An in vitro method and kit for determining, predicting, and / or monitoring fibrotoxicity of an agent and use thereof

Abstract

The invention is broadly in the field of medicine, more precisely in the field of drug screening. In particular, the invention concerns an in vitro method and kit for determining, predicting and / or monitoring fibrotoxicity of an agent and uses of such a kit.

Description

[0001] An in vitro method and kit for determining, predicting, and / or monitoring fibrotoxicity of an agent and use thereof.

[0002] FIELD OF THE INVENTION

[0003] The invention is broadly situated in the field of medicine, more precisely in the field of drug screening. In particular, the invention concerns an in vitro method and kit for determining, predicting and / or monitoring fibrotoxicity of an agent and use of such a kit.

[0004] BACKGROUND OF THE INVENTION

[0005] The pharmaceutical industry has introduced a myriad of drugs on the market to address a wide range of health concerns. For years, the adverse effects stemming from these compounds were often downplayed due to the belief that their therapeutic benefits outweighed the adverse effects. One of these adverse effects is drug-induced fibrosis in tissues, also known as fibrotoxicity. The potential of a drug to induce fibrosis in tissues has been largely unnoticed for many years, despite its potential for serious consequences, including tissue impairment with potentially devastating consequences for all tissues, including heart, lung, liver, kidney, and skin disorders, which can be lethal. Despite the potentially lethal outcomes of fibrosis, there is no standardized regulatory toxicological assessment for a drug candidate's potential to induce fibrosis (i.e. to assess the fibrotoxicity of a drug candidate). Regardless of the specific triggers, tissue fibrosis involves universal mechanisms across different organs: fibrosis arises from uncontrolled scarring processes driven by fibroblasts. Fibroblasts are typically located within the extracellular matrix (ECM). Although usually inactive or quiescent, fibroblasts exposed to pro-fibrotic compounds transform into myofibroblasts. While a transient activation of fibroblasts into myofibroblasts is essential for wound healing, their prolonged presence leads to excessive ECM synthesis and deposition, resulting in gradual and irreversible tissue stiffening. This damage impairs tissue function and can even lead to death. Lately, some progress has been achieved in identifying drug-induced fibrosis and various screening models are available, such as cell-based in vitro toxicology screening models. Recent advancements in these in vitro model systems have improved the ability to predict adverse effects in both animals and humans. Despite significant progress in cellbased in vitro models, reproducing the pathophysiology of fibrosis in a laboratory setting remains challenging. When cultured under standard conditions in two dimensions (2D) on rigid substrates, fibroblasts spontaneously transform into myofibroblasts permanently. To address this, three- dimensional (3D) models have been developed using collagen gels. These 3D-models enhance ECM remodelling and artificially instil myofibroblast characteristics. The known collagen gels also lack the inherent "reservoir" function of the ECM. The ECM serves as a storage site for various sequestered growth factors that play active roles in fibrosis. Consequently, maintaining the interaction between fibroblasts and a physiologically authentic extracellular matrix is crucial. Hence, these cell models fall short of fully replicating tissue microstructures.

[0006] While the cytotoxic dose (CC5o) is a regulatory requirement, and hence is documented for most agents used in e.g. medicine or food, the fibrotxicity dose (FT5o) of said agents for human use is not measured or evaluated prior to approval, although it may significantly impact the health. As will be shown herein, the fibrotoxic dose (FT5o) occurs at concentrations much lower than the cytotoxic doses (CC5o), potentially leading to dosages and amounts of agents being allowed for human use at which they can cause fibrotoxicity.

[0007] In view thereof, there remains a need in the art for more advanced and refined models to assess the potential fibrotoxicity of drugs and drug candidates.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention is at least in part based on the development of a method for determining, predicting, and / or monitoring fibrotoxicity of an agent. In particular, as evidenced by the examples which illustrate certain representative embodiments of the invention, the inventors have developed a method and a kit for determining, predicting, and / or monitoring fibrotoxicity of an agent. The method is an in vitro method that enables fibroblasts to revert back to their inactive or quiescent nature. Furthermore, the method allows fibroblasts to mimic and synthesize their own ECM reminiscing in vivo ECM while preserving the interplay between fibroblasts and the ECM.

[0010] In view of these advantages, an aspect of the invention provides a method and a kit for determining, predicting, and / or monitoring fibrotoxicity of an agent.

[0011] As will become clear form the experimental section below, the present invention provides for a method of assessing the the fibrotxicity dose (FT5o) of agents for human use which may significantly impact the health. As will be shown herein, the fibrotoxic dose (FT5o) occurs at concentrations much lower than the cytotoxic doses (CC5o), potentially leading to dosages and amounts of agents being allowed for human use at which they can cause fibrotoxicity and the present invention may hence provide a tool to better assess the safety of agents for human use and consumption that are not only not cytotoxic but also not fibrotoxic.

[0012] More specifically, the invention provides the following aspects: Aspect 1. An in vitro method for determining, predicting, and / or monitoring fibrotoxicity of a candidate agent, comprising: a. providing fibroblasts, preferably primary fibroblasts, obtained from a subject, in a cellculture environment; b. adding the candidate agent to the fibroblasts of step a), c. detecting the expression level of at least three biomarkers each independently selected from the group consisting of: transforming growth factor-pi receptor (TGFPR1), cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF) and Smooth Muscle a-Actin (SMA) in the fibroblasts, prior to (step a) and after adding the candidate agent to said fibroblasts (step b) and d. comparing the expression level of said at least three biomarkers to the expression level of said markers measured before and after said agent was added to said cell-culture environment, wherein an increase in total expression level of said at least three biomarkers indicates that the candidate agent is fibrotoxic.

[0013] In a preferred embodiment of Aspect 1, step a) of said method comprises growing fibroblasts in a fibrin gel that is uniaxially aligned in a recipient to obtain a tissue engineered 3D cell-derived matrix gel.

[0014] In a preferred embodiment of said aspect, said at least three biomarkers include the TGFPR1 biomarker.

[0015] The term "agent" as used herein has to be seen as broadly as possible and can be any type of agent that in one way or another comes into contact with the body of subject, either externally or internally, and could potentially lead to a reaction of tissue leading to fibrosis. Typical non-limiting examples are: a (candidate) drug, biologic, or medicine, in a single form or in combination, a pharmaceutical composition or formulation including active ingredient(s), or biologies (cell, antibodies, peptides, nucleotides (mRNA, cDNA, and excipients, or can be such an excipient as such, it can be a food or beverage component or additives such as a colorant, pigment, flavouring aid, sweetener, acidifier, thickener, binder, filler, and the like, or can be any agent present on a medical device for internal use, such as a component of a pacemaker, stent, stoma, any type of intubation means, tubing or needle, or it can be an agent for topical use or application on the body in general or on the skin, such as additives in creams, oils, emulsions, ointments, components of bandages, stickers, plasters, tampons and the like, or can be any agent used in food packaging, such as polymers, softeners, colorants, etc., or can be an agent present in cleaning, washing or sanitation products, etc. Aspect 2. The method according to aspect 1, comprising detecting at least four, preferably at least five, biomarkers in said cell culture environment.

[0016] Aspect 3. The method according to aspect 1, wherein the subject is a mammal, preferably a human.

[0017] Aspect 4. The method according to anyone of aspects 1 to 3, wherein said fibroblasts are primary fibroblasts.

[0018] Aspect 5. The method according to anyone of aspects 1 to 4, wherein said fibroblasts are grown in a culture medium comprising fibrinogen and thrombin, or in a fibrin gel, preferably resulting from the enzymatic reaction of fibrinogen with thrombin.

[0019] Aspect 6. The method according to anyone of aspects 1 to 5, wherein said fibrin gel containing said fibroblasts is aligned uniaxially in a recipient to obtain a tissue-engineered 3D cell-derived fibrin gel matrix. In certain embodiments, the fibrin gel is added to a removable mold in said recipient during polymerisation, wherein the mold can be removed after polymerisation of the fibrin gel. In certain embodiments, fibroblasts remodel the fibrin of the polymerised uniaxially aligned fibrin gel into a complex cell-derived extracellular matrix.

[0020] Aspect 7. The method according to anyone of aspects 1 to 6, wherein said recipient is produced through 3D printing, preferably by stereolithography.

[0021] Aspect 8. The method according to anyone of aspects 1 to 7, wherein said recipient is produced using biocompatible resins.

[0022] Aspect 9. The method according to anyone of aspects 1 to 8, wherein the fibroblasts are aligned for at least 3 days, preferably for at last 4 days.

[0023] Aspect 10. The method according to anyone of aspects 1 to 9, wherein said (primary) fibroblasts are in a quiescent state.

[0024] Aspect 11. The method according to anyone of aspects 1 to 10, wherein said fibroblasts have replaced the provisional fibrin matrix into a complex cell-derived extracellular matrix restoring both the mechanical and chemical properties of the extracellular matrix and have returned to quiescence.

[0025] Aspect 12. The method according to anyone of aspects 1 to 10, wherein the expression level of said at least three biomarkers is measured at least 2 days after addition of the agent or a combination of agents, preferably after at least 3 days, preferably after at least 4 days.

[0026] Aspect 13. The method according to anyone of aspects 1 to 10, wherein a fibrotoxicity index is calculated for said agent. Aspect 14. The method of aspect 11, wherein said agent is ranked according to its risk for inducing fibrosis.

[0027] Aspect 15. The method according to anyone of aspects 1 to 12, used for screening one or more agents for inducing fibrosis or fibrotic disease.

[0028] Aspect 16. The method according to aspects 13, wherein said fibrosis, tissue fibrosis or fibrotic disease is selected from the group comprising idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis, endomyocardial fibrosis, mediastinal fibrosis, bone marrow fibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, renal fibrosis, iatrogenic fibrosis, myocardial fibrosis, renal tubulointerstitial fibrosis, fibroids, liver fibrosis, fibrotic skin disease, keloidal scar, and oral submucous fibrosis.

[0029] Aspect 17. A kit for determining, predicting, and / or monitoring fibrotoxicity of an agent, the kit comprising:

[0030] - means for measuring the level of at least three biomarkers independently selected from the group consisting of: transforming growth factor-pi receptor (TGFPR1), cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF) and Smooth Muscle a-Actin (SMA); and

[0031] - a reference value or means for establishing said reference value, wherein said reference value represents a known value for determining, predicting and / or monitoring of fibrotoxicity. In a preferred embodiment, said at least three biomarkers include the TGFPR1 biomarker.

[0032] Aspect 18. The kit according to aspect 16, wherein said agent is a candidate drug and said kit is used to determine drug-induced fibrosis.

[0033] Aspect 19. The kit according to aspects 17 or 18, wherein said kit further comprises a recipient for uniaxially aligning fibroblasts.

[0034] Aspect 20. The kit according to any one of aspects 17 to 19, wherein said recipient comprises fibroblasts embedded in a fibrin gel, preferably resulting from the enzymatic reaction of fibrinogen with thrombin.

[0035] Aspect 21. The kit according to anyone of aspects 17 to 20, wherein said fibrin gel containing said fibroblasts are aligned uniaxially in said recipient to obtain a tissue-engineered 3D cell-derived matrix.

[0036] Aspect 22. The kit according to anyone of aspects 17 to 21, wherein said recipient is produced through 3D printing, preferably by stereolithography.

[0037] Aspect 23. The kit according to anyone of aspects 17 to 22, wherein said recipient is produced using biocompatible resins. Aspect 24. A computer-implemented method for assessing the fibrotoxicity of an agent comprising the ranking of agents according to their risk of fibrotoxicity according to aspect 14.

[0038] Aspect 25. The method according to any one of aspects 1 to 16, wherein said method is a high throughput screening.

[0039] Aspect 26. Use of the kit according to any one of aspects 17 to 23, for determining the fibrotoxicity of an agent or for determining whether or not a candidate agent could induce fibrosis or a fibrotic disease.

[0040] Aspect 27. The use according to aspect 26, wherein the fibrotoxicity dose (FT5o) of the agent is determined as the dose in which 50% of the fibroblasts undergo fibrosis, optionally wherein also the cytotoxicity dose (CC5o) of said agent is determined, i.e. the dose where cell viability is reduced by 50% as compared to untreated cells. Typically, said cells are fibroblasts, preferably primary fibroblasts.

[0041] The CC5o is determined as the dosage of agent needed to reduce viability of the cells in an assay with 50%, typically determined by reduction of the normalized Beta-actin expression of said cells to below 50% of the starting expression level (i.e. of untreated cells).

[0042] The FT50 is determined as the dosage of agent needed to induce fibrosis in 50% of the cells in an assay, typically defined as the dose at which a 50% or higher increase of the normalized TGFBR expression in said cells versus the starting expression level (i.e. of untreated cells) occurs.

[0043] Aspect 27. The use according to aspects 26 or 27, using the method according to any one of aspects 1 to 17.

[0044] The above and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of appended claims is hereby specifically incorporated in this specification.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 illustrates a heatmap for TGFBR1, CTGF, MMP2, SMA, and Cofilin for five agents: adenosine (lpM and lOpM), amiodarone (lOpM), cabergoline (lOpM), benfluorex (lOpM), and doxorubicin (lOOnM).

[0047] Figure 2 illustrates the FT50 for benfluorex (in nM) based on 1 fibrotic marker (SMA).

[0048] Figure 3 illustrates a fibrotoxicity index for SMA / CTGF / TGFBR1 (A), CTGF / TGFBR1 / MMP2 (B), SMA / CTGF / MMP2 (C), SMA / MMP2 / TGFBR1 (D), cofilin / TGFBRl / MMP2 (E), SMA / CTGF / cofilin (F), pSMA / TGFBRl / cofilin (G), SMA / MMP2 / cofilin (H), CTGF / TGFBRl / cofilin (I), and CTGF / cofilin / MMP2 (J).

[0049] Figure 4 illustrates a fibrotoxicity index for SMA / CTGF / TGFBR1 / MI\ / IP2 (A), SMA / CTGF / TGFBRl / cofilin (B), SMA / TGFBRl / MMP2 / cofilin (C), CTGF / TGFBRl / MMP2 / cofilin (D), and SMA / CTGF / MMP2 / cofilin (E).

[0050] Figure 5 illustrates a fibrotoxicity index defined as the average of 3 fibrotic markers (here TGFBR1, CTGF and SMA) for five agents : adenosine (lpM and 10pM), amiodarone (10pM), cabergoline (10pM), benfluorex (10pM), and doxorubicin (100nM).

[0051] Figure 6 illustrate the fibrotoxicity dose (FT5o) and the cytotoxicity dose (CC5o) for doxorubicin based on TGFBR1 and Beta-actin expression respectively, on primary cardiac fibroblast cells.

[0052] DETAILED DESCRIPTION OF THE INVENTION

[0053] As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0054] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass "consisting of" and "consisting essentially of", which enjoy well-established meanings in patent terminology.

[0055] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression "from... to..." or the expression "between... and..." or another expression.

[0056] The terms "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

[0057] Whereas the terms "one or more" or "at least one", such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g. any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0058] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0059] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0060] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e. also in the context of other aspects or embodiments of the invention, unless otherwise defined. For example, embodiments directed to products are also applicable to corresponding features of methods and uses.

[0061] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0062] Reference throughout this specification to "one embodiment", "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, alternative combinations of claimed embodiments are encompassed, as would be understood by those in the art.

[0063] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to standard handbooks as well as to the general background art referred to herein and to the further references cited therein.

[0064] A first aspect provides an in vitro method for determining, predicting, and / or monitoring fibrotoxicity of an agent, comprising: a. providing fibroblasts, preferably primary fibroblasts, in a cell-culture environment, b. adding the agent to the fibroblasts of step a), c. detecting the expression level of at least three biomarkers independently selected from the group consisting of: transforming growth factor-pi receptor (TGFPR1), cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF) and Smooth Muscle a-Actin (SMA) in the fibroblasts prior to and after adding the compound to said fibroblasts (step b) and d. comparing the expression level of said at least three biomarkers to the expression level of said markers measured before and after said agent was added to said cell-culture environment, wherein an increase in total expression level of said at least three biomarkers indicates that the agent is fibrotoxic. Preferably, at least TGFPR1 is part of the biomarker analysis.

[0065] Preferably, in step a) of said method, growing fibroblasts in a fibrin gel that is uniaxially aligned in a recipient to obtain a tissue engineered 3D cell-derived matrix gel.

[0066] As used herein, the term "fibrotoxicity" or "fibrotoxic" generally refers to an agent having the ability to cause or induce fibrosis in a subject.

[0067] In certain embodiments, in step c) the expression levels of at least four biomarkers, preferably at least five biomarkers are detected in said cell-culture environment, optionally obtained from a subject.

[0068] In a preferred embodiment, to assess the fibrotic potential of an agent as defined herein, it is first necessary to develop an assay or method providing in vivo-like data at an in vitro scale by recreating the in vivo 3D context of (preferably primary) fibroblasts which results in preserving their quiescent state. Said 3D context can be obtained by developing a coherent mechanical environment (in 3 dimensions) by growing said fibroblasts in a fibrin gel that is uniaxial ly aligned in a recipient to obtain a tissue engineered 3D cell-derived matrix gel. This creates and preserves the interplay between the fibroblasts and the extracellular matrix (ECM) and makes them return to, and / or stay in, a quiescent state.

[0069] As used herein, the term "determining" generally refers to determining the presence and / or measuring the quantity of any one or more of transforming growth factor-pi receptor (TGFPR1), cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF) or Smooth Muscle a-Actin (SMA) expressed or secreted by fibroblasts in vitro, or in the medium or the extracellular matrix of the gel. This determination may be performed by any existing, available or conventional detection and / or quantification method used to measure the presence or absence (e.g., readout being present vs. absent; or detectable amount vs. undetectable amount) and / or quantity (e.g., readout being an absolute or relative quantity) of a peptide, polypeptide, protein, or nucleic acid, in or on a fibroblast or fibroblast population, or in the medium or the extracellular matrix of the gel. For example, such methods may include western blot, ELISA, biochemical assay methods, immunoassay methods, mass spectrometry analysis methods, or combinations thereof. Preferably, at least TGFPR1 is part of the biomarker analysis.

[0070] In particular embodiments, measuring the presence and / or quantity of any one or more of said biomarkers comprises measuring the amount of peptides, polypeptides or proteins of said biomarkers, or mRNA levels of said biomarkers, or both.

[0071] In preferred embodiments, measuring the presence and / or quantity of any one or more of said biomarkers comprises measuring the amount of peptides, polypeptides or proteins of said biomarkers. The term "predicting" or "prediction" generally refer to a statement, declaration, indication or forecasting of a disease or condition in a subject not (yet) showing any, or a limited, clinical manifestation of said disease, condition, or (adverse) side effects. A prediction of a certain clinical disease manifestation, condition, or adverse effect in a subject may indicate a probability, chance, or risk that said subject will develop said clinical manifestation, condition, or (adverse) side effect, for example the prediction to develop or diagnose a subject with fibrosis or a fibrotic disease within a certain time period after treatment with said agent. Said probability, chance or risk may be indicated as any suitable qualitative or quantitative expression, wherein non-limiting examples of a quantitative expression include absolute values, ranges or statistics. Alternatively, probabilities, chances, or risks may be indicated relative to a suitable control subject or group of control subject (i.e. a control subject population (such as, e.g., relative to a general, normal or healthy subject or subject population)). Therefore, any probability, chance or risk may be advantageously indicated as increased or decreased, upregulated or downregulated, as fold-increased or fold-decreased relative to a suitable control subject or subject population, or relative to a baseline value which may be derived from either a control subject (population), textbook reference values. It is evident that when a population of subjects is used to define the baseline value, said baseline value will be a centre size of one or more values (parameters) of a population, such as the mean or median of said value. A skilled person further appreciates that monitoring of fibrosis or a fibrotic disease may allow to predict the progression, aggravation, alleviation or recurrence of the clinical image or severity of fibrosis or a fibrotic disease. Furthermore, monitoring may be applied in the course of a medical treatment of a subject.

[0072] The term "expressing" or "expression" as used throughout this specification generally encompasses the generation of any transcription or translation product, such as RNA, including microRNA, peptides, polypeptides and proteins including post-translationally modified peptides polypeptides and proteins, by a cell, such as a fibroblast, as well the presentation of peptides, polypeptides or proteins on the cell surface and secreted peptides, polypeptides or proteins.

[0073] By means of additional guidance, when a fibroblast or population of fibroblasts is said to be positive for or to express or comprise expression of a given gene, peptide, polypeptide or protein, such as cofilin, MMP2, CTGF, TGFPR1 or SMA, a skilled person would conclude the presence or evidence of a distinct signal for the gene, peptide, polypeptide or protein when carrying out a measurement capable of detecting or quantifying the gene, peptide, polypeptide or protein in or on the fibroblasts, the medium, the extracellular matrix, and / or the gel. Suitably, the presence or evidence of the distinct signal for the gene, peptide, polypeptide or protein would be concluded based on a comparison of a negative control, for example the measurement result obtained for the fibroblasts, the medium, the extracellular matrix, and / or the gel before the agent was added to the fibroblasts to a result of the same measurement carried out after exposing the fibroblasts to the agent. Exposing the fibroblasts to the agent is typically done by addition of the agent to the medium wherein the fibroblasts are present or grown.

[0074] In certain embodiments, in step c) and d) said at least three biomarkers can comprise any one of the following combinations: cofilin, MMP2, and CTGF; or cofilin, MMP2, and TGFPR1; or cofilin, MMP2, and SMA; or cofilin, CTGF, and TGFPR1; or cofilin, CTGF, and SMA; or cofilin, TGFPR1, and SMA; or MMP2, CTGF, and TGFPR1; or MMP2, CTGF, and SMA; or CTGF, TGFPR1, and SMA. In certain embodiments, said at least three biomarkers can comprise TGFPR1 and at least two biomarkers selected from the group consisting of: cofilin, MMP2, CTGF, and SMA. In certain embodiments said at least two biomarkers can comprise cofilin and MMP2; or cofilin and CTGF; or cofilin, and SMA; or MMP2 and CTGF; or MMP2 and SMA; or CTGF and SMA. In certain embodiments, in step c) and d) the expression levels of at least four biomarkers, preferably at least five biomarkers are compared in said cell-culture environment. In certain embodiments, in step c) and d) said at least four biomarkers can comprise cofilin, MMP2, CTGF, and TGFPR1; or cofilin, CTGF, TGFPR1, and SMA; or cofilin, CTGF, TGFPR1, and SMA; or MMP2, CTGF, TGFPR1, and SMA. In certain embodiments, said at least four biomarkers can comprise TGFPR1 and at least three biomarkers selected from the group consisting of: cofilin, MMP2, CTGF, and SMA. In certain embodiments said at least three biomarkers can comprise cofilin, MMP2, and CTGF; or cofilin, MMP2, and SMA; cofilin, CTGF, and SMA; or MMP2, CTGF, and SMA. In certain embodiments, in step c) and d) said at least 5 biomarkers can comprise TGFPR1, cofilin, MMP2, CTGF, and SMA.

[0075] As used herein, the term "biomarker", "marker" or "biological marker" refers to a naturally occurring indicator such as a molecule, gene, protein, cell type, trace element, feature or characteristic which can be measured accurately, objectively and reproducibly, by which a particular pathological or physiological condition, such as but not limited to a process, disease, syndrome or disorder, can be identified. The status of a biomarker refers to the outcome of measurement of said biomarker and comprises the presence, absence and / or amount of said biomarker, wherein the "amount" of said biomarker includes changes in the amount of said biomarker. In a preferred embodiment, the biomarker is a protein.

[0076] By means of additional guidance, cofilin is an actin-binding protein located in the cell. By means of an example, human cofilin gene is annotated under NCBI Genbank Gene ID 1072 and human cofilin protein is annotated under Uniprot under accession number E9PLJ3. mRNA and protein sequences of human cofilin are annotated under the following NCBI Genbank accession numbers: cofilin ( NM_005507.3), and cofilin protein (NP_005498.1). By further means of an example, mus musculus cofilin gene is annotated under NCBI Genbank Gene ID 12631 and human cofilin protein is annotated under Uniprot under accession number P18760. mRNA and protein sequences of human cofilin are annotated under the following NCBI Genbank accession numbers: cofilin (NM_007687.5), and cofilin protein (NP_031713.1).

[0077] By means of additional guidance, MMP2 (matrix metalloproteinase-2 or matrix metallopeptidase 2 or MMP-II) is also known in the art as 72kDA type IV collagenase, CLG4, 72kDa gelatinase, gelatinase A, CLG4A, MONA or TBE-1. MMP2 is a secretory protein that functions as a gelatinase and collagenase and is involved in degradation of extracellular matrix proteins and tissue repair. By means of an example, human MMP2 gene is annotated under NCBI Genbank Gene ID 4313 and human MMP2 protein is annotated under Uniprot under accession number P08253. mRNA and protein sequences of human MMP2 are annotated under the following NCBI Genbank accession numbers: MMP2 mRNA (NM_004530.6, NM_001127891.3, NM_001302508.1, NM_001302509.2, NM_001302510.2

[0078] NM_001127891.2, NM_001302508.1, NM_001302509.1, NM_001302510.1, NM_004530.5), and MMP2 protein (NP_001121363.1, NP_001289437.1, NP_001289438.1, NP_001289439.1, NP_004521.1).

[0079] By means of additional guidance, connective tissue growth factor (CTGF) is also known in the art as cellular communication network factor 2 (CCN2), hypertrophic chondrocyte-specific gene product 24 (Hcs24), NOV2 and insulin-like growth factor binding protein-8 (IGFBP8), IBP-8. CTGF is a secretory protein involved in cell adhesion. By means of an example, human CTGF gene is annotated under NCBI Genbank Gene ID 1490 and human CTGF protein is annotated under Uniprot under accession number P029279. mRNA and protein sequences of human CTGF are annotated under the following NCBI Genbank accession numbers: CTGF mRNA (NM_001901.4), and CTGF protein (NP_001892.2).

[0080] By means of additional guidance, transforming growth factor-pi receptor (TGFPR1) is also known in the art TGFR-1, tbetaR-l, AAT5; ALK5; ESSI; LDS1; MSSE; SKR4; TBRI; ALK-5; LDS1A; LDS2A; TBR-i;

[0081] ACVRLK4. TGFPR1 is a transmembrane serine / threonine kinase forming an heterodimer with TGFBR-2 for transducing TGFB1, TGFB2 an TGFB3 cytokine signal from the cell surface to the cytoplasm. . By means of an example, human TGFPR1 gene is annotated under NCBI Genbank Gene ID 7046 and human

[0082] TGFPRlprotein is annotated under Uniprot under accession number P36897. mRNA and protein sequences of human TGFPR1 are annotated under the following NCBI Genbank accession numbers:

[0083] TGFPR1 mRNA (NM_001407418.1, NM_001407423.1, NM_001407424.1, NM_001407425.1,

[0084] NM_001407434.1, NM_001407432.1, NM_001407419.1, NM_001407433.1, NM_001407422.1,

[0085] NM_001407426.1, NM_001407428.1, NM_001407420.1, NM_001407429.1, NM_001407416.1,

[0086] NM_001407417.1, NM_001407435.1, NM_001407436.1, NM_001407438.1, NM_001407437.1,

[0087] NM_001407430.1, NM_001407427.1, NR_176360.1, NM_001130916.3, NM_001306210.2,

[0088] NR_176362.1, NR_176361.1, NR_176363.1, and NM_004612.4), and TGFPR1 protein

[0089] (NP_001394347.1, NP_001394352.1, NP_001394353.1, NP_001394354.1, NP_001394363.1,

[0090] NP_001394361.1, NP_001394348.1, NP_001394362.1, NP_001394351.1, NP_001394355.1,

[0091] NP_001394357.1, NP_001394349.1, NP_001394358.1, NP_001394345.1, NP_001394346.1,

[0092] NP_001394364.1, NP_001394365.1, NP_001394366.1, NP_001394367.1, NP_001394359.1,

[0093] NP_001394356.1, NP_001293139.1, NP_004603.1, NP_001124388.1, NRJ_176360.1, NRJ_176361.1,

[0094] NRJ_176362.1, and NRJ_176363.1).

[0095] By means of additional guidance, smooth muscle a-actin (SMA) is also known in the art as actin alpha 2, smooth muscle (ACTA2). By means of an example, human SMA gene is annotated under NCBI Genbank Gene ID 59 and human SMA protein is annotated under Uniprot under accession number P62736 . mRNA and protein sequences of human SMA are annotated under the following NCBI Genbank accession numbers: SMA mRNA (NM_001406466.1, NM_001320855.2 NM_001406462.1, NM_001406468.1, NM_001406471.1, NM_001406463.1, NM_001141945.3) and SMA protein (NP_001393396.1 , NP_001393391.1, NP_001393397.1, NP_001393392.1, NP_001307784.1, and NP_001135417.1, NP_001393400.1)

[0096] As used herein, the cytotoxicity dose or CC5o is the dosage of agent needed to reduce viability of the cells in an assay with 50%, typically determined by reduction of the normalized Beta-actin expression of said cells to below 50% of the starting expression level (i.e. of untreated cells). In this assay typically fibroblasts, preferably primary fibroblasts as used.

[0097] As used herein, the fibrotoxicity dose or FT5o is the dosage of agent needed to induce fibrosis in 50% of the cells in an assay, typically determined by a 50% or higher increase of the normalized TGFBR expression in said cells versus the starting expression level (i.e. of untreated cells). In this assay typically fibroblasts, preferably primary fibroblasts as used.

[0098] In certain embodiments, both the cytotoxicity and fibrotoxicity dose can be determined for any given agent in order to assess the maximum amount or dosage that can be safely consumed or administered to a subject.

[0099] As used herein, the terms "subject" typically and preferably denotes humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, even more preferably mammals, such as, e.g. non-human primates, rodents, canines, felines, equines, ovines, porcines, and the like. The term "non-human animals" includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such as chicken, amphibians, reptiles, etc. In certain embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal. In some preferred embodiments of the methods and uses as taught herein, the subject is a human subject. In other embodiments, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex.

[0100] In particular embodiments, the agent causing fibrosis or having fibrotic potential show an increase of at least three biomarkers selected from the group consisting of: cofilin, MMP2, CTGF, TGFPR1 and / or SMA by at least about 20% (about 1.2-fold or more), or by at least about 30% (about 1.3-fold or more), or by at least about 40% (about 1.4-fold or more), or by at least about 50% (about 1.5-fold or more), or by at least about 60% (about 1.6-fold or more), or by at least about 70% (about 1.7-fold or more), or by at least about 80% (about 1.8-fold or more), or by at least about 90% (about 1.9-fold or more), or by at least about 100% (about 2-fold or more) after administration of the agent to fibroblasts, relative to the protein level of the respective biomarkers before addition of the agent or relative to a reference protein level of the respective biomarkers.

[0101] These biomarkers can be measured in the form of a peptide, polypeptide, protein, or nucleic acid, or a group of one, or two, or three or more biomarkers in the form of peptides, polypeptides, proteins, and / or nucleic acids. The term "measured" indicates the presence or absence and / or quantity of said biomarker or of said group of biomarkers, preferably before and after addition of the agent.

[0102] The terms "quantity", "amount" and "level" are synonymous and generally well-understood in the art. The terms as used herein may particularly refer to an absolute quantification of a peptide, polypeptide, protein, or nucleic acid, or to a relative quantification of a peptide, polypeptide, protein, or nucleic acid, i.e., relative to another value such as relative to a reference value as taught herein.

[0103] An absolute quantity of a peptide, polypeptide, protein, or nucleic acid may be advantageously expressed as weight or as molar amount, or more commonly as a concentration, e.g., weight per volume or mol per volume.

[0104] A relative quantity of a peptide, polypeptide, protein, or nucleic acid may be advantageously expressed as an increase or decrease or as a fold-increase or fold-decrease relative to said another value, such as relative to a reference value as described elsewhere herein. Performing a relative comparison between first and second parameters (e.g., first and second quantities) may but need not require determining first the absolute values of said first and second parameters. For example, a measurement method may produce quantifiable readouts (such as, e.g., signal intensities) for said first and second parameters, wherein said readouts are a function of the value of said parameters, and wherein said readouts may be directly compared to produce a relative value for the first parameter vs. the second parameter, without the actual need to first convert the readouts to absolute values of the respective parameters.

[0105] A relative quantity of a biomarker may be determined as a percentage (fraction) or a fold-increase or fold-decrease of the amount of each biomarker before and after addition of the agent or by comparison of the amount of each biomarker measured after addition of the agent to the reference value of each biomarker. Accordingly, measuring the quantity of two or more biomarkers selected from any two or more (e.g. two, three, four or all five) of cofilin, MMP2, CTGF, TGFPR1 or SMA as taught herein may typically involve (i) determining the amount (or presence) of cofilin, MMP2, CTGF, TGFPR1 and / or SMA in the cell-culture environment, in or on a fibroblast or fibroblast population, or in the medium or the extracellular matrix of the gel, after addition of the agent, optionally also before addition of the agent, and (ii) calculating the fraction or fold-increase or fold-decrease of the amount of said biomarkers. The quantity of said biomarkers may be measured by any means known in the art. There are several techniques known in the art for measuring the amount of specific proteins, for example, ELISA, Mass Spectrometry or, preferably western blot.

[0106] As used herein, the term "fibroblast" or "fibroblasts" generally refers to a type of connective tissue cell, typically with a spindle shape, that can contribute to the formation of the extracellular matrix. Fibroblasts can typically synthesize and secrete elastin, various proteoglycans and collagens, such as collagen I and III. In particular embodiments, the fibroblasts can be fibroblast cell lines or primary fibroblasts obtained from a tissue, such as dermal tissue, cardiac tissue, adipose tissue, umbilical cord tissue, lung tissue, kidney tissue, intestinal tissue.

[0107] In particular embodiments, fibroblasts are cultured in vitro under appropriate conditions allowing the fibroblasts to transition from myofibroblasts into quiescent fibroblasts and vice versa. In certain embodiments, fibroblasts are grown under appropriate conditions in a fibrin gel, preferably resulting from the enzymatic reaction of fibrinogen with thrombin. In certain embodiments, the fibrin gel containing said fibroblasts is aligned uniaxially in a recipient after polymerisation of the fibrin gel. In certain embodiments, the fibrin gel is added to a removable mold in the recipient during polymerisation, wherein the mold can be removed after polymerisation of the fibrin gel. In certain embodiments, the fibroblasts seeded into the fibrin gel remodel the fibrin of the polymerised uniaxially aligned fibrin gel into a complex cell-derived extracellular matrix (ECM) mimicking the in vivo 3D environment. Typically, the complex cell-derived extracellular matrix is obtained after at least four days. In certain embodiments, the fibroblasts have replaced the provisional fibrin matrix into a complex cell- derived extracellular matrix, thereby restoring both the mechanical and chemical properties of the extracellular matrix and the fibroblasts have returned to quiescence. In certain embodiments, the fibroblasts replacing the fibrin gel into a complex cell-derived extracellular matrix, are typically myofibroblasts. After replacement of the fibrin gel into the complex cell-derived extracellular matrix (recreating the 3D in vivo context of fibroblasts), the fibroblasts can go into a quiescent state.

[0108] As used herein, the term "myofibroblast" or "myofibroblasts" generally refers to fibroblasts that are metabolically active and produce collagen, mostly collagen I and III. These myofibroblasts typically strongly express SMA and have increased collagen mRNA levels.

[0109] As used herein, the term "quiescence" or "quiescent state" generally refer to a specialized cellular state wherein the cells are metabolically inactive. The quiescent state of cells is typically temporary and reversible, wherein cells can resume their metabolically active state and optionally re-enter in an active state and / or re-enter the cell cycle when certain signal or conditions prompt the cells. In certain embodiments, fibroblasts in a quiescent state have decreased SMA levels, do not secrete collagen I and III and optionally do not stabilize focal adhesion. As used herein, the term "the active state" of a biological cell, such as a fibroblast, refers to the condition in which the cell is functionally engaged in its physiological processes, exhibiting metabolic and cellular activities associated with its normal or specific functions. In the case of a fibroblast, an active state would signify its ability to carry out activities related to extracellular matrix production, tissue repair, and other functions typical of fibroblasts.

[0110] In certain embodiments, recipient, and optionally mold, is produced through 3D printing, preferably by stereolithography. In embodiments, said recipient, and optionally mold, is produced using biocompatible materials, such as biocompatible resins.

[0111] In certain embodiments, the agent is added upon establishment of the complex cell-derived extracellular matrix or when the fibroblasts have turned into the quiescent state.

[0112] In certain embodiments, the agent is added after the fibroblasts have been cultured for at least 3 days or at least 4 days or at least 5 days.

[0113] In certain embodiments, the expression level of said at least three biomarkers is measured before and after addition of the agent or a combination of agents, for example at least a day after addition of the agent, at least 2 days, at least 3 days , at least 4 days , at least 5 days, preferably at least 6 days, preferably at least 7 days after addition of the agent or a combination of agents.

[0114] In certain embodiments, a fibrotoxicity index is established for an agent or combination of agents. The fibrotoxicity index can be determined by calculating the average expression level of at least 3 biomarkers markers, optionally of at least four biomarkers, or at least five biomarkers, preferably the average protein expression level. In embodiments, an average (or mean) expression level of at least 3 biomarkers selected from cofilin, MMP2, CTGF, TGFPR1 or SMA, or an average (or mean) expression level of at least four biomarkers of which at least three biomarkers are selected from cofilin, MMP2, CTGF, TGFPR1 or SMA, or an average (or mean) expression level of at least five biomarkers of which at least three biomarkers are selected from cofilin, MMP2, CTGF, TGFPR1 or SMA can be calculated. In embodiments, the agent or combination of agents are ranked according to their risk for inducing fibrosis.

[0115] For example, an average or mean may encompass an increase of the value of total expression level of at least three biomarkers, preferably of at least four biomarkers, more preferably all cofilin, MMP2, CTGF, TGFPR1 or SMA by, without limitation, at least about 20% (about 1.2-fold or more), or by at least about 30% (about 1.3-fold or more), or by at least about 40% (about 1.4-fold or more), or by at least about 50% (about 1.5-fold or more), or by at least about 60% (about 1.6-fold or more), or by at least about 70% (about 1.7-fold or more), or by at least about 80% (about 1.8-fold or more), or by at least about 90% (about 1.9-fold or more), or by at least about 100% (about 2-fold or more), or by at least about 150% (about 2.5-fold or more), or by at least about 200% (about 3-fold or more), or by at least about 500% (about 6-fold or more), or by at least about 700% (about 8-fold or more), or like, relative to the reference value with which a comparison is being made. In embodiments, an increase of at least 20% of the average expression level indicates that the agent is a risk for inducing fibrosis. In embodiments, an increase of at least 50% of the average expression level indicates that the agent is a high risk for inducing fibrosis.

[0116] In an exemplary embodiment, the in vitro method for determining, predicting, and / or monitoring fibrotoxicity of an agent, comprising: a. growing fibroblasts in a fibrin gel uniaxially aligned in a recipient to obtain a tissue engineered 3D cell-derived matrix gel, al. said fibroblasts replace the provisional fibrin matrix into a complex cell-derived extracellular matrix, b. adding the agent to the fibroblasts , c. detecting the expression level of at least three biomarkers each independently selected from the group consisting of: transforming growth factor-pi receptor (TGFPR1), cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF) and Smooth Muscle a-Actin (SMA) in the fibroblasts prior to (step b) and after adding the candidate agent to said fibroblasts (step b) and d. comparing the expression level of said at least three biomarkers to the expression level of said markers measured before and after said agent was added to said cell-culture environment, wherein an increase in total expression level of said at least three biomarkers indicates that the agent is fibrotoxic.

[0117] In an exemplary embodiment, the in vitro method for determining, predicting, and / or monitoring fibrotoxicity of an agent, comprising: a. growing fibroblasts in a fibrin gel uniaxially aligned in a recipient to obtain a tissue engineered 3D cell-derived matrix gel, a2. said fibroblasts turning to a quiescent state, b. adding the agent to the fibroblasts, c. detecting the expression level of at least three biomarkers each independently selected from the group consisting of: transforming growth factor-pi receptor (TGFPR1), cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF) and Smooth Muscle a-Actin (SMA) in the fibroblasts prior to (step b) and after adding the candidate agent to said fibroblasts (step b) and d. comparing the expression level of said at least three biomarkers to the expression level of said markers measured before and after said agent was added to said cell-culture environment, wherein an increase in total expression level of said at least three biomarkers indicates that the agent is fibrotoxic.

[0118] In an exemplary embodiment, the in vitro method for determining, predicting, and / or monitoring fibrotoxicity of an agent, comprising: a. growing fibroblasts in a fibrin gel uniaxially aligned in a recipient to obtain a tissue engineered 3D cell-derived matrix gel, al. said fibroblasts replace the provisional fibrin matrix into a complex cell-derived extracellular matrix, a2. said fibroblasts turn to a quiescent state, b. adding the agent to the fibroblasts , c. detecting the expression level of at least three biomarkers each independently selected from the group consisting of: transforming growth factor-pi receptor (TGFPR1), cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF) and Smooth Muscle a-Actin (SMA) in the fibroblasts prior to (step b) and after adding the candidate agent to said fibroblasts (step b) and d. comparing the expression level of said at least three biomarkers to the expression level of said markers measured before and after said agent was added to said cell-culture environment, wherein an increase in total expression level of said at least three biomarkers indicates that the agent is fibrotoxic.

[0119] In embodiments, the fibroblasts of step a) are preferably primary fibroblasts. In embodiments, prior to step a), the method comprises a step of providing fibroblasts, preferably primary fibroblasts.

[0120] In embodiments, in step a) the fibroblasts can be uniaxially aligned in the fibrin gel. In embodiments, the fibroblasts turn to a quiescent state in the complex cell-derived extracellular matrix.

[0121] A further aspect of the invention provides a method for use in screening an agent for inducing fibrosis or fibrotic disease.

[0122] Non-limiting examples of fibrosis, tissue fibrosis, or fibrotic diseases may include idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis, endomyocardial fibrosis, mediastinal fibrosis, bone marrow fibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, renal fibrosis, iatrogenic fibrosis, myocardial fibrosis, renal tubulointerstitial fibrosis, fibroids, liver fibrosis, fibrotic skin disease, keloidal scar, and oral submucous fibrosis. In a preferred embodiment, the fibrotic diseases or fibrosis is idiopathic pulmonary fibrosis (IPF). In certain embodiments, the method is a high throughput screening method.

[0123] In an aspect, the invention provides a kit for determining, predicting, and / or monitoring fibrotoxicity of an agent, the kit comprising:

[0124] - means for measuring the level of at least three biomarkers independently selected from the group consisting of: cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF), transforming growth factor-pi receptor (TGFPR1) and Smooth Muscle a-Actin (SMA); and

[0125] - a reference value or means for establishing said reference value, wherein said reference value represents a known value for determining, predicting and / or monitoring of fibrotoxicity.

[0126] In certain embodiments, the kit can be used for performing the method as disclosed herein.

[0127] In particular embodiments, the means for measuring the level of at least three biomarkers independently selected from the group consisting of: cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF), transforming growth factor-pi receptor (TGFPR1) and Smooth Muscle a-Actin (SMA) comprises at least three antibodies for independently detecting cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF), transforming growth factor-pi receptor (TGFPR1) or Smooth Muscle a-Actin (SMA). In particular embodiments, the means for measuring the level of the at least three biomarkers comprises at least three antibodies independently selected from the group consisting of: at least one antibody detecting cofilin, at least one antibody detecting MMP2, at least one antibody detecting CTGF, at least one antibody detecting TGFPR1, and at least one antibody detecting SMA.

[0128] As used herein, the term "antibody" is used in the broadest sense and generally refers to an immunologic binding agent. The term encompasses whole immunoglobulin molecules, immunologically effective fragments of immunoglobulins, i.e., fragments displaying the ability to specifically bind the antigen recognised by the whole immunoglobulin molecule, as well as constructs comprising an antigen-binding portion comprised within a modified immunoglobulin-like framework, and constructs comprising an antigen-binding portion comprised within a non- immunoglobulin-like framework or scaffold. Antibody fragments comprise a portion of an intact antibody comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv, and single-domain sdFv (sdFv) antibodies, such as VL, VH or VHH single-domain antibodies. Fusions proteins of the heavy (VH) and light (VL) chain variable regions, commonly known as single chain Fv (scFv), are also included in antibody fragments. The term "antibody" thus includes without limitation intact monoclonal antibodies, intact polyclonal antibodies, multivalent (e.g., 2-, 3- or more-valent) antibodies and / or multi-specific (e.g., bi- or more-specific) antibodies formed from at least two intact antibodies, and further immunologically effective fragments of any of such antibodies as well as multivalent and / or multi-specific composites of such fragments (e.g., diabodies, triabodies, tetrabodies, multibodies). The term further encompasses without limitation intact antibodies and antibody fragments of non-human animal origin, as well as chimeric, humanised or chimeric / humanised forms of such antibodies or antibody fragments, and further encompasses fully human antibodies or antibody fragments. More broadly, grafting of at least one complementaritydetermining region (CDR) from an antibody of one origin onto a framework of another origin is contemplated. The term "antibody" also encompasses any fusion proteins, protein conjugates or protein complexes comprising an immunoglobulin molecule or an immunologically effective fragment thereof, as well as chemically and / or enzymatically modified or derivatised immunoglobulin molecules or immunologically effective fragments thereof. The term "antibody" is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide which is made to encompass at least one CDR capable of specifically binding to an epitope on a cognate antigen, regardless whether such molecules are produced in vitro, in cell culture, or in vivo. For example, antibodies produced by recombinant DNA techniques in cultured host cells (e.g., bacterial, yeast or fungal, plant or animal cells) or in non-human host organisms (e.g., in transgenic plants or transgenic animals) are also encompassed.

[0129] In particular embodiments, the reference value for the quantity of in vitro fibroblasts expressing of secreting any one or more of cofilin, MMP2, CTGF, TGFPR1 or SMA may be determined by determining the quantity of in vitro fibroblast expressing or secreting any one or more of cofilin, MMP2, CTGF, TGFPR1 or SMA, respectively, in reference fibroblasts, thereby producing a reference value. Preferably, the reference value for the quantity of any one or more of cofilin, MMP2, CTGF, TGFPR1 or SMA expressed or secreted by the in vitro fibroblasts may be determined by determining the quantity of any one or more of cofilin, MMP2, CTGF, TGFPR1 or SMA, respectively, in reference fibroblasts before the agent is added to the fibroblasts, thereby producing a reference value.

[0130] One or more reference values for cofilin, MMP2, CTGF, TGFPR1 or SMA obtained from one or more reference fibroblasts may be used to determine a threshold or cut-off value as generally known in the art to provide for a certain degree of fibrosis or fibrotic potential of the agent.

[0131] One or more reference values for a combination of at least three, four or all five of cofilin, MMP2, CTGF, TGFPR1 or SMA can be obtained from one or more reference fibroblasts to determine a threshold or cut-off value as generally known in the art to provide for a certain degree of fibrosis or fibrotic potential of the agent. In certain embodiments, a threshold or cut-off value may encompass an increase of the value of average expression level for a combination of any three or more, four or more, or all five biomarkers selected from cofilin, MMP2, CTGF, TGFPR1 or SMA, without limitation, at least about 20% (about 1.2-fold or more), or by at least about 30% (about 1.3-fold or more), or by at least about 40% (about 1.4-fold or more), or by at least about 50% (about 1.5-fold or more), or by at least about 60% (about 1.6-fold or more), or by at least about 70% (about 1.7-fold or more), or by at least about 80% (about 1.8-fold or more), or by at least about 90% (about 1.9-fold or more), or by at least about 100% (about 2-fold or more). In certain embodiments, an increase of at least about 20% (about 1.20-fold or more), or by at least about 21% (about 1.21-fold or more), or by at least about 22% (about 1.22-fold or more), or by at least about 23% (about 1.23-fold or more), or by at least about 24% (about 1.24-fold or more), or by at least about 25% (about 1.25-fold or more), or by at least about 26% (about 1.26-fold or more), or by at least about 27% (about 1.27-fold or more), or by at least about 28% (about 1.28-fold or more), or by at least about 29% (about 1.29-fold or more), or by at least about 30% (about 1.30-fold or more) of the average expression level for a combination of any three or more, four or more, or all five biomarkers selected from cofilin, MMP2, CTGF, TGFPR1 or SMA can indicated an intermediate risk for induction of fibrosis. In embodiments, an increase of at least 45% (about 1.45-fold or more), or by at least about 46% (about 1.46-fold or more), or by at least about 47% (about 1.47-fold or more), or by at least about 48% (about 1.48-fold or more), or by at least about 49% (about 1.49-fold or more), or by at least about 50% (about 1.50-fold or more), or by at least about 51% (about 1.51-fold or more), or by at least about 52% (about 1.52-fold or more), or by at least about 53% (about 1.53-fold or more), or by at least about 54% (about 1.54-fold or more), or by at least about 55% (about 1.55-fold or more), or by at least about 56% (about 1.56-fold or more), or by at least about 57% (about 1.57-fold or more), or by at least about 58% (about 1.58-fold or more), or by at least about 59% (about 1.59-fold or more), or by at least about 60% (about 1.6-fold or more), ), or by at least about 61% (about 1.61-fold or more), or by at least about 62% (about 1.62-fold or more), or by at least about 63% (about 1.63-fold or more), or by at least about 64% (about 1.64-fold or more), or by at least about 65% (about 1.65-fold or more), or by at least about 66% (about 1.66-fold or more), or by at least about 67% (about 1.67-fold or more), or by at least about 68% (about 1.68-fold or more), or by at least about 69% (about 1.69-fold or more), or by at least about 70% (about 1.70-fold or more) of the average expression level indicates that the agent is a high / major risk for inducing fibrosis.

[0132] In particular embodiments, the values, measured after administration of the agent or combination of agents to the fibroblast, for any three or more biomarker selected from cofilin, MMP2, CTGF, TGFPR1 or SMA, preferably at least four or more biomarker selected from cofilin, MMP2, CTGF, TGFPR1 or SMA, more preferably all biomarker selected from cofilin, MMP2, CTGF, TGFPR1 an SMA or compared to a reference value or cut-off value representing fibroblasts having no fibrotoxic capacity.

[0133] For example, an average or mean may encompass an increase of the value of average expression level of at least three biomarkers, preferably of at least four biomarkers, more preferably all cofilin, MMP2, CTGF, TGFPR1 or SMA by, without limitation, at least about 20% (about 1.2-fold or more), or by at least about 30% (about 1.3-fold or more), or by at least about 40% (about 1.4-fold or more), or by at least about 50% (about 1.5-fold or more), or by at least about 60% (about 1.6-fold or more), or by at least about 70% (about 1.7-fold or more), or by at least about 80% (about 1.8-fold or more), or by at least about 90% (about 1.9-fold or more), or by at least about 100% (about 2-fold or more), or by at least about 150% (about 2.5-fold or more), or by at least about 200% (about 3-fold or more), or by at least about 500% (about 6-fold or more), or by at least about 700% (about 8-fold or more), or like, relative to the reference value with which a comparison is being made. In embodiments, an increase of at least 20% of the average expression level indicates that the agent is a risk for inducing fibrosis. In embodiments, an increase of at least 50% of the average expression level indicates that the agent is a high / major risk for inducing fibrosis.

[0134] Preferably, a deviation may refer to a statistically significant observed alteration. For example, a deviation may refer to an observed alteration, which falls outside of error margins of reference values in given reference cells (as expressed, for example, by standard deviation or standard error, or by a predetermined multiple thereof, e.g., ±lxSD or ±2xSD or ±3xSD, or ±lxSE or ±2xSE or ±3xSE). Deviation may also refer to a value falling outside of a reference range defined by values in given reference cells (for example, outside of a range which comprises >40%, > 50%, >60%, >70%, >75% or >80% or >85% or >90% or >95% or even >100% of values in given reference cells).

[0135] In certain preferred embodiments, said agent is a (candidate) drug, medicine or pharmaceutical composition and said kit is used to determine drug-induced fibrosis.

[0136] In certain embodiments, the kit can comprise a recipient, and optionally a mold, for uniaxially aligning fibroblasts. In embodiments said recipient comprises fibroblasts embedded in a fibrin gel, preferably resulting from the enzymatic reaction between fibrinogen and thrombin. In certain embodiments said fibrin gel containing said fibroblasts is aligned uniaxially in said recipient to obtain a tissue-engineered 3D cell-derived fibrin gel matrix. In certain embodiments said recipient is produced through 3D printing, preferably by stereolithography. In certain embodiments said recipient is produced using biocompatible resins.

[0137] A further aspect of the invention provides a computer-implemented method for assessing the fibrotoxicity of an agent comprising the ranking of agents according to their risk of fibrotoxicity.

[0138] In certain embodiments, an agent or combination of agents having an increased average expression level of at least three biomarkers, or at least four biomarkers or at least five biomarkers by at least about 20% (about 1.2-fold or more), or by at least about 30% (about 1.3-fold or more), or by at least about 40% (about 1.4-fold or more), or by at least 45% (about 1.45-fold or more) after administration of the agent, e.g., to fibroblasts, relative to the reference value or the average value of these biomarkers before administration of the agent or combination thereof, can be ranked as moderate risk for inducing fibrosis. In certain embodiments, an agent or combination of agents having an increased average expression level of at least three biomarkers, or at least four biomarkers or at least five biomarkers or by at least about 50% (about 1.5-fold or more), or by at least about 60% (about 1.6-fold or more), or by at least about 70% (about 1.7-fold or more), or by at least about 80% (about 1.8-fold or more), or by at least about 90% (about 1.9-fold or more), or by at least about 100% (about 2-fold or more) after administration of the agent, e.g., to fibroblasts, relative to the reference value or the average value of these biomarkers before administration of the agent or combination thereof. In embodiments, at least three of at least three biomarkers, or at least four biomarkers or at least five biomarkers are selected from cofilin, MMP2, CTGF, TGFPR1 or SMA, preferably at least TGFPR1, SMA and CTGF.

[0139] A further aspect of the invention provides, a use of the kit for determining the fibrotoxicity of an agent or for determining whether or not a candidate agent could induce fibrosis or a fibrotic disease.

[0140] A further aspect of the invention provides, the use of the method as described herein for determining fibrotoxicity of a drug or candidate drug.

[0141] EXAMPLES

[0142] Example 1

[0143] Freshly isolated primary fibroblasts cultured for a maximum of 2 passages, to preserve the mechanical memory, were embedded in a fibrin gel. Primary fibroblasts were isolated either from mice or humans using a lysis buffer containing a mixture of 0.25% trypsin without EDTA, collagenase IV or A respectively (4mg / ml) and calcium chloride (0.3mg / mL) up to 2 hours at 37°C. After mechanical dissociation, cells were passed through a 100pm cell strainer. Cells were centrifuged at 1800 rpm for 5 minutes and resuspended in DMEM F-12 medium supplemented with 10% Fetal Bovine Serum (FBS), Antibiotics and anti-mycotic (Penicillin, Streptomycin, Amphotericin B), 1% L-glutamine. Cells were cultured in standard conditions (37 °C in 5% carbon dioxide). Fibroblasts adhere within 3h, at which time the culture medium was changed, removing non-adherent cells. Then, the medium was changed every other day until cells reached ~80% confluence. Primary fibroblasts were then trypsinized and then suspended in fibrin gels. This fibrin clot results from the enzymatic reaction of the thrombin with the fibrinogen. Between 1.5 x 105and 2.5 x 105cells were re-suspended in 300pL of complete DMEM F12 containing 4mg / ml of fibrinogen, 8U / mL of thrombin and 0.2mM of L-ascorbate-2-phosphate. Cell suspensions were incubated for 20 minutes at 37°C to induce polymerization of the fibrin. This gel was anchored uniaxial ly around an anchoring piece, described in Figure 1, 3D-printed by stereolithography using biocompatible resins (BioMedclear Formlabs). Using this uniaxial anchoring, primary fibroblasts return to quiescence over time. To avoid contamination, plates were exposed to ultraviolet irradiation in a biological safety cabinet for at least 30 minutes. Matrix constructs were cultured at 37 °C in 5% carbon dioxide with complete DMEM F-12 supplemented with 0.2mM of L-ascorbate-2- phosphate to promote matrix assembly. The culture medium was changed every other day for a maximum of 7 days. After several days, the myofibroblasts had replaced the provisional fibrin matrix into a complex cell- derived extracellular matrix restoring both the mechanical and chemical properties of the extracellular matrix (reservoir function) and getting back to quiescence. Some drugs are known to promote or maintain the persistence of myofibroblasts and therefore induce fibrotoxicity. To test the potential fibrotoxicity of drug candidates, matrix constructs were treated on day 4 and fibrotic markers expression was measured on day 7 by western blot (or other proteomic technics). To detect fibrotoxicity, a combination of intracellular and extracellular markers (TGFBR1, CTGF, MMP2, SMA, Cofilin) were used and a fibrotoxicity index was established.

[0144] Example 2 - Fibrotoxicity heat map

[0145] A heatmap was determined for all five biomarkers (TGFBR1 (ALK5), CTGF, MMP2, SMA, Cofilin) for four agents: adenosine (lpM and 10pM)(MedChemExpress, New Jersey, USA), amiodarone (lOpM) (MedChemExpress, New Jersey, USA), cabergoline (10pM)(MedChemExpress, New Jersey, USA), and benfluorex (lOpM) (MedChemExpress, New Jersey, USA). These agents are known to induce fibrosis, particularly iatrogenic fibrosis. The increase in protein expression level of each biomarker for each agent is shown in figure 1.

[0146] Example 3 - FT5o of benfluorex

[0147] The FT5o was determined for benfluorex. The FT50, represents a fibrotoxic dose 50, the concentration of the agent tested that generates 50% of the maximum expression of any marker. As shown in figure 2 the FTsofor benfluorex is 694.1nM.

[0148] Example 4 - Fibrotoxicity index

[0149] The fibrotoxicity index is defined as the average of several fibrotic markers. After 4 days of development, 3D-tissue-engineered-cell-derived matrix gels were treated with lpM Adenosine, lOpM Adenosine, lOpM cabergoline, lOpM Amiodarone, lOpM Benfluorex, or lOOnM doxorubicin - agents known to induce iatrogenic fibrosis. Proteins were extracted at day 4 (prior treatment) and day 7 (3 days after treatment). For this protein extraction, frozen matrix samples were thawed at 4°C for 5 minutes and disrupted at 4°C using a bullet blender (Next Advance) with 1.6mm steel beads in protein extraction buffer containing 1.1% Sodium dodecyl sulfate (Sigma, L3771) , 0.3% sodium deoxycholate (Sigma, D6750), 25mM dithiothreitol (Sigma, 1114740001), complete anti-protease and antiphosphatase (Roche, 4693159001, 4906837001) in a 25mM ammonium bicarbonate (Sigma, A6141) buffer, pH 9.2. After centrifugation, the supernatants were mixed in Bolt LDS Sample Buffer (ratio 3:1 v / v, Invitrogen, B0007) and heated for 10 minutes at 95°C. Samples were loaded onto Bolt™ 4-12% BisTris gels (Invitrogen, NW04120BOX) for electrophoresis in MOPS SDS Running Buffer (Invitrogen, B0001) complemented with antioxidants (Invitrogen, BT0005). The transfer was performed using Tris- Glycine in Methanol buffer (respectively 3g / L - 14,3g / L in 1:5 (v:v) methanol: distilled water) (Merck, 1083870500 ; Sigma, G8126 ; Sigma, 179957) at 100V for lh on nitrocellulose membrane (Amersham™ Protran™0.45pm NC, GE Healthcare Life Science, 10600002). Then, membranes were blocked with Odyssey PBS blocking buffer (Li-cor Biosciences, 927-70003 ) and incubated with primary antibodies overnight at 4°C. Fibrotic markers were assayed by immunoblotting using p-actin as a loading control. Fluorescence intensity was measured by a fluorescent scanner (a Sapphire Biomolecular imager)and quantified. Data were normalized on p-actin (Sigma, A5441)for each sample. For the purpose of the example, the fibrotoxicity index presented is the average of 3 markers selected from the group consisting of SMA (Sigma, A2547), CTGF (Proteintech, 23936), TGFBR1 (Abeam, ab31013) , MMP2 (Proteintech, 66366), and cofilin (Proteintech, 66057), as shown in Figure 3; the average of 4 markers selected from the group consisting of SMA, CTGF, TGFBR1, MMP2, and cofilin, shown in Figure 4, and the average of 5 markers (SMA, MMP2, CTGF, Cofilin, TGFBR1), shown in Figure 5. The benfluorex (Mediator), withdrawn from the market in 2009 due to the unexpected development of severe valvular fibrosis, generated a high fibrotoxicity index score as shown in figures 3 to 5. The values of major, intermediate and minor risks are arbitrarily defined for the purpose of the example. As an example, the situation at day 4, prior to any treatment (the fibroblasts are still activated in myofibroblasts) was considered as 100% and the situation at day 7 (the fibroblasts are back to quiescence) was considered as 0%. The percentage presented in the examples is where the average of the marker combination is between those 2 control situations.

[0150] In Figure 6, the difference in dose of doxorubicin leading to cytotoxicity (CC5o) is compared to the dose of doxorubicin leading to fibrotxicity (FT5o).

[0151] This assay was performed on cardiac fibroblasts using the same protocol as the fibrotoxic assessment (heatmap / fibrotoxic index) above. Primary cardiac fibroblasts were embedded in a fibrin gel anchored uniaxially. The artificial tissue, also called cell-derived matrix model, was treated on day 4 for a duration 1 of three days. After this treatment period, the cell-derived matrix constructs were snap-frozen, and proteins were extracted for western blot analysis.

[0152] Beta-actin is a housekeeping gene, and a significant decrease in Beta-actin expression indicates cell death. Therefore, this marker was used to determine the CC5o- TGF-beta-receptor 1 (TGFBR1) expression was determined for the (FT5o).

[0153] Such a measurement currently does not exist, although it may significantly impact patient health. While the cytotoxic dose (CC5o) is a regulatory requirement, and hence is documented for most agents used in medicine or food, the fibrotoxic dose (FT5o) occurs at concentrations much lower than the cytotoxic doses (see figure 6), potentially leading to dosages of agents being allowed for human use at which they can cause fibrotoxicity.

[0154] The molecular signature presented in the heatmap has been designed to identify any fibrotoxic events based on various pathway activations (cf. e.g. Figure 1). Consequently, the FibroToxic index is defined as the average of at least three fibrotic markers. However, the fibrotoxic dose 50 (cf. Figure 6) is determined from any individual fibrotic marker depending on the pathway affected.

Claims

CLAIMS1. An in vitro method for determining, predicting, and / or monitoring fibrotoxicity of an agent, comprising: a. growing fibroblasts in a fibrin gel that is uniaxially aligned in a recipient to obtain a tissue engineered 3D cell-derived matrix gel, b. adding the agent to the fibroblasts of step a), c. detecting the expression level of at least three biomarkers each independently selected from the group consisting of: transforming growth factor-pi receptor (TGFPR1), cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF) and Smooth Muscle a-Actin (SMA) in the fibroblasts prior to (step b) and after adding the candidate agent to said fibroblasts (step b) and d. comparing the expression level of said at least three biomarkers to the expression level of said markers measured before and after said agent was added to said cell-culture environment, wherein an increase in total expression level of said at least three biomarkers indicates that the agent is fibrotoxic.

2. The method according to claim 1, comprising detecting at least four, preferably at least five, biomarkers in said cell-culture environment.

3. The method according to claim 1 or 2, wherein said fibroblasts are primary fibroblasts.

4. The method according to anyone of claims 1 to 3, wherein in step a) said fibroblasts are grown in a culture medium containing fibrinogen and thrombin, or in a fibrin gel resulting from the enzymatic reaction of fibrinogen with thrombin.

5. The method according to anyone of claims 1 to 4, wherein the fibrin gel containing said fibroblasts is added to a removable mold in said recipient during polymerisation, wherein the mold can be removed after polymerisation of the fibrin gel.

6. The method according to anyone of claims 1 to 5, wherein said recipient is produced through 3D printing, preferably by stereolithography, and / or wherein said recipient is produced using biocompatible resins.

7. The method according to anyone of claims 1 to 6, wherein the fibroblasts are aligned for at least 3 days, preferably for at last 4 days.

8. The method according to anyone of claims 1 to 7 , wherein said fibroblasts are in a quiescent state.

9. The method according to anyone of claims 1 to 8, wherein said fibroblasts replace the provisional fibrin matrix into a complex cell-derived extracellular matrix and said fibroblasts turning to a quiescent state .

10. The method according to anyone of claims 1 to 9, wherein the expression level of said at least three biomarkers is measured at least 3 days after addition of the agent or a combination of agents, preferably after at least 4 days, preferably after at least 5 days.

11. A method for screening an agent for inducing fibrosis or fibrotic disease according to anyone of claims I to 10, preferably selected from the group consisting of: idiopathic pulmonary fibrosis (I PF), pulmonary fibrosis, endomyocardial fibrosis, mediastinal fibrosis, bone marrow fibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, renal fibrosis, iatrogenic fibrosis, myocardial fibrosis, renal tubulointerstitial fibrosis, fibroids, liver fibrosis, fibrotic skin disease, keloidal scar, and oral submucous fibrosis.

12. A kit for determining, predicting, and / or monitoring fibrotoxicity of an agent, using the method according to anyone of claims 1 to 11, the kit comprising:- means for measuring the level of at least three biomarkers each independently selected from the group consisting of: transforming growth factor-pi receptor (TGFPR1), cofilin, Matrix Metalloproteinase 2 (MMP2), connective tissue growth factor (CTGF) and Smooth Muscle a-Actin (SMA); and- a reference value or means for establishing said reference value, wherein said reference value represents a known value for determining, predicting and / or monitoring of fibrotoxicity.

13. The kit according to claim 12, wherein said kit further comprises a recipient for uniaxially aligning fibroblasts, preferably wherein said recipient comprises fibroblasts embedded in a fibrin gel, preferably resulting from the enzymatic reaction between fibrinogen and thrombin.

14. The kit according to claim 13, wherein said fibrin gel containing said fibroblasts is aligned uniaxially in said recipient to obtain a tissue engineered 3D cell-derived fibrin gel matrix, preferably wherein said recipient is produced through 3D printing, preferably by stereolithography, more preferably wherein said recipient is produced using biocompatible resins.

15. Use of the kit according to any one of claims 13 or 14, for determining the fibrotoxicity of an agent or for determining whether or not a candidate agent could induce fibrosis or a fibrotic disease.

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