chondroitin sulfate) and mixed with type I rat collagen. The solutions were aliquoted in transwell. After 45 minutes of incubation at 37° C. to allow for collagen polymerization, DMEM-F12 (further comprising with heat-inactivated foetal bovine serum, penicillin/streptomycin, L-glutamine and amphotericin B) was added inside each transwell on top of the collagen matrix. Keratinocytes were added the following day. Before being added, the keratinocytes were carefully detached using Accutase (Thermo Fisher Scientific), centrifuged and 0.5·106 keratinocytes resuspended in E2 medium (EpiLife basal medium supplemented with Human Keratinocyte Growth Supplement, penicillin/streptomycin and CaCl2). The medium in the transwell was also replaced with E2 medium. On the next 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 to expose the keratinocyte layer to air. From day 3 on, the E3 medium was changed every other day. In a subset of samples, TGFβ (10 ng/mL) and antibodies (BB7 or control antibodies, both at 1000 nM) were added with every medium change.
TGFβ1 assay: Primary cultures of cells derived from 4 patients with dcSSC were grown for 48 hours while being treated with either 1000 nM of BB7 or control IgG. The media was subsequently removed and the level of active TGFβ1 was measured using the mink lung cell bioassay overnight (according to standard protocol).
Scratch-wound assay: Wound closure assays were performed by injuring the monolayer of fibroblasts from either WT mice or TG2KO mice to produce cell-free lines on confluent cell monolayers with sterile plastic pipette tips. Migration of cells into the clearing space was then monitored for 48 hours and photographed as per standard protocols. Cells were treated with 0.5% BSA (negative control, no FBS) or 10% FBS (positive control), either alone or with 0.1% 2 ng/ml TGFβ1.
3-D collagen matrix contraction: Twenty-four-well tissue culture plates were precoated with sterile 2% bovine serum albumin in PBS (2 ml/well) to prevent the gels from binding to the plastic. Gel contraction assays were performed by allowing 2 ml of DMEM with 10% fetal bovine serum (FBS), 1.0 mg/ml bovine type I collagen, and 8×104 fibroblasts/pericytes from WT or TG2KO mice to form a gel for 3 hours at 37° C. before releasing the gels from the dish. Contraction of the gel was quantified by loss of gel weight and decrease in gel diameter throughout a 48-hour period. The gels were then treated with DMEM containing 10% fetal calf serum (FCS) or 2 ng/ml TGFβ1 and then maintained for a period of 48 hours, allowing mechanical tension to develop. To initiate contraction, the gels were gently released from the culture dish using a sterile pipette tip and the contraction monitored throughout a 48-hour period.
Statistical analysis: Statistical significance to compare groups was calculated by one way ANOVA or unpaired student two-tailed t-test, using Microsoft Excel or GraphPad Prism V8.43. A value of p<0.05 was considered significant.
The objective of this study was to determine if the presence of collagen and TG2 expression were correlated with scleroderma and its severity.
Presence of TG2 in serum samples from 200 SSc subjects and 26 healthy volunteers was assessed. TG2 was detectable in only 29 out of 200 SSc subjects and in 2 out of 26 healthy individuals (
In addition to the serum samples above analysed, presence of TG2 in plasma samples from 76 SSc subjects and 20 healthy volunteers was assessed. These samples showed a clear increase in TG2 in the plasma of SSc patients compared to controls (
Then, urine samples from 30 SSc subjects and 20 healthy volunteers were analysed for detecting the presence of TG2. The protein was detectable in none of those samples.
As no clear clinical subtype was identified based on serum, plasma or urine samples, subjects were classified according to recognised clinical phenotypes, in order to include subjects:
For staining, antibody IA12 was used. Sections were scored with 0 for absent staining, 1 for moderate staining or 2 for intense staining by an experienced researcher in a blinded manner. Skin sections from 56 SSc subjects and 13 controls were stained and observed for semiquantitative evaluation (
TG2 needs to be outside of the cell to both be active and be targetable with an antibody. This can be assessed using immunofluorescence for TG2 (antigen) and TG2 ISA (activity). Staining of skin cryosections from 18 healthy individuals demonstrated either no TG2 staining or a TG2 staining that was restricted to the epidermal layer of the skin with the exception of weak-to-modest staining in two subjects (
TG2 antigen was measured in both dermal, and lung fibroblasts from SSc patients and healthy volunteers by western blot.
These results demonstrate that TG2 is highly expressed in scleroderma lesional tissue and that within the skin TG2 is widely distributed and associated with many cells types including cell in the epidermis, and in the fibrotic dermis, fibroblasts-like cells, inflammatory cells and also closely associated with the microvasculature.
Initially, TG2 antigen was assessed in dermal and lung fibroblasts from dcSSc patients, and in dermal fibroblasts from IcSSc patients, and was shown to be elevated in patient cells compared to cells from a healthy control (
TG2 activity was then assessed in cultured human dermal fibroblasts from a second site. An increased activity of TG2 was detected in SSc fibroblasts as compared to fibroblasts from healthy individuals (
Although the numbers were too small for fully accurate statistical evaluation, fibroblasts from subjects with pulmonary involvement tended to have higher levels of TG2 (
The total levels of TG2 in dermal fibroblasts derived from SSc subjects and healthy volunteers were quantified by Western blot (
Further, the inhibitory activity of the inhibitory TG2 antibody BB7 was verified in six dermal fibroblasts lines with high levels of TG2 activity. A TG2 inhibitory antibody dose response was performed with the following antibody concentrations: 1000, 750, 500, 250, 100, 50, 25, 10 and 5 nM, in order to assess the effect said antibody on TG2 activity in the six fibroblasts lines. As demonstrated in
Analyses of individual fibroblast “lines” showed that TG2 inhibition strongly reduced fibronectin and collagen I/Ill deposition (
The most anti-TG2 responsive cell line was used for dose titration studies and for measuring changes in phosphorylated SMAD 2/3 and cell production of SMA, Collagen 1 and CTGF (
Although no strong effect was observed at the mRNA level, the results highlighted in
Treatment with BB7 also resulted in a reduction in the expression of Col-1 and α-SMA when dermal fibroblasts have been cultured with TGFβ1 (
These surprising findings were indicative of a change in TGFβ1 driven SMAD signalling by blocking TG2 activity. To confirm this the levels of active TGFβ1 were measured in the media of cells exposed to BB7 using the mink lung bioassay and shown to be reduced by >80% (
Individual ECM components were stained for using immunofluorescence techniques and quantified using automated image analysis. Fibronectin, collagens 1&2 (analysed together) and collagen IV were all reduced with application of BB7 in SSc fibroblasts, but not in healthy fibroblasts (
Experiments were then reproduced on a skin composite model. Primary cultures from an identified clinical phenotype where TG2 appears to play a significant role were used for such a modelling. In this so called full-thickness skin model, fibroblasts are typically grown in a three-dimensional collagen matrix. The dermis-like part is then overlaid by epidermal keratinocytes, which are induced to undergo differentiation and polarization, which generates within a week a fully polarized epidermis that is separated from the dermis by a functional basal membrane. SSc fibroblasts as well as fibroblasts from healthy individuals can be used. A major advantage of this model besides the opportunity to study crosstalk between the two major cell populations in the skin is that fibroblasts are not per se pre-activated by the stiff plastic surfaces of conventional culture dishes.
Given the mode of action of TG2 as a cross linking enzyme, it was hypothesized that this mode of action may be more relevant in a 3-dimensional culture setting that better resembles the physiologic environment of fibroblasts in the skin than standard 2D culture of fibroblasts on stiff plastic surfaces. Four different fibroblast “lines” were tested. Fibroblast “lines” were pretested in 2D and only lines with at least some mild response to TG2 inhibition were selected for further validation in the full thickness skin model. These four “lines” included the two fibroblast “lines” reported above with mild-to-moderate responses, plus two additional “lines” that were identified by screening of another six SSc “lines” (4 out of 11 screened lines). A significant antifibrotic effects was observed in presence of anti-TG2 antibody (at 1000 nM), with statistically significant decreases in TGFβ1-induced gel thickening, dermal thickening, myofibroblast counts and collagen I deposition was compared to untreated skin equivalents and compared to skin equivalents incubated with control antibodies at 1000 nM (
Targeting TG2 inhibition was found promising with regard to ECM deposition in a subset of SSc fibroblast “lines” in conventional 2D culture systems and in full-thickness 3D skin models. The effects were more pronounced in the full-thickness skin model, indicating that standard cell culture approaches may not be optimal to evaluate the therapeutic potential of TG2 inhibition. These antifibrotic effects seem to be independent of the TGFβ1/SMAD signalling.
To investigate the effect of TG2 deletion on the development of dermal fibrosis, the extent of bleomycin-induced skin remodelling was examined in global TG2 KO mice. For that purpose, wildtype (WT) or transglutaminase 2 knockout (TG2KO) mice were injected with either saline (Control) or 50 μL of 2 U/mL bleomycin (Bleo) intradermally into a 1 cm×1 cm patch of skin on their back, every other day for 28 days. After 28 days the animals were terminated and the skin taken for; histology, collagen assay, TG2 crosslink quantification.
Sections of skin were stained with Masson's Trichrome and Picrosirius Red staining to visualise ECM and collagens respectively. Dermal thickness measurements were then taken, with an average of three measurements across the section. The sections stained with Picrosirius Red were then placed under polarized light to visualise collagen thickness.
WT animals—those with a normal level of TG2—had a significant increased skin thickness (as represented by the collagen staining) in response to the bleomycin injury (
When viewed under polarized light, thick collagen fibers appear red, and then progressively thinner through orange, yellow to the thinnest collagen fibers appearing green. The percentage distribution of collagen fiber thickness was calculated (
The effect of TG2 deletion was also functionally examined using a scratch test to study the migration of fibroblasts and the response to TGFβ1 (
The ability of dermal WT and TG2KO fibroblast populations to contract was explored using the 3-D type I collagen gel contraction assay (
The absence of TG2 expression by fibroblasts resulted in several functional deficits crucial to scarring and fibrosis, as demonstrated by the impact on cell migration and the ability of TG2 null cells to remodel type I collagen 3-D matrices. The significantly reduced migration of these fibroblasts following scratch wounding and inability to effectively contract collagen gels suggest alterations in cell adhesion, attachment and motility, and an impairment in the transition of fibroblasts to their activated contractile myofibroblasts counterparts. These surprising findings increase the understanding of the role of TG2 in fibrogenesis and scleroderma.
These studies provide good evidence for a link between TG2 expression and dermal fibrosis and some insight into the potential molecular mechanism(s) involved. Together they suggest value in the TG2 inhibition, such as with anti-TG2 antibodies, as shown herein, as a promising avenue for effective therapy for connective tissue fibrosis. The data adds evidence to support the association of TG2 with fibrotic pathologies and provides further clues to the potential role of TG2 in promoting tissue fibrosis in scleroderma.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2116665.7 | Nov 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/082293 | 11/17/2022 | WO |
