Specific antibody for detecting human cardiac myosin binding protein C

Abstract

The invention discloses specific antibodies against cardiac myosin binding protein C. Specifically, the invention discloses a monoclonal antibody aiming at cardiac myosin binding protein C. The antibody has obvious binding activity to a cardiac myosin binding protein C antigen.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular, relates to a specific antibody targeting cardiac myosin binding protein C and an application thereof. Background Art

[0002] Acute myocardial infarction (AMI) increases the risk of death and recurrent ischemia and must be rapidly recognized and differentiated from other causes of chest pain. Cardiac necrosis biomarkers are essential to confirm or exclude AMI in suspected non-ST-segment elevation acute coronary syndrome (NSTE-ACS) and the diagnosis must be made in the appropriate clinical context.

[0003] Currently, cardiac troponin (cTn) has become the gold standard biomarker, however, cTn has potential defects: 1) The concentration of cTn in the blood reaches its peak only 16-18 hours after myocardial infarction; 2) The detectable concentration of cTn in the blood is not high; 3) Most normal people can also detect the presence of cTn in the blood, but about 25% of myocardial infarction patients cannot detect the difference in content from normal patients. Therefore, the specificity of cTn diagnosis is not strong.

[0004] AMI patients have a rapid morbidity and high mortality rate. Currently, classic detection markers such as troponin and myoglobin appear late, and more rapid and sensitive detection methods are needed.

[0005] Therefore, it is urgent to find new biomarkers for the diagnosis of myocardial infarction and develop specific detection kits. The ideal biomarker for AMI is to be rapidly released from myocardial cells into the blood in large quantities after myocardial infarction, which can quickly shorten the diagnosis time and save lives. Summary of the invention

[0006] The purpose of the present invention is to provide a cardiac myosin binding protein C monoclonal antibody and its application to solve the problems existing in the above-mentioned prior art.

[0007] In a first aspect of the present invention, there is provided a heavy chain variable region of an antibody, wherein the heavy chain variable region has a complementary determining region CDR selected from the following group: (i) V shown in SEQ ID NO.2 H -CDR1, V shown in SEQ ID NO.3 H -CDR2, and V shown in SEQ ID NO.4 H -CDR3; preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.8; or (ii) V shown in SEQ ID NO.10 H -CDR1, V shown in SEQ ID NO.11H -CDR2, and V shown in SEQ ID NO.12 H -CDR3; preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.16.

[0008] In another preferred embodiment, the antibody V H The CDR region of the chain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity to any one of SEQ ID NOs: 2, 3 and 4, SEQ ID NOs: 10, 11 and 12.

[0009] In another preferred embodiment, the antibody V H The CDR region of the chain comprises one or more amino acid substitutions compared to any one of SEQ ID NOs: 2, 3 and 4, SEQ ID NOs: 10, 11 and 12, preferably conservative amino acid substitutions.

[0010] In another preferred embodiment, the antibody V H The chain comprises an amino acid sequence having a sequence similarity of at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% to any one of SEQ ID NOs: 8 and 16.

[0011] In another preferred embodiment, the antibody V H The chain comprises one or more amino acid substitutions compared to any of SEQ ID NOs: 8 and 16, preferably conservative amino acid substitutions.

[0012] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, and more preferably 1) amino acid and can retain the ability to specifically bind to cardiac myosin binding protein C.

[0013] In another preferred embodiment, the cardiac myosin binding protein C is human cardiac myosin binding protein C.

[0014] The second aspect of the present invention provides an antibody heavy chain, wherein the heavy chain has the heavy chain variable region and heavy chain constant region described in the first aspect of the present invention.

[0015] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0016] In a third aspect of the present invention, there is provided a light chain variable region of an antibody, wherein the light chain variable region has a complementary determining region CDR selected from the following group: (i) V shown in SEQ ID NO.5 L -CDR1, V shown in SEQ ID NO.6 L -CDR2, V shown in SEQ ID NO.7 L -CDR3, preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO.9, or (ii) V shown in SEQ ID NO.13 L -CDR1, V shown in SEQ ID NO.14 L -CDR2, V shown in SEQ ID NO.15 L -CDR3, preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO.17.

[0017] In another preferred embodiment, the antibody V L The CDR region of the chain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity to any one of SEQ ID NOs: 5, 6 and 7, SEQ ID NOs: 13, 14 and 15.

[0018] In another preferred embodiment, the antibody V L The CDR region of the chain comprises one or more amino acid substitutions compared to any one of SEQ ID NOs: 5, 6 and 7, SEQ ID NOs: 13, 14 and 15, preferably conservative amino acid substitutions.

[0019] In another preferred embodiment, the antibody V L The chain comprises an amino acid sequence having a sequence similarity of at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% to any one of SEQ ID NOs: 9 and 17.

[0020] In another preferred embodiment, the antibody V L The chain comprises one or more amino acid substitutions compared to any of SEQ ID NOs: 9 and 17, preferably conservative amino acid substitutions.

[0021] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, and more preferably 1) amino acid and can retain the ability to specifically bind to cardiac myosin binding protein C.

[0022] The fourth aspect of the present invention provides a light chain of an antibody, wherein the light chain has the light chain variable region and light chain constant region described in the third aspect of the present invention.

[0023] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0024] The fifth aspect of the present invention provides an antibody having: (1) a heavy chain variable region as described in the first aspect of the present invention; and / or (2) a light chain variable region as described in the third aspect of the present invention; or, the antibody having: a heavy chain as described in the second aspect of the present invention; and / or a light chain as described in the fourth aspect of the present invention.

[0025] In another preferred embodiment, the antibody is an antibody specific against cardiac myosin binding protein C.

[0026] In another preferred embodiment, the antibody includes: single-chain antibody (scFv), double-chain antibody, monoclonal antibody, chimeric antibody (such as human-mouse chimeric antibody), mouse antibody, or humanized antibody.

[0027] In another preferred embodiment, the antibody is a fully human antibody.

[0028] In another preferred embodiment, the antibody is a full-length antibody or an antibody fragment having immunological activity.

[0029] In another preferred embodiment, the antibody fragment having immunological activity is Fab or (Fab") 2.

[0030] In another preferred embodiment, the antibody recognizes a linear epitope of cardiac myosin binding protein C.

[0031] In another preferred embodiment, the antibody recognizes a conformational epitope of cardiac myosin binding protein C.

[0032] The sixth aspect of the present invention provides a recombinant protein, which has: (i) the sequence of the heavy chain variable region as described in the first aspect of the present invention, the sequence of the heavy chain as described in the second aspect of the present invention, the sequence of the light chain variable region as described in the third aspect of the present invention, the sequence of the light chain as described in the fourth aspect of the present invention, or the sequence of the antibody as described in the fifth aspect of the present invention; and (ii) an optional tag sequence that assists expression and / or purification.

[0033] In another preferred embodiment, the tag sequence is selected from the following group: 6×His tag, GGGS sequence, FLAG tag.

[0034] In another preferred embodiment, the recombinant protein includes bispecific antibodies and chimeric antibodies.

[0035] In another preferred embodiment, the recombinant protein specifically binds to cardiac myosin binding protein C.

[0036] The seventh aspect of the present invention provides a polynucleotide encoding a polypeptide selected from the following groups: (1) the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention; or (2) the recombinant protein as described in the sixth aspect of the present invention.

[0037] In another preferred embodiment, the polynucleotide has a sequence shown in any one of SEQ ID NOs. 18, 19, 20 and 21.

[0038] In another preferred embodiment, the polynucleotide has a nucleotide sequence with at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to any one of SEQ ID NOs. 18, 19, 20 and 21.

[0039] The eighth aspect of the present invention provides a vector comprising the polynucleotide described in the seventh aspect of the present invention.

[0040] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0041] The ninth aspect of the present invention provides a genetically engineered host cell, which contains the vector described in the eighth aspect of the present invention or the polynucleotide described in the seventh aspect of the present invention integrated into its genome.

[0042] The tenth aspect of the present invention provides an immunoconjugate, which contains: (a) the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, or the recombinant protein as described in the sixth aspect of the present invention; and (b) a coupling portion selected from the following group: a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0043] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0044] In another preferred embodiment, the detectable marker is selected from the following group: biotin, fluorescein, chemiluminescent group, chemical fluorescent group, fluorescent protein, enzyme, colloidal gold, colored magnetic beads, latex particles, biotin label, radionuclide, antibody, ligand, antigen, receptor, nanoparticle or a combination thereof.

[0045] In another preferred embodiment, the nanoparticles are selected from the group consisting of nanogold, nanosilver, quantum dots, or a combination thereof.

[0046] In another preferred embodiment, the enzyme is selected from the following group: horseradish peroxidase, acid phosphatase, or a combination thereof.

[0047] The eleventh aspect of the present invention provides a pharmaceutical composition comprising: (i) the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, or the recombinant protein as described in the sixth aspect of the present invention, or the immunoconjugate as described in the tenth aspect of the present invention; and (ii) a pharmaceutically acceptable carrier.

[0048] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0049] In another preferred embodiment, the pharmaceutical composition is a tablet or a capsule.

[0050] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for treating heart disease.

[0051] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for treating myocardial infarction or myocardial ischemia.

[0052] The twelfth aspect of the present invention provides the use of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, or the immunoconjugate as described in the tenth aspect of the present invention for preparing a medicament, a reagent, a test plate or a kit.

[0053] In another preferred embodiment, the reagent, detection plate or kit is used to detect myocardial ischemia.

[0054] In another preferred embodiment, the reagent, detection plate or kit is used to detect myocardial infarction.

[0055] In another preferred embodiment, the medicament is used for treating or preventing myocardial infarction.

[0056] In another preferred embodiment, the reagents include chips and immune particles coated with antibodies.

[0057] The thirteenth aspect of the present invention provides a detection product, the detection product comprising: (1) the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, or the conjugate according to the tenth aspect of the present invention; and (2) Optional buffer solution or buffering agent.

[0058] In another preferred embodiment, the detection product is used to detect cardiac myosin binding protein C.

[0059] In another preferred embodiment, the detection products include: detection reagents, lateral flow sheets, chips, test strips, detection plates, and test sheets.

[0060] In another preferred embodiment, the test piece comprises: a detection area, wherein another antibody (antibody 2) against cardiac myosin binding protein C is fixed to the detection area, and the antibody 2 is used to capture the cardiac myosin binding protein C.

[0061] In another preferred embodiment, the second antibody is a capture antibody.

[0062] In another preferred embodiment, the second antibody is selected from the antibody or fragment thereof as described in the fifth aspect of the present invention.

[0063] In another preferred embodiment, the test piece includes: a quality control area, in which an antibody (secondary antibody) that binds to an antibody (primary antibody) against cardiac myosin binding protein C is fixed, and the secondary antibody is used to capture the antibody (primary antibody) against cardiac myosin binding protein C.

[0064] In another preferred embodiment, the primary antibody is a detection antibody.

[0065] In another preferred embodiment, the primary antibody is selected from the antibody or fragment thereof as described in the fifth aspect of the present invention.

[0066] In another preferred embodiment, the primary antibody is a biotin-labeled antibody against cardiac myosin binding protein C.

[0067] In another preferred embodiment, the primary antibody is selected from the antibodies or fragments thereof as described in the fifth aspect of the present invention and is a biotin-labeled antibody against cardiac myosin binding protein C.

[0068] In another preferred embodiment, the second antibody and the first antibody are both selected from the antibodies or fragments thereof as described in the fifth aspect of the present invention.

[0069] In another preferred embodiment, the detection sheet is selected from: a multi-well plate, preferably a 96-well plate, and a PVDF membrane.

[0070] In another preferred embodiment, the antibody (secondary antibody) that binds to the antibody (primary antibody) against cardiac myosin binding protein C is selected from: HRP-labeled goat anti-mouse IgG secondary antibody.

[0071] The fourteenth aspect of the present invention provides a detection kit, which comprises the antibody described in the fifth aspect of the present invention, the recombinant protein described in the sixth aspect of the present invention, or the conjugate described in the tenth aspect of the present invention or the detection product described in the thirteenth aspect of the present invention.

[0072] In another preferred embodiment, the kit includes a detection product and instructions.

[0073] In a fifteenth aspect, the present invention provides a method for detecting cardiac myosin binding protein C in a sample, the method comprising the steps of: (a) providing samples to be tested; (b) mixing the sample with the antibody described in the fifth aspect of the present invention, the recombinant protein described in the sixth aspect of the present invention, the conjugate described in the tenth aspect of the present invention, or the detection preparation described in the thirteenth aspect of the present invention to form a mixture; (c) detecting the presence or absence of the "antibody-cardiac myosin binding protein C complex" in the mixture, wherein if the complex exists, it indicates that cardiac myosin binding protein C exists in the sample; if the complex does not exist, it indicates that cardiac myosin binding protein C does not exist in the sample.

[0074] A sixteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising: (a) culturing the host cell according to the ninth aspect of the present invention under conditions suitable for expression; (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.

[0075] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 A schematic diagram of the protein structure of Mybpc1, Mybpc2, and Mybpc3 is shown, among which Mybpc1 is a slow skeletal muscle type, Mybpc2 is a fast skeletal muscle type, and Mybpc3 is a cardiac muscle type.

[0077] Figure 2 A schematic diagram of the process of using antibodies for detection is shown, using a double antibody sandwich method for sample detection and ultimately developing color through an enzymatic method.

[0078] Figure 3 The non-reduced and reduced SDS-PAGE electrophoresis images of SEQ NO.1 supernatant after purification are shown. The results show that the protein was purified by Ni column, and it can be seen that the purity after purification is high, SDS-PAGE electrophoresis purity ≥ 90%, and the electrophoresis size is between 55-70KDa.

[0079] Figure 4 The non-reduced and reduced SDS-PAGE electrophoresis images of SEQ NO.1 after digestion and recovery are shown. It can be seen that the protein purity after digestion is high, with SDS-PAGE electrophoresis purity ≥90%, and the electrophoresis size after tag removal is between 40-55KDa.

[0080] Figure 5 The results of hybridoma cell fusion experiments are shown. The marker concentration is 0.1 mg / ml, and the size is marked in the figure. The mouse polyclonal antibody is diluted four times, and the antibody 2F11 2D7, 4C5 3C5, 3C1 2A8 stock solution is loaded, and the loading volume is 5 ul. The SDS results show that the purity of the ascites after purification is above 90%, the antibody heavy chain size is about 55KDa, and the light chain size is about 25KDa.

[0081] Figure 6 The EC of the antibody of the present invention is shown 50 The EC values ​​of 4C53C5 were obtained by analyzing the ELISA results using GradPad Prism software. 50 =0.0067 μg / ml, EC of 2F112D750 =0.0069μg / ml.

[0082] Figure 7 The results of ELISA detection after the antibodies 2F112D7 and 4C53C5 of the present invention were labeled were shown. The diluted primary antibody 2F112D7 was coated in equal amounts in the ELISA detection plate as a capture antibody. After blocking, capturing the gradient diluted antigen standard, adding the HRP-labeled secondary antibody for incubation, TMB color development and other steps, the OD450 value was finally read. DETAILED DESCRIPTION

[0083] The inventors have unexpectedly obtained a high-affinity monoclonal antibody (e.g., mouse monoclonal antibody) that recognizes and binds to cardiac myosin binding protein C through extensive and in-depth research and a large number of screenings. The experimental results show that the antibody of the present invention can specifically bind to two different antigenic epitopes of cardiac myosin binding protein C (cMyBP-C) and does not interact with related isoforms of MyBP-C from fast skeletal muscle and slow skeletal muscle. The antibody of the present invention can bind to cMyBP-C well after biotin is modified at the C position of IgG. This antibody (especially the antibody pair) is suitable as a capture antibody and a detection antibody for ELISA (enzyme-linked immunosorbent assay) to quantitatively determine cMyBP-C in serum, plasma, whole blood or other body fluids, and is suitable for the development of detection reagents, kits, etc. for early diagnosis of acute myocardial infarction (AMI). In addition, the antibody of the present invention can also be used in antibody chip reactions, immunoprecipitation, and Western Blotting for research on the protein.

[0084] Based on the characteristics of MYBPC3, the present invention prepares MYBPC3-C0 antibody, anti-MYBPC3 antibody that specifically binds to humans, the capture antibody and the detection antibody bind to different antigenic epitopes, and develops a kit for detecting MYBPC3 using human c MYBPC3.

[0085] Specifically, the present invention utilizes MYBPC3-C0 recombinant protein antigen to produce MYBPC3-C0 monoclonal antibody, constructs a tag-MYBPC3-C0C1f recombinant protein vector, expresses the tag-C0C1f protein using bacteria, yeast, insect cells, animal or human cells, and purifies the tag-C0C1f protein as a MYBPC3 calibrator.

[0086] On this basis, the present invention has been completed.

[0087] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terminology used herein is intended only to describe specific embodiments, and is not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0089] As used herein, the term "optional" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a specific sequence may have but need not have, and may be 1, 2 or 3.

[0090] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0091] the term MyBP-C

[0092] There are three isoforms of MyBP-C in adult muscle - fast skeletal and slow skeletal (encoded by mybpc1 and mybpc2 genes on chromosomes 12q23.3 and 19q33.3, respectively) and cardiac isoform (gene MYBPC311p11.2 on chromosome). The amino acid sequences of the three MyBP-C isoforms are highly homologous. Uniquely, the cardiac isoform contains an additional immunoglobulin-like domain (C0) at the N-terminus, a phosphorylation site (M motif) between C1 and C2, and a 28-amino acid insertion in the C5 domain. It also has a specific antigenic epitope, and the cardiac type MYBPC3 can be specifically determined by immunological methods. The entire protein consists of 12 domains, including 8 immunoglobulin (IgC2)-like domains, 3 fibronectin (FN3) domains, plus the above-mentioned M domain (see Appendix). Figure 1 . Schematic diagram of the protein structure of Mybpc1, Mybpc2, and Mybpc3).

[0093] MYBPC3 (Mybpc3 or mybpc-3) has 1274 amino acids (UniprotKB: Q14896) and a molecular weight of 140KDa. MYBPC3 belongs to the type III fibronectin superfamily and the immunoglobulin superfamily, and contains three type III fibronectin domains (Fibronectintype3, FN3) and eight immunoglobulin-like domains (Immunoglobulin, Ig), a Pro-Ala domain (PAdomain), and a Myosin (S2) binding region (Mdomain).

[0094] MYBPC3 is involved in the structure and function of sarcomere proteins. The C10 domain of MYBPC3 binds to myosin fibers, while the C8-C10 structural region binds to titin, which stabilizes the sarcomere structure. MYBPC3 has a specific region between the C1 and C2 domains. The M domain binds to the neck of myosin, which is also the phosphorylation site of MYBPC3. As the action site of cAMP-dependent protein kinase and calmodulin-dependent protein kinase, it phosphorylates specific myocardial motifs to regulate myocardial contractility. MYBPC3 is not only involved in the maintenance of myocardial structure, but also in intracellular information transmission, affecting the contraction and relaxation of myofilaments and protecting the myocardium from ischemic damage. These functions are mainly mediated by phosphorylation at sites such as Ser-273, Ser-282 and Ser-302. Dephosphorylation of these specific sites will lead to MYBPC3 degradation, releasing a 30-40KDa MYBPC3-N-terminal fragment (C0C1f) into the blood circulation, thereby increasing the level of C0C1f in the blood.

[0095] Antibody As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0096] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-folded configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0097] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of the different classes of immunoglobulins are well known in the art.

[0098] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a homogeneous antibody population, and should not be construed as requiring any special method to produce the antibody.

[0099] The present invention also includes monoclonal antibodies having the corresponding amino acid sequence of the anti-cardiac myosin binding protein C monoclonal antibody, monoclonal antibodies having the variable region chain of the anti-cardiac myosin binding protein C monoclonal antibody, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing a hypervariable region (complementarity determining region, CDR), as long as the hypervariable region is identical to the hypervariable region of the light chain and heavy chain of the present invention or has at least 90% homology, preferably at least 95% homology.

[0100] As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the anti-cardiac myosin binding protein C monoclonal antibody or its fragment to form a conjugate. The present invention also includes cell surface markers or antigens combined with the anti-cardiac myosin binding protein C monoclonal antibody or its fragment.

[0101] The present invention includes not only complete monoclonal antibodies, but also antibody fragments (antigen-binding fragments) having immunological activity, such as Fab or (Fab") 2 fragments; antibody heavy chains; and antibody light chains.

[0102] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment having antigen-binding activity. The Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of the antibody, but has no constant region, and is the smallest antibody fragment with all antigen-binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding.

[0103] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0104] As used herein, the term "heavy chain variable region" is synonymous with "V H ” can be used interchangeably.

[0105] As used herein, the term "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0106] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody has a complementary determining region CDR selected from the following group: V H-CDR1, whose amino acid sequence is SEQ ID NO.2; V H -CDR2, the amino acid sequence of which is SEQ ID NO.3; V H -CDR3, whose amino acid sequence is SEQ ID NO.4.

[0107] In another preferred embodiment, the heavy chain variable region (V H ) has an amino acid sequence of SEQ ID NO.8.

[0108] In another preferred embodiment of the present invention, the heavy chain variable region of the antibody has a complementary determining region CDR selected from the following group: V H -CDR1, whose amino acid sequence is SEQ ID NO.10; V H -CDR2, the amino acid sequence of which is SEQ ID NO.11; V H -CDR3, whose amino acid sequence is SEQ ID NO.12.

[0109] In another preferred embodiment, the heavy chain variable region (V H ) has an amino acid sequence of SEQ ID NO. 16.

[0110] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the above-mentioned heavy chain variable region and a heavy chain constant region, and the heavy chain constant region may be of murine or human origin.

[0111] As used herein, the terms "light chain variable region" and "V L ” can be used interchangeably.

[0112] In a preferred embodiment of the present invention, the light chain variable region of the antibody has a complementary determining region CDR selected from the following group: V L -CDR1, the amino acid sequence of which is SEQ ID NO. 5; V L -CDR2, the amino acid sequence of which is SEQ ID NO.6; V L -CDR3, the amino acid sequence of which is SEQ ID NO.7; In another preferred embodiment, the light chain variable region (V L ) has an amino acid sequence of SEQ ID NO.9.

[0113] In another preferred embodiment of the present invention, the light chain variable region of the antibody has a complementary determining region CDR selected from the following group: V L -CDR1, the amino acid sequence of which is SEQ ID NO. 13; V L -CDR2, the amino acid sequence of which is SEQ ID NO. 14; V L -CDR3, the amino acid sequence of which is SEQ ID NO. 15; In another preferred embodiment, the light chain variable region (V L ) has an amino acid sequence of SEQ ID NO. 17.

[0114] In a preferred embodiment of the present invention, the light chain of the antibody comprises the above-mentioned light chain variable region and a light chain constant region, and the light chain constant region may be of murine or human origin.

[0115] In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, and all refer to antibodies that specifically bind to cardiac myosin binding protein C, such as proteins or polypeptides having a heavy chain variable region (such as the amino acid sequence shown in SEQ ID NO.8 or SEQ ID NO.16) and / or a light chain variable region (such as the amino acid sequence shown in SEQ ID NO.9 or SEQ ID NO.17). They may or may not contain an initial methionine.

[0116] In another preferred embodiment, the antibody is a mouse or human-mouse chimeric monoclonal antibody against cardiac myosin binding protein C, and its heavy chain constant region and / or light chain constant region can be a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or light chain constant region of human IgG1, IgG2, etc.

[0117] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having heavy and light chains containing variable regions, as long as the variable regions are identical or at least about 90% homologous, preferably at least about 95% homologous, to the variable regions of the heavy and light chains of the antibodies of the present invention.

[0118] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0119] The variable regions of the heavy and / or light chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having monoclonal antibody light and heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0120] The present invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0121] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0122] The antibody of the present invention refers to a polypeptide having cardiac myosin binding protein C binding activity and comprising the above CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and comprising the above CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0123] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0124] The present invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0125] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0126] Table 1

[0127] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0128] The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO. 18, 19, 20 and 21 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding an amino acid sequence identical to the polypeptide of the present invention, but different from the coding region sequence shown in SEQ ID NO. 18, 19, 20 and 21.

[0129] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0130] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0131] The present invention also relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, and more preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptides shown in SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.16, and SEQ ID NO.17.

[0132] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0133] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0134] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0135] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0136] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0137] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl 2 Another method is to use MgCl 2 If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0138] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0139] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0140] The antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0141] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0142] Detectable labels for detection purposes include, but are not limited to, biotin, fluorescein, chemiluminescent groups, chemifluorescent groups, fluorescent proteins, enzymes, colloidal gold, colored magnetic beads, latex particles, biotin labels, radionuclides, antibodies, ligands, antigens, receptors, nanoparticles, or combinations thereof.

[0143] Typically, the nanoparticles are selected from the group consisting of gold nanoparticles, silver nanoparticles, quantum dots or a combination thereof.

[0144] Typically, the enzyme is selected from the group consisting of horseradish peroxidase, acid phosphatase, or a combination thereof.

[0145] Therapeutic agents that may be conjugated include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.

[0146] In addition, therapeutic agents that can be combined or coupled with the antibodies of the present invention include, but are not limited to: 1. radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. biological toxins (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 (Gillies et al., 1992, Proceedings of the National Academy of Sciences (PNAS) 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al., 2003, Cancer Research 63, 3202); 4. gold nanoparticles / nanorods (Lapotko et al., 2005, Cancer Communications (Cancer Research) 63, 3202); letters) 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 5. Virus particles (Peng et al., 2004, Gene therapy 11, 1234); 6. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 7. Nanomagnetic particles; 8. Prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0147] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

[0148] The pharmaceutical composition of the present invention can be directly used to bind to cardiac myosin binding protein C molecules, and thus can be used to prolong the half-life of the drug. In addition, other therapeutic agents can also be used simultaneously.

[0149] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 10 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0150] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 8 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 1 milligram / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0151] Hybridoma cell lines The present invention also provides a hybridoma cell line capable of producing the monoclonal antibody against cardiac myosin binding protein C of the present invention; preferably, the present invention provides a hybridoma cell line with a high titer of the monoclonal antibody against cardiac myosin binding protein C.

[0152] After obtaining the hybridoma producing the cardiac myosin binding protein C monoclonal antibody of the present invention, the skilled person can conveniently use the hybridoma cell line to prepare the antibody. In addition, the skilled person can also conveniently know the structure of the antibody of the present invention (such as the heavy chain variable region and light chain variable region of the antibody), and then can prepare the monoclonal antibody of the present invention by a recombinant method.

[0153] Preparation of monoclonal antibodies

[0154] The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, the antigens of the present invention can be administered to animals to induce the production of monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6: 511, 1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981) or can be prepared by a recombinant DNA method (U.S. Pat. No. 4,816,567), obtained by a phage display system; obtained from transgenic humanized mice by immunizing the mice.

[0155] Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium (HAT medium medium), and include myeloma cell lines, such as murine myeloma cell lines, including myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, California, USA) and SP-2, NZ0 or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, Maryland, USA).

[0156] Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibodies Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)].

[0157] The culture medium in which the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies with the desired specificity, such as, by in vitro binding analysis, for example, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The location of cells expressing the antibody can be detected by FACS. Then, the hybridoma clones can be subcloned by limiting dilution steps and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) 59-103 pages). Suitable culture media used to achieve this purpose include, for example, DMEM or RPMI-1640 culture media. In addition, hybridoma cells can be grown as ascites tumors in animals.

[0158] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0159] The present invention provides a monoclonal antibody against cardiac myosin binding protein, in particular a monoclonal antibody against cardiac myosin binding protein C. In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells. The supernatant of the hybridoma cell culture is taken, IgG is crudely extracted by saturated ammonium sulfate precipitation, and the crudely extracted antibody is purified by affinity chromatography column (Protein G-Sephrose).

[0160] In a preferred embodiment of the present invention, the monoclonal antibody is prepared by the method of producing monoclonal antibodies using the ascites of Balb / C mice. The hybridoma cells are inoculated into the abdominal cavity of sensitized mice, and the abdomen is obviously swollen in about 10 days. The ascites is extracted, and the crude antibody is purified by affinity chromatography column (Protein G-Sephrose) after crude extraction by saturated ammonium sulfate precipitation method.

[0161] The main advantages of the present invention are: (1) cMyBPC is rapidly released into the blood after a myocardial infarction occurs. It has a high concentration and a fast rate, making it suitable as a marker for diagnosing acute myocardial infarction.

[0162] (2) The present invention provides a pair of cMyBPC detection antibodies with better sensitivity, which can buy more time for diagnosis and treatment for patients.

[0163] (3) This antibody can be used in other detection methods based on the double antibody sandwich method.

[0164] (4) Better binding to target antigens in human samples.

[0165] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where detailed conditions are not specified are usually carried out according to conventional conditions such as the conditions described in "Molecular Cloning Laboratory Guide" by Sambrook.J et al. (Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0166] Materials and methods Unless otherwise specified, the experimental materials used in the examples of the present invention can be obtained from commercial channels.

[0167] The present invention mainly includes 1. expression of recombinant protein; 2. immunization and antibody screening; 3. ELISA affinity detection (see the schematic diagram of the detection process Figure 2 ); 4. Human blood sample testing.

[0168] 1. Design and expression of in vitro recombinant antigens: >MYBPC3-C0C1f (SEQ NO.1): MPEPGKKPVSAFSKKPRSVEVAAGSPAVFEAETERAGVKVRWQRGGSDISASNKYGLATEGTRHTLTVREVGPADQGSYAVIAGSSKVKFDLKVIEAEKAEPMLAPAPAPAEATGAPGEAPAPAAELGESAPSPKGSSSAALNG PTPGAPDDPIGLFVMRPQDGEVTVGGSITFSARVAGASLLKPPVVKWFKGKWVDLSSKVGQHLQLHDSYDRASKVYLFELHITDAQPAFTGSYRCEVSTKDKFDCSNFNLTVHEAMTGDLDLLSAFRRTSLAGGGG(1-280aa) After the amino acid sequence was codon-optimized, it was constructed into the modified pCDNA3.4 vector (donated by Fudan University). The N-terminal of the vector contains a His-sumom tag. The corresponding plasmid was constructed and transfected into Expi293F cells (A14527, Gbico). After 3 days of expression, the supernatant was collected by centrifugation and purified with Ni Smart Beads (SA035005, Smart-Lifescience). The purified protein was added with ULPM enzyme digestion (HE001, Oritop Biotech), and the purity of the obtained protein was analyzed after SDS-PAGE electrophoresis. The antigen bands obtained after purification are shown in Figure 3 and Figure 4 .

[0169] 2. Mouse Immunization 1) Antigen emulsification: The antigen was diluted with PBS and then mixed with the adjuvant at a volume ratio of 1:1 and fully emulsified.

[0170] 2) Freund's complete adjuvant was used for the first immunization and Freund's incomplete adjuvant was used for the remaining immunizations.

[0171] 3) After emulsification, the mice were immunized by multiple subcutaneous injections, with 0.05-0.1 ml injected at each point.

[0172] 4) After the immunization is completed, write the immunization time and quantity on the mouse cage and keep a record of the experiment.

[0173] The detailed steps are shown in Table 2.

[0174] Table 2

[0175] 3. Serum titer detection - ELISA method 1) Antigen coating: C0C1f antigen coating was diluted with 1X CBS to a concentration of 10ug / ml. 100ul of the diluted antigen was added to each well using a dispenser (the amount of antigen per plate was 10ug) and reacted at 37℃ for 2h.

[0176] 2) Washing: After shaking off the liquid, pat dry on absorbent paper. Add 200ul of washing solution PBST to each well with a dispenser, shake gently, shake off the washing solution, pat dry on absorbent paper, and repeat washing twice.

[0177] 3) Blocking: Add 200ul of blocking solution (PBST + 5% skim milk powder) to each well and block at 37℃ for 1h.

[0178] 4) Washing: shake off the liquid and pat dry on absorbent paper. Use a dispenser to add 200ul of PBST to each well, shake gently, shake off the washing solution, pat dry on absorbent paper, repeat washing 3 times, and store at 4℃ for later use.

[0179] 5) Serum collection: Collect blood from the eye corner of the mouse using a capillary blood collection tube, and blow the blood into a 1.5 ml EP tube using an ear blower. Mark the EP tube and the corresponding mouse. After all the blood is collected, let it stand at 4°C for half an hour.

[0180] 6) Sample preparation: Let stand for half an hour, then take out and centrifuge at 8000rpm for 15 minutes.

[0181] 7) Primary antibody incubation: Add the centrifuged serum to the reaction wells, dilute with blocking solution, 100ul per well, cover with tin foil, and incubate at 37℃ for 1h.

[0182] 8) Washing: shake off the liquid and pat dry on absorbent paper. Add 200ul of washing solution PBST to each well with a dispenser, shake gently, shake off the washing solution, pat dry on absorbent paper, and repeat washing 3 times.

[0183] 9) Secondary antibody incubation: Dilute goat anti-mouse IgG-HRP with blocking solution at 1:10000, add 100ul to each well with a pipette, cover with tin foil, and incubate at 37℃ for 1h.

[0184] 10) Washing: shake off the liquid and pat dry on absorbent paper. Add 200ul of washing solution PBST to each well with a dispenser, shake gently, shake off the washing solution, pat dry on absorbent paper, and repeat washing 4 times.

[0185] 11) Color development: Add 100ul of TMB color development solution to each well using a dispenser, cover with tin foil, and react at room temperature for 5 minutes.

[0186] 12) Stop: Add stop solution and pipette 50ul into each well.

[0187] After five immunizations, the mice completed the animal immunization experiment, the serum titer was qualified, and the OD value displayed on the ELISA plate was as shown in Table 3: Table 3 Serum titers of mice after five immunizations

[0188] 4. Hybridoma cell fusion experiment After immunization, the spleen of the immunized animal was prepared and fused with myeloma cells (Ag8), and the obtained hybridoma cells were cultured. The reactivity of the cell supernatant after fusion to mybpc-3 was tested by ELISA, and the cell lines with higher titer affinity were selected for subcloning. After culture, the reactivity of the subcloned cell culture supernatant to mybpc-3 was tested again, and the cell lines with higher titer affinity were selected for the second subcloning. The cell culture supernatant of the second subclone was detected by indirect ELISA, and the positive cell lines were expanded and cultured. The supernatant was collected and subtype identification was performed. After the cells were collected, they were injected into mice to prepare ascites. After the ascites was purified, the purity was verified by SDS to be above 90%. The results are shown in Figure 5 .

[0189] 5. Antibody titer and sensitivity testing Antigen: C0C1f after enzyme digestion, concentration: 0.92mg / ml, 300ng / well, 32 wells; Primary Antibody: C0C1f -2F11 2D7, batch number: D240408C05, concentration: 0.91mg / ml, start with 5ug in the first well (5.49ul antibody is supplemented to 100ul with PBST), dilute 5 times from the second well to the 15th well, and the 16th well is a negative control; HM134-4C5 3C5, batch number: D240408C03, concentration: 2.85mg / ml, start with 5ug in the first well (1.75ul antibody is supplemented to 100ul with PBST), dilute 5 times from the second well to the 15th well, and the 16th well is a negative control; Secondary antibody: goat anti-mouse (1:5000) Results Figure 6 :EC 50 : C0C1f-4C53C5 is 0.0067μg / ml, and C0C1f-2F112D7 is 0.0069μg / ml.

[0190] 6. The above 2F112D7 and 4C53C5 were labeled and then used for ELISA detection.

[0191] Primary Antibody (Coating Antibody): 2F11 2D7, batch number: D240408C05, concentration: 0.91mg / ml, start with 5ug of the coated antibody in the first well (5.49ul of antibody is supplemented to 100ul with PBST), dilute 5 times from the second well to the 15th well, and the 16th well is a negative control; Secondary antibody (labeled antibody): 4C5 3C5, batch number: D240408C03, concentration: 2.85mg / ml, the labeled antibody is directly labeled with horseradish peroxidase (HRP, Suzhou subfamily), and the labeling method is sodium periodate oxidation method. After the aldehyde-modified HRP binds to the antibody, it is blocked by ethanolamine and ultrafiltration to replace the buffer. After recovery, the concentration is set at 2~3mg / ml. The antibody recovery rate results are as follows: Experimental process: 1) Primary antibody coating: 2F11 2D7 antibody coating, diluted with 1X CBS to a concentration of 2ug / ml, add 100ul of the diluted antibody to each well with a dispenser (2ug of antibody per plate), and incubate at 37℃ for 2h.

[0192] After incubation, shake off the liquid in the wells and pat dry on absorbent paper.

[0193] 2) Blocking: Add 200ul of blocking solution (1.5% BSA solution) to each well and block at 37℃ for 2h.

[0194] 3) Washing: shake off the liquid and pat dry on absorbent paper. Add 200ul of washing solution TBST to each well using a dispenser, shake gently, shake off the washing solution, pat dry on absorbent paper, repeat washing 3 times, and store at 4℃ for later use.

[0195] 4) Preparation of antigen calibrators: Dilute the antigen concentration to 20,000 pg / ml with 1X PBS, and then perform a 2-fold gradient dilution to obtain 8 gradient concentrations of antigen calibrators.

[0196] 5) Sample loading: Add 100ul of the diluted antigen calibrator to each well using a pipette and incubate at 37℃ for 2h.

[0197] After incubation, shake off the liquid in the wells and pat dry on absorbent paper.

[0198] 6) Secondary antibody incubation: dilute the labeled antibody at 1:2000 with 1X PBS, add 100ul to each well with a pipette, cover with tin foil, and incubate at 37℃ for 1h.

[0199] 7) Washing: shake off the liquid and pat dry on absorbent paper. Add 200ul of washing solution PBST to each well with a dispenser, shake gently, shake off the washing solution, pat dry on absorbent paper, and repeat washing 4 to 5 times.

[0200] 8) Color development: Add 100ul of TMB color development solution to each well using a dispenser, cover with tin foil, and react at room temperature for 5 minutes.

[0201] 9) Stop: Add stop solution and pipette 50ul into each well.

[0202] 10) Reading and data processing: Place the ELISA plate at the designated position on the ELISA reader, select the reading area and wavelength as 450nm, and read the OD of each well. 450 Data. Make a fitting curve based on the data and calculate the correlation coefficient.

[0203] The experimental results are as follows Figure 7 As shown, there is a good correlation between the concentration of the antigen standard and the final color development number, which proves that the antibody used in our method can be used for antigen detection needs.

[0204] discuss In the existing technical solutions, troponin cannot accurately determine the occurrence of AMI due to the time lag after the onset of AMI patients and the low concentration in patients and in healthy people. The existing cmybp-c antibody has poor sensitivity and specificity in detecting the presence of human protein, and cannot solve the problem of early diagnosis of acute myocardial infarction patients.

[0205] Myosin-Binding Protein C (Myosin-Binding Protein C, Cardiac-Type, MYBPC3 or cMyBP-C) is more in line with this standard. The reasons are as follows: 1) MYBPC3 is rapidly released into the blood after myocardial infarction, which helps shorten the diagnosis time. 3 hours after AMI, the level of MYBPC3 in the blood has increased, reaching a peak in 6 hours, and lasting until 12 hours after AMI, when MYBPC3 returns to the baseline level; while the level of cTn in the blood will not increase until 6-12 hours later. Studies have shown that an increase in cTn levels can be detected when 3-9 mg of myocardial necrosis is required, while an increase in MYBPC3 levels can be detected when only 0.07 mg of myocardial tissue is necrotic. 2) The concentration of MYBPC3 in the blood of AMI patients is high. cMyC is mainly a thick filament-related protein, one of the most abundant proteins in myocardial proteins, ranking 19th among more than 2,300 quantified proteins. In the ultra-early stage of AMI, intact MYBPC3 and its hydrolysis products are continuously released into the blood in large quantities, which can be several times or even dozens of times higher than cTnT. It is relatively easy to measure the concentration of MYBPC3 in the blood. Therefore, MYBPC3 can become a sensitive and specific indicator for the ultra-early diagnosis of AMI.

[0206] Cmbpc can be used as an alternative detection indicator, but there is no good detection antibody to develop ELISA detection kit. At present, there is no reliable kit for detecting cMyBPC in patients with acute myocardial infarction in the clinic. The possible reason is that there is no antibody with high affinity and good specificity, and new antibodies with specificity need to be developed.

[0207] The present invention is based on the following points: (1) recombinant protein antigen sequence specificity and antigen-antibody binding epitope specificity; (2) antibodies have good affinity with natural human proteins; (3) antigen-antibody binding epitope specificity, forming a pair of specific binding antibodies for sandwich detection with high affinity; a pair of specific detection antibodies has been developed with high sensitivity and specificity, suitable for the development of detection kits.

[0208] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0209] Sequence information of the present invention Sequences involved in the first monoclonal antibody 2F112D7: V H -CDR1: Its amino acid sequence is SANTFH (SEQ ID NO. 2) V H -CDR2: Its amino acid sequence is GFNPYNGVSNINQNFKG (SEQ ID NO. 3) V H -CDR3: Its amino acid sequence is NYDPFDY (SEQ ID NO. 4) V L -CDR1: Its amino acid sequence is RSSQNIVHSNGNTYLE (SEQ ID NO. 5) V L -CDR2: Its amino acid sequence is KVSNRFS (SEQ ID NO. 6) V L -CDR3: Its amino acid sequence is FQGSHVPLT (SEQ ID NO. 7) 2F112D7 heavy chain variable region (V H )Amino acid sequence EVQLQQSGPELVKPGASVKISCKTSGFTFTANTFHWMKQSHGKSLEWIGGFNPYNGVSNINQNFKGKATLTVDKSSSTAYMELRSLTSDDSAVYYCARNYDPFDYWGQGTTLTVSS (SEQ ID NO.8) Encoding 2F112D7 heavy chain variable region (V H ) amino acid nucleotide sequence GAGGTTCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATTCACATTCACTGCAAACACCTTTCACTGGATGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTTTTAATCCTTACAATGGTGTTTCTAACATCAACCAGAATTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAACTCCGCAGCCTGACATCTGACGATTCTGCAGTCTATTACTGTGCAAGGAATTACGACCCTTTTGACTACTGGGGCCAAGGCACCACTCTCACCGTCTCGAGC (SEQ ID NO.18) Amino acid sequence of the variable region of the light chain (VL) of 2F112D7 DVVMTQSPLSLTVSLGDHASISCRSSQNIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK (SEQ ID NO.9) Nucleotide sequence encoding the variable region of the light chain (V L ) amino acids of GATGTTGTGATGACCCAAAGTCCACTCTCCCTGACTGTCAGTCTTGGAGATCACGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTGCATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTTGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO.19) Sequences related to the second monoclonal antibody 4C53C5: VH -CDR1: Its amino acid sequence is DYYIH (SEQ ID NO. 10) V H -CDR2: Its amino acid sequence is WIDPENGDTEYVPKFQG (SEQ ID NO. 11) V H -CDR3: Its amino acid sequence is RGVVAHYYAMDY (SEQ ID NO. 12) V L -CDR1: Its amino acid sequence is KSSQSLLNSGDQKNKLA (SEQ ID NO. 13) V L -CDR2: Its amino acid sequence is GASTRES (SEQ ID NO. 14) V L -CDR3: Its amino acid sequence is QNDHSYPLT (SEQ ID NO. 15) 4C53C5 heavy chain variable region (V H )Amino acid sequence QVQLQQSGAELVRSGASVKLSCTASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGDTEYVPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNTRGVVAHYYAMDYWGQGTSVTVSS (SEQ ID NO.16) Encoding 4C53C5 heavy chain variable region (V H ) amino acid nucleotide sequence CAGGTTCAACTGCAGCAGTCTGGGGCAGAGCTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTATATACACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTGATACTGAATATGTCCCGAAATTCCAGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTATTGTAATACAAGGGGGGTAGTAGCTCATTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCGAGC (SEQ ID NO.20) 4C53C5 light chain variable region (V L ) Amino acid sequence DVVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGDQKNKLAWYQQKPGQSPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLELK (SEQ ID NO.17) Nucleotide sequence encoding the 4C53C5 light chain variable region (V L ) Amino acids GATGTTGTGATGACCCAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAGATCAAAAAAACAAGTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGTTGATCTACGGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACCGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATCATAGTTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO.21)

Claims

1. A heavy chain variable region of an antibody, characterized in that The heavy chain variable region has a complementarity determining region (CDR) selected from the group consisting of: (i) V shown in SEQ ID NO.2 H -CDR1, V shown in SEQ ID NO.3 H -CDR2, and V shown in SEQ ID NO.4 H -CDR3; preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.8; or (ii) V shown in SEQ ID NO.10 H -CDR1, V shown in SEQ ID NO.11 H -CDR2, and V shown in SEQ ID NO.12 H -CDR3; preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.

16.

2. A heavy chain of an antibody, characterized in that The heavy chain has the heavy chain variable region and heavy chain constant region according to claim 1.

3. A light chain variable region of an antibody, characterized in that: The light chain variable region has a complementarity determining region CDR selected from the group consisting of: (i) V shown in SEQ ID NO.5 L -CDR1, V shown in SEQ ID NO.6 L -CDR2, V shown in SEQ ID NO.7 L -CDR3, preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO.9, or (ii) V shown in SEQ ID NO.13 L -CDR1, V shown in SEQ ID NO.14 L -CDR2, V shown in SEQ ID NO.15 L -CDR3, preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO.

17.

4. A light chain of an antibody, characterized in that The light chain has the light chain variable region and light chain constant region according to claim 3.

5. An antibody, characterized in that The antibody has: (1) the heavy chain variable region of claim 1; and / or (2) the light chain variable region of claim 3; or, the antibody has: the heavy chain of claim 2; and / or the light chain of claim 4.

6. A recombinant protein, characterized in that The recombinant protein has: (i) the sequence of the heavy chain variable region of claim 1, the sequence of the heavy chain of claim 2, the sequence of the light chain variable region of claim 3, the sequence of the light chain of claim 4, or the sequence of the antibody of claim 5; and (ii) an optional tag sequence that facilitates expression and / or purification.

7. An immunoconjugate, characterized in that: The immunoconjugate contains: (a) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5, or the recombinant protein of claim 6; and (b) a conjugated portion selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

8. Use of the heavy chain variable region as claimed in claim 1, the heavy chain as claimed in claim 2, the light chain variable region as claimed in claim 3, the light chain as claimed in claim 4, the antibody as claimed in claim 5, the recombinant protein as claimed in claim 6, or the immunoconjugate as claimed in claim 7 for preparing a medicament, a reagent, a test plate or a kit.

9. A detection product, characterized in that: The detection product comprises: (1) the antibody according to claim 5, the recombinant protein according to claim 6, or the conjugate according to claim 7; and (2) Optional buffer solution or buffering agent.

10. A detection kit, characterized in that: The detection kit comprises the antibody according to claim 5, the recombinant protein according to claim 6, or the conjugate according to claim 7 or the detection product according to claim 9.

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