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WO2009118185A1 - Peptides dérivés de la troponine cardiaque i et leur utilisation à titre de marqueurs pronostiques et diagnostiques pour l'insuffisance cardiaque - Google Patents

Peptides dérivés de la troponine cardiaque i et leur utilisation à titre de marqueurs pronostiques et diagnostiques pour l'insuffisance cardiaque Download PDF

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Publication number
WO2009118185A1
WO2009118185A1 PCT/EP2009/002237 EP2009002237W WO2009118185A1 WO 2009118185 A1 WO2009118185 A1 WO 2009118185A1 EP 2009002237 W EP2009002237 W EP 2009002237W WO 2009118185 A1 WO2009118185 A1 WO 2009118185A1
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Prior art keywords
peptide
seq
ctnl
antibody
troponin
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PCT/EP2009/002237
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English (en)
Inventor
Ziya Kaya
Hugo Albert Katus
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Universitätsklinikum Heidelberg
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Publication of WO2009118185A1 publication Critical patent/WO2009118185A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin

Definitions

  • the present invention relates to peptides derived from cardiac troponin I (cTnl) which are the antigenic determinant(s) or pathogenic epitope(s) of cardiac troponin I (cTnl) and antibodies and antibody fragments specific for these peptides.
  • the present invention furthermore relates to the use of these peptides, antibodies and antibody fragments for the prognosis and/or diagnosis of heart insufficiency, in particular of an inflammation of the myocardium and/or fibrosis.
  • the present invention further relates to methods for the prognosis and/or diagnosis of heart insufficiency, in particular of an inflammation of the myocardium and/or fibrosis, utilizing the peptides.
  • Heart failure has become an increasingly prevalent disorder with considerable morbidity and mortality. While many causal mechanisms such as inherited cardiomyopathies, ischemic cardiomyopathy or muscular overload are easily identified in clinical practice, the molecular mechanisms that determine the progression of heart failure or ventricular remodeling are largely unknown. There is compelling evidence that inflammatory mechanisms may contribute to progressive heart failure. Thus, myocardial infiltration of lymphocytes and mononuclear cells, increased expression of pro-inflammatory chemokines and cytokines and circulating autoantibodies are frequently observed in myocarditis and DCM (dilated cardiomyopathy). The antibodies identified in DCM patients are directed against various myocardial constituents (1, 2).
  • Cardiac troponins in blood are the preferred biomarkers of myocardial injury.
  • the fact that they are strictly intracellular proteins which are not found in the circulation of healthy individuals provides a high level of clinical sensitivity and specificity even when cardiac lesions are small. Thus, any significantly detectable troponin in circulation is considered a sign of acute myocardial cell damage (3, 4).
  • Nishimura et al. reported that PD-I receptor deficient mice developed autoantibodies against cardiac troponin I (cTnl) and -as a consequence- severe dilated cardiomyopathy (5). They further found that administration of monoclonal anti-cTnl antibodies induced myocardial dysfunction highly likely by facilitating Ca 2+ influx into cardiomyocytes (6).
  • the present invention aims to provide means and methods for the prognosis and diagnosis of heart insufficiency, including inflammation, fibrosis and heart failure, in particular autoimmune myocarditis.
  • this object is solved by providing peptides derived from cardiac troponin I (cTnl) which are antigenic determinants or pathogenic epitopes of cardiac troponin I (cTnl).
  • the peptide of the present invention comprises an amino acid sequence of SEQ ID NO. 9 or
  • variants comprising modified amino acid(s), unnatural amino acid(s) or peptidomimetic(s) ;
  • this object is furthermore solved by providing an antibody or antibody fragment specific for a peptide of the present invention.
  • this object is furthermore solved by providing an antibody or antibody fragment specific for cardial troponin I (cTnl), which does not bind to a region or an epitope of cTnl which comprises or corresponds to a peptide of the present invention.
  • cTnl cardial troponin I
  • this object is furthermore solved by providing the peptide, the antibody or antibody fragment according to the present invention for use in medicine.
  • this object is furthermore in particular solved by providing the peptide, the antibody or antibody fragment according to the present invention for the prognosis and/or diagnosis of heart insufficiency, in particular of an inflammation of the myocardium and/or fibrosis.
  • this object is furthermore solved by a method for the prognosis and/or diagnosis of heart insufficiency, in particular of an inflammation of the myocardium and/or fibrosis.
  • the method of the present invention comprises a) providing a patient sample, preferably a bodily fluid or tissue, b) providing at least one peptide according to the present invention, c) determining whether the patient has an immune reaction against said peptide(s), wherein said immune reaction is determined by determining the presence or absence of antibodies against said peptide(s) or determining the presence or absence of T cell response in said patient sample, d) optional, determining the presence or absence of further markers in said patient sample.
  • the present invention provides peptides derived from cardiac troponin I
  • an “antigenic determinant” or “pathogenic epitope” of cardiac troponin I refers to a amino acid sequence or sequence motif of the protein sequence of cTnl that
  • the peptide is derived from murine cTnl, human cTnl, rat cTnl or other cTnl homologues, more preferably from murine cTnl.
  • Murine cTnl, human cTnl, rat cTnl or other cTnl homologues as well as their amino acid sequences and the nucleotide sequences of the genes encoding them are known in the art.
  • For an amino acid sequence of murine cTnl see Genbank Accession No. NP 033432 (SEQ ID NO. 28); for an amino acid sequence of human cTnl see Genbank Accession No. CAA62301 (SEQ ID NO. 29); for an amino acid sequence of rat cTnl see Genbank Accession No. CAA41402 (SEQ ID NO. 30).
  • a preferred amino acid sequence of murine cTnl is [SEQ ID NO. 28]
  • a preferred amino acid sequence ofhuman cTnl is [SEQ ID NO.29]
  • a preferred amino acid sequence ofrat cTnl is [SEQ ID NO.30]
  • a peptide derived from cardiac troponin I comprises an amino acid sequence of SEQ ID NO. 9 or SEQ ID NO. 11 or variants thereof.
  • SEQ ID NO. 9 refers to amino acid residues 105 to 122 of murine cTnl VDKVDEERYDVEAKVTKN
  • SEQ ID NO. 11 refers to amino acid residues 131 to 148 of murine cTnl QKIYDLRGKFKRPTLRRV. More preferably, the peptide has the amino acid sequence of SEQ ID NO. 9 or consists of it.
  • variants of the peptide are selected from
  • N-terminally and/or C-terminally truncated or elongated peptides comprising at least 7 contiguous amino acids of SEQ ID NO. 9 or SEQ ID NO. 11 ; preferably N-terminally and/or C-terminally truncated or elongated peptides comprising at least 10 contiguous amino acids of SEQ ID NO. 9 or SEQ ID NO. 11
  • the N-terminally and/or C-terminally truncated or elongated peptides can comprise any number of amino acid residues from SEQ ID NO. 9 or 11 in the range from 7 to 18 amino acids, such as 7, 8, 9, 10, ..., 17, 18 amino acids);
  • variants comprising modified amino acid(s), unnatural amino acid(s) or peptidomimetic(s) ;
  • a peptide sequence is considered a variant of the peptides of the present invention when it
  • a peptide sequence is considered a variant of the peptides of the present invention when it shows at least 80%, preferably 90% activity of the peptide with SEQ ID NO. 9, wherein "activity” refers to (i) induction of inflammation and/or fibrosis to the heart of mice immunized with the respective peptide tested; and/or (ii) induction of an immune response of mice immunized with the respective peptide (antibody titer against cTnl and the peptide tested).
  • Amino acid substitution variants comprise conservative or non conservative replacement by other amino acids.
  • Conservative amino acid substitutions typically relate to substitutions among amino acids of the same class. These classes include, for example,
  • amino acids having basic side chains such as lysine, arginine, and histidine ;
  • - amino acids having nonpolar side chains such as glycine, alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan, and cysteine.
  • the variants have an amino acid substitution or deletion (only) at one amino acid position.
  • amino acid substitution variant comprises or has amino acid residues 104- 121 of human cTnl: VDKVDEERYDIEAKVTKN [SEQ ID NO. 17] .
  • the variants do not comprise or have the following sequences:
  • VEVVDEERYDIEAKCLHN [SEQ ID NO. 18]
  • variants that are N-terminally and/or C-terminally elongated peptides comprise or have amino acid residues of both peptides of SEQ ID NO. 9 and SEQ ID NO. 11 or at least 7 (preferably 10) contiguous amino acids of both SEQ ID NO. 9 and SEQ ID NO. 11, such as a peptide that comprises or has amino acid residues of murine cTnl from the start of peptide 9/SEQ ID NO. 9 to the end of peptide 11/SEQ ID NO. 11 (i.e. amino acid residues 105 to 148 of murine cTnl): VDKVDEERYDVEAKVTKNITEIADLTOKIYDLRGKFKRPTLRRV [SEQ ID NO. 21 ]
  • variants of the peptide are selected from
  • N-terminally and/or C-terminally truncated or elongated peptides of SEQ ID NO. 21 preferably N-terminally and/or C-terminally truncated or elongated peptides comprising at least 10, preferably 12 contiguous amino acids of SEQ ID NO. 21;
  • the N-terminally and/or C-terminally truncated or elongated peptides can comprise any number of amino acid residues of SEQ ID NO. 21 in the range from 10 to 44 amino acids, such as 10, 11, 12 ..., 43, 44 amino acids);
  • variants comprising modified amino acid(s), unnatural amino acid(s) or peptidomimetic(s) (as described above);
  • the peptide of the present invention comprises 10 contiguous amino acids of SEQ ID NO. 9 or SEQ ID NO. 11, preferably selected from
  • ERYDVEAKVT (SEQ ID NO.26).
  • the peptides according to the present invention have a length of at least 7 amino acids, preferably have a length from about 10 to about 50 amino acids, wherein at least 7, preferably at least 10 amino acids are contiguous amino acid residues of SEQ ID NO. 9 or SEQ ID NO. 11 (or wherein at least 10, preferably 12 amino acids are contiguous amino acid residues of SEQ ID NO. 21).
  • the peptides consist of any length in the range from 7 to about 100 amino acids, preferably from 7 to about 50 amino acids, such as 7, 8, 9, 10, ..., 44, 45, 46, 47, 48, 49, 50 amino acids. It should be noted that "" denotes every integer in the respective range.
  • the peptides according to the present invention do not comprise the full-length protein cardiac troponin I (cTnl), such as murine cTnl (SEQ ID NO. 28), human cTnl (SEQ ID NO. 29), rat cTnl (SEQ ID NO. 30) or other cTnl homologies.
  • the peptides according to the present invention comprise N- terminal and/or C-terminal modifications.
  • hydrophobic modification such as acylation (e.g. with carboxylic acids, fatty acids, C8 to C22 fatty acids, amino acids with lipophilic side chains); addition of hydrophobic moieties (e.g. with cholesterol, derivatives of cholesterol, phospholipids, glycolipids, glycerol esters, steroids, ceramids, isoprene derivatives, adamantane, farnesol, aliphatic groups, polyaromatic compounds);
  • acylation e.g. with carboxylic acids, fatty acids, C8 to C22 fatty acids, amino acids with lipophilic side chains
  • hydrophobic moieties e.g. with cholesterol, derivatives of cholesterol, phospholipids, glycolipids, glycerol esters, steroids, ceramids, isoprene derivatives, adamantane, farnesol, aliphatic groups, polyaromatic compounds
  • a modification with a moiety that protects from degradation e.g. amide, D-amino acid, modified amino acid, cyclic amino acid; natural and synthetic polymer, such as PEG, glycane;
  • a moiety that boosts the immune response e.g. bovine serum albumin, keyhole limpet hemocyanin.
  • Acylation is preferably selected from acylation with carboxylic acids, fatty acids, amino acids with lipophilic side chains.
  • Preferred fatty acids are saturated or unsaturated fatty acids, branched or unbranched fatty acids, preferably with 8 to 22 carbon atoms (C8 to C22).
  • the hydrophobic modification by acylation can be selected from acylation with myristoyl (C 14), palmitoyl (C 16) or stearoyl (C 18).
  • hydrophobic moieties is preferably selected from addition of cholesterol, derivatives of cholesterol, phospholipids, glycolipids, glycerol esters, steroids, ceramids, isoprene derivatives, adamantane, farnesol, aliphatic groups, polyaromatic compounds.
  • the attachment of the hydrophobic moieties is preferably by covalent binding, which can be achieved via carbamate, amide, ether, disulfide or any other linkage that is within the skill of the person skilled in the art.
  • the acylation or addition of hydrophobic moieties is preferably N-terminal, wherein "N- terminal" refers to the hydrophobic modification at the N-terminus, i.e.
  • the respective first amino acid residue but comprises also the hydrophobic modification in close proximity to the N-terminus.
  • the hydrophobic modification can furthermore be obtained by an attachment of a hydrophobic moiety at a site close to the N-terminus of the peptide of the invention.
  • Such a modification is preferably C-terminal, wherein "C-terminal” refers to the modification at the C-terminus, i.e. the respective last amino acid residue, but comprises also the modification in close proximity to the C-terminus, such as the last but one amino acid residue, the last but two amino acid residue or more amino acid residues (e.g. introduction of one D- amino acid that protects the carrier from en2ymatic degradation e.g. by the action of carboxypeptidases) .
  • C-terminal refers to the modification at the C-terminus, i.e. the respective last amino acid residue, but comprises also the modification in close proximity to the C-terminus, such as the last but one amino acid residue, the last but two amino acid residue or more amino acid residues (e.g. introduction of one D- amino acid that protects the carrier from en2ymatic degradation e.g. by the action of carboxypeptidases) .
  • Preferred moieties that protect from degradation are selected from amides, D-amino acids, modified amino acids, cyclic amino acids; natural and synthetic polymers, such as PEG, glycane.
  • Preferred moieties that boost the immune response are selected from bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • the peptides according to the present invention comprise
  • - label such as fluorescent dye(s), radioisotope(s) and contrast agent(s), and/or
  • the label(s) are preferably suitable for an in vivo medical use in diagnostics and prognostics, as known to the skilled artisan.
  • Preferred radioisotopes are 131 1, 125 1, 99m Tc, 18 F, 68 Ga, 111 In, 90 Y, 177 Lu.
  • Preferred fluorescent dyes are Alexa dyes, derivatives of rhodamine and fluorescein, Cy-dyes, green fluorescent protein (GFP) and its derivatives (EGFP, CFP, ECFP, YFP, EYFP, etc), zoanFP or the red fluorescent protein drFP583 and their derivates.
  • Preferred contrast agents are Gadolinium (Gd) complexes, supramagnetic iron (Fe) complexes and particles, compounds containing atoms of high atomic number, i.e. iodine for computer tomography (CT), microbubbles and carriers such as liposomes that contain these contrast agents.
  • peptides of this invention can be prepared by a variety of procedures readily known to those skilled in the art, in general by synthetic chemical procedures and/or genetic engineering procedures.
  • Synthetic chemical procedures include more particularly the solid phase sequential and block synthesis (Erickson and Merrifield, 1976).
  • the solid phase sequential procedure can be performed using established automated methods such as by use of an automated peptide synthesizer.
  • the peptides of the present invention may also be obtained by coupling (poly)peptide fragments that are selectively protected, this coupling being effected e.g. in a solution.
  • the peptides can further be produced by genetic engineering techniques as known to the skilled artisan, utilizing eukaryotic and/or prokaryotic expression systems.
  • the present invention provides antibodies and antibody fragments.
  • an antibody or antibody fragment of the present invention is specific for a peptide of the present invention as defined herein, i.e. it specifically binds thereto.
  • An “antibody” refers to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, genetically engineered antibodies, such as humanized antibodies, chimeric antibodies etc.
  • antibody fragment refers to a fragment of antibody that specifically binds to the respective epitope/antigen and comprises Fab, single chain Fv, genetically engineered antibody fragments (such as diabodies), etc.
  • a further antibody or antibody fragment of the present invention is specific for cardial troponin I (cTnl) and does not bind to a region or an epitope of cTnl which comprises or corresponds to a peptide of the present invention as defined herein.
  • the antibodies and/or antibody fragments of this invention can be prepared by a variety of procedures known to those skilled in the art, preferably by genetic engineering procedures, but also synthetic chemical procedures.
  • the present invention provides the peptide(s), antibody(ies) and antibody fragment(s) of the present invention for use in medicine.
  • the present invention provides the first medical use of the peptide(s), antibody(ies) and antibody fragment(s) of the present invention.
  • the present invention also provides second medical use(s) of the peptide(s), antibody(ies) and antibody fragment(s) of the present invention.
  • the present invention provides the peptide(s), antibody(ies) and antibody fragment(s) of the present invention for the prognosis and/or diagnosis of heart insufficiency.
  • Heart insufficiency or “heart failure” refers to a condition that can result from any structural or functional cardiac disorder that impairs the ability of the heart to fill with or pump a sufficient amount of blood throughout the body.
  • causes and contributing factors to congestive heart failure include the following (with specific reference to left (L) or right (R) sides): genetic family history of CHF, ischemic heart disease/myocardial infarction (coronary artery disease), thyrotoxicosis (hyperthyroidism), hypothyroidism, anemia, arrhythmia, hypertension, infection, cardiac fibrosis, coarctation of the aorta (L), aortic stenosis/regurgitation (L), mitral regurgitation (L), pulmonary stenosis/pulmonary hypertension/cor pulmonale/pulmonary embolism (R), mitral valve disease (L), cardiomyopathy, including noncompaction cardiomyopathy (L&R) and inflammation.
  • L&R noncompaction cardiomy
  • the peptide(s), antibody(ies) and antibody fragment(s) of the present invention are in particular suitable for the prognosis and/or diagnosis of an inflammation of the myocardium and/or fibrosis.
  • the peptide, the antibody or the antibody fragment of the present invention is a suitable marker for the prognosis and/or diagnosis of heart insufficiency, in particular of an inflammation of the myocardium and/or fibrosis,.
  • the peptide, the antibody or the antibody fragment of the present invention is used for determining a patient group, in particular a patient risk group, with an inflammation of the myocardium and/or fibrosis.
  • the inflammation of the myocardium and/or fibrosis is autoimmune myocarditis.
  • the peptide, the antibody or the antibody fragment of the present invention is used for risk stratification in acute cardiac disorders of patients.
  • “Risk assessment” or “risk stratification” of subjects with heart failure refers to the evaluation of factors, such as biomarkers, in order to predict the risk of future events or even death and in order to decide about the type, manner, dosis, regimen of therapy and treatment for the individual subject.
  • the present invention demonstrates the use of the peptides of the invention (preferably peptide 9 and 11 sequences; SEQ ID NO. 9 and 11) instead of the whole troponin molecule in testing patient samples (preferably sera) to identify patients at risk for myocardial damage induced by autoimmune inflammation.
  • the use of the peptides of the invention in tests is more specific for identifying the risk of inflammation than using the whole troponin protein (TnI) since the inventors demonstrated that immunization with the other peptide sequences of troponin I (namely SEQ ID NOs. 1-8, 10, 12-16) did not induce an inflammation or fibrosis in the myocardium.
  • TnI whole troponin protein
  • the use of whole troponin protein will identify all antibodies directed against different sequences of troponin, including those that might have no pathogenic effect. For further details see below.
  • Troponin autoantibodies are present in patients and can produce false negative results by blocking the binding of troponin antibodies used in analytical assays to the target protein.
  • the presence of autoantibodies against cTnl in patients with acute coronary syndrome points to an early induction of an autoimmune response to cTnl in these patients.
  • troponin I autoantibodies may have clinical consequences.
  • Troponin autoantibodies induce dilation and cardiac dysfunction by the chronic stimulation of calcium influx in cardiomyocytes. Provoking an autoimmune response to cardiac troponin I induces severe inflammation in the myocardium followed by fibrosis and heart failure with increased mortality in mice.
  • the present invention identifies one major and one minor epitope of troponin I (peptide 9 and 11, respectively) that are responsible for disease induction. Using these newly identified epitope sequences of troponin I instead of the whole troponin molecule; a more specific screening test of patients with high risk for progressive autoimmune inflammatory heart failure is possible.
  • the above uses preferably comprises i. determining if a patient has an immune reaction against peptide(s) of the present invention, wherein said immune reaction is determined by o determining the presence or absence of antibodies against said peptide(s) or o determining the presence or absence of T cell response in a patient sample or the patient, ii. determining the presence or absence of o auto-antibodies against a peptide according to the present invention and/or o auto-antibodies against cardial troponin I (cTnl) in a patient sample or the patient, iii. determining cell proliferation and/or production of cytokine(s) and/or chemokine(s) and/or changes in expression of chemokine/cytokine receptors by different cells after stimulation with the peptide(s).
  • cell proliferation and/or production of cytokine(s) and/or chemokine(s) and/or changes in expression of chemokine/cytokine receptors by different cells is determined in cells of the immune system, such as (all subsets of) T cells, (all subsets of) B cells, monocytes/macrophages, endothelial cells, (all subsets of) dendritric cells, NK cells, mast cells, thrombocytes and (all subsets of) stem cells.
  • cytokine(s) such as Interferon-alpha, Interferon-beta,
  • Interferon-gamma TNF- ⁇ , IL-I, IL-2, IL-6, IL-5, IL-4, IL-IO, IL- 13, IL- 17, and others and/or chemokine(s), such as RANTES, MCP-I, MIP-Ia, MlP-l ⁇ , MIP-2, TCA-3, eotaxin, is determined.
  • the change in expression of chemokine receptors such as CCRl, CCR2, CCR5 is determined.
  • the present invention provides a method for the prognosis and/or diagnosis of heart insufficiency or heart failure.
  • the method of the present invention is in particular suitable for the prognosis and/or diagnosis of an inflammation of the myocardium and/or fibrosis, more particularly of autoimmune myocarditis.
  • the method of the present invention comprises the following steps: a) providing a patient sample, b) providing at least one peptide according to the present invention, c) determining whether the patient has an immune reaction against said peptide(s), wherein said immune reaction is determined by determining the presence or absence of antibodies against said peptide(s) or determining the presence or absence of T cell response in said patient sample, d) optional, determining the presence or absence of further markers in said patient sample.
  • the method of the present invention comprises determining cell proliferation and/or production of cytokine(s) and/or chemokine(s) and/or changes in expression of chemokine/cytokine receptors (such as upregulation) by different cells after stimulation with the peptide(s).
  • cell proliferation and/or production of cytokine(s) and/or chemokine(s) and/or changes in expression of chemokine/cytokine receptors by different cells is determined in cells of the immune system, such as (all subsets of) T cells, (all subsets of) B cells, monocytes/macrophages, endothelial cells, (all subsets of) dendritric cells, NK cells, mast cells, thrombocytes and (all subsets of) stem cells.
  • cytokine(s) such as Interferon-alpha, Interferon-beta,
  • Interferon-gamma TNF- ⁇ , IL-I, IL-2, IL-6, IL-5, IL-4, IL-IO, IL- 13, IL- 17, and others and/or chemokine(s), such as RANTES, MCP-I, MIP- l ⁇ , MIP- l ⁇ , MIP-2, TCA-3, eotaxin is determined.
  • chemokine(s) such as RANTES, MCP-I, MIP- l ⁇ , MIP- l ⁇ , MIP-2, TCA-3, eotaxin is determined.
  • the change in expression of chemokine receptors such as CCRl, CCR2, CCR5 is determined.
  • the patient sample is preferably a bodily fluid or tissue, more preferably whole blood, serum, a biopsy (preferably of the heart).
  • step c) preferably, the presence or absence of auto-antibodies against cardial troponin I (cTnl) and/or of auto-antibodies against a peptide according to the present invention is determined.
  • the method comprises the use of an antibody or antibody fragment according to the present invention.
  • the method according to the present invention is for determining a patient group, in particular a patient risk group, with an inflammation of the myocardium and/or fibrosis, preferably with autoimmune myocarditis.
  • the method according to the present invention is for risk stratification in acute cardiac disorders of patients.
  • markers for heart failure or heart insufficiency are known in the art, such as brain natiuretic peptide (BNP), NT-pro-BNP, ANP, troponin I, troponin T, hsCRP, heart-type fatty acid binding protein (H-FABP), myosin light chain- 1(MLC- l),creatinine, soluble CD40 ligand (sCD40L), PAPP-A, MPO, VEGF or PlGF.
  • BNP brain natiuretic peptide
  • NT-pro-BNP ANP
  • troponin I troponin T
  • hsCRP heart-type fatty acid binding protein
  • H-FABP heart-type fatty acid binding protein
  • MLC- l myosin light chain- 1(MLC- l),creatinine
  • sCD40L soluble CD40 ligand
  • mice developed significant autoantibody titers against all injected peptides by day 28. However, total IgG autoantibody titers were significantly higher in mice immunized with the peptides 7, 8, 10 and 14 [SEQ ID NOs. 7, 8, 10 and 14] (data not shown).
  • the inventors measured the mRNA-expression levels of the chemokines Ltn, RANTES, MIP- l ⁇ , MIP-Ia, MIP-2, IP-10, MCP-I, TCA-3, eotaxin and of the chemokine receptors CCRl, CCR2, CCRIb, CCR3, CCR4 and CCR5 in the myocardium of the immunized mice.
  • the inventors were able to detect mRNA levels for RANTES, MIP- l ⁇ , MIP- l ⁇ , MIP-2, MCP-I, TCA-3, eotaxin only in mice immunized with the residues 105-122 of mcTnl (peptide 9; SEQ ID NO. 9) but not in mice immunized with the other peptides or control buffer and adjuvant alone ( Figure 5A).
  • the inventors found that the mRNAs for the chemokine receptors CCRl, CCR2 and CCR5 were solely expressed in the myocardium of mice immunized with the residues 105-122 of mcTnl (peptide 9, SEQ ID NO. 9) ( Figure 5B).
  • mice immunized with mcTnl developed not only antibody against the whole mcTnl protein but also against the residues 105-122 of mcTnl (peptide 9 SEQ ID NO. 9) ( Figure 5C).
  • immunization with the residues 105-122 of mcTnl induced production of antibodies reacting with the complete protein mcTnl ( Figure 5D).
  • the next protein with amino acid sequences similar to both mcTnl epitopes was skeletal troponin I (residues 73-90 and 99-116 in slow skeletal troponin and residues 98-115 in fast skeletal troponin). Residues 73-90 of slow skeletal troponin were already different in 6 amino acids to the main epitope of mcTnl (105-122; peptide 9 SEQ ID NO. 9) ( Figure 6).
  • Residues 98-115 of fast skeletal troponin I were different in 3 amino acids and residues 99-116 of slow skeletal troponin I were different in 4 amino acids to the minor epitope of mcTnl (131-148; peptide 11).
  • mice were immunized with either hcTnl (104-121, SEQ ID NO. 17), or slow skeletal troponin I (residues 73-90 or 99-116, SEQ ID ON. 18 or SEQ ID NO. 19), or fast skeletal troponin I (residues 98-115, SEQ ID NO. 20).
  • Residues 105-122 peptide 9, SEQ ID NO.
  • mcTnl were the strongest inductor of inflammation and fibrosis in the myocardium which is accompanied by increased expression of inflammatory chemokines RANTES, IP-IO, MCP-I, MIP- l ⁇ , MIP- l ⁇ , MIP-2, TCA-3 and eotaxin, and of the chemokine receptors CCRl, CCR2, CCR5.
  • McTnI residues 131-148 (peptide 11, SEQ ID NO. 11) were a minor epitope inducing milder inflammation.
  • mice immunized with skeletal troponin I showed significant signs of inflammation.
  • mice immunized with mcTnl also developed antibodies directed against the residues 105-122 of mcTnl (peptide 9, SEQ ID NO. 9).
  • immunization with the residues 105-122 of mcTnl induced production of antibodies reacting with the complete protein mcTnl.
  • residues 105-122 of mcTnl SEQ ID NO.
  • mice pre- immunized with TnI prior to LAD ligation showed greater infarct size, more fibrosis, higher inflammation scores and reduced fractional shortening than mice without pre-immunization (8).
  • Proinflammatory cytokines are important in the development of autoimmune myocarditis.
  • the inventors previously showed that administration of either IL-I or TNF- ⁇ promoted virus- and myosin-induced myocarditis in genetically resistant B 10.
  • a mice (17).
  • the inventors demonstrated that the presence of myocarditis is associated with increased levels of TNF- ⁇ from cardiac myosin-stimulated splenocytes in culture (18).
  • myocardial injury was diminished (19).
  • IL-I and TNF- ⁇ are clearly critical in the pathogenesis of autoimmune myocarditis.
  • mice immunized with peptide sequences 7, 8 ,10 and 14 showed higher total IgG antibody titers compared to the other peptides used in the experiments only mice immunized with peptide 9 and 11 (SEQ ID NO. 9 and 11) showed inflammation and fibrosis in the myocardium.
  • mice When mice were immunized with the whole troponin protein (mcTnl) the inventors expect to induce an immune response (cellular and humoral) directed against different peptide sequences (epitopes) of this protein.
  • an immune response against peptide 9 can be induced not only by immunization with the synthesized peptide 9 alone but also when the whole troponin protein is used for immunization. This demonstrates that the peptide 9 sequences are immunodominant in the whole troponin protein.
  • mice were immunized with peptide 9 alone the inventors would expect initially an immune response directed only against the amino acid sequences of peptide 9. These antibodies are able to bind to the whole protein TnI as well. This major finding suggests that these amino acid sequences can be bound by antibodies even in the complex structure of the whole protein.
  • the peptide 9 sequence is not cryptic (i.e. buried in the core of the whole troponin protein complex). That was confirmed with the actual 3-D structure of troponin I (data not shown).
  • mice immunized with peptide 9 show at day 21 only antibodies binding itself (peptide 9) and the whole troponin protein TnI but do not have antibodies binding the other peptides used in our experiments.
  • the utility of the present peptide study indicated the use of peptide 9 (+11) sequences (SEQ ID NO. 9 and 11) instead of the whole troponin molecule in testing patient sera to identify patients at risk for myocardial damage induced by autoimmune inflammation.
  • the use of the peptide 9 (+11) sequences in tests would be more specific for identifying the risk of inflammation than using the whole troponin protein (TnI) since the inventors demonstrated that immunization with the other peptide sequences of troponin I did not induce an inflammation or fibrosis in the myocardium.
  • the use of whole troponin protein will identify all antibodies directed against different sequences of troponin including those that might have no pathogenic effect.
  • Chemokines such as RANTES, MCP-I, MIP 1- ⁇ and their major receptors, CCR2 and CCR5, play important roles in the pathogenesis of many inflammatory diseases (21-23).
  • MCP-I mRNA expression has been shown in endomyocardial biopsy specimens from patients with dilated cardiomyopathy and suggested a significant role of this chemokine in the regulation of inflammatory cell infiltration into the myocardium (23).
  • troponin I peptide 9 and 11, respectively
  • the inventors identified one major and one minor epitope of troponin I (peptide 9 and 11, respectively) that seem to be responsible for disease induction. Using these newly identified epitope sequences of troponin I instead of the whole troponin molecule; a more specific screening test of patients with high risk for progressive autoimmune inflammatory heart failure is possible. Furthermore, the inventors demonstrated the important role of troponin-specific T-cells in inducing the autoimmune inflammation.
  • the inventors furthermore showed that the absence of auto-antibodies against cardiac troponin I predicts improvement of left ventricular function after acute myocardial infarction. For further details, see Example 2.
  • anti-cTnl and anti-cTnT antibody titers were measured in sera from 272 patients with dilated- (DCM) and 185 with ischemic- (ICM) cardiomyopathy.
  • DCM dilated-
  • ICM ischemic-
  • AMD acute myocardial infarction
  • TnI-Ab negative patients showed a significant increase in LVEF and stroke volume 6-9 months after AMI. In contrast, there was no significant increase in LVEF and stroke volume in TnI-Ab positive patients.
  • mice were immunized with mcTnl or adjuvant alone.
  • T-cells were isolated from splenocytes and re-stimulated in vitro in the presence of dendritic cells and monocytes with lO ⁇ g/ml of mcTnl (mcTnl/mcTnl or no/mcTnl) for 48h or were not re-stimulated with mcTnl (mcTnl/no or CFA/no).
  • 10 6 -10 7 T-cells per mouse were transferred to WT mice irradiated with 600 rad.
  • FIG. 4 Histological examination of the hearts.
  • Figure 5 RNA protection assay and antibody titers.
  • Antibody titers directed against the whole mcTnl protein and against the peptide 9 were measured in mice immunized with either mcTnl ((C), 10,000 ⁇ 500 and 1,110 ⁇ 285) or peptide 9 ((D), 4,320 ⁇ l,098 and 4,676 ⁇ 1,143).
  • Antibody endpoint titers for each individual mouse were calculated as the greatest positive dilution of antibody above normal mouse serum levels for day 21.
  • mice were immunized with either mcTnl (residues 105-122), or hcTnl (residues 104-121), or skeletal troponin (residues 73-90). Effects on severity of inflammation and fibrosis on day 28 (Kruskal-Wallis test and U test).
  • B-G Histological examination of the hearts (stain with haematoxylin and eosin). Representative heart section of mice immunized with mcTnl (105-121) (peptide 9) with the inflammation scores of 3,5 (B and D) and 2,5 (C and E).
  • mice immunized with hcTnl (104-121) and inflammation score of 1 (F) and with perivascular inflammation (G) on day 28.
  • B and C 1Ox magnification
  • D-G 2Ox magnification
  • mice Female A/J mice (5-6 weeks of age) obtained from Harlan Winkelmann GmbH (33176 Borchen, Germany), female SCID mice (5-6 weeks of age) obtained from Charles River (Sulzfeld, Germany) were maintained in the animal facility at the University of Heidelberg and used in all experiments. The animal work was approved by the Animal Care and Use Committee of the University of Heidelberg.
  • the murine cardiac troponin subunit mcTnl was expressed in E.coli and purified as previously described 9 .
  • mcTnl was applied to a cardiac troponin C affinity column as second purification step (10). Isolated mcTnl-fractions were dialysed extensively against 1 mM HCl, then lyophilised and stored at - 80 °C. Cell sorting
  • CD90+, CD8+ and CD4+ T-cells were enriched to 90% purity from the spleen by magnetically activated cell sorting using anti-CD90, anti-CD8, anti-CD4- conjugated microbeads (Miltenyi-Biotec, Auburn, CA).
  • mice For the transfer experiments four groups of mice treated differently were used. Two groups of mice were first immunized with mcTnl on days 0 and 7. On day 21 purified T-cells from one group of mice were re-stimulated in vitro in the presence of dendritic cells and monocytes with lO ⁇ g/ml of mcTnl for 48h whereas T-cells from the second group were not re-stimulated with mcTnl. Additionally, two other groups of mice were immunized first with adjuvant alone on days 0 and 7.
  • T-cells from one group of mice were re-stimulated in vitro in the presence of dendritic cells and monocytes with lO ⁇ g/ml of mcTnl for 48 h whereas T-cells from the second group were not re-stimulated with mcTnl.
  • 10 6 -10 7 of stimulated T-cells were injected intraperitoneally (i.p.) to WT recipient mice irradiated with 600 rad or to non irradiated SCID mice.
  • three additional groups of WT recipient mice irradiated with 600 rad were injected i.p.
  • CD4+ and CD8+ subsets were isolated from the spleens of immunized mice and were re-stimulated in vitro in the presence of lO ⁇ g/ml of mcTnl for 48 h whereas CD8+ T-cells were re-stimulated in the presence of additional 50 IU/ml IL-2 (R&D Systems, 65205 Wiesbaden-Nordenstadt, Germany).
  • Antibody titers were essentially determined as described before (11). In brief, to measure serum anti— peptide or troponin I titers, plates were coated either with lOO ⁇ l/well of each peptide or cardiac troponin I (5 ⁇ g/ml) in bicarbonate buffer (pH 9.6) and incubated overnight. Anti -mouse secondary antibody diluted to 1 :5000 for IgG (Sigma) was used for detection. Serum samples from test mice were diluted to 1 :100, 1 :500, 1 :2500, and 1 :12500. Normal mouse serum was used as control. Optical densities were determined at 450nm. Antibody endpoint titers for each individual mouse were calculated as the greatest positive dilution of antibody yielding a positive signal. Cardiac-troponin I dependent cytokine production by splenocytes
  • splenocytes were cultured at 5x10 6 per well in RPMI 1640 complete medium in the presence of 10 ⁇ g/ml of either cTnl or medium alone for 48 h. Supernatant was collected, aliquoted and frozen at -20 °C. Cytokines (IL- l ⁇ , IL-2, IL-4, IL-6, IL-10, IL- 13, IL- 17, IFN- ⁇ , and TNF- ⁇ ) were measured by DuoSet ELISA Development Systems (R&D Systems, 65205 Wiesbaden-Nordenstadt, Germany), according to the manufacturer's instructions.
  • mice were sacrificed on day 21 after transfer of T-cells and on day 28 after immunization with peptides respectively. Sections of 5 ⁇ m thickness were cut at various depths in the myocardial tissue section and stained with haematoxylin and eosin to determine the level of inflammation and with Masson's Trichrome to detect collagen deposition.
  • Evidence of myocarditis and fibrosis was evaluated in a blinded manner by two independent investigators who used light microscopy, according to a scoring system: grade 0, no inflammation; grade 1, cardiac infiltration in up to 5% of the cardiac sections; grade 2, 6-10%; grade 3, 11-30%; grade 4, 31-50%; and grade 5, >50%.
  • the mCR-5 cytokine receptor multi-probe template set (BD Biosciences Pharmingen, Heidelberg, Germany) was used to measure mouse mRNAs encoding CCRl, CCR2, CCRIb, CCR3, CCR4, and CCR5.
  • the mCK-5c multi-probe template set (BD Biosciences Pharmingen, Heidelberg, Germany) was used to measure mouse mRNAs encoding Ltn, RANTES, MIP-Ib, MIP-Ia, MIP-2, IP-10, MCP-I, TCA-3, and eotaxin. The measurement was done according to the manufacturer's guidelines.
  • Transthoracic echocardiography was performed as previously described (8). The investigator who conducted the echocardiography was unaware of the treatment status.
  • VDKVDEERYDVEAKVTKN [SEQ ID NO. 9]
  • VDKVDEERYDIEAKVTKN [SEQ ID NO. 17]
  • VEVVDEERYDIEAKCLHN [SEQ ID NO. 18]
  • the purity of the peptides was >90%.
  • mice were injected subcutaneously with an emulsion of 120 ⁇ g of one of the peptides, or troponin I as positive control, or control buffer and adjuvant as negative control.
  • the peptide/protein was supplemented with CFA containing 5 mg/ml of Mycobacterium tuberculosis H37Ra (Sigma, St. Louis, MO, USA) on days 0, 7 and 14.
  • the mice were sacrificed on day 28 for histopathological evaluation, RNA protection assay analysis, and measurement of cytokines and antibody titers.
  • Results are expressed as mean ⁇ SEM. Data were analyzed with the Kruskal-Wallis test followed by the Mann- Whitney U test to explore the significance between the treatment groups. P values of ⁇ 0.05 were considered significant. The statistical software SPSS (ver 15.0) was used for all calculations.
  • mice receiving T-cells from mcTnl immunized mice that have been re-stimulated in vitro with mcTnl showed high mcTnl-specific total IgG antibody titers (Figure IA), severe inflammation (histoscore: 1.8 ⁇ 0.4) and fibrosis (histoscore: 1.9 ⁇ 0.4) in myocardium ( Figure 1C), and increased mcTnl-induced production of the cytokines IL-I ⁇ , IL-4, IL- 13, IL- 17, and TNF- ⁇ ( Figure IB) compared to mice receiving T-cells from mcTnl immunized mice that have not been re-stimulated in vitro with mcTnl or receiving T-cells from mice given adjuvant alone irrespective of their in vitro re-stimulation.
  • Figure IA mcTnl-specific total IgG antibody titers
  • Figure 1C severe inflammation
  • fibrosis histoscore
  • the inventors measured the mRNA expression levels of the chemokines Ltn, RANTES, MIP-I ⁇ , MIP- l ⁇ , MIP -2, IP-IO, MCP-I, TCA-3 and eotaxin as well as the levels of the chemokine receptors CCRl, CCR2, CCRIb, CCR3, CCR4 and CCR5 in the myocardium of the recipient mice.
  • the inventors were able to detect mRNA levels for RANTES, MIP- l ⁇ , MIP- l ⁇ , MIP-2, MCP-I, TCA-3 and eotaxin only in mice receiving mcTnl-specific T-cells unlike the otherwise treated mice ( Figure IG).
  • the inventors found that the mRNAs for CCRl, CCR2 and CCR5 were expressed only in the myocardium of mice receiving mcTnl- specific T-cells but not in the other groups of mice ( Figure IG).
  • CD4+ and CD8+ T-cells were isolated from the spleens of immunized mice and were re-stimulated in vitro with mcTnl or in the case of CD8+ T-cells were re-stimulated with mcTnl and supplemental IL-2.
  • Transfer of whole T- cells (CD90+; histoscore: 2.1 ⁇ 0.5) and transfer of CD4+ subset of T-cells (histoscore: 2.4 ⁇ 0.4) induced moderate to severe disease, whereas transfer of CD8+ subset of T-cells alone did not induce any signs of inflammation in the recipient mice (Figure 2B).
  • the peptide 9 sequences (SEQ ID NO. 9) was localized in an actual 3-D structure of troponin I in association to troponin T (with kind permission of 'Nature Publishing Group', data not shown) (12).
  • cTnl-Ab cardiac troponin I
  • DCM dilated-
  • ICM ischemic-
  • ICM ischemic cardiomyopathy
  • the Badwater marathon is a continuous 216 km (134 miles) race, which takes place in July under extreme heat in Death Valley, California, USA.
  • the start line is at Badwater, Death Valley, which marks the lowest elevation in the Western hemisphere at 282 feet (85,5 m) below sea level.
  • the race finishes at the Mt. Whitney Portals at 8,360 feet (2,533 m).
  • Blood samples (whole blood, EDTA-blood) were kept on crushed ice in the supporters' car until the shuttle service took over the specimens transferring them to the stationary laboratories for preanalytical preparation.
  • One EDTA-blood sample was transferred to a laboratory in Las Vegas on a daily basis on crushed ice for blood-count testing.
  • the samples were transferred by WorldCourier® that documented an uninterrupted cold chain to the Laboratory in Heidelberg, Germany. No sample showed any sign of haemolysis.
  • Peripheral venous blood samples were collected from all patients. 96-well plates were coated with anti-cTnl or anti-cTnT diluted in coating buffer (0,1 M NaHCO 3 /34mM Na 2 CO 3 , pH 9,5) and then incubated over night at 4°C. All washing steps were performed with lxPBS/0.05% Tween 20 three times each. 1% Gelatine (Cold Water Fish, Sigma)/lxPBS was used for blocking. After 2h incubation at 37°C half of the plate was coated with human cTnl or cTnT for another 2h at room temperature (RT) while the other half served as control, thus, only coated with lxPBS/l%BS A/0.1%T ween 20.
  • coating buffer 0.,1 M NaHCO 3 /34mM Na 2 CO 3 , pH 9,5
  • All washing steps were performed with lxPBS/0.05% Tween 20 three times each. 1% Gelatine (Cold Water Fish, Sigma)/
  • the dilution series of the serum samples were as follows: 1 :40, 1 :80, 1 :160, 1 :320.
  • HRP anti- human IgG or anti-human IgM (diluted 1 :7500 with lxPBS/l%BSA/0.1% Tween 20, Ih incubation at RT) was used as detection-antibody.
  • Blue Star HRP-Substrate from Diarect was applied for 45 min at RT. The reaction was stopped with 0.3M H 2 SO 4 . Finally, the absorbance was measured at 450 run.
  • MRI examinations were performed with a 1,5 Tesla whole-body MRI system (Philips Medical Systems, 1,5 Tesla). Functional and morphological cardiac assessment was adequately executed in all 108 Mi-patients. Duration of MRI scan was approximately 30 to 60 minutes. They were examined for several parameters such as the left ventricular ejection fraction (LVEF), the end-systolic volume (ESV), the end-diastolic volume (EDV) and the stroke volume (SV). Infarct size was calculated as previously described (27). All data were provided by independent examiners, on hospital admission and 6-9 months after MI.
  • LVEF left ventricular ejection fraction
  • ESV end-systolic volume
  • SV stroke volume
  • the McNemar test was used to evaluate the relationship between autoantibodies against cTnl and cTnT, separately for each group. As for the patients undergoing MRI investigations, they were compared with respect to their initial demographic data including cardiovascular risk factors and oral medication. The two- sided Mann- Whitney U test was adopted to compare the continuous parameters, while the two-sided Chi-square test was applied for the categorical data. The main criterion for the MRI examination was the LVEF. For this parameter a confirmatory comparison controlling the ⁇ - error was carried out to compare the difference of the MRI data on day 3-4 and 6-9 months later between the titer positive and negative group.
  • Table 3 Baseline data of patients, risk factors and medication at discharge of both cTnl-Ab negative and positive patients
  • ASA Acetylsalicylic acid
  • ACEI/ATl-bl ACE-inhibitor/ATl -blocker
  • MRI magnetic resonance imaging
  • MI myocardial infarction
  • the present follow-up study discloses several limitations. Our study is limited by its relatively small number of anti-cTnl positive patients. The inventors used a cut-off of 1:160 for the anti- cTnl antibody (total IgG) titers and classified the patients accordingly as cTnl-antibody negative or positive. Therefore only 10 persons were found to be anti-cTnl antibody positive. This group of patients showed no improvement of their heart function, while lower titers don't seem to have any significant clinical relevance. Further research is required to clarify the role of the humoral immune system in the post-MI phase, as data on the potential effect of cTnT autoantibodies or IgM subclasses on the myocardial function are lacking. Moreover, studies with larger number of patients with a longer follow-up are needed for more conclusive data towards the patient's prognosis in relation to their antibody titers.
  • MCP-I Monocyte chemoattractant protein 1
  • Antman EM Anbe DT, Armstrong PW, Bates ER, Green LA, Hand M, Hochman JS, Krumholz HM, Kushner FG, Lamas GA, Mullany CJ, Ornato JP, Pearle DL, Sloan MA, Smith SC, Jr., Alpert JS, Anderson JL, Faxon DP, Fuster V, Gibbons RJ, Gregoratos G, Halperin JL, Hiratzka LF, Hunt SA, Jacobs AK.

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Abstract

Cette invention concerne des peptides dérivés de la troponine cardiaque I (cTnl) qui sont le(s) déterminant(s) antigénique(s) ou épitope(s) pathogène(s) de la troponine cardiaque I (cTnl) et des anticorps et fragments d'anticorps spécifiques de ces peptides. Elle concerne, en outre, l'utilisation de ces peptides, anticorps et fragments d'anticorps pour le pronostic et/ou le diagnostic de l'insuffisance cardiaque, en particulier, d'une inflammation du myocarde et/ou d'une fibrose. Des procédés pour pronostiquer et/ou diagnostiquer l'insuffisance cardiaque, en particulier, une inflammation du myocarde et/ou une fibrose au moyen des peptides selon l'invention sont également décrits.
PCT/EP2009/002237 2008-03-27 2009-03-26 Peptides dérivés de la troponine cardiaque i et leur utilisation à titre de marqueurs pronostiques et diagnostiques pour l'insuffisance cardiaque WO2009118185A1 (fr)

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US20140112912A9 (en) * 2007-04-12 2014-04-24 Myra A. Lipes Diagnosis and treatment of autoantibody-mediated heart disease
US20150247851A1 (en) * 2007-04-12 2015-09-03 Myra A. Lipes Diagnosis and treatment of autoantibody-mediated heart disease
US20140255956A1 (en) * 2010-06-10 2014-09-11 Myra A. Lipes Diagnosis of myocardial autoimmunity in heart disease
US20130121998A1 (en) * 2010-06-16 2013-05-16 Myra A. Lipes Diagnosis of Myocardial Autoimmunity in Heart Disease
US8420330B2 (en) * 2011-07-15 2013-04-16 Myra A. Lipes Diagnosis and treatment of cardiac troponin 1 (cTn1) autoantibody-mediated heart disease
US10670611B2 (en) 2014-09-26 2020-06-02 Somalogic, Inc. Cardiovascular risk event prediction and uses thereof
CN110272502A (zh) * 2019-07-12 2019-09-24 深圳市亚辉龙生物科技股份有限公司 免疫原、分泌抗心肌肌钙蛋白i单克隆抗体的杂交瘤细胞及制备方法、单克隆抗体及应用
CN114966045A (zh) * 2022-05-07 2022-08-30 浙江大学 检测抗肌球蛋白轻链1-IgG自身抗体的试剂在制备检测血管内皮损伤的试剂盒中的应用
WO2025162496A1 (fr) * 2024-02-03 2025-08-07 菲鹏生物股份有限公司 Anticorps anti-ctni et son utilisation

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