Anti-beta1-AR-ECII antibody and application thereof

Abstract

The invention belongs to the technical field of biological medicines, particularly relates to application of beta1-AR-ECII or an autoantibody thereof as a marker or a target spot in diagnosis and / or treatment of cardiovascular diseases, and particularly provides an anti-beta1-AR-ECII antibody or an antigen binding fragment thereof and application thereof. Firstly, two anti-beta1-AR-ECII antibodies or antigen binding fragments thereof are newly screened, the two antibodies are respectively combined with N-terminal peptide and C-terminal peptide of beta1-AR-ECII, detection of beta1-AR-ECII is realized, the cost is low, and rapidness and effectiveness are realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-β1-AR-ECII antibody and a preparation method and application thereof. Background Art

[0002] β1-adrenoceptor autoantibody (β1-AA), as a product of immune system disorder, is an autoantibody targeting β1-adrenoceptor (β1-AR). It exists in various cardiovascular diseases such as cardiomyopathy (such as hypertensive cardiomyopathy, dilated cardiomyopathy or rheumatic cardiomyopathy), arrhythmia and heart failure, and is related to the pathogenesis.

[0003] Normal human blood may contain low levels of autoantibodies, which will not cause disease. However, if the titer of autoantibodies exceeds a certain level, it may damage the body and eventually induce disease. High positive rates of β1-AA are also present in the serum of patients with heart failure. The long-term presence of this high positive rate of β1-AA can lead to changes in cardiac morphology and function. Studies have confirmed that the long-term presence of β1-AA can lead to the occurrence of heart failure, and the production of β1-AA can also be induced in the process of myocardial remodeling leading to heart failure. The two are mutually causal and mutually reinforcing (see non-patent literature: Li Xiao, Du Yunhui, Zuo Qian, Liu Huirong, et al. β1 adrenergic receptor autoantibodies and their role in the occurrence and development of heart failure [J]. Progress in Physiological Sciences, 2013(05):67-71.). Further studies have shown that an imbalance between β1-AAs and β2-AAs in patients with heart failure is a mechanism for the progression of HF, and an elevated β1-AAs / β2-AAs ratio should be considered a clinical assessment factor for worsening cardiac function in patients with heart failure (see: Ning Cao, β2-adrenergic receptor autoantibodies alleviated myocardial damage induced by β1-adrenergic receptor autoantibodies in heart failure, [J]., 2018: 114(11)). However, the mechanism of β1-AA generation is still unclear at this stage. Summary of the Invention

[0004] To fill the gaps in the existing technology, this application studies the generation mechanism of β1-AA through β1-AR-ECII, and unexpectedly finds that β1-AR-ECII is directly related to cardiovascular disease (especially acute myocardial infarction) and can be used as a marker or target for the diagnosis and treatment of cardiovascular disease. In addition, two new anti-β1-AR-ECII antibodies or antigen-binding fragments thereof were newly screened. These two antibodies bind to the N-terminal peptide and C-terminal peptide of β1-AR-ECII, respectively, to achieve accurate, rapid and effective detection of β1-AR-ECII. The specific plan is as follows:

[0005] The first aspect of the present invention provides the use of β1-AR-ECII or its autoantibody as a marker or target in the preparation of products for diagnosing and / or treating cardiovascular diseases.

[0006] The second aspect of the present invention provides the use of a reagent for detecting β1-AR-ECII or its autoantibodies in the preparation of a product for diagnosing and / or treating cardiovascular diseases.

[0007] The β1-AR-ECII or its autoantibody is the β1-AR-ECII or its autoantibody in body fluids or cells.

[0008] Preferably, the β1-AR-ECII or its autoantibody is β1-AR-ECII or its autoantibody in blood, serum, plasma or myocardial cells.

[0009] The product includes a reagent for detecting β1-AR-ECII.

[0010] Preferably, the reagent for detecting β1-AR-ECII comprises an anti-β1-AR-ECII antibody or an antigen-binding fragment thereof.

[0011] The product can be a detection kit.

[0012] Preferably, the diagnosis of cardiovascular disease includes detecting the presence or content of β1-AR-ECII.

[0013] Preferably, the treatment of cardiovascular disease comprises administering a β2-AR / Gi pathway agonist, such as ICI118551, to a subject in need thereof.

[0014] Preferably, the cardiovascular disease includes acute myocardial infarction, cardiomyopathy (such as hypertensive cardiomyopathy, dilated cardiomyopathy or rheumatic cardiomyopathy), arrhythmia or heart failure.

[0015] Preferably, the diagnosis and / or treatment of cardiovascular disease may be the diagnosis and / or treatment of cardiovascular disease in humans or non-human animals.

[0016] In a third aspect of the present invention, an anti-β1-adrenergic receptor second extracellular loop (β1-AR-ECII) antibody or an antigen-binding fragment thereof is provided, wherein the anti-β1-AR-ECII antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0017] I) a heavy chain variable region comprising: a VHCDR1 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 1; a VHCDR2 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 2; a VHCDR3 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 3; and a light chain variable region comprising: a VLCDR1 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 4; a VLCDR2 comprising or as shown in SEQ ID NO: 5; a VLCDR3 comprising or as shown in SEQ ID NO: 6;

[0018] or,

[0019] II) the heavy chain variable region comprises: VHCDR1 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 7; VHCDR2 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 8; VHCDR3 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 9; and the light chain variable region comprises: VLCDR1 comprising or as shown in SEQ ID NO: 10; VLCDR2 comprising or as shown in SEQ ID NO: 11; VLCDR3 comprising or as shown in SEQ ID NO: 12.

[0020] Preferably, the anti-β1-AR-ECII antibody or antigen-binding fragment thereof further comprises a FR region. Further preferably, the FR region is derived from or is a FR region of a human or non-human animal (e.g., a non-human mammal), preferably derived from or is a FR region of a human, rodent or primate.

[0021] Preferably, the anti-β1-AR-ECII antibody or antigen-binding fragment thereof comprises VHFR1 (such as SEQ ID NO: 19), VHFR2 (SEQ ID NO: 20), VHFR3 (SEQ ID NO: 21), VHFR4 (SEQ ID NO: 22) of the heavy chain variable region, and VLFR1 (SEQ ID NO: 23), VLFR2 (SEQ ID NO: 24), VLFR3 (SEQ ID NO: 25), VLFR4 (SEQ ID NO: 26) of the light chain variable region.

[0022] In a specific embodiment of the present invention, the heavy chain variable region includes VHFR1, VHCDR1, VHFR2, VHCDR2, VHFR3, VHCDR3, and VHFR4 from N-terminus to C-terminus.

[0023] In a specific embodiment of the present invention, the light chain variable region includes VLFR1, VLCDR1, VLFR2, VLCDR2, VLFR3, VLCDR3, and VLFR4 from N-terminus to C-terminus.

[0024] Preferably, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13, or comprises an amino acid sequence having more than 90%, more than 95% or more than 99% homology to the amino acid sequence shown in SEQ ID NO: 13, or comprises an amino acid sequence having at least one, two or three or more, or at most ten, nine, eight, seven, six, five, four, three or two amino acid substitutions, deletions or mutations in SEQ ID NO: 13, and has the same or similar activity as SEQ ID NO: 13 (e.g., binding activity to β1-AR-ECII).

[0025] Preferably, the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 14, or includes an amino acid sequence having more than 90%, more than 95% or more than 99% homology to the amino acid sequence shown in SEQ ID NO: 14, or includes an amino acid sequence having at least one, two or three or more, or at most ten, nine, eight, seven, six, five, four, three or two amino acid substitutions, deletions or mutations in SEQ ID NO: 14, and has the same or similar activity as SEQ ID NO: 14 (e.g., binding activity to β1-AR-ECII).

[0026] Preferably, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or comprises an amino acid sequence having more than 90%, more than 95% or more than 99% homology to the amino acid sequence shown in SEQ ID NO: 15, or comprises an amino acid sequence having at least one, two or three or more, or at most ten, nine, eight, seven, six, five, four, three or two amino acid substitutions, deletions or mutations in SEQ ID NO: 15, and has the same or similar activity as SEQ ID NO: 15 (e.g., binding activity to β1-AR-ECII).

[0027] Preferably, the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or comprises an amino acid sequence having more than 90%, more than 95% or more than 99% homology to the amino acid sequence shown in SEQ ID NO: 16, or comprises an amino acid sequence having at least one, two or three or more, or at most ten, nine, eight, seven, six, five, four, three or two amino acid substitutions, deletions or mutations in SEQ ID NO: 16, and has the same or similar activity as SEQ ID NO: 16 (e.g., binding activity to β1-AR-ECII).

[0028] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 14.

[0029] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 16.

[0030] In a specific embodiment of the present invention, the anti-β1-AR-ECII antibody or antigen-binding fragment thereof is an IgG antibody.

[0031] Preferably, the anti-β1-AR-ECII antibody or antigen-binding fragment thereof is a mouse antibody.

[0032] Preferably, the anti-β1-AR-ECII antibody or antigen-binding fragment thereof includes Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or Fd.

[0033] The anti-β1-AR-ECII antibody or antigen-binding fragment thereof specifically binds to human or non-human animal β1-AR-ECII protein or fragment thereof.

[0034] In a fourth aspect, the present invention provides an antigen receptor, wherein the antigen receptor comprises the above-mentioned anti-β1-AR-ECII antibody or an antigen-binding fragment thereof.

[0035] Preferably, the antigen receptor can be a chimeric antigen receptor (CAR) or a synthetic T cell antigen receptor (STAR).

[0036] In a fifth aspect, the present invention provides an antibody-drug conjugate, wherein the antibody-drug conjugate comprises the anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to the third aspect and a cytotoxic agent.

[0037] In the sixth aspect of the present invention, a nucleic acid is provided, which encodes the anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to the third aspect, or the antigen receptor according to the fourth aspect, or the antibody-drug conjugate (ADC) according to the fifth aspect.

[0038] The seventh aspect of the present invention provides a cell, wherein the cell expresses the anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to the third aspect, or the cell expresses the antigen receptor according to the fourth aspect, or the cell contains the nucleic acid according to the sixth aspect.

[0039] The cells can be eukaryotic cells or prokaryotic cells.

[0040] Eukaryotic cells include animal and plant cells, such as T cells, yeast cells, HEK293 cells, or CHO cells.

[0041] Prokaryotic cells such as Escherichia coli.

[0042] Preferably, the cell may be a hybridoma cell.

[0043] In an eighth aspect, the present invention provides a method for preparing the anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to the third aspect, or the antigen receptor according to the fourth aspect, the preparation method comprising introducing the nucleic acid according to the sixth aspect into a host cell and then inducing its expression.

[0044] In the ninth aspect of the present invention, a method for detecting β1-AR-ECII in a sample is provided, the method comprising using the anti-β1-AR-ECII antibody or antigen-binding fragment thereof described in the third aspect, the nucleic acid described in the sixth aspect, the cell described in the seventh aspect, and the anti-β1-AR-ECII antibody or antigen-binding fragment thereof obtained by the preparation method described in the eighth aspect.

[0045] Preferably, the method comprises contacting an anti-β1-AR-ECII antibody or an antigen-binding fragment thereof with a sample, and detecting a complex formed between the antibody and β1-AR-ECII.

[0046] Further preferably, the method comprises:

[0047] 1) linking the anti-β1-AR-ECII antibody or antigen-binding fragment 1 to a solid support;

[0048] 2) Add the sample to be tested;

[0049] 3) further adding anti-β1-AR-ECII antibody or antigen-binding fragment thereof 2;

[0050] 4) Add labeled secondary antibody;

[0051] 5) Develop color and detect OD450 value.

[0052] Preferably, the sample comprises body fluid or cells. Further preferably, the body fluid comprises blood, plasma or serum. Further preferably, the cells comprise cardiomyocytes.

[0053] Preferably, the sample is serum, and the added volume is 1-1000 μl, for example, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 200 μl, 500 μl or 1000 μl, etc.

[0054] Preferably, if the sample is cells, the volume of cell culture supernatant added is 10-1000 μl, for example, 10 μl, 15 μl, 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 200 μl, 500 μl or 1000 μl, etc.

[0055] Preferably, the sample can be body fluid or cells of a human or non-human animal.

[0056] Preferably, the final concentration of the anti-β1-AR-ECII antibody or its antigen-binding fragment 1 in the reaction system is 1 μg / ml-100 μg / ml, for example, 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, 50 μg / ml, 80 μg / ml or 100 μg / ml, etc.

[0057] Preferably, the final concentration of the anti-β1-AR-ECII antibody or its antigen-binding fragment 2 in the reaction system is 1 μg / ml-100 μg / ml, for example, 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, 50 μg / ml, 80 μg / ml or 100 μg / ml, etc.

[0058] The anti-β1-AR-ECII antibody or antigen-binding fragment 1 thereof binds to amino acids 14-26 of β1-AR-ECII (SEQ ID NO: 17),

[0059] The anti-β1-AR-ECII antibody or antigen-binding fragment 2 thereof binds to amino acids 1-13 of β1-AR-ECII (SEQ ID NO: 18); or

[0060] The anti-β1-AR-ECII antibody or antigen-binding fragment 1 thereof binds to amino acids 1-13 of β1-AR-ECII (SEQ ID NO: 18),

[0061] The anti-β1-AR-ECII antibody or antigen-binding fragment 2 thereof binds to amino acids 14-26 of β1-AR-ECII (SEQ ID NO: 17).

[0062] The detection of β1-AR-ECII in a sample refers to the presence or content of β1-AR-ECII in the sample, wherein the presence refers to the presence or absence, and the content may be the expression level or protein concentration.

[0063] In a tenth aspect, the present invention provides a β1-AR-ECII detection kit, which comprises the anti-β1-AR-ECII antibody or antigen-binding fragment thereof as described in the third aspect.

[0064] Preferably, the detection kit further comprises a solid phase carrier, a buffer, a blocking solution, a diluent, an enzyme-labeled secondary antibody, and a colorimetric reagent. The buffer includes but is not limited to a substrate buffer and a washing buffer.

[0065] Preferably, the detection kit further comprises a standard.

[0066] In the eleventh aspect of the present invention, there is provided a use of the anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to the third aspect in studying the generation mechanism of β1-adrenoceptor autoantibody (β1-AA).

[0067] The twelfth aspect of the present invention provides a use of the anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to the third aspect in the preparation of a product for diagnosing and / or treating cardiovascular diseases.

[0068] Preferably, the cardiovascular disease includes acute myocardial infarction, cardiomyopathy (such as hypertensive cardiomyopathy, dilated cardiomyopathy or rheumatic cardiomyopathy), arrhythmia or heart failure.

[0069] In a thirteenth aspect, the present invention provides a method for diagnosing cardiovascular disease, wherein the method comprises detecting the presence or content of β1-AR-ECII or its autoantibodies in a sample from a subject.

[0070] The subject can be a human or a non-human animal.

[0071] Preferably, the sample comprises body fluid or cells. Further preferably, the body fluid comprises blood, plasma or serum. Further preferably, the cells comprise cardiomyocytes.

[0072] In a specific embodiment of the present invention, the sample is cell culture supernatant or serum.

[0073] Preferably, the sample may be derived from a human or a non-human animal.

[0074] The "antigen-binding fragment" described in the present invention is a part of an antibody that retains the specific binding activity of the antibody, that is, any part of the antibody can specifically bind to the epitope on the target molecule of the antibody. It includes, for example, Fab, Fab', F(ab')2, Fv, scFv, Fd, Fab'-SH and variants of these fragments. For example, the heavy chain and / or light chain of an antibody, the heavy chain variable region and / or light chain variable region of an antibody, or a single or more CDRs (Complementarity Determining Region) from the heavy chain or light chain of an antibody. Among them, Fab is a monovalent fragment composed of VL, VH, CL and CH1 domains. F(ab')2 is a divalent fragment containing two Fab fragments connected by a disulfide bond in the hinge region. Fd is an Fd fragment composed of VH and CH1 domains. Fv is an Fv fragment composed of the VL and VH domains of a single arm of an antibody. Fab' is a Fab fragment with one or more cysteine ​​residues at the C-terminus of the CH1 domain. Fab'-SH refers to a Fab' in which the cysteine ​​residues in the constant domains bear at least one free thiol group. scFv, or single-chain antibody fragment, consists of the heavy and light chain variable regions of an antibody, which can be connected by a short peptide linker, typically about 15-20 amino acids in length. VH represents the heavy chain variable region, VL represents the light chain variable region, and CL represents the light chain.

[0075] The "CDR" of the present invention refers to the complementary determining region within the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy chain and light chain, which are called CDR1, CDR2 and CDR3. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat et al. (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to antibody variable regions, but also provides residue boundaries that define the three CDRs. These CDRs can be referred to as Kabat CDRs. Each complementary determining region can include amino acid residues from a "complementarity determining region" as defined by Kabat. Chothia et al. (Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (-1989)) found that Kabat In some embodiments, the CDRs are CDRs that have similar peptide backbone conformations, although they have large diversity at the amino acid sequence level. These CDRs are referred to as L1, L2, and L3, or H1, H2, and H3, respectively, where "L" and "H" represent light chain and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, which have overlapping boundaries with Kabat CDRs. Other CDR boundary definitions may not strictly follow one of the above-mentioned systems, but will still overlap with Kabat CDRs, and the methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments utilize CDRs defined by Kabat or Chothia.

[0076] The terms "comprising" or "including" as used in the present invention are open-ended. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity or function as the original sequence.

[0077] The "homology" mentioned in the present invention refers to the fact that when using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the sequences used have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 1%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity and retain the same or similar activity or function.

[0078] The "non-human animals" mentioned in the present invention include non-human mammals, including but not limited to wild animals, zoo animals, commercial animals, pets, experimental animals, etc. Preferably, the non-human mammals include but are not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.

[0079] The method described in the present invention can be used for the purpose of diagnosing or treating a disease or for the purpose of diagnosing or treating a non-disease.

[0080] The present invention has the following beneficial effects:

[0081] (1) This application newly screened two anti-β1-AR-ECII antibodies or their antigen-binding fragments. These two antibodies bind to the N-terminal peptide and C-terminal peptide of β1-AR-ECII, respectively, and achieve rapid, effective, economical, and batch detection of β1-AR-ECII.

[0082] (2) The newly screened anti-β1-AR-ECII antibody or its antigen-binding fragment is used to detect β1-AR-ECII. When detecting β1-AR-ECII in serum, the detection range is 1 ng / ml-1400 ng / ml, which covers most of the short peptides present in the serum; it is particularly suitable for detecting the β1-AR-ECII content in the serum of patients with myocardial infarction, the serum of actively immunized mice or rats, and the primary cultured cardiomyocytes of neonatal mice subjected to ischemia and hypoxia.

[0083] (3) This application can further explore the generation and intervention mechanism of β1-adrenoceptor autoantibody (β1-AA) by detecting β1-AR-ECII. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0085] Figure 1 Shown is the Coomassie brilliant blue purity identification graph of β1-AR-mAb1 and β1-AR-mAb2 antibodies;

[0086] Figure 2 Shown is the mass spectrum of CDR1 of the heavy chain variable region of β1-AR-mAb1;

[0087] Figure 3 Shown is the mass spectrum of CDR2 of the heavy chain variable region of β1-AR-mAb1;

[0088] Figure 4 Shown is the mass spectrum of CDR3 of the heavy chain variable region of β1-AR-mAb1;

[0089] Figure 5 Shown is the mass spectrum of CDR1 of the light chain variable region of β1-AR-mAb1;

[0090] Figure 6 Shown is the mass spectrum of CDR2 of the light chain variable region of β1-AR-mAb1;

[0091] Figure 7 Shown is the mass spectrum of CDR3 of the light chain variable region of β1-AR-mAb1;

[0092] Figure 8 Shown is the mass spectrum of CDR1 of the heavy chain variable region of β1-AR-mAb2;

[0093] Figure 9 Shown is the mass spectrum of CDR2 of the heavy chain variable region of β1-AR-mAb2;

[0094] Figure 10Shown is the mass spectrum of CDR3 of the heavy chain variable region of β1-AR-mAb2;

[0095] Figure 11 Shown is the mass spectrum of CDR1 of the light chain variable region of β1-AR-mAb2;

[0096] Figure 12 Shown is the mass spectrum of CDR2 of the light chain variable region of β1-AR-mAb2;

[0097] Figure 13 Shown is the mass spectrum of CDR3 of the light chain variable region of β1-AR-mAb2;

[0098] Figure 14 Shown are the EC50 and R of β1-AR-mAb1 (Panel a) and β1-AR-mAb2 (Panel b). 2 result;

[0099] Figure 15 Shown is the schematic diagram of the double-antibody sandwich ELISA method for detecting β1-AR-ECII;

[0100] Figure 16 The results are based on the concentration of β1-AR-ECII standard and OD 450 The standard curve was drawn based on the values;

[0101] Figure 17 Figure 1 shows elevated serum β1-AA levels in patients with ST-segment elevation acute myocardial infarction (STEMI) and a negative correlation with cardiac function. Panel a represents the OD values ​​of serum β1-AA in patients with non-coronary artery disease (NCHD, n = 50) and STEMI patients (n = 160) detected by ELISA. Data are presented as mean ± standard error; unpaired t-test, *** represents P < 0.001. Panel b represents the positive rate of serum β1-AA in patients with NCHD and STEMI, chi-square test, *** represents P < 0.001. Panels c and d represent the correlation between serum β1-AA OD values ​​in STEMI patients and left ventricular stroke volume (LVSV) and left ventricular ejection fraction (LVEF) as shown by echocardiography. In the Pearson correlation analysis, "r" represents the correlation coefficient, and P < 0.05 indicates statistical significance.

[0102] Figure 18Figure 1 shows the presence of β1-AR-ECII in serum from patients with primary STEMI and in culture supernatants of ischemic and hypoxic cardiomyocytes. Figure a shows the experimental flow chart: first, peptides were purified from serum from patients with primary STEMI and from culture supernatants of neonatal rat cardiomyocytes cultured in ischemic and hypoxic conditions using mouse monoclonal β1-AA. Time-of-flight mass spectrometry was then used to identify whether the purified peptide sequences contained the β1-AR-ECII sequence. Figure b shows that mass spectrometry revealed that the sera of three selected patients with primary STEMI all contained the β1-AR-ECII sequence (n = 3). Figure c shows that mass spectrometry revealed the presence of the β1-AR-ECII sequence in culture supernatants of primary neonatal rat cardiomyocytes (NRCMs) cultured in the absence of serum and oxygen (n = 3 or 1).

[0103] Figure 19 Figure 1 shows the abnormal exposure of β1-AR-ECII caused by acute myocardial infarction, which leads to the production of β1-AA. Figure a represents the experimental flow chart of the active immunization of mice with the supernatant of cultured cardiomyocytes under ischemia and hypoxia to produce β1-AA. 6 The culture supernatant of NRCMs cultured for 24 hours under ischemia and hypoxia was mixed with Freund's complete adjuvant in a 1:1 ratio to prepare an emulsified antigen, which was intraperitoneally injected once a week for 2 weeks. Panel b represents the removal of β1-AR-ECII from the ischemia and hypoxia culture supernatant using a β1-AA monoclonal antibody combined with immunoprecipitation. The β1-AA content in the mouse serum was detected by ELISA at the second week after active immunization. n = 5. Data are presented as mean ± standard error. One-way analysis of variance combined with Tukey's post-test. *** represents P < 0.001. NS (No Significance), indicating no statistical significance. Panel c represents the collection of venous serum from STEMI patients at the onset and at readmission two months later. The β1-AA level was detected by ELISA. n = 30. Data are presented as mean ± standard error. Paired t-test. *** P < 0.001;

[0104] Figure 20 This figure shows that activation of the β2-AR / Gi pathway reduces β1-AR-ECII levels in the supernatant of NRCMs cultured under ischemic hypoxia. NRCMs were pretreated with 1 μM ICI118551, a β2-AR / Gi pathway-biased agonist, for 30 minutes and then cultured in the serum-free and oxygen-free state for 24 hours. β1-AR-ECII levels in the cell supernatant were assessed by ELISA. Data are presented as mean ± standard error. One-way ANOVA with Tukey's post-hoc test; *** indicates P < 0.001, ** indicates P < 0.01.

[0105] Figure 21This figure shows β1-AR-ECII-induced cardiac injury in mice. C57BL / 6 mice were injected with 5 mg / kg / day of β1-AR-ECII or 50% of β1-AR-ECI / ECIII for 3 consecutive days. One week later, cardiac ultrasound was performed to assess systolic and diastolic function (a). Mmode represents M-mode echocardiography, PWD represents pulsed Doppler echocardiography, and TD represents tissue Doppler echocardiography. Picrosirius red staining was used to assess the extent of cardiac fibrosis (b). LVEF represents left ventricular ejection fraction, E / E' represents early diastolic velocity at the mitral cusp / early diastolic velocity at the mitral annulus, and collagen area represents collagen area. N = 5. Data are presented as mean ± standard error. Scale bar: 50 μm. One-way analysis of variance with Tukey's post-hoc test was performed. *** represents P < 0.001; ** represents P < 0.01; * represents P < 0.05. DETAILED DESCRIPTION

[0106] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0107] It should be noted that the methods used in the present invention, unless otherwise specified, are all conventional methods, and the reagents used in the present invention, unless otherwise specified, are all commercially available products.

[0108] Animals used in the examples: BALB / c mice: SPF (Specific Pathogen Free) BALB / c mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., female, 56-64 days old.

[0109] The main reagents used in the examples are shown in Table 1.

[0110] Table 1

[0111]

[0112]

[0113] Some experimental materials used in Examples 3 and 4:

[0114] High-affinity ELISA plate (FST015-200 pcs, Beyotime); substrate buffer (R21957, ThermoFisher), antibody diluent (Zhongshan Jinqiao, ZLI-9028); ELISA blocking buffer (C04-01002, Bioss); washing buffer 0.5% PBST (Prilai, B1019); goat anti-mouse IgG / HRP secondary antibody (ZB-2305, Zhongshan Jinqiao); ABTS (A1888-2G, Sigma); 0.5% hydrogen peroxide solution (AE4828A / ES, Chuangshi).

[0115] Accuracy calculation formula: average concentration of mass spectrometry / average concentration of this application detection method * 100%

[0116] STEMI inclusion criteria:

[0117] Inclusion criteria were: ① STEMI patients met the diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Acute ST-segment Elevation Myocardial Infarction (2015)"; ② STEMI patients undergoing emergency PCI had to undergo emergency PCI within 12 hours of onset at Beijing Friendship Hospital, Capital Medical University. Other included patients underwent coronary angiography during their hospitalization at Beijing Friendship Hospital and, if necessary, PCI (for patients with chest pain who presented with negative angiography, the coronary angiography showed no abnormalities); ③ Enrolled patients were able to complete echocardiography; ④ agreed to participate in the trial and signed informed consent. Exclusion criteria were: ① Previous myocardial infarction or revascularization; ② Congestive heart failure; ③ Atrial fibrillation; ④ Renal insufficiency; ⑤ Acute infectious disease within the past 3 months; ⑥ Rheumatologic immune system disease; ⑦ Malignant tumor; ⑧ Claustrophobia; ⑨ Patients who did not agree to participate in the trial.

[0118] Example 1: Mouse immunization and mouse polyclonal antiserum detection

[0119] 1. Preparation of Immunogen

[0120] The β1-AR-ECII-1 peptide (HWWRAESDEARRC (SEQ ID NO: 18)) and the β1-AR-ECII-2 peptide (YNDPKCCDFVTNR (SEQ ID NO: 17)) were provided by Gill Biochemical (Shanghai) Co., Ltd. and dissolved in DMSO to a final concentration of 5 mg / mL. 2 mg of the β1-AR-ECII-1 and β1-AR-ECII-2 peptides were coupled to the KLH carrier protein using the EDC method to obtain the β1-AR-ECII-1-KLH and β1-AR-ECII-2-KLH coupled proteins.

[0121] β1-AR-ECII-1-KLH and β1-AR-ECII-2-KLH coupled proteins were used to prepare immunogen 1 and immunogen 2, respectively. The coupled proteins were mixed with Freund's complete adjuvant in equal volumes and then emulsified by ultrasonication to prepare immunogen 1 and immunogen 2.

[0122] 2. Immunization of mice

[0123] A group of mice (3 mice), animal numbered 1-3#, was immunized with β1-AR-ECII-1-KLH, and another group of mice (3 mice), animal numbered 4-6#, was immunized with β1-AR-ECII-2-KLH. The two groups of mice were immunized with the prepared immunogens 1 and 2, respectively. The mouse immunization process was as follows: the mice were immunized with immunogen 1 and immunogen 2 for the first time; once a day, for a total of 4 times, and the immunization sites were the subcutaneous tissue of the back and the leg muscles.

[0124] 3. Serological testing

[0125] On the 14th day after immunization, the tail blood of the mice was collected and the serum antibody titer was detected using the indirect ELISA method. The steps are as follows:

[0126] (1) Coating: Add 100 μL of 1 μg / mL β1-AR-ECII-1 or β1-AR-ECII-2 to each well for coating and react at 4°C overnight; (2) Washing: Wash the plate three times with PBS solution; (3) Blocking: Block with 5% milk-PBS at room temperature for 1 hour and then wash the plate once with PBS solution; (4) Adding primary antibody: Add gradient diluted mouse tail blood 1 / 500, 1 / 1000, 1 / 5000, 1 / 10000, and 1 / 50000 to the ELISA plate for reaction at room temperature for 1 hour; (5) Washing: Then wash the plate three times with PBS solution and pat dry; (6) Adding secondary antibody: Add HRP-labeled goat anti-mouse IgG (Fc) secondary antibody diluted 1:2000 and react at room temperature for 1 hour; (7) Washing: Wash the plate five times with PBS solution and pat dry; (8) Color development: Add TMB for color development and react at room temperature for 20 minutes in the dark. Then add 50 μL of stop solution, mix well and read the OD on a microplate reader. 450 The tail blood was evaluated based on the test results. The test results are shown in Table 2 (coating antigen β1-AR-ECII-1, NC is a pre-immune negative serum control) and Table 3 (coating antigen β1-AR-ECII-2, NC is a pre-immune negative serum control).

[0127] Table 2: ELISA evaluation results of tail blood of mice in the β1-AR-ECII-1 immunization group on day 14

[0128] No. 1 / 500 1 / 1,000 1 / 5,000 1 / 10,000 1 / 50,000 NC 1# 1.267 0.523 0.136 0.056 0.011 0.006 2# 1.880 1.013 0.303 0.094 0.036 0.012 3# 1.443 0.660 0.165 0.070 0.016 0.007

[0129] Table 3: ELISA evaluation results of tail blood of mice in the β1-AR-ECII-2 immunization group on day 14

[0130] No. 1 / 500 1 / 1,000 1 / 5,000 1 / 10,000 1 / 50,000 NC 4# 0.790 0.520 0.290 0.126 0.019 0.006 5# 0.321 0.175 0.031 0.015 0.007 0.008 6# 0.165 0.051 0.009 0.007 0.007 0.007

[0131] It can be seen from the tail blood evaluation results in Table 2-3 that the antibody titer of the tail blood on D14 in the β1-AR-ECII-1 and β1-AR-ECII-2 immunization groups was relatively high, reaching 1 / 50,000. After further boosting the immunization, the tail blood was evaluated again.

[0132] Example 2: Cell fusion and hybridoma screening and confirmation

[0133] According to the ELISA evaluation results of tail blood in Example 1, mice 1# and 4# were selected, and spleen cells of the mice were taken for cell fusion with myeloma cells SP2 / 0. The specific steps of the fusion were as follows: SP2 / 0 cells in good growth state were blown down and centrifuged at 1500 rpm for 3 min; the supernatant was discarded and the cells were resuspended in 30 mL of preheated DMEM culture medium; centrifuged at 1500 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in an appropriate amount of preheated DMEM culture medium and the cells were counted; the corresponding mouse spleen cells and SP2 / 0 cells were taken at a ratio of 10:1. After mixing the cells in a 50mL centrifuge tube, centrifuge at 1500 rpm for 3 minutes and discard the supernatant. Then, slowly add 1mL of preheated PEG and incubate in a 37°C water bath for 25 minutes. Slowly add 5mL of preheated DMEM containing 10% fetal bovine serum along the tube wall, gradually increasing the speed to add 15mL of preheated DMEM containing 10% fetal bovine serum, until the total volume reaches 40mL. Centrifuge at 1200 rpm for 5 minutes, discard the supernatant, and add 25mL of recovery medium. After culturing for 48 hours, transfer the cells to a selective semisolid medium. After 10 days of culture, transfer the cells to a 96-well plate for further culture. After 7 days of culture in the 96-well plate, remove the supernatant and analyze to select positive cell lines. The cell culture supernatant (after 1:1 dilution) in a 96-well plate was evaluated by indirect ELISA (same steps as in Example 1) to screen monoclonal cell lines that could secrete monoclonal antibodies recognizing β1-AR-ECII-1 and β1-AR-ECII-2 antigens.

[0134] Positive hybridoma cells were transferred and expanded to 48-well plates. After culturing for 2 days, the cell supernatant (after 1:1 dilution) was taken and further screened and verified using the indirect ELISA method. The results are shown in Table 4 below (NC is a negative control, which is a diluent of 5% milk-PBS; PC is a positive control, which is a final dilution of 1:500 with the mouse bled).

[0135] Table 4: Rescreening and verification of positive clones

[0136]

[0137] From the results in Table 4, three positive clones with stable antigen recognition were obtained from the seven positive clones. After expanded culture, competition confirmation experiments were performed on the three monoclonal antibodies. ELISA detection was performed. The primary antibody of the normal group was used to dilute the cell supernatant in a gradient of 1:1, 1:10, 1:100, 1:1000, and 1:10000. 3 μg / mL of β1-AR-ECII-1 or β1-AR-ECII-2 was added to the cell supernatant of the competition group. The results are shown in Tables 5 and 6 below (NC is a negative control, which is a diluent of 5% milk-PBS; PC is a positive control, and the mice were finally bled and diluted 1:500).

[0138] Table 5: Positive clone competition experiment (β1-AR-ECII-1)

[0139]

[0140]

[0141] Table 6: Positive clone competition experiment (β1-AR-ECII-2)

[0142]

[0143] From the results in Tables 5-6, it can be seen that all three monoclonal antibodies have obvious competitive effects. The second strain targeting β1-AR-ECII-1 and the third strain targeting β1-AR-ECII-2 were selected for subsequent antibody extraction experiments.

[0144] Example 3: Antibody purification and validation

[0145] 1. Purification

[0146] Using a kit (Melon TMβ1-AR-mAb1 and β1-AR-mAb2 were separated and purified using a Gel Monoclonal IgG Purification Kit, Thermo Fisher, #45214. The purified IgGs were then analyzed by SDS-PAGE (4 μg loading). After staining with Coomassie Brilliant Blue, the grayscale values ​​of the light and heavy chains of β1-AR-mAb1 IgG accounted for 95.7% of the total grayscale value (i.e., purity) and 97.4% of the total grayscale value (i.e., purity) of the grayscale values ​​of the light and heavy chains of β1-AR-mAb1 and β1-AR-mAb2, respectively (Image J, v1.8.0.345), compared with those of Commercial antibody-1 (97.1%) and Commercial antibody-2 (97.1%). antibody-2 (96.4%), Rabbit IgGs control (98.3%), see Figure 1 The above results indicate that the β1-AR-mAb1 and β1-AR-mAb2 separated and purified in this example have reached the purity of commercial antibodies.

[0147] 2. Sequencing

[0148] The variable region sequences of the two antibodies were sent to Bio-Tech for detection by time-of-flight mass spectrometry. The variable region sequence of the β1-AR-mAb1 is shown in Table 7, and the variable region sequence of the β1-AR-mAb2 is shown in Table 8. The mass spectrum of CDR1-3 of the heavy chain variable region of β1-AR-mAb1 is shown in Figure 2-4 The mass spectrum of CDR1-3 of the light chain variable region of β1-AR-mAb1 is shown in FIG. Figure 5-7 The mass spectrum of CDR1-3 of the heavy chain variable region of β1-AR-mAb2 is shown in FIG. Figure 8-10 The mass spectrum of CDR1-3 of the light chain variable region of β1-AR-mAb2 is shown in FIG. Figure 11-13 .

[0149] Table 7

[0150]

[0151]

[0152] Table 8

[0153]

[0154]

[0155] 3. Detection of binding activity with β1-AR-ECII

[0156] (1) Incubation of β1-AR-mAb1 or β1-AR-mAb2: dilute β1-AR-mAb1 or β1-AR-mAb2 into substrate buffer at a final concentration of 1:1-1:10^6, add 100 μl / well to the ELISA plate, and incubate at 4°C overnight.

[0157] (2) Washing: Discard the liquid in the ELISA plate. Add 200 μl of 0.5% PBST and let it stand for 5 minutes. Repeat 3 times.

[0158] (3) Blocking: Add 5% ELISA blocking solution, incubate at 37°C for 1 h, and seal the plate with sealing film to prevent evaporation.

[0159] (4) Washing: Discard the liquid in the ELISA plate. Add 200 μl of 0.5% PBST and let it stand for 5 minutes. Repeat 3 times.

[0160] (5) Adding antigen: Add 100 μl of β1-AR-ECII standard at a concentration of 100 ng / ml, incubate at 37°C for 1.5 h, and seal the ELISA plate with sealing film to prevent evaporation.

[0161] (6) Washing: Discard the liquid in the ELISA plate. Add 200 μl of 0.5% PBST and let it stand for 5 minutes. Repeat 3 times.

[0162] (7) Incubation with secondary antibody: HRP-labeled goat anti-mouse secondary antibody was diluted 1:2000 and added to the ELISA plate, 100 μl per plate, incubated at 37°C for 1 h, and sealed with sealing film to prevent evaporation.

[0163] (8) Washing: Discard the liquid in the ELISA plate. Add 200 μl of 0.5% PBST and let it stand for 5 minutes. Repeat 3 times.

[0164] (9) Color development: Dissolve 1.1 mg / ml ABTS in 100 ml of 0.5% hydrogen peroxide solution, add 100 μl of the color development solution to each well of the ELISA plate, and incubate at 37°C for 0.5 h.

[0165] (10) Read the OD value and read the OD value of the microplate in the microplate reader. 450 Numerical values, calculation of EC50 and R 2 .

[0166] The results are as follows Figure 14As shown, the EC50 value of β1-AR-mAb1 was 3.482, and the EC50 value of β1-AR-mAb2 was 3.560, indicating that the purified antibodies had strong antigen binding ability.

[0167] Example 4: Detection of β1-AR-ECII (see Figure 15 )

[0168] 1. Antibodies and Antigens Used in This Example

[0169] Purified β1-AR-mAb1 (variable region sequence shown in Table 7, 50 μg / ml), β1-AR-mAb2 (variable region sequence shown in Table 8, 50 μg / ml), and the β1-AR-ECII full-length peptide (HWWRAESDEARRCYNDPKCCDFVTNR (SEQ ID NO: 27), synthesized by Gill Biochemical) obtained in Example 3

[0170] 2. Experimental steps:

[0171] (1) Incubation of β1-AR-mAb1: dilute β1-AR-mAb1 in substrate buffer to a final concentration of 1:100, add 100 μl / well to the ELISA plate, and incubate in a 4°C refrigerator overnight.

[0172] (2) Washing: Discard the liquid in the ELISA plate. Add 200 μl of 0.5% PBST and let it stand for 5 minutes. Repeat 3 times.

[0173] (3) Blocking: Add 5% ELISA blocking solution, incubate at 37°C for 1 h, and seal the plate with sealing film to prevent evaporation.

[0174] (4) Washing: Discard the liquid in the ELISA plate. Add 200 μl of 0.5% PBST and let it stand for 5 minutes. Repeat 3 times.

[0175] (5) Add samples: add different concentrations of β1-AR-ECII standard (0, 1 ng / ml, 10 ng / ml, 50 ng / ml, 100 ng / ml, 500 ng / ml, 1000 ng / ml, 2000 ng / ml), or 1:100 diluted patient / rat / mouse serum, or ischemic hypoxic cultured neonatal mouse cardiomyocyte supernatant, 100 μl each; dilute the standard and serum with PBS, incubate at 37°C for 1.5 h, and seal the ELISA plate with sealing film to prevent evaporation.

[0176] (6) Washing: Discard the liquid in the ELISA plate. Add 200 μl of 0.5% PBST and let it stand for 5 minutes. Repeat 3 times.

[0177] (7) Incubation of β1-AR-mAb2: dilute β1-AR-mAb2 into antibody buffer at a final concentration of 1:1000, add 100 μl / well to the ELISA plate, incubate at 37°C for 1.5 h, and seal the plate with sealing film to prevent evaporation.

[0178] (8) Washing: Discard the liquid in the ELISA plate. Add 200 μl of 0.5% PBST and let it stand for 5 minutes. Repeat 3 times.

[0179] (9) Incubation with secondary antibody: HRP-labeled goat anti-mouse secondary antibody was diluted 1:2000 and added to the ELISA plate, 100 μl per plate, incubated at 37°C for 1 h, and sealed with sealing film to prevent evaporation.

[0180] (10) Washing: Discard the liquid in the ELISA plate. Add 200 μl of 0.5% PBST and let it stand for 5 minutes. Repeat 3 times.

[0181] (11) Color development: Dissolve 1.1 mg / ml ABTS in 100 ml of 0.5% hydrogen peroxide solution, add 100 μl of the color development solution to each well of the ELISA plate, and incubate at 37°C for 0.5 h.

[0182] (12) Read the OD value and read the OD value of the microplate in the microplate reader. 450 The concentration of β1-AR-ECII in serum or culture supernatant was calculated using a standard curve prepared with standard samples.

[0183] 3. Experimental results

[0184] The determination results of β1-AR-ECII standards at different concentrations are shown in Table 9:

[0185] Table 9

[0186] Concentration (ng / ml) 2000 1000 500 100 50 10 1 0 <![CDATA[OD 450 (Average)]]> 1.291 0.727 0.4215 0.2555 0.195 0.1845 0.1385 0.0955

[0187] Draw a standard curve based on the determination results of the standard Figure 16 ).

[0188] The results of the determination of human serum β1-AR-ECII are shown in Table 10.

[0189] Table 10

[0190]

[0191] Compared with the gold standard mass spectrometry test results, the detection accuracy of this ELISA kit for myocardial infarction patients can reach 90.87%.

[0192] The results of the determination of β1-AR-ECII in rat serum are shown in Table 11, wherein the concentration of the immunogen applied in active immunizations 1, 2, and 3 was 500 ng / kg.

[0193] Table 11

[0194]

[0195]

[0196] Compared with the mass spectrometry detection results as the gold standard, the detection accuracy of this ELISA kit for actively immunized rats can reach 91.47%.

[0197] The results of the determination of β1-AR-ECII in mouse serum are shown in Table 12, wherein the concentration of the immunogen applied in active immunizations 1, 2, and 3 was 500 ng / kg.

[0198] Table 12

[0199]

[0200] Compared with the gold standard mass spectrometry detection results, the detection accuracy of this ELISA kit for actively immunized mice can reach 92.83%.

[0201] According to the above results, the detection method of the present application can effectively detect the content of β1-AR-ECII in the serum of patients with myocardial infarction, the serum of mice actively immunized with β1-AR-ECII, and the serum of rats actively immunized with β1-AR-ECII.

[0202] Example 5: Elevated serum β1-AA levels in patients with acute myocardial infarction are associated with poor cardiac function

[0203] In order to evaluate the OD value and positive rate of β1-AA in patients with ST-segment elevation acute myocardial infarction (STEMI), this example selected 160 STEMI patients (regardless of whether they were newly diagnosed) and 50 patients with non-coronary heart disease chest pain (NCHD) with negative coronary angiography according to the inclusion and exclusion criteria. The patient information is shown in Table 13 (values ​​are expressed as mean ± SD, median (upper quartile, lower quartile), n (%); p-values ​​were obtained by Student's t test, Mann-Whitney U test and chi-square test). The level of β1-AA in serum was detected by ELISA, and patients with a P / N value > 2.1 were defined as positive patients. The results showed that the OD value of serum β1-AA in STEMI patients was significantly higher than that in NCHD patients ( Figure 17a), and the positive rate of β1-AA in STEMI patients was 34.4%, which was also higher than 6.0% in NCHD patients ( Figure 17 b), the above results suggest that acute myocardial infarction can cause increased production of β1-AA. This example also explored the correlation between the level of serum β1-AA and cardiac function in STEMI patients. The results showed that the OD value of serum β1-AA in STEMI patients was negatively correlated with left ventricular-stroke volume (LVSV). Figure 17 c, r = -0.198, P = 0.014;), and negatively correlated with left ventricular ejection fraction (LVEF) ( Figure 17 d, r = -0.170, P = 0.032;), indicating that the increase of serum β1-AA caused by acute myocardial infarction was associated with the patients' poor cardiac function.

[0204] Table 13

[0205]

[0206]

[0207] Among them, pNT-proBNP: peak N-terminal pro-B-type natriuretic peptide; pCK-MB: peak creatine kinase-MB; pTNI: peak troponin I; HsCRP: high-sensitivity C-reactive protein; WBC: white blood cell; FBG: fasting blood glucose; GLU: blood glucose level on admission; eGFR: estimated glomerular filtration rate; HbA1c: glycated hemoglobin; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol.

[0208] Example 6: Abnormal exposure of β1-AR-ECII during acute myocardial infarction

[0209] To clarify the abnormal exposure of β1-AR-ECII in myocardial cells during myocardial infarction, this example used β1-AA affinity purification to purify the serum of three β1-AA-positive patients with initial STEMI and the supernatant of primary myocardial cells from one SD rat cultured for 24 hours under ischemic hypoxia. The presence of β1-AR-ECII was then identified by time-of-flight mass spectrometry (see experimental procedures for details). Figure 18 a). The results showed that amino acid fragments containing the β1-AR-ECII sequence were found in the serum of STEMI patients and the supernatant of myocardial cells cultured in ischemic hypoxia ( Figure 18bc), whereas no amino acid fragments of the β1-AR-ECII sequence were found in the supernatant of patients with negative chest pain and normal cultured cardiomyocytes, suggesting that ischemic and hypoxic damage caused by myocardial infarction can lead to abnormal exposure of cardiomyocyte β1-AR-ECII to the blood.

[0210] Example 7: Acute myocardial infarction causes abnormal exposure of β1-AR-ECII in the blood, which can lead to the production of β1-AA

[0211] To clarify whether abnormal exposure to β1-AR-ECII in the blood can lead to the production of β1-AA, in this example, the supernatant of primary neonatal rat cardiomyocytes cultured for 24 hours under ischemia and hypoxia was mixed with Freund's complete adjuvant in a 1:1 ratio and emulsified. The mixture was then injected intraperitoneally into 10-week-old male C57BL / 6N mice once a week using active immunization. The results showed that two weeks after active immunization with the supernatant of cells cultured under ischemia and hypoxia, the β1-AA level in the mouse serum was significantly increased. After β1-AR-ECII was removed by β1-AA, active immunization with the supernatant of cardiomyocytes cultured under ischemia and hypoxia did not cause an increase in β1-AA ( Figure 19 This example also tested the serum β1-AA levels of 30 patients with initial STEMI who were readmitted to the hospital two months later. The results showed that compared with the initial myocardial infarction, the OD values ​​of serum β1-AA in these STEMI patients were significantly increased two months later ( Figure 19 c) The above results suggest that abnormal exposure of β1-AR-ECII in the blood caused by acute myocardial infarction can lead to the production of β1-AA.

[0212] Example 8: Activation of the β2-AR / Gi pathway reduces abnormal exposure of β1-AR-ECII in ischemic and hypoxic cardiomyocytes

[0213] Primary neonatal rat cardiomyocytes cultured under ischemic hypoxia were pretreated with 1 μM β2-AR / Gi pathway-biased agonist ICI118551 hydrochloride (Merrck, #72795-01-8) for 30 minutes, and the level of β1-AR-ECII in the culture supernatant was detected using the detection method of this application (see the principle for details). Figure 15 The results showed that activation of the β2-AR / Gi pathway could partially reverse the increase in β1-AR-ECII levels in the cell supernatant caused by ischemia and hypoxia ( Figure 20 ).

[0214] Example 9: β1-AR-ECII directly causes damage to the mouse heart

[0215] To determine whether β1-AR-ECII directly affects cardiac injury and decreased cardiac function in mice, this study injected β1-AR-ECII peptide or β1-AR-ECI / ECIII peptide (5 mg / kg / day, ip) into the upper abdominal cavity of 10-week-old C57 mice for 3 consecutive days. One week later, cardiac function was assessed using echocardiography. Figure 21 a), Sirius red staining to detect the degree of cardiac fibrosis in mice ( Figure 21 b) At this stage, β1-AA has not yet formed (antibody formation requires 2 weeks). The results showed that β1-AR-ECII alone significantly impaired the contractile and diastolic function of the mouse heart, caused an increase in cardiac collagen area, and led to cardiac fibrosis. However, the use of other β1-AR extracellular cyclic peptide fragments (β1-AR-ECI + β1-AR-ECIII) did not affect the function and structure of the mouse heart. These results suggest that β1-AR-ECII directly affects the mouse heart function and causes damage to the heart structure.

Claims

1. Use of β1-AR-ECII or its autoantibodies as markers or targets in the preparation of products for the diagnosis and / or treatment of cardiovascular diseases.

2. The use according to claim 1, characterized in that The product includes a reagent for detecting β1-AR-ECII. Preferably, the reagent for detecting β1-AR-ECII includes an anti-β1-AR-ECII antibody or an antigen-binding fragment thereof.

3. An anti-β1-AR-ECII antibody or an antigen-binding fragment thereof, characterized in that: The anti-β1-AR-ECII antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein I) a heavy chain variable region comprising: a VHCDR1 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 1; a VHCDR2 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 2; a VHCDR3 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 3; and a light chain variable region comprising: a VLCDR1 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 4; a VLCDR2 comprising or as shown in SEQ ID NO: 5; a VLCDR3 comprising or as shown in SEQ ID NO: 6; or, II) the heavy chain variable region comprises: VHCDR1 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 7; VHCDR2 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 8; VHCDR3 comprising an amino acid sequence comprising or as shown in SEQ ID NO: 9; and the light chain variable region comprises: VLCDR1 comprising or as shown in SEQ ID NO: 10; VLCDR2 comprising or as shown in SEQ ID NO: 11; VLCDR3 comprising or as shown in SEQ ID NO:

12.

4. The anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to claim 3, characterized in that in: I) the amino acid sequence of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 13, or comprises an amino acid sequence having greater than 90% homology to the amino acid sequence of SEQ ID NO: 13; the amino acid sequence of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14, or comprises an amino acid sequence having greater than 90% homology to the amino acid sequence of SEQ ID NO: 14; or II) the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or comprises an amino acid sequence having more than 90% homology to the amino acid sequence shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or comprises an amino acid sequence having more than 90% homology to the amino acid sequence shown in SEQ ID NO:

16.

5. The anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to claim 3 or 4, characterized in that The anti-β1-AR-ECII antibody or antigen-binding fragment thereof includes Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or Fd.

6. A nucleic acid or a cell comprising a nucleic acid, characterized in that The nucleic acid encodes the anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to any one of claims 3 to 5.

7. A method for detecting β1-AR-ECII in a sample, characterized in that: The method comprises using the anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to any one of claims 3 to 5; Preferably, an anti-β1-AR-ECII antibody or an antigen-binding fragment thereof is contacted with a sample, and a complex formed between the anti-β1-AR-ECII antibody or an antigen-binding fragment thereof and β1-AR-ECII is detected.

8. The method according to claim 7, characterized in that The method includes: 1) linking the anti-β1-AR-ECII antibody or antigen-binding fragment 1 to a solid support; 2) Add the sample to be tested; 3) further adding anti-β1-AR-ECII antibody or antigen-binding fragment thereof 2; 4) Add labeled secondary antibody; 5) Color development and OD detection 450 value; Preferably, the sample includes body fluid or cells; further preferably, the body fluid includes blood, plasma or serum; further preferably, the cells include cardiomyocytes; Preferably, the final concentration of the anti-β1-AR-ECII antibody or antigen-binding fragment thereof 1 and / or the anti-β1-AR-ECII antibody or antigen-binding fragment thereof 2 in the reaction system is 1 μg / ml-10 μg / ml.

9. Use of the anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to any one of claims 3 to 5 in the preparation of a product for diagnosing and / or treating cardiovascular diseases.

10. A β1-AR-ECII detection kit, characterized in that: The detection kit comprises the anti-β1-AR-ECII antibody or antigen-binding fragment thereof according to any one of claims 3 to 5; Preferably, the detection kit further comprises one or more of a solid phase carrier, a buffer, a blocking solution, a diluent, an enzyme-labeled secondary antibody or a colorimetric reagent.

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