Antibody against skeletal troponin t and diagnostic uses thereof

Abstract

The present invention refers, inter alia, to an antibody against skeletal Troponin T (skTnT), wherein the anti-skTnT antibody is not capable of binding to cardiac Troponin T (cTnT). The present invention also relates to uses of said antibodies for determining a skeletal muscle damage, methods of determining the amount of skTnT, wherein the method comprises contacting a sample with at least one of said antibodies, and determining the amount of skTnT. The invention further refers to corresponding polynucleotides encoding for and compositions comprising said antibody. The invention also relates to a computer-implemented method of determining an amount of skTnT using said antibody.

Description

ANTIBODY AGAINST SKELETAL TROPONIN T AND DIAGNOSTIC USES THEREOF

[0001] The present application claims priority of the European Patent application No. 23219580.0, filed December 22, 2023, the disclosure of which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to an antibody against skeletal Troponin T, as well as to uses thereof. The invention further relates to a composition comprising at least two of the antibodies, a polynucleotide encoding the antibody, and a method for determining an amount of a skeletal Troponin T comprising contacting a sample with at least one of the antibodies, and determining the amount of skeletal Troponin T. The invention also relates to a computer implemented method for determining an amount of skeletal Troponin T.BACKGROUND OF THE INVENTION

[0003] Troponins are regulatory protein complexes that are involved in the contraction mechanism of striated muscle by forming complexes with tropomyosin and actin. Troponins are built of three monomeric subunits, namely troponin I (Tnl), troponin C (TnC), and troponin T (TnT). TnT has a comparable high binding affinity to tropomyosin, Tnl and TnC, which can bind calcium ions in addition to the other two troponin monomers Tnl and TnT. When calcium ions are released from the sarcoplasmic reticulum, said ions can bind TnC, which leads to a conformational change of Tnl. This results in an exposure of myosin binding sites on the actin filaments previously blocked by tropomyosin, while binding of myosin heads to actin is now possible. Thus, in the presence of calcium ions, tropomyosin, which obscures the myosin-binding site on actin, is displaced via conformational change and displacement of the troponin complex on the actin filament, thereby triggering muscle contraction. Thus, troponins are integral to the contraction of muscles with TnT anchoring the troponin complex onto the actin filament and transducing calcium-dependent conformational changes induced by binding of calcium to TnC and conformational change of Tnl to configure the actin filament and regulate muscle contraction and relaxation.

[0004] In case of the troponin subunits Tnl and TnT different isoforms exist in humans, which can show, e.g., tissue-specific expression. Accordingly, Tnl and TnT isoforms with cardiac or skeletal muscle restricted expression may also referred to as skeletal Tnl and cardiac Tnl as well as skeletal TnT (skTnT) and cardiac TnT (cTnT), respectively. Furthermore, Tnl and TnT isoforms can be distinguished based on the muscle type, wherein the respective isoform is expressed. Muscles like skeletal muscles comprise slow-twitch and / or fast-twitch muscle fibers and may also be referred to as slow-twitch or fast-twitch muscles depending on their respective predominant fiber type. While for example human skeletal muscles in the back or the back of the lower legs are primarily built of slow-twitch fibers, fast-twitch muscle fibers can be found for example as primary fiber type in smaller muscles in hands and around eyes enabling rapid movements. Fast-twitch muscle fibers can also be found in skeletal muscles as these fibers have the ability to contract quickly and thus, enable high-intensity activities for short periods, like sprinting and jumping. In case of TnT, vertebrates exhibit three genes that encode for fiber-type-specific TnT genes with TNNT1 encoding slow skeletal muscle TnT (sskTnT), TNNT2 encoding cTnT, and TNNT3 encoding fast skeletal muscle TnT (fskTnT). Moreover, alternative splicing adds further complexity to the biological function of troponins.

[0005] Troponins can offer valuable insights into biological processes and deviations thereof. For example, cardiac troponin T is released into the blood upon heart muscle damage, necrosis and / or degeneration. Thus, cTnT release into the blood is considered indicative for cardiac diseases such as, e.g., myocarditis, acute coronary syndrome and acute myocardial infarction (AMI). Especially cardiac troponins represent important biomarkers, e.g., for the diagnosis of AMI due to their specificity for myocardial cell necrosis and a superior sensitivity than creatine kinase muscle brain (CKMB), which has been used as indicator of AMI before the development of cardiac troponin assays. Furthermore, elevated cardiac troponin concentrations may also be observed, e.g., in cases of non-ischemic cardiac diseases. Accordingly, serum levels of cTnT are commonly considered as important biomarkers, e.g., for detection, (differential) diagnosis and / or prognosis of heart diseases, for predicting pre- operatively a perioperative risk of major adverse cardiac events in the context of non-cardiac surgeries as well as in diagnosis of perioperative myocardial infarction and myocardial injuries after non-cardiac surgeries.

[0006] In comparison with coronary artery diseases, skeletal muscle diseases (SMDs) are rare diseases. In particular, SMD relates to a poorly defined, heterogeneous group of comparably rare disorders affecting skeletal musculature. For example, while coronary artery disease may affect more than 1 ,500 subjects out of 100,000, less than 100 subjects are affected by SMD. Earlier studies have reported that not only cardiac Troponin levels are - in principle - detectable in blood samples, but that also skeletal Troponin is detectable e.g. in patients suffering from a SMD (Simpson et al., Clinical Chemistry, Volume 51 , Issue 6, 1 June 2005). However, elevated serum concentrations of skeletal troponins may not only known to be caused by SMD, but have also been reported in case of skTnl, e.g., after statin treatment, maximal eccentric contractions and after marathons (cf. e.g. Chapman et al., Science and Medicine in Sport 2013; Trentini et al., BiochemMed (Zagreb), 2019.; Paana et al., International Journaly of Cardiology, 2019).

[0007] Accordingly, there is a need for providing reliable, quantitative detection of skeletal Troponin in human subjects.SUMMARY OF THE INVENTION

[0008] The present invention addresses the above mentioned need by inter alia providing the antibodies, the uses, the methods, the compositions, the polynucleotides, the vectors and the kits having the features of the respective independent claims.

[0009] In a first aspect, the invention provides an antibody against skeletal Troponin T (skTnT), wherein the anti-skTnT antibody is not capable of binding to cardiac Troponin T (cTnT).

[0010] Thus, by the present invention, for the first time, specific anti-skTnT antibodies are provided having the capacity of distinguishing between skTnT and cTnT polypeptides. The inventors surprisingly found that despite the high degree of conservation between skTnT and cTnT polypeptides it is possible to generate antibodies highly specific for skTnT polypeptides.

[0011] Advantageously, these antibodies are suitable for reliable quantification of skeletal Troponin in human subjects. For example, the antibodies as herewith provided thus enable determination of skeletal muscle damages. SMDs such as myopathy, myositis, skeletal myopathy, muscular dystrophy such as Duchenne muscular dystrophie, rhabdomyolysis, neuropathy, myasthenic syndrome and autosomal dominant (AD) with muscle symptoms, myotonic dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, mitochondrial disease, and glycogen storage disease; dermatomyositis, polymyositis, sporadic inclusion body myositis, hereditary inclusion body myositis, or immune-mediatednecrotizing myositis may be accurately detected and / or a respective disease progression may be monitored. It is further envisaged that even predictions of a further disease course may be more accurately made using the antibodies as herein described. A further advantage of the antibody as herewith provided is the possibility of determining e.g. a SMD in a sample of a subject that may - at the same time - suffer from e.g. a cardiac disease. In particular, since the antibody is not capable of binding to cTnT, the risk of false positive outcomes is avoided when determining a SMD in these situations.

[0012] A further advantage is that the specific antibodies as herewith provided, which distinguish between cTnT and skTnT polypeptides, are suitable for e.g. determining a level of a physical fitness of a subject such as a human subject._The terms “specificity” or “specific” with respect to an antibody as herein provided, means that the antibody may specifically bind to and immunologically recognize its target, e.g., skTnT, with high avidity. In the context of the present application, “specificity” of an antibody for skTnT is furthermore understood to mean that there is no crossreactivity of the antibody with cTnT polypeptides.

[0013] An additional benefit of the antibodies as herein provided is their improved suitability for accurate monitoring diagnostics during treatment with drugs such as e.g. medical drugs. As described above, it has been reported earlier that e.g. after statin treatment plasma levels of skeletal Troponin are increased. Similarily, skeletal muscle damage may be induced in cancer patients treated with immune checkpoint inhibitors (ICI) or other treatments. Thus, the provision of the antibodies as described herein is a powerful tool for e.g. monitoring diagnostics during treatment with pharmaceuticals.

[0014] In a second aspect, the invention provides a use of an antibody as described herein for determining a skeletal muscle damage.

[0015] In a third aspect, the invention provides a method of determining the amount of skeletal Troponin T (skTnT), the method comprising a) Contacting a sample with at least one antibody as herein described, and b) Determining the amount of skT nT.

[0016] In a fourth aspect, the invention provides a polynucleotide encoding an antibody as provided by this invention.

[0017] In a fifth aspect, the invention provides a vector comprising the polynucleotide.

[0018] In a sixth aspect, the invention provides a composition comprising at least two antibodies against skTnT as described herein, wherein(i) the at least two antibodies are capable of simultaneously binding to fskT nT and at least one of said at least two antibodies is not capable of binding to sskTnT; or(ii) the at least two antibodies are capable of simultaneously binding to sskTnT and at least one of said at least two antibodies is not capable of binding to fskTnT; or(iii) the at least two antibodies are capable of simultaneously binding to sskTnT; and wherein the at least two antibodies are capable of simultaneously binding to fskTnT.

[0019] An advantage of this composition according to (i) is e.g. that it may be used in a sandwich assay (which is described in more detail below) for (e.g. quantitative) detection of fskTnT, since the use of the antibody not capable of binding to sskTnT e.g. as capture or detection antibody, may assure specific detection of fskTnT.

[0020] An advantage of this composition according to (ii) is e.g. that it may be used in a sandwich assay (which is described in more detail below) for (e.g. quantitative) detection of sskTnT, since the use of the antibody not capable of binding to fskTnT e.g. as capture or detection antibody, may assure specific detection of sskTnT.

[0021] An advantage of this composition according to (iii) is e.g. that it may be used in a sandwich assay (which is described in more detail below) for detection of total skTnT, since both antibodies may be capable of detecting both sskTnT and fskTnT, and thus use of one of these antibodies as capture antibody and the other antibody as detection antibody may assure specific detection of total skT nT.

[0022] If two of the above described compositions (i), (ii) and (iii) are combined (e.g. by performing two independent sandwich assays), by using a method as provided according to the seventh aspect below, it may be possible to determine (quantitatively) an amount of sskTnT, fskTnT and total skTnT in a sample of a subject e.g. by only making use of two antibodies as herein provided.

[0023] In a seventh aspect, the invention provides a computer-implemented method of determining an amount of skeletal Troponin T (skTnT), said method comprising(a) Receiving a first signal indicative of fast skTnT (fskTnT) and a second signal indicative of slow skTnT (sskTnT), or receiving a first signal indicative of fskTnT and a second signal indicative of both sskTnT and fskTnT; or receiving a first signal indicative of (sskTnT) and a second signal indicative of both sskTnT and fskTnT; and(b) Determining the amount of skTnT based on the received first and secondsignals, if the first signal is indicative of fast skTnT (fskTnT) and the second signal is indicative of slow skTnT (sskTnT); or the amount of sskTnT if the first signal is indicative of fskTnT and a second signal indicative of both sskTnT and fskTnT, or the amount of fskTnT if the first signal indicative of sskTnT and the second signal indicative of both sskTnT and fskTnT; wherein at least one of the received signals corresponds to the binding of at least one antibody as herein described to skTnT.

[0024] In an eighth aspect, the invention provides a computer-readable medium having stored thereon a program code for executing the computer-implemented method as herein provided when run on a computer.

[0025] All aspects of the invention provide the above described advantages and improvements related to the provision of the highly specific antibodies against skTnT, being able to distinguish between cTnT and skTnT polypeptides.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the drawings, in which:

[0027] Figs. 1 shows a Sequence alignment of human cTnT, sskTnT and fskTnT. Shown are canonical sequences.

[0028] Fig. 2 shows a kinetic screening with exemplary kinetic signatures of antibody / TnT interactions for monoclonal Antibodies (mAbs) 15B5 (top), 16B9 (middle) and 16F11 (bottom) according to the present invention. Shown is the binding to (A) sskTnT (B) fskTnT (C) cTNT to with 150 nM TnT concentrations, Interactions with significant target binding are overlaid with a Langmuir 1:1 dissociation fitting model (grey).

[0029] Fig. 3 exemplary shows obtained binding signatures of deselected antibodies from the kinetic screening due to no binding, artefact binding, too slow complex formation velocities, fast dissociation or biphasic dissociation, or a combination of slow complex formation and biphasic dissociation.

[0030] Fig. 4 shows a kinetic screening with exemplary kinetic signatures of antibody / TnT interactions for mAbs 1A6, 2H2, 4C8 and 2A10 (top to bottom) according to the present invention. Shown is the binding to binding to (A) sskTnT and (B) fskTnT with 150 nM TnT concentrations, Interactions with significant target binding are overlaid with a Langmuir1 :1 dissociation fitting model (grey).

[0031] Fig. 5 shows results of the detailed kinetic characterization of mAbs 15B5, 16B9 and 16F11 (left to right) binding fskTnT according to the present invention. Shown are series of fskTnT-concentrations, c = 3-240 nM, with duplicates for concentration 26.7 nM, wherein Figure 5 A) shows measured kinetic profiles (black) being overlaid with the Langmuir 1 :1 binding Model, Rl =0, Rmax global with allowed correction for mass transport limitation (grey), and wherein Figure 5 B) shows measured kinetic profiles (grey) overlaid with visualized MTL- corrected profiles (black).

[0032] Fig. 6 shows results of the detailed kinetic characterization of mAbs 2H2, 4C8 and 2A10 (left to right) binding fskTnT according to the present invention. Shown are series of fskTnT-concentrations, c = 3-240 nM, with duplicates for concentration 26.7 nM, wherein Figure 6 A) shows measured kinetic profiles (black) being overlaid with the Langmuir 1 :1 binding Model, Rl =0, Rmax global with allowed correction for mass transport limitation (grey), and wherein Figure 6 B) shows measured kinetic profiles (grey) overlaid with visualized MTL- corrected profiles (black).

[0033] Fig. 7 shows results of the detailed kinetic characterization for monoclonal antibodies (mAbs) 15B5, 16B9 and 16F11 binding helical peptides at 37°C. Shown are the binding signatures (left) for peptide concentration-series, c = 1.2-300 nM, 1.2-100 nM or 1.2- 33.3 nM, depicted in black and overlays (right) with a Langmuir 1 :1 binding Model, Refractive Index (Rl) set 0 and Rmax global, depicted grey, wherein Figure 7A shows the kinetic signature for mAb 15B5 binding to peptides TNNT3b (115-176), TNNT3c (106-146) and TNNT3d (106- 186), (top to bottom); wherein Figure 7B shows mAb 16F11 binding to peptides TNNT3b (IIS- 176), TNNT3c (106-146) and TNNT3d (106-186) (top to bottom); wherein Figure 7C shows mAb 16B9 binding to peptides TNNT3b (115-176), TNNT3c (106-146) and TNNT3d (106-186), TNNTI b (40-132) and TNNTIc (102-141), (top to bottom). The interactions for monoclonal Abs 16F11 and 16B9 are corrected for the occurred mass transport limitation.

[0034] Fig. 8 shows results of the detailed kinetic characterization for mAbs 2H2, 4C8, 2A10 and 1A6 binding helical peptides at 37°C. Shown are the binding signatures (left) for peptide concentration-series, c = 1.2-300 nM, 1.2-100 nM or 1.2-33.3 nM, depicted in black and overlays (right) with a Langmuir 1 :1 binding Model, Refractive Index (Rl) set 0 and Rmax global, depicted grey, wherein Figure 8A) shows mAb 2H2 binding to helical peptides TNNT3 (115-176), TNNT3 (148-186) and TNNT3 (106-186 (top to bottom); wherein Figure 8B) shows mAb 4C8 binding to helical peptides TNNT3 (115-176), TNNT3 (148-186) and TNNT3 (106- 186 (top to bottom); These fittings are corrected for the occurred mass transport limitation; and wherein Figure 8C) shows mAb 2A10 binding to helical peptide TNNT3 (38-76); andwherein Figure 8D) shows mAb 1A6 binding to helical peptide TNNT1 (148-184).DETAILED DESCRIPTION OF THE INVENTION

[0035] As explained above, in a first aspect the invention is directed to an antibody against skeletal Troponin T (skTnT), wherein the anti-skTnT antibody is not capable of binding to cardiac Troponin T (cTnT).

[0036] In plasma / blood of human subjects, in principle, both skTnT polypeptides and cTnT polypeptides may be present, depending on e.g. a health condition and / or a physical constitution of the subject. The high specificity of the provided anti-skTnT antibodies thus e.g. allows for a determination of skTnT levels in a subject without risk of detecting false positive poylpeptides due to cross-reactivity of the antibody with the highly conserved cTnT polypeptide. As also skTnl determination may lead to inaccurate results e.g. due to crossreactivity of the antibody, or in situations wherein skeletal Troponin T polypeptide concentration in blood / plasma more accurately reflects a skeletal muscle damage than skeletal Troponin I polypeptide concentrations.

[0037] According to an embodiment, the anti-skTnT antibody may be capable of binding to a skTnT peptide region conserved between skTnT and cTnT (conserved skTnT peptide sequence).

[0038] Sequence conservation as described herein may refer to identical or similar sequences in nucleic acids (DNA and / or RNA) or proteins across species and thus, across orthologous sequences, or within a genome. A sequence position and / or a region (subsequence) within a sequence may be conserved due to selective pressure. Accordingly, conserved sequences like conserved peptide sequences may be identified and / or defined based on estimates of selective pressure like the ratio of non-synonymous substitution rate to synonymous substitution rate. Additionally or alternatively, (e.g. pairwise or multiple, preferably multiple) sequence alignments may be used to determine sequence position(s) and / or region(s) conserved between said sequences used for the alignment based on sequence identity, similarity and / or substitution (matrix). Examples of software suitable for performing a sequence alignment may be software programs like Clustal Omega and the like (cf. e.g. services provided by EMBL-EBI).

[0039] As troponin protein sequences may exhibit evolutionarily conserved sites, for example, Shakur et al. (NPJ Genom Med. 2021 Jun 14;6(1):47. doi: 10.1038 / s41525-021- 00204-w) studied the conservation of amino acids in TnC, TnT and Tnl across species andfound that the protein sequences were highly conserved except in their N-termini. As regards the protein sequence of cTnT it was concluded that compared to the rest of TnT, the 75 N- terminal amino acids appeared to be least conserved across species. This sequence is also the least conserved sequence between the different TnT forms of homo sapiens, which are fast skeletal Troponin T, slow skeletal Troponin T and cardiac Troponin T. Taking a closer look at the details given in the article’s Supplementary Figure 1c it can be seen that indeed the 73 N-terminal amino acids of TnT appear to be least conserved across species. Accordingly, it is preferred that the conserved cTnT peptide sequence consists of amino acids 74 to 298 of SEQ ID NO: 1. Preferably, the anti-skTnT antibody is not capable of binding to the conserved cTnT peptide sequence consisting of amino acids 74 to 298 of SEQ ID NO: 1.

[0040] Surprisingly, the inventors found that it is possible to generate functional anti- skTnT antibodies that are capable of distinguishing between skTnT and cTnT polypeptides, which bind to a skTnT peptide region that is highly conserved between skTnT and cTnT. This approach has several advantages. For example, an anti-skTnT antibody for skTnT quantification may be designed to be capable of binding to a skTnT peptide sequence and / or epitope positioned in a region within the skTnT peptide sequence that is less affected by mutation, degradation and / or modification. The underlying consideration is that such a region is considered more stable compared to other regions within the skTnT sequence.

[0041] As regards skeletal Troponin T, according to an embodiment, skTnT refers herein to human skTnT. Human skTnT may refer to the canonical full length slow and fast skTnT encoded by the gene TN NT 1 and TNNT3, respectively. In the context of the present invention it is preferred that skTnT has the sequence set forth in SEQ ID NO: 2 and / or 3. Said sequences are given in Table 1. In particular, the sequence set forth in SEQ ID NO: 2 has a total length of 278 amino acids, and its sequence corresponds to the canonical full length slow skTnT (sskTnT) sequence as laid down in the UniProt database entry P13805 at the time of the filing of the present application. The sequence set forth in SEQ ID NO: 3 has a total length of 269 amino acids, and its sequence corresponds to the canonical full length fast skTnT (fskTnT) sequence as laid down in the UniProt database entry P45378 at the time of the filing of the present application. Nevertheless, it is understood by the skilled artisan that herein term “skTnT” encompasses also isoforms of skTnT, e.g. in case of human sskTnT the 3 isoforms laid down in the UniProt database entry P13805 at the time of the filing of the present application and / or derivatives thereof as well as in case of human fskTnT the 7 isoforms laid down in the UniProt database entry P45378 at the time of the filing of the present application and / or derivatives thereof. Derivatives may encompass mutated sequences as e.g. found naturally in samples under study, e.g. in case of samples obtained from human subjects, as well as (e.g. post-translationally) modified and / or degraded forms of the respective canonicalfull length skTnT sequence or any of its isoforms. While positions and (e.g. conserved) peptide sequences of skTnT are given herein for the canonical full length sskTnT and fskTnT sequence as the preferred sskTnT and fskTnT reference sequence, respectively, the skilled artisan is well aware of means and methods to transfer knowledge about said positions and peptide sequences to sskTnT isoforms and derivatives thereof and fskTnT isoforms and derivatives thereof, respectively.

[0042] According to an embodiment, the conserved skTnT peptide sequence consists of i) amino acids 40 to 278 of SEQ ID NO: 2 (sskTnT), and / or ii) amino acids 44 to 269 of SEQ ID NO: 3 (fskTnT). In this embodiment, the conserved cTnT peptide sequence consists of amino acids 74 to 298 of SEQ ID NO: 1 as the conserved skTnT peptide sequence thus (substantially) corresponds to said conserved cTnT peptide sequence. In this case, said conserved peptide sequences refer to peptide sequences being conserved in homologous skTnT and cTnT. Accordingly, the anti-skTnT antibody is preferably capable of binding to i) the conserved skTnT peptide sequence consisting of amino acids 40 to 278 of SEQ ID NO: 2 (sskTnT), and / or ii) the conserved skTnT peptide sequence consisting of amino acids 44 to 269 of SEQ ID NO: 3 (fskTnT).

[0043] According to an embodiment, the anti-skTnT antibody may be capable of binding to both the conserved sskTnT peptide sequence consisting of amino acids 40 to 278 of SEQ ID NO: 2 and to the conserved fskTnT peptide sequence consisting of amino acids 44 to 269 of SEQ ID NO: 3.

[0044] For example, the antibodies as herein provided may be used as scavenger antibodies in, e.g. commercially available, cTnT quantification assay like the Elecsys® cTnT- hs assay. Furthermore, by quantifying cTnT in the presence of the a anti-skTnT antibody as herein provided in a concentration that is preferably at least close to or within said anti-skTnT antibody’s saturation range, even concentration dependent interference effects may be overcome. A further advantage of the antibody as herein described is that they may be easily added to existing cTnT quantification assays either by “updating” current assay formulations by adding said anti-skTnT antibody as herein provided as scavenger antibody before packaging the respective formulation or by providing an additional formulation comprising the anti-skTnT antibody, wherein said additional formulation can be added to the sample before the sample’s amount of cTnT is quantified - if desired and / or required. Thus, in case a cTnT quantification is envisioned in a cost-sensitive manner and / or in the absence of any hint towards a (potentially) elevated skTnT level in a sample, current assays may be performed as usual, whereas the use of anti-skTnT antibody as herein provided may be considered especially whenever it is impossible to securely rule out any (potentially) elevated skTnT levelin the sample. Consequently, the anti-skTnT antibody as herein described used as scavenger antibody can ensure reliable, reproducible and high-sensitive cTnT quantification using currently available cTnT quantification assays without requiring information about a sample's background.

[0045] Alternatively, the antibodies as herein provided may also be used to purify a sample of a patient in which a cTnT concentration is to be determined. For example, it may also be envisioned to perform a pre-treatment of a Patient’s sample in which cTnT concentration is to be determined with the anti-skTnT antibody as herein provided in order to perform a purification step. In this case, the sample may be contacted with the anti-skTnT antibody, wherein the anti-skTnT scavenger antibody is immobilized on a surface and / or capable of being immobilized on a surface, thereby enabling immobilization of skTnT in the sample. The scavenger antibody may be (e.g. capable of being) immobilized on a surface by various means and methods, e.g. by labelling the scavenger antibody with a biotin label that can be immobilized on a streptavidin-coated surface like a streptavidin-coated microparticle surface or using a purification method e.g. based on centrifugation, precipitation, (e.g. column based) chromatography etc. Thus, by removing skTnT from the sample, using the anti-skTnT antibody as herein provided, the sample can be cleared in view of potentially interfering skTnT in advance before performing a cTnT quantification using an anti-cTnT antibody using an available cTnT quantification assay. If the anti-skTnT antibody is capable of binding to a skTnT peptide region conserved between skTnT and cTnT, each skTnT polypeptide may be refused from the sample, which may - in a potential cross-reactivity reaction - be recognized as false positive by an anti-cTnT antibody in a cTnT detection assay.

[0046] According to an embodiment, the anti-skTnT antibody may be capable of binding to a skTnT peptide region conserved between skTnT and cTnT (conserved skTnT peptide sequence), the anti-skTnT antibody may be capable of specifically binding to an epitope within an amino acid sequence as set forth in any one of SEQ-ID- No. 4 - 9.

[0047] For example, the conserved skTnT peptide sequence may comprise i) a slow skTnT (sskTnT) peptide sequence consisting of the sequence set forth in SEQ ID NO: 4, and / or ii) a fast skTnT (fskTnT) peptide sequence consisting of the sequence set forth in SEQ ID NO: 5. Said conserved peptide sequences refer to peptide sequences being conserved in (e.g. homologous) skTnT and cTnT. Accordingly, the anti-skTnT antibody is preferably capable of binding to the i) sskTnT peptide sequence consisting of the sequence set forth in SEQ ID NO: 4, and / or ii) fskTnT peptide sequence consisting of the sequence set forth in SEQ ID NO: 5.

[0048] According to an embodiment, the anti-skTnT antibody may be capable of bindingboth to the sskTnT peptide sequence consisting of the sequence set forth in SEQ ID NO: 4 and to the fskTnT peptide sequence consisting of the sequence set forth in SEQ ID NO: 5. The respective sequences are given in Table 1.

[0049] Additionally or alternatively, the conserved skTnT peptide sequence may comprise i) an sskTnT peptide sequence consisting of the sequence set forth in SEQ ID NO: 6, and / or ii) a fskTnT peptide sequence consisting of the sequence set forth in SEQ ID NO: 7.

[0050] Additionally or alternatively, the conserved skTnT peptide sequence may comprise i) an sskTnT peptide sequence consisting of the sequence set forth in SEQ ID NO: 8, and / or ii) a fskTnT peptide sequence consisting of the sequence set forth in SEQ ID NO: 9.

[0051] According to a further embodiment, the anti-skTnT antibody may be capable of binding to fast skeletal Troponin T (fskTnT), and further wherein said antibody is capable of binding to slow skeletal Troponin T (sskTnT).

[0052] An advantage of providing an antibody according to this embodiment is e.g. that such antibody is suitable for determining a total amount of skTnT. In particular, both fskTnT and sskTnT polypeptides are detectable without cross-reactivity with cTnT polypeptides.

[0053] For example, an antibody according to this embodiment, when bound to sskTnT, binds to at least one of the following residues: E at aa 57 of SEQ ID No. 2; and further wherein, when bound to fskTnT, the antibody binds to at least one of the following residues: E at amino acid residue (aa) 61 of SEQ ID No. 3; or when bound to sskTnT, the antibody binds to at least one of the following residues: K at aa 120; R at aa 122 and K at aa 132 of SEQ ID No. 2; and further wherein, when bound to fskTnT, the antibody binds to at least one of the following residues: K at aa 124; R at aa 126 and K at aa 136 of SEQ ID No. 3; or when bound to sskTnT, the antibody binds to at least one of the following residues: G at aa 158; A at aa 159, L at aa 165, Q at aa position 170 and R at aa 172 of SEQ ID No. 2; and further wherein, when bound to fskTnT, the antibody binds to at least one of the following residues: G at aa 162; A at aa 163, L at aa position 169, Q at aa position 174 and R at aa 176 of SEQ I D No. 3.

[0054] According to another embodiment, the antibody may be capable of binding to fast skeletal Troponin T (fskTnT), and further the antibody maybe not capable of binding to slow skeletal Troponin T (sskTnT).

[0055] An advantage of providing an antibody according to this embodiment is e.g. that such antibody is suitable for determining specifically fskTnT polypeptides. In particular, fskTnT may be determined without cross-reactivity of the antibody with sskT nT polypeptides and cT nTpolypeptides.

[0056] For example, an antibody according to this embodiment, when bound to fskTnT, binds to at least one of the following residues: P at aa position 50, R at aa position 51 , K at aa position 53, T at aa position 55 and A at aa position 56 of SEQ ID No. 3; or wherein when bound to fskTnT, the antibody binds to at least one of the following residues: A at aa position 122, of SEQ ID No. 3; or when bound to fskTnT, the antibody binds to at least one of the following residues: N at aa position 164, Y at aa position 165, S at aa position 166, S at position 166, A at aa position 169, and D at aa position 172 of SEQ I D No. 3.

[0057] According to another embodiment, an antibody according to the present invention antibody may be capable of binding to slow skeletal Troponin T (ssk TnT), and further the antibody may not be capable of binding to fast skeletal Troponin T (fskTnT),

[0058] An advantage of providing an antibody according to this embodiment is e.g. that such antibody is suitable for determining specifically sskTnT polypeptides. In particular, sskTnT may be determined without cross-reactivity of the antibody with fskTnT polypeptides and cTnT polypeptides.

[0059] For example, an antibody according to this embodiment, when bound to sskTnT, binds to at least one of the following residues: the antibody binds to at least one of the following residues: P at aa position 40, K at aa position 41 , P at aa position 42, D at aa position 43, V at aa position 45, V at aa position 46, P at aa position 48 and I at aa position 50 of SEQ ID No. 2; or when bound to sskTnT, the antibody binds to at least one of the following residues: F at aa position 117, T at aa position 119, A at aa position 126, and K at aa position 127, of SEQ ID No. 2; or when bound to sskTnT, the antibody binds to at least one of the following residues: V at aa position 166 of SEQ ID No. 2.

[0060] According to some embodiments, the anti-skTnT antibody may be a monoclonal anti-skTnT antibody. For example, additionally or alternatively, the anti-skTnT antibody may be an anti-skTnT antibody that has been affinity maturated, humanized and / or stabilized. Preferably, the anti-skTnT antibody may be a monoclonal anti-skTnT antibody that has been affinity maturated, humanized and / or stabilized.

[0061] According to some embodiment, the anti-skTnT antibody may be capable of binding to an epitope within skTnT as set forth in SEQ ID No. 2 (sskTnT) and / or as set forth in SEQ ID No. 3 (fskTnT) with a KD of 10'7M or less, or with a KD of 10'8M or less, or with a KD of 10'9M or less, or with a KD of 5x 10'9or less, or with a KD of 10'1° or less.

[0062] Additionally or alternatively, the anti-skTnT antibody may described by defining its variable regions. Commonly, an antibody comprises a heavy chain variable region, comprising three CDRs, and a light chain variable region, comprising three CDRs. A “CDR” refers to a complementarity-determining region and may be understood as a hypervariable domain that may determine and / or influence the antibodies specific binding.

[0063] For example, the anti-skTnT antibody may comprise a heavy chain variable region (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 and a light chain variable region (VL) comprising CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of CDR-H1 as depicted in SEQ ID NO: 10, CDR-H2 as depicted in SEQ ID NO: 11 , CDR-H3 as depicted in SEQ ID NO: 12, CDR-L1 as depicted in SEQ ID NO: 13, CDR-L2 as depicted in SEQ ID NO: 14 and CDR-L3 as depicted in SEQ ID NO: 15. Respective sequences are given in Table 1.

[0064] For example, the anti-skTnT antibody may comprise a heavy chain variable region (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 and a light chain variable region (VL) comprising CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of CDR-H1 as depicted in SEQ ID NO: 16, CDR-H2 as depicted in SEQ ID NO: 17, CDR-H3 as depicted in SEQ ID NO: 18, CDR-L1 as depicted in SEQ ID NO: 19, CDR-L2 as depicted in SEQ ID NO: 20 and CDR-L3 as depicted in SEQ ID NO: 21. Respective sequences are given in Table 1.

[0065] For example, the anti-skTnT antibody may comprise a heavy chain variable region (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 and a light chain variable region (VL) comprising CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of CDR-H1 as depicted in SEQ ID NO: 22, CDR-H2 as depicted in SEQ ID NO: 23, CDR-H3 as depicted in SEQ ID NO: 24, CDR-L1 as depicted in SEQ ID NO: 25, CDR-L2 as depicted in SEQ ID NO: 26 and CDR-L3 as depicted in SEQ ID NO: 27. Respective sequences are given in Table 1.

[0066] Accordingly, the anti-skTnT antibody may comprise i) a VH region selected from the group consisting of those depicted in SEQ ID NO: 28, 29 and 30 and / or ii) a VL region selected from the group consisting of those depicted in SEQ ID NO: 31 , 32 and 33. Preferably, the anti-skTnT antibody comprises i) a VH region as depicted in SEQ ID NO: 28 or 30 and / or ii) a VL region as depicted in SEQ ID NO: 31 or 33. Respective sequences are given in Table 1.

[0067] Preferably, the anti-skTnT antibody comprises a) a VH region as depicted in SEQ ID NO: 28 and a VL region as depicted in SEQ ID NO: 31 , or b) a VH region as depicted in SEQ ID NO: 29 and a VL region as depicted in SEQ ID NO: 32, or c) a VH region as depicted in SEQ ID NO: 30 and a VL region as depicted in SEQ ID NO: 33. Preferably, the anti-skTnT antibody comprises a VH region as depicted in SEQ ID NO: 28 and a VL region as depictedin SEQ ID NO: 31 , or a VH region as depicted in SEQ ID NO: 30 and a VL region as depicted in SEQ ID NO: 33.

[0068] As described above, in accordance with a second aspect of the present invention, there is provided herein a use of an antibody as herein described for determining a skeletal muscle damage.

[0069] For example, the skeletal muscle damage may be a skeletal muscle disorder (SMD), a skeletal muscle damage induced by physical exercise and / or a skeletal muscle damage induced by a drug treatment such as medical drug treatment.

[0070] According to an embodiment, the use may comprise using the antibody for determining a level of a physical fitness of a subject, if the skeletal muscle damage is induced by a physical exercise. For example, it may be possible to determine sskTnT, and / or fskTnT, and or a total amount of skTnT in order to determine the fitness level of a subject. It is further envisaged that a ratio between sskTnT and fskTnT may be calculated in order to determine the fitness level of the subject. In exemplary embodiments, e.g. the determined value for sskTnT, fskTnT, total skTnT and / or the ratio between sskTnT and fskTnT may be compared with a reference value. For example, the reference value may be indicative for a particular fitness level of the subject. For example, the reference value may be associated with a determination of the level of physical fitness of the subject using the antibody as herein described before start of a physical exercise period, e.g. a training period. Comparison between the reference value and the determined value may then be indicative for a training effect. However, other reference values and or / comparisons of the determined value for sskTnT, fskTnT, total skTnT and / or the ratio between sskTnT and fskTnT that the skilled person would consider in order to determine the fitness level of the subject are contemplated.

[0071] According to an embodiment, SMD may be selected from the group consisting of myopathy, myositis, skeletal myopathy, muscular dystrophy, rhabdomyolysis, neuropathy, myasthenic syndrome and autosomal dominant (AD) with muscle symptoms, preferably the SMD may be selected from the group consisting of myopathy and myositis; and / or the myopathy may be selected from the group consisting of myotonic dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, mitochondrial disease, and glycogen storage disease; and the myositis may be selected from the group consisting of dermatomyositis, polymyositis, sporadic inclusion body myositis, hereditary inclusion body myositis, immune-mediated necrotizing myositis, myositis with overlap with collagenous disease, myositis induced by physical exercise, myositis induced by drug treatment such as medical drug treatment, preferably statin-induced myositis, or a myositis induced by treatment with a check point inhibitor (ICI), by a treatment with an inhibitor of avascular endothelial growth factor (VEGFI) or combined treatment, and vasculitis.

[0072] In accordance with a further aspect, there is provided a method of determining the amount of skeletal Troponin T (skTnT), the method comprising a) Contacting a sample with at least one antibody as herein provided, and b) Determining the amount of skT nT.

[0073] For example, skTnT may be i) fast skTnT (fskTnT), ii) slow skTnT (sskTnT), or iii) fskT nT and sskT nT.

[0074] According to an embodiment, the method may further comprise c) Comparing the determined amount of skTnT to a reference.

[0075] For example, the determined amount of skTnT compared to a reference may or may not be indicative of a disease associated with skTnT, preferably of a skeletal muscle damage and / or of a risk of skeletal muscle damage.

[0076] In particular, the reference may be e.g. a reference for a healthy condition and / or for skeletal muscle damage.

[0077] According to an embodiment, the determined amount of skTnT compared to a reference may or may not be indicative for a change of a disease associated with skTnT, preferably of a change in skeletal muscle damage, over time.

[0078] It is herewith envisaged that the reference may be a reference skTnT amount determined at an earlier point in time.

[0079] In exemplary embodiments, an increased amount of skTnT in the sample compared to the reference may be indicative for skeletal muscle damage.

[0080] For example, the skeletal muscle damage may be a skeletal muscle disorder (SMD), a skeletal muscle damage induced by physical exercise and / or a skeletal muscle damage induced by a drug treatment such as a medical drug treatment, wherein the SMD may be selected from the group consisting of myopathy, myositis, skeletal myopathy, muscular dystrophy, rhabdomyolysis, neuropathy, myasthenic syndrome and autosomal dominant (AD) with muscle symptoms, preferably the SMD is selected from the group consisting of myopathy and myositis.

[0081] According to an embodiment, the myopathy may be a non-inflammatory myopathy, preferably selected from the group consisting of myotonic dystrophy,facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, mitochondrial disease, and glycogen storage disease; and / or the myositis may be selected from the group consisting of dermatomyositis, polymyositis, sporadic inclusion body myositis, hereditary inclusion body myositis, immune-mediated necrotizing myositis, myositis with overlap with collagenous disease, myositis induced by physical exercise, myositis induced by drug treatment such as a medical drug treatment, preferably statin-induced myositis or a myositis induced by treatment with ICI or VEGFI or combined treatment, and vasculitis.

[0082] In accordance with an embodiment, the sample may be a human sample.

[0083] For example, the sample may be a fluid sample and / or the sample may be selected from the group consisting of serum, plasma, whole blood, interstitial fluid, urine and skeletal muscle tissue, preferably wherein the sample may be a serum or plasma sample.

[0084] According to a further aspect, there is provided a polynucleotide encoding an antibody as herein provided.

[0085] According to a further aspect, there is provided a vector comprising the polynucleotide.

[0086] According to a further aspect, there is provided a host cell transformed or transfected or transduced with a polynucleotide as herein described, or with the vector as herein described.

[0087] According to a further aspect, there is also provided a method of producing the antibody against anti-skeletal troponin T (skTnT) as herein described, the method comprising culturing host cells as herein described and isolating said antibody.

[0088] According to a further aspect, there is provided a composition comprising at least two antibodies against skTnT as herein provided, wherein(i) the at least two antibodies are capable of simultaneously binding to fskT nT and at least one of said at least two antibodies is not capable of binding to sskTnT; or(ii) the at least two antibodies are capable of simultaneously binding to sskTnT and at least one of said at least two two antibodies is not capable of binding to fskTnT; or(iii) the at least two antibodies are capable of simultaneously binding to sskTnT; and wherein the at least two antibodies are capable of simultaneously binding to fskTnT.

[0089] For example, skTnT quantification assays may be based on the detection of a signal indicative for the amount of skTnT, wherein said signal may be obtained from a labelled anti-skTnT antibody when bound to an immobilized skTnT. In particular, skTnT comprised in asample may be immobilized on a surface using an anti-skTnT immobilization antibody (also referred to, e.g., as “catcher” antibody or “capture” antibody). A labelled anti-skTnT antibody (also referred to, e.g., as “detector” antibody) may bind to the immobilized skTnT and after a washing step a signal obtained from the label of the detector antibody may be indicative for the amount of skTnT in the sample. Such approaches may also be referred to as “sandwich assays” (as employed, e.g., in case of the Elecsys® cTnT-hs assay) as the target skTnT is bound by two anti-skTnT antibodies for signal read-out, namely the anti-skTnT immobilization antibody and the labelled anti-skTnT antibody, used for signal read-out. Herein, the term “anti- skTnT antibody” may refer to an anti-skTnT immobilization antibody or a labelled anti-skTnT antibody in a sandwich assay setting, preferably to the labelled anti-skTnT antibody (used for skTnT detection, and thus the “detector” antibody).

[0090] For example, in the above embodiment, an antibody as herein described that specifically binds to sskTnT, or fskTnT, or both sskTnT and fskTnT may be used as capture antibody. If an sskTnT or an fskTnT antibody is used as capture antibody, the detection antibody may also be a sskTnT antibody or fskTnT antibody, respectively. Sensitivity of the assay for determining skTnT in a sample may thus be increased. If the capture antibody is an antibody that binds to both sskTnT and fskTnT, the detection antibody may either be a further antibody that binds to both sskTnT and fskTnT, or e.g an antibody specific for fskTnT, or an antibody specific for sskTnT. If the detection antibody detects both sskTnT and fskTnT, a total amount of skTnT may be determined with high sensitivity. If the detection antibody is sskTnT or fskTnT, sskTnT or fskTnT, respectively, may be determined with high sensitivity.

[0091] According to a further aspect, there is provided a computer-implemented method of determining an amount of skeletal Troponin T (skTnT), said method comprising(a) Receiving a first signal indicative of fast skTnT (fskTnT) and a second signal indicative of slow skTnT (sskTnT), or receiving a first signal indicative of fskTnT and a second signal indicative of both sskTnT and fskTnT; or receiving a first signal indicative of (sskTnT) and a second signal indicative of both sskTnT and fskTnT; and(b) Determining the amount of skTnT based on the received first and second signals, if the first signal is indicative of fast skTnT (fskTnT) and the second signal is indicative of slow skT nT (sskT nT); or the amount of sskT nT if the first signal is indicative of fskTnT and a second signal indicative of both sskTnT and fskTnT, or the amount of fskTnT if the first signal indicative of sskTnT and the second signal indicative of both sskT nT and fskT nT ;wherein at least one of the received signals corresponds to the binding of at least one antibody as herein described to a skTnT polypeptide.

[0092] An advantage of this method is that only two signals are required in order to determine three different amounts of skTnT. In particular, e.g. by receiving signals for fskTnT and sskTnT, it is possible to determining the amount of fskTnT, sskTnT and total skTnT in the sample. Alternatively, if e.g. signals are achieved for total skTnT and fskTnT, it is also possible to determining the amount of fskTnT, sskTnT and total skTnT in the sample. Alternatively, also when receiving signals for total skTnT and sskTnT, the amount of fskTnT, sskTnT and total skTnT in the sample may be determined.

[0093] In accordance with an embodiment, step (b) may comprise(b1) Determining an amount of fskTnT based on the received first signal, and(b2) Determining an amount of sskTnT based on the received second signal.

[0094] According to an embodiment, in step(b1) The amount of fskTnT may be determined by comparing the received first signal to a first calibration curve and / or a first reference curve, and(b2) The amount of sskTnT may be determined by comparing the received second signal to a second calibration curve and / or a second reference curve.

[0095] According to an embodiment, in step (b) the amount of skTnT may be determined by combining the amount of fskTnT determined in step (b1) and the amount of sskTnT determined in step (b2).

[0096] According to an embodiment, the computer-implemented method may further comprise(c) Comparing the determined amount of skTnT with a reference.

[0097] According to an embodiment, the amount of skTnT may be the combined amountof fskT nT and of sskT nT.

[0098] According to an embodiment, the received first signal may be a signal obtained from a first signal detecting device, wherein said first signal detecting device may be capable of detecting a signal of an anti-skTnT antibody as herein described, and wherein said anti- skTnT antibody comprises a label detectable by said signal detecting device.

[0099] According to an embodiment, the received first signal may be a signal obtained from a first signal detecting device and wherein the received second signal may be a signal obtained from the first or a second signal detecting device, wherein said first and / or second signal detecting device may be capable of detecting a signal of an anti-skTnT antibody as herein described, and wherein said anti-skTnT antibody comprises a label detectable by said signal detecting.

[0100] The invention further relates to a computer program product comprising a program code for executing the computer-implemented method as herein provided when run on a computer.

[0101] According to a further aspect, there is provided a computer-readable medium having stored thereon a program code for executing the computer-implemented method herein described.

[0102] Each publication, patent, patent application or other document cited herein is hereby incorporated by reference in its entirety.

[0103] The invention will be further illustrated by the following non-limiting Experimental Examples.

[0104] Sequences as used herein are depicted in below Table 1.

[0105] Table 1. Sequences as used herein.Experimental Examples

[0106] The inventors developed highly specific antibodies against skTnT that are capable of recognizing epitopes of skTnT, without showing cross-reactivity to the highly homologous cTnT. The design of the antibody epitopes was based on the M11.7 anti-cTnT antibody used in cardiac Troponin T assays (Elecsys® cTnT-hs assay and POC test). The binding site of the M11.7 antibody in cTnT was aligned to fast and skeletal Troponin T sequences. Based on these alignments, the inventors designed various immunogens and screening peptides to target both fast and slow skTnT and to ensure no cross-reactivity to cTnT by counter screening with cTnT peptides. Due to the high homology of cTnT, sskTnT and fskTnT proteins, antibodies were designed that are able to recognize differences of only one or two amino acids. The antibodies were found to be suitable scavenger antibodies for diagnostic immunoassays.

[0107] Antibody generation against human skeletal Troponin T

[0108] Rational: For generating specific antibodies against skTnT, New Zealand white rabbits were immunized with selected skTnT peptides and screened for skTnT peptide and protein binding capability. To rule out recognition of cTnT, counter screenings with cTnT peptides and recombinant cTnT were performed. Peptides for screening and immunization were generated in-house by peptide synthesis.

[0109] Peptides were synthesized by means of fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis on a multiple peptide synthesizer, e.g. from Protein Technologies, Inc. For this, 4.0 equivalents of each amino acid derivative were used. Amino acid derivatives were dissolved in dimethylformamide containing 1 equivalent of 1-hydroxy-7-azabenzotriazol. Peptides were synthesized on Tentagel R resin. Coupling reactions were carried out for 5 minutes in dimethylformamide as a reaction medium with 4 equivalents HATLI (1- [Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) and 8 equivalents of N,N-diisopropylethylamine relative to resin loading. The Fmoc group was cleaved in 8 minutes after each synthesis step using 25% piperidine in dimethyl formamide. Release of the peptide from the synthesis resin and the cleavage of the acid-labile protecting groups were achieved in 3 hours at room temperature with 9.5 ml trifluoroacetic acid, 0.25 ml triisopropylsilane, and 0.25 ml water. The reaction solution was subsequently mixed cooled diisopropyl ether to precipitate the peptide. The precipitate was filtered, washed again with diisopropyl ether, dissolved in a small amount of aqueous acetic acid and lyophilized. The crude material obtained was purified by preparative reversed phase chromatography (RP-HPLC) using a gradient of acetonitrile / water containing 0.1% trifluoroacetic acid. The identity of the purified material was checked by means of ionspray mass spectrometry.

[0110] As immunogens, human fast and slow skTnT peptides (slow skTnT: sskTnT / TNNT1 , fast skTnT: fskTnT / TNNT3) were synthesized. Therefore, 3- (maleimido)propionic acid N-hydroxysuccinimide ester was added to a solution of keyhole limpet hemocyanin (KLH) in phosphate buffer (20 mM , pH 7.2). The reaction was incubated for 5 hours at room temperature and then dialyzed against phosphate buffer (0.1 M, pH 7.0). Cysteine containing glycopeptide was dissolved in DMSO and added to a solution of maleimide-activated KLH containing 0.1 M ethylenediaminetetraacetic acid (EDTA). The solution was agitated for 5 hours at room temperature and then dialyzed against phosphate buffer (0.1 M, pH 7.0) to yield KLH-peptide conjugate. The resulting immunogen peptides had the sequences as set forth in SEQ ID NOs 4, 5, 6, 7, 8, and 9 with an additional “pAla-Ahx- pAla-Cys” at the respective C-terminus (resulting in the sequences as set forth in SEQ ID No 48, 49, 46, 47, 50 and 51 , respectively) ; all immunogens were coupled to KLH. For screening, biotinylated human fast and slow skTnT peptides were used with human cTnT (TNNT2) peptides for counter screening to rule out cross-reactivity to cTnT. The resulting screening peptides comprised the sequences as set forth in SEQ ID Nos 4, 5, 6, 7, 8, 9, 34, 35 and 36 including a pAla at the N-terminus (resulting in SEQ ID No 56, 57, 53, 54, 59, 60, 55, 52, and 58, respectively) with an additional “Biotin-PEG3” label at the respective N-terminus; peptides were used as biotin conjugates for ELISA and SPR screenings.

[0111] The immunization procedure resulted in various rabbit IgG clones reacting specifically with human skTnT, but not with cTnT (de-selection of cTnT binders with cTnT screening peptides). Specificity of these antibodies for skTnT peptides and the full-length protein was demonstrated by enzyme-linked immunosorbent assay (ELISA) screenings of the B cell supernatant (not shown). Next, candidates identified via the ELISA were analysed for their kinetic binding properties at 37°C via plasmon resonance spectroscopy (SPR). All experiments were conducted with recombinant human slow skeletal Troponin T (sskTnT), molecular mass 33 kDa, recombinant human fast skeletal Troponin TnT (fskTnT), molecular mass 31 kDa and recombinant human cardiacTnT (cTnT), molecular mass 35 kDa. Interactions were analyzed using the system buffer HBS-ET+ pH 7.4, 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 0.05% (w / v) Tween 20, pH 7.4 The system buffer was supplemented with 1 mg / mL Carboxymethyldextran (Fluka) and used as sample buffer.

[0112] Kinetic screening was performed at 37 °C on GE Healthcare BIAcore™ 8K +, 8K and B4000 instruments. A Biacore CM5 Series S was functionalized at 25°C with a rabbit species-specific antibody capture system. A polyclonal goat anti-rabbit IgG Fc capture antibody GARbFcy (111-005-046, Jackson ImmunoResearch) was amine coupled usingEDC / NHS chemistry according to the manufacturer’s instructions. Finally, ligand densities between 10000 RU-15000 RU were obtained. Rabbit antibody (IgG 150 kDa) solutions were diluted in sample buffer and were injected at 5 pl / min or 10 pL / min for 2 minutes. The antibody Capture Level (CL) in resonance units (RU) was monitored. 150 nM analyte sskTnT, cTNT and fskTnT was injected to the precaptured antibodies. The analyte association phases were monitored for 5 minutes. The dissociation phases were monitored for 5 minutes. After each measurement cycle, the capture system was regenerated by subsequent injections of 10 mM Glycine buffers pH 2.0 and pH 2.25 at 20 pL / min for 60 seconds. Kinetic signatures were monitored by the BIAcore™ 8K Control-SW V3.0.11.15423 and evaluated by the BIAcore™ Insight Evaluation SW V3.0.11.15423, respectively B4000 Control SW V1.1 and Evaluation SW V1.1. Kinetic data was interpreted by report point evaluations. Two report points, the recorded response signal shortly before the end of the protein analyte injection, analyte Binding Late (BL), and the signal shortly before the end of the dissociation phase, Stability Late (SL), were used to compare the antibody / antigen complex stabilities. The dissociation rate constant kd (s-1) was calculated according to a Langmuir model and the antibody / antigen complex half-life was calculated in minutes according to the formula t / 2 diss = ln(2) / ( / 60). The Molar Ratio, the binding stoichiometry was calculated by the formula MR = B(antigen)* MW(antibody) / (MW(antigen)* CL (antibody)). In total, 1095 antibodies from culture supernatants were analyzed. Candidates were identified with specific binding for sskTnT or fskTnT, or binding both, sskTnT and fskTnT and no detectable binding to cTnT were identified. Further, the obtained sensorgrams of the identified candidates were analyzed by visual inspection for fastest complex formation into Ab-equilibrium. The antibodies majority was deselected due to slow complex formation or artefact binding. Finally, 75 antibodies were identified with different specificity patterns (specificity for only fskTnT, specificity for only sskTnT, or specificity for both, sskTnT and fskTnT). Based on the different binding patterns in combination with superior kinetic properties, i.e. either with fastest complex formation, or fast complex formation combined with highest complex half-life times clones 15B5, 16B9, 16F11 , 2H2, 4C8, 2A10 and 1A6 were selected for further, detailed kinetic characterization. The candidates 15B5, 16B9, 16F11 , 1A6, 2H2, 4C8 and 2A10 were kinetically characterized in detail for the binding of fskTnT protein to determine the kinetic rate constants kaand kd and the resulting affinity KD. AS described under section

[0105] for the kinetic screening, a multi cycle kinetic instead of a single concentration was performed by injecting a series of fskTnT concentrations, c = 3-240 nM with duplicates for concentration 26.7 nM. The dissociation was monitored for 10 minutes. The association rate constant ka(M'1s_1), dissociation rate constant kd (s-1) and the dissociation equilibrium constant KD, known as affinity, was calculated according to a 1 :1 Langmuir model Rl =0, Rmax global using the SW Scrubber V 2.0c. In case of occurred mass transport limitation (MTL), the interaction were corrected for MTL using thetwo-compartment model. Additionally, the monoclonal Abs 15B5, 16B9, 16F11 , 1A6, 2H2, 4C8 and 2A10 were analyzed for the binding to a selection of 9 helical peptides to determine the kinetic rate constants kaand fe and the resulting affinity KD. Tested peptides: TNNT3a (38-76) (SEQ ID No. 37), TNNT3b (115-176) (SEQ ID No. 38), TNNT3c (106-146) (SEQ ID No. 39), TNNT3d (106-186) (SEQ ID No. 40), TNNT3e (148-186) (SEQ ID No. 41), TNNTIa (31-70) (SEQ ID No. 42), TNNTIb (40-132) (SEQ ID No. 43), TNNTIc (102-141) (SEQ ID No. 44) and TNNTId (148-184) (SEQ ID No. 45). All peptides were analyzed with concentrations c = 1.2- 300 nM, 1.2-100 nM or 1.2-33.3 nM, dissociation was monitored for 20 minutes.

[0113] Sequence alignments of skTnT and cTnT

[0114] As shown in Figure 1 , sequence alignments of human skTnT and human cTnT revealed stretches of (near) perfect sequence identity indicative for conserved sequence regions. More specifically, the upper part of Figure 1 shows amino acid sequences for cTnT (first row) as well as different slow-twich (s) and fast-twitch (f) skTnT sequences. The level of amino acid conservation is indicated in the lower part of Figure 1 , wherein the consensus sequence is shown as determined across the investigated sequences. The size of the letters representing the amino acids being is indicative for the level of conservation of the respective amino acid with comparatively small letters indicating comparatively low levels of conservation in contrast to comparatively large letters indicating comparatively strong amino acid conservation across the investigated sequences. Furthermore, a star below the three aligned sequences indicate conservation of the respective amino acid in all three sequences. Two points and 1 point below the sequences indicate, respectively, conservation among two of the three sequences at this position.

[0115] Results of Kinetic screening of generated antibodiesFig. 2 demonstrates the results of a kinetic screening with exemplary kinetic signatures of antibody / TnT interactions for mAbs 15B5 (top), 16B9 (middle) and 16F11 (bottom) according to the present invention. Shown is the binding to (A) sskTnT (B) fskTnT (C) cTNT to with 150 nM TnT concentrations, Interactions with significant target binding are overlaid with a Langmuir 1 :1 dissociation fitting model (grey).Fig. 3 demonstrates exemplary obtained binding signatures of deselected of antibodies from the kinetic screening due to no binding, artefact binding, too slow complex formation velocities, fast dissociation or biphasic dissociation, or a combination of slow complex formation and biphasic dissociation.Fig. 4 demonstrates the results of a kinetic screening with exemplary kinetic signatures of antibody / TnT interactions for mAbs 1A6, 2H2, 4C8 and 2A10 (top to bottom) according to the present invention. Shown is the binding to (A) sskTnT and (B) fskTnT with 150 nM TnT concentrations, Interactions with significant target binding are overlaid with a Langmuir 1 :1 dissociation fitting model (grey).

[0116] Results of detailed Kinetic Characterization of generated antibodies binding fskTnT

[0117] Fig. 5 demonstrates the results of a kinetic characterization for mAbs 15B5, 16B9 and 16F11 binding fskTnT according to the present invention. Specifically, Fig. 5 shows kinetic signatures of antibody / fskTnT interactions for mAbs 15B5, 16B9 and 16F11 (left to right) according to the present invention.Shown are series of fskTnT-concentrations, c = 3-240 nM, with duplicates for concentration 26.7 nM. Fig. 5A) Measured kinetic profiles (black) are overlaid with the Langmuir 1 :1 binding Model, Rl =0, Rmax global with allowed correction for mass transport limitation, depicted grey (right). Fig. 5B) Measured kinetic profiles (grey) overlaid with Langmuir 1 :1 binding Model, Rl =0, Rmax global with fitting, here visualizing the binding signatures, which are corrected for the MTL (black).

[0118] Fig. 6 demonstrates the results of a kinetic characterization for mAbs 2H2, 4C8 and 2A10 binding fskTnT according to the present invention. The binding signatures for mabs 2H2, 4C8, 2A10 show a fast complex formation into the antibody saturation or coming close to it. The dissociation for the interactions vary. mAb 1A6 did not bind to fskTnT, data not shown. Specifically, Fig. 6 shows Kinetic signatures of antibody / TnT interactions for mAbs 2H2, 4C8, 2A10 (left to right) according to the present invention. Shown are series of fskTnT- concentrations, c = 3-240 nM, with duplicates for concentration 26.7 nM.Fig. 6A) Measured kinetic profiles (black) are overlaid with the Langmuir 1 :1 binding Model, Rl =0, Rmax global with allowed correction for mass transport limitation, depicted grey (right).Fig. 6B) Measured kinetic profiles (grey) overlaid with Langmuir 1 :1 binding Model, Rl =0, Rmax global with fitting, here visualizing the profiles, which are corrected for the MTL (black).

[0119] The obtained binding parameters are summarized in table 2. mAbs 15B5, 16B9,16F11 , 2H2, 4C8 and 2A10 show high affine binding of fskTnT with affinities ranging between KD = 3.9 nM for mAb 4C8 and 100 nM for 16F11. All interactions are characterized by a fast association rate constant ka > 7.0E+05 M-1s-1 . The half-life times vary between 3 minutes for clones 16B9 and 28 minutes for clone 2H2. Clone 1A6 did not bind to fskTnT, in line with the data from the kinetic screening, data not shown.

[0120] Table 2Binding parameters for mAbs 15B5, 16B9, 16F1 , 2H2, 4C8 and 2A10 binding fast skTnT at 37°C. SE - Standard error of mean of the SPR-fit in comparison to the measured data points. Stated values are corrected for the occurred mass transport limitation via the Scrubber SW Version 2.0c using a two-compartment model.

[0121] Results of detailed kinetic characterization of generated antibodies binding to a selection of helical TNNT1 and TNNT3-peptidesMAbs 15B5 and 16F11 show specific binding to 3 of the 5 analyzed helical TNNT3-peptides TNNT3b (115-176), TNNT3c (106-146) and TNNT3d (106-186). No detectable binding occurred for TNNT3-peptides TNNT3a (38-76) and TNNT3e (148-186) and for all TNNT1- peptides (TNNT1a-d). These data confirm the results of the SPR-based kinetic screening analyzing the Ab-binding to skTnT proteins. The binding signatures for 15B5 and 16F11 differ from each other: mAb 16F11 shows a faster association and faster dissociation than mAb15B5, when binding to TNNT3b (115-176) and TNNT3c (106-146), see Fig. 7 and Table 3. mAb 16B9 shows binding to peptides to TNNT3b (115-176), TNNT3c (106-146) and TNNT3d (106-186) and additionally shows binding to TNNTI b (40-132) and TNNTIc (102-141), confirming the specificity pattern of the kinetic screening.

[0122] Fig. 7 demonstrates the results of a kinetic characterization for mAbs 15B5, 16B9 and 16F11 binding to a selection of helical TNNT-peptides

[0123] Specifically, Fig. 7 shows kinetic signatures for mAbs 15B5, 16F11 and 16B9 binding helical peptides at 37°C. Shown are the binding signatures (left) for peptide concentration-series, c = 1.2-300 nM, 1.2-100 nM or 1.2-33.3 nM, depicted in black and overlays (right) with a Langmuir 1 :1 binding Model, Refractive Index (Rl) set 0 and Rmax global, depicted grey. Fig. 7A) mAb 15B5 binding to peptides TNNT3 (115-176), TNNT3 (106-146) and TNNT3 (106-186), (top to bottom). Fig. 7B) mAb 16F11 binding to peptides TNNT3 (115- 176), TNNT3 (106-146) and TNNT3 (106-186) (top to bottom). Fig. 7C) mAb 16B9 binding to peptides TNNT3 (115-176), TNNT3 (106-146) and TNNT3 (106-186), TNNT1 (40-132) and TNNT1 (102-141), (top to bottom). The interactions for mAbs 16F11 and 16B9 are corrected for the occurred mass transport limitation. The obtained results are summarized in table 3.

[0124] Table 3Binding parameters for mAbs 15B5, 16F11 and 16B9 binding helical peptides at 37°C. SE - Standard error of mean of the SPR-fit in comparison to the measured data points.The obtained SPR-data are fitted with the Langmuir 1 :1 binding model, Rl set to zero and Rmax global. Stated values of mabs 16F11 and 16B9 are corrected for the occurred mass transport limitation.

[0125] Fig. 8_demonstrates Kinetic signatures for mAbs 2H2, 4C8, 2A10 and 1A6 binding to helical peptides at 37°C

[0126] mAbs 2H2 and 4C8 show binding to helical peptides TN NT3b (115-176), TNNT3e (148-186) and TNNT3d (106-186), see Fig. 8 A) and B). Both mabs, 2H2 and 4C8 do not bind to helical TNNT3a (38-76) or peptides TNNTIc (102-141), TNNTId (148-184), TNNTIa (SI- 70), TNNTIb (40-132), data not shown.The interactions for mAb 4C8 are slightly mass transport limited. Therefore, the interactions are corrected for the mass transport limitation (MTL) using the two compartment model of the Scrubber SW Version 2.0c. mAb 2A10 has a deviating binding pattern compared to mAbs 2H2 and 4C8: The mAb 2A10 binds to helical peptide TNNT3a (38-76), see Fig. 8 C). There is no binding detectable for the other TNNT3-peptides, nor to the TN NT1 -peptides, data not shown. mAb 1A6 shows specific binding to helical peptide TNNTId (148-184), see Fig. 8 D), and no detectable binding to the other TNNT1 or TNNT3 peptides.

[0127] Specifically, Fig. 8 shows kinetic signatures for mAbs 2H2, 4C8, 2A10 and 1A6 binding to helical peptides at 37°C. Shown are series of peptide concentrations, c = 1.2-300 nM, depicted in black (left) and overlays with the Langmuir 1 :1 binding Model, Rl =0, Rmax global, depicted grey (right). Fig 8 A) mAb 2H2 binding to helical peptides TNNT3 (115-176), TNNT3 (148-186) and TNNT3 (106-186 (top to bottom). Fig 8 B) mAb 4C8 binding to helical peptides TNNT3 (115-176), TNNT3 (148-186) and TNNT3 (106-186 (top to bottom). These fittings are corrected for the occurred mass transport limitation. Fig 8 C) mAb 2A10 binding to helical peptide TNNT3 (38-76). Fig. 8 D) mAb 1A6 binding to helical peptide TNNT1 (148- 184).

[0128] The kinetic binding constants and affinities for the Langmuir law obeying interactions of mAbs 2H2, 4C8, 2A10 and 1A6 are stated in table 4. mAb 2H2 shows high affine binding with KD = 250 pM and 70 pM for binding the TNNT3 peptides b (115-176) and e (148-186). mAb 4C8 shows affine binding with KD = 5.0 nM and 2.3 nM for binding the TNNT3 peptides TNNT3b (115-176) and TNNT3e (148-186). mAb 2A10 shows affine binding with KD = 9.1 nM TNNT3a (38-76) peptide. mAb 1A6 shows high affine binding with with KD < 24 pM to TNNTId (148-184) with a complex half-life time of t / 2 diss > 115 minutes.

[0129] Table 4

[0130] Binding parameters for mAbs 2H2, 4C8, 2A10 and 1A6 binding helical peptides at 37°CSE - Standard error of mean of the SPR-fit in comparison to the measured data points.

[0131] The obtained SPR-data are fitted with the Langmuir 1 :1 binding model, Rl set to zero and Rmax global. Stated values of mabs 4C8 are corrected for the occurred mass transport limitation via the Scrubber SW Version 2.0c using a two-compartment model.Interaction of mAb 1A6 binding to helical peptide TNNT1 (148-184) shows slightly complex binding behavior, therefore, stated constants represent apparent values.

[0132] These results demonstrate the high specificity of the generated antibodies for skTnT, as none of the antibodies show cross-reactivity with cTnT. Furthermore, the datademonstrate that the inventors could generate highly specific antibodies which recognize either both sskTnT and fskTnT, or only sskTnT, or only fskTnT.

[0133] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0134] All patents, patent applications and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0135] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments of the invention will become apparent from the following claims.

[0136] Equivalents: Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0137] The invention is further characterized by the following items:1. An antibody against skeletal Troponin T (skTnT), wherein the anti-skTnT antibody is notcapable of binding to cardiac Troponin T (cTnT). The antibody according to item 1 , wherein the anti-skTnT antibody is capable of binding to a skTnT peptide region being included in a polypeptide region conserved between skTnT and cTnT (conserved skTnT peptide sequence). The antibody according to item 1 or item 2, wherein the anti-skTnT antibody is capable of specifically binding to an epitope within an amino acid sequence as set forth in any one of SEQ-ID- No. 4-9. The antibody according to any one of items 1-3, wherein said antibody is capable of binding to fast skeletal Troponin T (fsk TnT), and further wherein said antibody is capable of binding to slow skeletal Troponin T (sskTnT); he antibody according to item 4, wherein, when bound to sskTnT, the antibody binds to at least one of the following residues: E at aa 57 of SEQ ID No. 2; and further wherein, when bound to fskTnT, the antibody binds to at least one of the following residues: E at aa 61 of SEQ ID No. 3; or when bound to sskTnT, the antibody binds to at least one of the following residues: K at aa 120; R at aa 122 and K at aa 132 of SEQ ID No. 2; and further wherein, when bound to fskT nT, the antibody binds to at least one of the following residues: K at aa 124; R at aa 126 and K at aa 136 of SEQ ID No. 3; or when bound to sskTnT, the antibody binds to at least one of the following residues: G at aa 158; A at aa 159, L at aa 165, Q at aa position 170 and R at aa 172 of SEQ ID No. 2; and further wherein, when bound to fskTnT, the antibody binds to at least one of the following residues: G at aa 162; A at aa 163, L at aa position 169, Q at aa position 174 and R at aa 176 of SEQ ID No. 3. The antibody according to any one of items 1-3, wherein said antibody is capable of binding to fast skeletal Troponin T (fskTnT), and further wherein said antibody is not capable of binding to slow skeletal Troponin T (sskTnT); he antibody according to item 5, preferably wherein, when bound to fskTnT, the antibody binds to at least one of the following residues: P at aa position 50, R at aa position 51 , K at aa position 53, T at aa position 55 and A at aa position 56 of SEQ ID No. 3; or wherein when bound to fskTnT, the antibody binds to at least one of the following residues: A ataa position 122, of SEQ ID No. 3; or when bound to fskTnT, the antibody binds to at least one of the following residues: N at aa position 164, Y at aa position 165, S at aa position 166, S at position 166, A at aa position 169, and D at aa position 172 of SEQ ID No. 3.7. The antibody according to any one of items 1-3, wherein said antibody is capable of binding to slow skeletal Troponin T (ssk TnT), and further wherein said antibody is not capable of binding to fast skeletal Troponin T (fskTnT),8. The antibody according to item 7, preferably wherein, when bound to sskTnT, the antibody binds to at least one of the following residues: P at aa position 40, K at aa position 41 , P at aa position 42, S at aa position 43, P at aa position 45, V at aa position 46, V at aa position 47, P at aa position 49 and I at aa position 51 of SEQ ID No. 2; or when bound to sskTnT, the antibody binds to at least one of the following residues: F at aa position 117, T at aa position 119, A at aa position 126, and K at aa position 127, of SEQ ID No. 2; or when bound to sskTnT, the antibody binds to at least one of the following residues: V at aa position 166 of SEQ ID No. 2.9. The antibody according to any one of the items 1-8, wherein the anti-skTnT antibody may comprise a heavy chain variable region (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 and a light chain variable region (VL) comprising CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of CDR-H1 as depicted in SEQ ID NO: 10, CDR-H2 as depicted in SEQ ID NO: 11 , CDR-H3 as depicted in SEQ ID NO: 12, CDR-L1 as depicted in SEQ ID NO: 13, CDR- L2 as depicted in SEQ ID NO: 14 and CDR-L3 as depicted in SEQ ID NO: 15.10. The antibody according to any one of the items 1-8, wherein the anti-skTnT antibody may comprise a heavy chain variable region (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 and a light chain variable region (VL) comprising CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of CDR-H1 as depicted in SEQ ID NO: 16, CDR-H2 as depicted in SEQ ID NO: 17, CDR-H3 as depicted in SEQ ID NO: 18, CDR-L1 as depicted in SEQ ID NO: 19, CDR- L2 as depicted in SEQ ID NO: 20 and CDR-L3 as depicted in SEQ ID NO: 21.11. The antibody according to any one of items 1-8, wherein the anti-skTnT antibody may comprise a heavy chain variable region (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 and a light chain variable region (VL) comprising CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of CDR-H1 as depicted in SEQ ID NO: 22, CDR-H2 as depicted in SEQ ID NO: 23, CDR-H3 as depicted in SEQ ID NO: 24, CDR-L1 as depicted in SEQ ID NO: 25, CDR- L2 as depicted in SEQ ID NO: 26 and CDR-L3 as depicted in SEQ ID NO: 27.12. The antibody according to any one of items 1-8, wherein the antibody may comprise a VH region selected from the group consisting of those depicted in SEQ ID NO: 28, 29 and 30 and / or ii) a VL region selected from the group consisting of those depicted in SEQ ID NO: 31 , 32 and 33. Preferably, the anti-skTnT antibody comprises i) a VH region as depicted in SEQ ID NO: 28 or 30 and / or ii) a VL region as depicted in SEQ ID NO: 31 or 33.13. The antibody according to item 13, wherein the anti-skTnT antibody comprises a) a VH region as depicted in SEQ ID NO: 28 and a VL region as depicted in SEQ ID NO: 31 , or b) a VH region as depicted in SEQ ID NO: 29 and a VL region as depicted in SEQ ID NO: 32, or c) a VH region as depicted in SEQ ID NO: 30 and a VL region as depicted in SEQ ID NO: 33. Preferably, the anti-skTnT antibody comprises a VH region as depicted in SEQ ID NO: 28 and a VL region as depicted in SEQ ID NO: 31 , or a VH region as depicted in SEQ ID NO: 30 and a VL region as depicted in SEQ ID NO: 33.14. The antibody according to any one of items 1-13, wherein the anti-skTnT antibody may be a monoclonal anti-skTnT antibody.15. The antibody according to any one of items 1-14, wherein the anti-skTnT antibody may be an anti-skTnT antibody that has been affinity maturated, humanized and / or stabilized.16. The antibody according to any one of items 1-15, wherein the anti-skTnT antibody is capable of binding to an epitope within skTnT as set forth in SEQ ID No. 2 (sskTnT) and / or as set forth in SEQ ID No. 3 (fskTnT) with a KD of 10'7M or less, or with a KD of 10'8M or less, or with a KD of 10'9M or less, or with a KD of 5x 10'9or less, or with a KD of 10'1° or less.17. Use of an antibody according to any one of items 1-16 for determining a skeletal muscle damage.18. The use according to item 17, wherein the skeletal muscle damage is a skeletal muscle disorder (SMD), a skeletal muscle damage induced by physical exercise and / or a skeletal muscle damage induced by a drug treatment such as medical drug treatment.19. The use according to item 17 or 18, wherein the use comprises using the antibody for determining a level of a physical fitness of a subject.20. The use of item 18, wherein the SMD is selected from the group consisting of myopathy, myositis, skeletal myopathy, muscular dystrophy, rhabdomyolysis, neuropathy, myasthenic syndrome and autosomal dominant (AD) with muscle symptoms, preferably the SMD is selected from the group consisting of myopathy and myositis; and / or wherein the myopathy is selected from the group consisting of myotonic dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, mitochondrial disease, and glycogenstorage disease; and wherein the myositis is selected from the group consisting of dermatomyositis, polymyositis, sporadic inclusion body myositis, hereditary inclusion body myositis, immune-mediated necrotizing myositis, myositis with overlap with collagenous disease, myositis induced by physical exercise, myositis induced by drug treatment such as medical drug treatment, preferably statin-induced myositis, or a myositis induced by treatment with a check point inhibitor (ICI), by a treatment with an inhibitor of a vascular endothelial growth factor (VEGFI) or combined treatment, and vasculitis.21 . The use of any one of items 17-20, comprising use of the antibody for determining a drug treatment such as a medical drug treatment.22. A method of determining the amount of skeletal Troponin T (skTnT), the method comprising a) Contacting a sample with at least one antibody according to any one of items 1-16, and b) Determining the amount of skT nT.23. The method of item 22, further comprising c) Comparing the determined amount of skTnT to a reference.24. The method of item 21 or 22, wherein the determined amount of skTnT compared to a reference is or is not indicative of a disease associated with skTnT, preferably of a skeletal muscle damage and / or of a risk of skeletal muscle damage.25. The method of item 24, wherein the reference is a reference for a healthy condition and / or for skeletal muscle damage.26. The method of any one of items 21-25, wherein the determined amount of skT nT compared to a reference is or is not indicative for a change of a disease associated with skTnT, preferably of a change in skeletal muscle damage, over time.27. The method of item 26, wherein the reference is a reference skTnT amount determined at an earlier point in time.28. The method of any one of items 21-27, wherein an increased amount of skTnT in the sample compared to the reference is indicative for skeletal muscle damage.29. The method of any one of items 21-28, wherein the skeletal muscle damage is a skeletal muscle disorder (SMD), a skeletal muscle damage induced by physical exercise and / or a skeletal muscle damage induced by a medical treatment.30. The method of item 29, wherein the SMD is selected from the group consisting of myopathy, myositis, skeletal myopathy, muscular dystrophy, rhabdomyolysis, neuropathy, myasthenic syndrome and autosomal dominant (AD) with muscle symptoms, preferably the SMD is selected from the group consisting of myopathy and myositis.31. The method of item 30, wherein the myopathy is a non-inflammatory myopathy, preferably selected from the group consisting of myotonic dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, mitochondrial disease, and glycogen storage disease; and wherein the myositis is selected from the group consisting of dermatomyositis, polymyositis, sporadic inclusion body myositis, hereditary inclusion body myositis, immune-mediated necrotizing myositis, myositis with overlap with collagenous disease, myositis induced by physical exercise, myositis induced by medical treatment, preferably statin-induced myositis, or a myositis induced by treatment with a check point inhibitor (ICI), by a treatment with an inhibitor of a vascular endothelial growth factor (VEGFI) or combined treatment, and vasculitis.32. The method according to any one of items 21-31 , wherein the skTnT is i) fast skTnT (fskTnT), ii) slow skTnT (sskTnT), or iii) fskTnT and sskTnT.33. A polynucleotide encoding an antibody as defined in any one of items 1-16.34. A vector comprising the polynucleotide of item 33.35. A composition comprising at least two antibodies against skTnT according to any one of items 1-16, wherein(i) the at least two antibodies are capable of simultaneously binding to fskT nT and at least one of said at least two antibodies is not capable of binding to sskTnT; or(ii) the at least two antibodies are capable of simultaneously binding to sskTnT and at least one of said at least two two antibodies is not capable of binding to fskTnT; or(iii) the at least two antibodies are capable of simultaneously binding to sskTnT; and wherein the at least two antibodies are capable of simultaneously binding to fskTnT. A computer-implemented method of determining an amount of skeletal Troponin T (skTnT), said method comprising(a) Receiving a first signal indicative of fast skTnT (fskTnT) and a second signal indicative of slow skTnT (sskTnT), or receiving a first signal indicative of fskTnT and a second signal indicative of both sskTnT and fskTnT; or receiving a first signal indicative of (sskTnT) and a second signal indicative of both sskTnT and fskTnT; and(b) Determining the amount of skTnT based on the received first and second signals, if the first signal is indicative of fast skTnT (fskTnT) and the second signal is indicative of slow skTnT (sskTnT); or the amount of sskTnT if the first signal is indicative of fskTnT and a second signal indicative of both sskTnT and fskTnT, or the amount of fskTnT if the first signal indicative of sskTnT and the second signal indicative of both sskT nT and fskT nT ; wherein at least one of the received signals corresponds to the binding of at least one antibody according to any one of items 1-16 to skTnT. The computer-implemented method of item 36, wherein step (b) comprises(b1) Determining an amount of fskTnT based on the received first signal, and(b2) Determining an amount of sskTnT based on the received second signal. The computer-implemented method of item 36 or 37, wherein in step(b1) The amount of fskTnT is determined by comparing the received first signal to a first calibration curve and / or a first reference curve, and(b2) The amount of sskTnT is determined by comparing the received second signal to a second calibration curve and / or a second reference curve. The computer-implemented method of item 36 or 37, wherein in step (b) the amount of skTnT is determined by combining the amount of fskTnT determined in step (b1) and the amount of sskTnT determined in step (b2).40. The computer-implemented method of any one of items 36-39, further comprising(c) Comparing the determined amount of skTnT with a reference.41. The computer-implemented method of any one of items 36-40, wherein the amount of skTnT is the combined amount of fskTnT and of sskTnT.42. The computer-implemented method of any one of claims 36-41 , wherein the received first signal is a signal obtained from a first signal detecting device, wherein said first signal detecting device is capable of detecting a signal of an anti-skTnT antibody as defined in items 1-16 and wherein said anti-skTnT antibody comprises a label detectable by said signal detecting device.43. The computer-implemented method of any one of items 36-42, wherein the received first signal is a signal obtained from a first signal detecting device and wherein the received second signal is a signal obtained from the first or a second signal detecting device, wherein said first and / or second signal detecting device is / are capable of detecting a signal of an anti-skTnT antibody of as defined in items 1-16 and wherein said anti-skTnT antibody comprises a label detectable by said signal detecting .44. A computer program product comprising a program code for executing the computer- implemented method of any one of items 36-43 when run on a computer.45. A computer-readable medium having stored thereon a program code for executing the computer-implemented method of any one of items 36-43 when run on a computer.46. A device for carrying out a method of determining skeletal T roponin T (skT nT), the device comprising means for receiving a signal indicative of skTnT in a sample in the presence or suspected presence of an anti-skTnT antibody according to any one of items 1-16 and determining skTnT based on the received signal.The device of item 46, wherein the device further comprises means for comparing the determined skTnT to a reference, wherein the determined skTnT compared to the skTnT reference is or is not indicative for a skeletal muscle damage.

Claims

Claims1. An antibody against skeletal Troponin T (skTnT), wherein the anti-skTnT antibody is not capable of binding to cardiac Troponin T (cTnT).

2. The antibody according to claim 1 , wherein the anti-skT nT antibody is capable of binding to a skTnT peptide sequence comprised in a conserved sequence region between skTnT and cTnT (conserved skTnT peptide sequence).

3. The antibody according to claim 1 or claim 2, wherein the anti-skTnT antibody is capable of specifically binding to an epitope within a skTnT peptide sequence as set forth in any one of SEQ-ID- No. 4-9.

4. The antibody according to any one of claims 1-3, wherein said antibody is capable of binding to fast skeletal T roponin T (fsk T nT), and further wherein said antibody is capable of binding to slow skeletal Troponin T (sskTnT); preferably wherein, when bound to sskTnT, the antibody binds to at least one of the following residues: E at aa 57 of SEQ ID No. 2; and further wherein, when bound to fskTnT, the antibody binds to at least one of the following residues: E at aa 61 of SEQ ID No. 3; or when bound to sskTnT, the antibody binds to at least one of the following residues: K at aa 120; R at aa 122 and K at aa 132 of SEQ ID No. 2; and further wherein, when bound to fskTnT, the antibody binds to at least one of the following residues: K at aa 124; R at aa 126 and K at aa 136 of SEQ ID No. 3; or when bound to sskTnT, the antibody binds to at least one of the following residues: G at aa 158; A at aa 159, L at aa 165, Q at aa position 170 and R at aa 172 of SEQ ID No. 2; and further wherein, when bound to fskTnT, the antibody binds to at least one of the following residues: G at aa 162; A at aa 163, L at aa position 169, Q at aa position 174 and R at aa 176 of SEQ ID No. 3.

5. The antibody according to any one of claims 1-3, wherein said antibody is capable of binding to fast skeletal Troponin T (fskTnT), and further wherein said antibody is not capable of binding to slow skeletal Troponin T (sskTnT); preferably wherein, when bound to fskTnT, the antibody binds to at least one of the following residues: P at aa position 50, R at aa position 51 , K at aa position 53, T at aa position 55 and A at aa position 56 of SEQ ID No. 3; or wherein when bound to fskTnT,the antibody binds to at least one of the following residues: A at aa position 122, of SEQ ID No. 3; or when bound to fskTnT, the antibody binds to at least one of the following residues: N at aa position 164, Y at aa position 165, S at aa position 166, S at position 166, A at aa position 169, and D at aa position 172 of SEQ ID No. 3.

6. The antibody according to any one of claims 1-3, wherein said antibody is capable of binding to slow skeletal Troponin T (ssk TnT), and further wherein said antibody is not capable of binding to fast skeletal Troponin T (fskTnT), preferably wherein, when bound to sskTnT, the antibody binds to at least one of the following residues: P at aa position 40, K at aa position 41 , P at aa position 42, S at aa position 43, P at aa position 45, V at aa position 46, V at aa position 47, P at aa position 49 and I at aa position 51 of SEQ ID No. 2; or when bound to sskTnT, the antibody binds to at least one of the following residues: F at aa position 117, T at aa position 119, A at aa position 126, and K at aa position 127, of SEQ ID No. 2; or when bound to sskTnT, the antibody binds to at least one of the following residues: V at aa position 166 of SEQ ID No. 2.

7. The antibody according to any one of the claims 1-3, wherein the anti-skTnT antibody may comprise a heavy chain variable region (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 and a light chain variable region (VL) comprising CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of CDR-H1 as depicted in SEQ ID NO: 10, CDR-H2 as depicted in SEQ ID NO: 11 , CDR-H3 as depicted in SEQ ID NO: 12, CDR-L1 as depicted in SEQ ID NO: 13, CDR- L2 as depicted in SEQ ID NO: 14 and CDR-L3 as depicted in SEQ ID NO: 15.

8. The antibody according to any one of the claims 1-3, wherein the anti-skTnT antibody may comprise a heavy chain variable region (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 and a light chain variable region (VL) comprising CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of CDR-H1 as depicted in SEQ ID NO: 16, CDR-H2 as depicted in SEQ ID NO: 17, CDR-H3 as depicted in SEQ ID NO: 18, CDR-L1 as depicted in SEQ ID NO: 19, CDR- L2 as depicted in SEQ ID NO: 20 and CDR-L3 as depicted in SEQ ID NO: 21.

9. The antibody according to any one of items 1-3, wherein the anti-skTnT antibody may comprise a heavy chain variable region (VH) comprising CDR-H1 , CDR-H2 and CDR-H3 and a light chain variable region (VL) comprising CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of CDR-H1 as depicted in SEQ ID NO: 22, CDR-H2 as depicted in SEQ ID NO: 23, CDR-H3 as depicted in SEQ ID NO: 24, CDR-L1 as depicted in SEQ ID NO: 25, CDR- L2 as depicted in SEQ ID NO: 26 and CDR-L3 as depicted in SEQ ID NO: 27.

10. Use of an antibody according to any one of claims 1-9 for determining a skeletal muscle damage.11 . The use according to claim 10, wherein the skeletal muscle damage is a skeletal muscle disorder (SMD), a skeletal muscle damage induced by physical exercise and / or a skeletal muscle damage induced by a drug treatment such as medical drug treatment; preferably wherein the use comprises using the antibody for determining a level of a physical fitness of a subject, if the skeletal muscle damage is induced by a physical exercise.

12. The use of claim 11 , wherein the SMD is selected from the group consisting of myopathy, myositis, skeletal myopathy, muscular dystrophy, rhabdomyolysis, neuropathy, myasthenic syndrome and autosomal dominant (AD) with muscle symptoms, preferably the SMD is selected from the group consisting of myopathy and myositis; and / or wherein the myopathy is selected from the group consisting of myotonic dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, mitochondrial disease, and glycogen storage disease; and wherein the myositis is selected from the group consisting of dermatomyositis, polymyositis, sporadic inclusion body myositis, hereditary inclusion body myositis, immune-mediated necrotizing myositis, myositis with overlap with collagenous disease, myositis induced by physical exercise, myositis induced by drug treatment such as medical drug treatment, preferably statin-induced myositis, or a myositis induced by ICI, VEGFI or combined treatment, and vasculitis.

13. A method of determining the amount of skeletal Troponin T (skTnT), the method comprising a) Contacting a sample with at least one antibody according to any one of claims 1- 6, and b) Determining the amount of skT nT.

14. The method of claim 13, wherein skTnT is i) fast skTnT (fskTnT), ii) slow skTnT (sskTnT), or iii) fskTnT and sskTnT.

15. A polynucleotide encoding an antibody as defined in any one of claims 1-9.

16. A composition comprising at least two antibodies against skTnT according to claims 1- 9, wherein(i) the at least two antibodies are capable of simultaneously binding to fskT nT and at least one of said at least two antibodies is not capable of binding to sskTnT; or(ii) the at least two antibodies are capable of simultaneously binding to sskTnT and at least one of said at least two two antibodies is not capable of binding to fskT nT ; or(iii) the at least two antibodies are capable of simultaneously binding to sskTnT; and wherein the at least two antibodies are capable of simultaneously binding to fskT nT.

17. A computer-implemented method of determining an amount of skeletal Troponin T (skTnT), said method comprising(a) Receiving a first signal indicative of fast skTnT (fskTnT) and a second signal indicative of slow skTnT (sskTnT), or receiving a first signal indicative of fskTnT and a second signal indicative of both sskTnT and fskTnT; or receiving a first signal indicative of (sskTnT) and a second signal indicative of both sskTnT and fskT nT ; and(b) Determining the amount of skTnT based on the received first and second signals, if the first signal is indicative of fast skTnT (fskTnT) and the second signal is indicative of slow skT nT (sskT nT); or the amount of sskT nT if the first signal is indicative of fskTnT and a second signal indicative of both sskTnT and fskT nT, or the amount of fskT nT if the first signal indicative of sskT nT and the second signal indicative of both sskTnT and fskTnT; wherein at least one of the received signals corresponds to the binding of at least one antibody according to any one of claims 1-9 to skTnT.

18. A computer-readable medium having stored thereon a program code for executing the computer-implemented method of claim 15 when run on a computer.

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