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CN-122029193-A - Assay for MYBPC3

CN122029193ACN 122029193 ACN122029193 ACN 122029193ACN-122029193-A

Abstract

The present invention relates to monoclonal antibodies or fragments thereof that bind to MYBPC3 (myosin binding protein C, cardiac). Further, the present invention relates to a kit comprising said monoclonal antibody of the present invention. In some embodiments, the kit is provided as a sandwich assay. The invention further relates to the in vitro use of said monoclonal antibody or fragment thereof of the invention, or said kit for the detection of MYBPC3 in a sample. Furthermore, the invention relates to diagnostic methods, such as assessment of cardiac disorders, e.g. myocardial infarction.

Inventors

  • H. Duffer
  • A. MORO
  • A. Spear
  • C. WAGNER
  • M. A. Abel
  • A. M. Engel
  • M. GEIGER
  • L. Hilllinghouse
  • T. Yesco
  • U. Zakhnyshek
  • KASTNER PETER
  • T. MAYER

Assignees

  • 罗氏诊断有限公司

Dates

Publication Date
20260512
Application Date
20240730
Priority Date
20230731

Claims (16)

  1. 1. A monoclonal antibody or fragment thereof that binds to MYBPC3 (myosin binding protein C, cardiac type), wherein the monoclonal antibody or fragment thereof is selected from the group consisting of I) A first monoclonal antibody or fragment thereof comprising a first light chain variable domain and/or a first heavy chain variable domain, (A) The first light chain variable domain comprises (A1) First light chain CDR1, which hybridizes with a light chain having SEQ ID NO. 1 [ ] ) Said first light chain CDR1 of the indicated amino acid sequence differs by no more than a total of three amino acid additions, substitutions and/or deletions, (A2) First light chain CDR2, which hybridizes with a light chain having SEQ ID NO. 2 # ) Said first light chain CDR2 of said amino acid sequence differs by no more than a total of three amino acid additions, substitutions and/or deletions, and/or (A3) First light chain CDR3, which hybridizes with a light chain having SEQ ID NO. 3 [ ] ) Said first light chain CDR3 of the amino acid sequence shown differs by no more than a total of three amino acid additions, substitutions and/or deletions, (B) The first heavy chain variable domain comprises (B1) First heavy chain CDR1, which is identical to a heavy chain CDR having SEQ ID NO. 4 # ) Said first heavy chain CDR1 of the indicated amino acid sequence differs by no more than a total of three amino acid additions, substitutions and/or deletions, (B2) First heavy chain CDR2, which is complementary to a heavy chain having SEQ ID NO. 5 # ) Said first heavy chain CDR2 of said amino acid sequence differs by no more than a total of three amino acid additions, substitutions and/or deletions, and/or (B3) First heavy chain CDR3, which hybridizes with a polypeptide having SEQ ID NO. 6 [ ] ) Said first heavy chain CDR3 of the indicated amino acid sequence differs by no more than a total of three amino acid additions, substitutions and/or deletions, Or (b) Ii) a second monoclonal antibody or fragment thereof comprising a second light chain variable domain and/or a second heavy chain variable domain, (A) The second light chain variable domain comprises (A1) A second light chain CDR1, which hybridizes a light chain having SEQ ID NO. 9 # ) The second light chain CDR1 of the amino acid sequences shown differ by no more than a total of three amino acid additions, substitutions and/or deletions, (A2) A second light chain CDR2 which hybridizes with a light chain having SEQ ID NO. 10 # ) The second light chain CDR2 of the amino acid sequences shown differ by no more than a total of three amino acid additions, substitutions and/or deletions, and/or (A3) A second light chain CDR3, which hybridizes with a light chain having SEQ ID NO. 11 # ) The second light chain CDR3 of the amino acid sequences shown differ by no more than a total of three amino acid additions, substitutions and/or deletions, (B) The second heavy chain variable domain comprises (B1) A second heavy chain CDR1, which hybridizes a polypeptide having SEQ ID NO. 12 [ ] ) Said second heavy chain CDR1 of the indicated amino acid sequence differs by no more than a total of three amino acid additions, substitutions and/or deletions, (B2) A second heavy chain CDR2, which hybridizes with a polypeptide having SEQ ID NO. 13 [ ] ) Said second heavy chain CDR2 of said amino acid sequence differs by no more than a total of three amino acid additions, substitutions and/or deletions, and/or (B3) A second heavy chain CDR3, which hybridizes with a polypeptide having SEQ ID NO. 14 [ ] ) The second heavy chain CDR3 of the amino acid sequences shown differ by no more than a total of three amino acid additions, substitutions and/or deletions.
  2. 2. The monoclonal antibody or fragment thereof according to claim 1, wherein The first light chain variable structural domain has SEQ ID NO. 7 # ) The light chain variable domains of the sequences shown are at least 85%, such as at least 90% or at least 95% identical, and The first heavy chain variable structural domain has SEQ ID NO. 8deg.C ) The heavy chain variable domains of the indicated sequences are at least 85%, such as at least 90% or at least 95% identical, Or (b) The second light chain variable structural domain has SEQ ID NO. 15 # ) The light chain variable domains of the sequences shown are at least 85%, such as at least 90% or at least 95% identical, and The second heavy chain variable structural domain has SEQ ID NO. 16 # WGQGTSVTVSS) are at least 85%, such as at least 90% or at least 95% identical.
  3. 3. The monoclonal antibody or fragment of claim 1 or 2, wherein I) The first light chain variable domain comprises the sequence set forth in SEQ ID NO.7 and the first heavy chain variable domain comprises the sequence set forth in SEQ ID NO. 8, Ii) the second light chain variable domain comprises the sequence set forth in SEQ ID NO. 15 and the second heavy chain variable domain comprises the sequence set forth in SEQ ID NO. 16.
  4. 4. A monoclonal antibody or fragment thereof according to any one of claims 1 to 3, wherein I) The first monoclonal antibody or fragment thereof comprises (A) A first light chain variable domain comprising (A1) First light chain CDR1, which hybridizes with a light chain having SEQ ID NO. 4 # ) Said first light chain CDR1 of the amino acid sequence differs by no more than one amino acid addition, substitution or deletion, (A2) First light chain CDR2, which hybridizes with a light chain having SEQ ID NO. 5 # ) Said first light chain CDR2 of said amino acid sequence differs by no more than one amino acid addition, substitution or deletion, and/or (A3) First light chain CDR3, which hybridizes with a light chain having SEQ ID NO. 6 # ) Said first light chain CDR3 of the amino acid sequence differs by no more than one amino acid addition, substitution or deletion, (B) A first heavy chain variable domain comprising (B1) First heavy chain CDR1, which is identical to a heavy chain having SEQ ID NO. 7 # ) The first heavy chain CDR1 of the amino acid sequence shown differs by no more than one amino acid addition, substitution or deletion, (B2) First heavy chain CDR2, which hybridizes with a polypeptide having SEQ ID NO. 8 # ) The first heavy chain CDR2 of the amino acid sequence shown differs by no more than one amino acid addition, substitution or deletion, and/or (B3) First heavy chain CDR3, which hybridizes with a polypeptide having SEQ ID NO. 9 @ ) The first heavy chain CDR3 of the amino acid sequence shown differs by no more than one amino acid addition, substitution or deletion, Ii) the second monoclonal antibody or fragment thereof comprises (A) A second light chain variable domain comprising (A1) A second light chain CDR1, which hybridizes a light chain having SEQ ID NO. 9 # ) The second light chain CDR1 of the amino acid sequence shown differs by no more than one amino acid addition, substitution or deletion, (A2) A second light chain CDR2 which hybridizes with a light chain having SEQ ID NO. 10 # ) The second light chain CDR2 of the amino acid sequence shown differs by no more than one amino acid addition, substitution or deletion, and/or (A3) A second light chain CDR3, which hybridizes with a light chain having SEQ ID NO. 11 # ) The second light chain CDR3 of the amino acid sequence shown differs by no more than one amino acid addition, substitution or deletion, (B) A second heavy chain variable domain comprising (B1) A second heavy chain CDR1, which hybridizes a polypeptide having SEQ ID NO. 12 [ ] ) The second heavy chain CDR1 of the amino acid sequence shown differs by no more than one amino acid addition, substitution or deletion, (B2) A second heavy chain CDR2, which hybridizes with a polypeptide having SEQ ID NO. 13 [ ] ) The second heavy chain CDR2 of the amino acid sequence shown differs by no more than one amino acid addition, substitution or deletion, and/or (B3) A second heavy chain CDR3, which hybridizes with a polypeptide having SEQ ID NO. 14 [ ] ) The second heavy chain CDR3 of the amino acid sequences shown differ by no more than one amino acid addition, substitution or deletion.
  5. 5. The monoclonal antibody or fragment thereof according to any one of claims 1 to 4, wherein I) The first monoclonal antibody or fragment thereof comprises (A) A first light chain variable domain comprising (A1) The first light chain CDR1 having SEQ ID NO.1 # ) The sequence of the sequence shown in the figures, (A2) The first light chain CDR2 has SEQ ID NO. 2 # ) The sequence shown, and (A3) The first light chain CDR3 having SEQ ID NO. 3 # ) The sequence of the sequence shown in the figures, And (B) A first heavy chain variable domain comprising (B1) First heavy chain CDR1 having SEQ ID NO. 4 # ) The sequence of the sequence shown in the figures, (B2) First heavy chain CDR2 having SEQ ID NO.5 # ) The sequence shown, and (B3) First heavy chain CDR3 having SEQ ID NO.6 # ) The sequence of the sequence shown in the figures, Ii) the second monoclonal antibody or fragment thereof comprises (A) A second light chain variable domain comprising (A1) A second light chain CDR1 having SEQ ID NO. 9 # ) The sequence of the sequence shown in the figures, (A2) A second light chain CDR2 having SEQ ID NO. 10 # ) The sequence shown, and (A3) A second light chain CDR3 having SEQ ID NO. 11 # ) The sequence of the sequence shown in the figures, And (B) A second heavy chain variable domain comprising (B1) A second heavy chain CDR1 having SEQ ID NO. 12 # ) The sequence of the sequence shown in the figures, (B2) Second chain CDR2 having SEQ ID NO. 13 [ ] ) The sequence shown, and (B3) A second heavy chain CDR3 having SEQ ID NO. 14 # ) The sequence shown.
  6. 6. The monoclonal antibody or fragment thereof according to any one of claims 1-5, wherein the first antibody or fragment thereof, and/or the second antibody or fragment thereof is labeled, and/or Wherein the fragment is a Fab fragment, a Fab ' fragment, a Facb fragment, a F (ab ') 2 fragment, an scFv fragment or an Fv fragment, in particular wherein the fragment is a F (ab ') 2 fragment.
  7. 7. A kit comprising I) A primary antibody or fragment thereof as defined in any one of the preceding claims, and Ii) a secondary antibody or fragment thereof as defined in any one of the preceding claims.
  8. 8. The kit of claim 7, comprising I. A first monoclonal antibody or antigen-binding fragment thereof that binds to MYBPC3 (myosin binding protein C, cardiac), wherein the first monoclonal antibody or antigen-binding fragment thereof comprises a first light chain variable domain and a first heavy chain variable domain, wherein A) The first light chain variable domain comprises SEQ ID NO. 7deg.C ) The sequence shown, and B) The first heavy chain variable domain comprises SEQ ID NO. 8deg.C ) The sequence shown And A second monoclonal antibody or antigen-binding fragment thereof that binds to MYBPC3 (myosin binding protein C, cardiac), wherein the second monoclonal antibody or antigen-binding fragment thereof comprises a second light chain variable domain and a second heavy chain variable domain, wherein A) The second light chain variable domain comprises SEQ ID NO. 15 # ) The sequence shown, and B) The second heavy chain variable domain comprises SEQ ID NO. 16 # ) The sequence shown.
  9. 9. The kit of claim 7 or 8, wherein the kit comprises i) a biotinylated F (ab') 2 fragment of the first monoclonal antibody and ii) the second monoclonal antibody, preferably wherein the second antibody is ruthenized.
  10. 10. Use of the monoclonal antibody or fragment thereof according to any one of claims 1 to 6, or the kit according to any one of claims 7 to 9, in vitro for the detection of MYBPC3 in a sample, for example wherein the detection is a quantitative detection.
  11. 11. The in vitro use according to claim 10, wherein the sample is a body fluid, such as a blood, serum or plasma sample, in particular wherein the sample is a human blood, serum or plasma sample.
  12. 12. A method for detecting MYBPC3 in a sample, the method comprising (A) Contacting a sample comprising MYBPC3 with the first and/or second monoclonal antibody or fragment thereof of any one of claims 1-6, thereby forming a complex comprising MYBPC3 and the monoclonal antibody or fragment thereof, and (B) Detecting the complex formed in step (a), thereby detecting MYBPC3 in the sample.
  13. 13. A complex, comprising I) MYBPC3, and Ii) a first monoclonal antibody or fragment thereof and/or a second monoclonal antibody or fragment thereof according to any one of claims 1 to 6.
  14. 14. Use of a monoclonal antibody or fragment thereof according to any one of claims 1 to 6, or a kit according to any one of claims 7 to 9, in vitro for assessing a cardiac disorder.
  15. 15. The in vitro use according to claim 14, wherein the cardiac disorder is heart failure, or wherein the cardiac disorder is myocardial injury or myocardial infarction.
  16. 16. The in vitro use of claim 15, wherein the cardiac disorder is myocardial infarction, and wherein ● The myocardial infarction is eliminated and the method can eliminate the myocardial infarction, ● Differentiating between type 1 and type 2 myocardial infarction, and/or ● A patient suspected of suffering from Acute Coronary Syndrome (ACS) is predicted to be at risk of myocardial infarction and/or cardiovascular death, for example 30 days.

Description

Assay for MYBPC3 Technical Field The present invention relates to monoclonal antibodies or fragments thereof that bind to MYBPC3 (myosin binding protein C, cardiac). Further, the present invention relates to a kit comprising the monoclonal antibody of the present invention. In some embodiments, the kit is provided as a sandwich assay. The invention further relates to the in vitro use of the monoclonal antibody or fragment thereof of the invention, or the kit for the detection of MYBPC3 in a sample. Furthermore, the invention relates to diagnostic methods, such as assessment of heart conditions, e.g. myocardial infarction. Background The goal of modern medicine is to provide personalized treatment regimens. These are treatment regimens that take into account the individual needs or risks of the patient. For emergency measures that require a decision of a possible treatment regimen in a short time, even personalized treatment regimens should be considered. Under such circumstances, measuring circulating biomarkers with a blood test can help diagnose disease states (such as with a troponin test to help diagnose cardiomyocyte injury). According to the unified definition of Acute Myocardial Infarction (AMI) (THYGESEN K et al, circulation 2018;138: e618-e651. Doi: 10.1161/CIR.0000000000000617.) five different types of AMI, such as type 1 and type 2 myocardial infarction, are defined based on the different mechanism pathways behind AMI. Diagnostic tests for patients suspected of suffering from AMI (acute myocardial infarction) require access to an emergency room, recording a 12-lead Electrocardiogram (ECG), conducting blood tests to diagnose or rule out myocardial damage, assessing clinical symptoms and medical history, conducting physical examination, and other diagnostic tests for diagnosis of AMI or differential diagnosis. Diagnosis and management of ACS (acute coronary syndrome) remains challenging for patients not enrolled or excluded with hypersensitive troponin. Using the 1 hour algorithm of highly sensitive troponin, between 20% and 25% of patients suspected of having AMI (acute myocardial infarction) remain in the observation area, including patients that were not classified as inclusion/exclusion after two consecutive measurements of T0 and T1. Using a rapid protocol (according to the 0 hour algorithm in the ESC (european cardiology institute) guidelines), even up to 70% of patients suspected of having AMI remain in the waiting area (patients not classified by inclusion/exclusion after a single measurement at T0). Cardiac myosin binding protein C (MYBPC 3, alternative abbreviation: cMyBPC, cMyc or MYBPC3, cMyBP-C) is the cardiac isoform of MyBP and is the structural myoprotein of cardiomyocytes. MYBPC3 has high heart specificity and is released into the blood stream after myocardial necrosis. MYBPC3 is a highly specific cardiac protein associated with myocardial contractility. Its specific role and high abundance in the myocardium makes it an excellent biomarker candidate for circulatory central muscle injury. It is described that the concentration of myocardial necrosis biomarker is increased in blood samples of patients suffering from acute myocardial infarction. Furthermore, differences in the concentration of MYBPC3 were observed in type 1MI versus type 2 MI patients (see, e.g., nestelberger et al, 2021 JAMA Cardiol. Doi: 10.1001/jamacardio.2021.0669). Kozhuharov et al disclose that MYBPC3 can be used for diagnosis and risk stratification of acute Heart failure (Eur J Heart Fail.2021, 5 months; 23 (5): 716-725. Doi: 10.1002/ejhf.2094. Epub 2021, 2, 5 days. PMID: 33421273). Kaier et al disclose an algorithm for early diagnosis of myocardial infarction using cardiac myosin binding protein C (European Heart journal. Act Cardiovascular Care, vol.11, 4 th, 2022, 4 th month, pages 325-335). WO 2015/110114 and WO2015/110115 disclose monoclonal antibodies that bind MYBPC 3. Marjot et al describe the development and use of a high sensitivity immunoassay for cardiac myosin binding protein C. TRANSL RES 2016:2016, 170:17-25.e5. There remains a need for MYBPC3 assays that can detect biomarkers with high sensitivity. It is therefore an object of the present invention to provide means and methods that allow reliable detection of MYBPC 3. The inventors have identified two antibodies, designated mAB "7B5" and mAB "4.1.1", which are particularly useful for detection of MYBPC 3. Antibodies 7B5 and M-4.1.1 recognize the nonlinear conformational epitope presented by the naturally folded MYBPC3 protein (see tables 7 and 8 of the examples section). In particular, antibodies can be used as antibody pairs in sandwich assays. The sandwich assays of the present invention allow assessment of heart conditions such as myocardial infarction or heart failure. Disclosure of Invention The present invention relates to monoclonal antibodies or fragments thereof that bind to MYBPC3 (myosin binding protein C, cardiac). In a first aspect