Anti-cTnI antibodies and uses thereof

Abstract

The invention discloses an anti-cTnI antibody and application thereof, and relates to the field of antibodies. The anti-cTnI antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for cTnI detection, and has excellent affinity or activity.

Description

[0001] Cross - reference to related applications

[0002] This disclosure claims priority to a Chinese patent application with application number 202311806118.9, titled "Anti - cTnI Antibody and Its Use", filed with the Chinese Patent Office on December 26, 2023, the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention belongs to the technical field of antibodies. Specifically, the present invention relates to a cTnI antibody and its use. More specifically, the present invention relates to an antibody, nucleic acid molecule, vector, cell or host, method for preparing an antibody, conjugate, reagent or kit and its use, method for detecting cTnI, method for screening cTnI antibody and mutant library of cTnI. Background art

[0004] Before the 1980s, the World Health Organization (WHO) had been using the activity of myocardial enzyme spectrum as one of the diagnostic criteria for acute myocardial infarction (AMI). In the late 1980s, researchers found that the sensitivity and specificity of troponin (Tn) were higher than those of biomarkers such as creatine phosphokinase (CK), creatine phosphokinase isoenzyme (CK - MB), lactate dehydrogenase, and aspartate aminotransferase. Cardiac troponin I (cTnI) is only present in the myocardium and is a marker of myocardial cells. Its abnormal changes can affect the systolic and diastolic functions of the heart and can be used for diagnosing myocardial necrosis and judging myocardial injury, etc. It has become one of the most sensitive and specific markers for myocardial cell injury and is a recognized main biochemical marker for rapid diagnosis of AMI and acute coronary syndromes (ACS), as well as assisting in ACS risk stratification and reflecting its prognosis.

[0005] In normal people, the content of cTnI in blood is generally lower than 0.3 μg / L. When the integrity of the myocardial cell membrane is damaged due to ischemia or hypoxia, etc., free cTnI can quickly penetrate the cell membrane and enter the bloodstream. Therefore, rapid, sensitive and accurate determination of cTnI in human blood and its change trend at the initial stage of the disease have important clinical significance for the diagnosis of acute myocardial infarction, risk stratification of acute coronary syndrome, and monitoring of myocardial injury caused by various factors. Clinically, methods for detecting cTnI levels include enzyme - linked immunosorbent assay (ELISA), chemiluminescence, colloidal gold, etc. Different methods have their own advantages and disadvantages, but all require specific monoclonal antibodies against cTnI. Therefore, there is a strong demand in this field for antibodies that can effectively bind to cTnI and detect it. Summary of the invention

[0006] The present invention aims to provide an antibody against cTnI, a reagent or a kit for detecting cTnI.

[0007] In one aspect of the present invention, an antibody against cTnI is proposed, which includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3. The amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are as follows:

[0008] HCDR 1: DYNLH;

[0009] HCDR 2: YIYPYNGX1TGYNQKFKS, where X1 is I or V;

[0010] HCDR 3: DAYX2YDYLTD, where X2 is D or W;

[0011] LCDR 1: RTX3KNX4GTNIX5, where X3 is S or R, X4 is V or M, and X5 is H, F or Y;

[0012] LCDR 2: YASERLP;

[0013] LCDR 3: QQSNNWPYT.

[0014] In the second aspect of the present invention, an antibody against cTnI is proposed, which includes a heavy chain variable region and / or a light chain variable region. The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 17 or its variant. Compared with the amino acid sequence shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 includes at least one of the following site mutations: I57V, D102W. The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 18 or its variant. Compared with the amino acid sequence shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 includes at least one of the following site mutations: S26R, V29M, H34Y / F.

[0015] In the third aspect of the present invention, an antibody against cTnI is proposed, which includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3. The HCDR1, HCDR2, HCDR3 are the HCDR1, HCDR2, HCDR3 of the heavy chain variable region defined by the antibody in the second aspect. The LCDR1, LCDR2, LCDR3 are the LCDR1, LCDR2, LCDR3 of the light chain variable region defined by the antibody in the second aspect.

[0016] In a fourth aspect of the present invention, there is provided a nucleic acid molecule encoding the antibody described in the first, second or third aspect.

[0017] In a fifth aspect of the present invention, there is provided a vector comprising the nucleic acid molecule described in the fourth aspect.

[0018] In a sixth aspect of the present invention, there is provided a cell or host comprising: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expressing the antibody described in the first, second or third aspect.

[0019] In a seventh aspect of the present invention, there is provided a method for preparing the antibody described in the first, second or third aspect, the method comprising culturing the cell or host described in the sixth aspect.

[0020] In an eighth aspect of the present invention, there is provided a conjugate comprising: the antibody described in the first, second or third aspect and a conjugate moiety conjugated thereto.

[0021] In a ninth aspect of the present invention, there is provided a reagent or kit comprising: the antibody described in the first, second or third aspect or the conjugate described in the seventh aspect.

[0022] In a tenth aspect of the present invention, there is provided the use of the antibody described in the first, second or third aspect, the conjugate described in the eighth aspect, the reagent or kit described in the ninth aspect in detecting cTnI, preparing a product for detecting cTnI or preparing a product for indicating a cTnI-related disease.

[0023] In an eleventh aspect of the present invention, there is provided a method for detecting cTnI, the method comprising: contacting a sample to be detected with the antibody described in the first, second or third aspect, the conjugate described in the eighth aspect or the reagent or kit described in the ninth aspect to form an immune complex.

[0024] In a twelfth aspect of the present invention, there is provided a method for screening a cTnI antibody, the method comprising: a) designing primers for amino acid substitution at the sites of X1, X2, X3, X4 and X5 defined in the antibody described in the first aspect, or at the mutated sites defined in the antibody described in the second aspect; b) constructing a mutant library with the primers in a) using the nucleic acid molecule described in the fourth aspect, the vector described in the fifth aspect or the cell described in the sixth aspect as a template; c) screening for cTnI antibodies from the mutant library.

[0025] In a thirteenth aspect of the present invention, there is provided a mutant library, which comprises the antibody described in the first aspect, the second aspect or the third aspect.

[0026] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. Detailed Embodiments

[0027] Embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention.

[0028] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0029] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0030] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0031] In this document, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects.

[0032] In this document, the terms "optionally", "optional", "optionally", "optional" or "option" generally mean that the subsequent event or condition may or may not occur, and this description includes the case where the event or condition occurs and the case where the event or condition does not occur.

[0033] As used herein, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, without being limited to a specific structure, as long as they exhibit the required antigen-binding ability.

[0034] As used herein, the terms "full-length antibody", "full-length monoclonal antibody", or "full-length mAb" are all composed of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0035] As used herein, the terms "polyclonal antibody" and "multispecific antibody" are synonymous and both refer to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, abbreviated as bisAbs), antibodies that can recognize three antigenic epitopes, or antibodies that can recognize four antigenic epitopes. This is a broad understanding, and the specific structure is not limited as long as they can recognize multiple antigenic epitopes. In the present invention, at least one of the multiple antigenic epitopes is derived from cTnI.

[0036] As used herein, the term "antigen-binding fragment" is a fragment that contains a part or all of an antibody, lacks at least some of the amino acids present in the full-length chain, but still has the functional activity of specifically binding to an antigen. For example, the fragment may contain a part or all of the antibody CDR. Such fragments bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments include, but are not limited to, Fab, Fab', F(ab)2, F(ab')2, Fv, scFv, scFv-Fc fusion proteins, scFv-Fv fusion proteins, single-domain antibodies, or minimal recognition units. Such fragments can be produced by recombinant nucleic acid techniques or can be produced by enzymatic cleavage or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0037] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment that contains only the Fab molecule, which is composed of VH and CH1 of the heavy chain and the intact light chain, and the light chain and the heavy chain are linked by a disulfide bond.

[0038] As used herein, the term "F(ab’)2 antibody" or "F(ab’)2 fragment" has two antigen-binding F(ab’) parts linked together by disulfide bonds.

[0039] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment composed only of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) linked by non-covalent bonds, which is the smallest functional fragment of the antibody molecule that retains the complete antigen-binding site.

[0040] In this text, the terms "single-chain antibody" and "scFv fragment" refer to an antibody or fragment formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody through a short peptide.

[0041] In this text, the terms "minimal recognition unit" and "MRU" both refer to an antibody or fragment consisting of only one CDR, and its molecular weight is very small, only about 1% of the complete antibody.

[0042] In this text, the terms "complementary determining region", "CDR" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, which refer to the regions containing one or more or even all of the main amino acid residues that play a role in the binding affinity of the antibody to the antigen or epitope it recognizes.

[0043] In this text, the heavy-chain complementary determining regions (CDRs of the heavy-chain variable region) are denoted by "HCDRs" or "HCDR", which include HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2), and HCDR3 (also known as CDR-H3); the light-chain complementary determining regions (CDRs of the light-chain variable region) are denoted by "LCDRs" or "LCDR", which include LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2), and LCDR3 (also known as CDR-L3). Commonly used CDR definition systems in the art include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" can be found in Chothia et al., J Mol Biol 196: 901-917 (1987). Exemplary defined CDRs are listed in Table 1 below. The definitions in different literatures are slightly different. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that the CDRs defined in the present invention include, but are not limited to, the CDRs defined by the methods in Table 1. CDRs defined by other methods disclosed in the art based on the heavy-chain variable region and light-chain variable region disclosed in this application also fall within the scope of protection of this disclosure.

[0044] Table 1: CDR Definitions 1

[0045] CDR Kabat <![CDATA[AbM 2 > IMGT Chothia HCDR1 <![CDATA[H31~H35 3 > <![CDATA[H26~H35 3 > <![CDATA[H26~H33..35 5 > <![CDATA[H26~H32..34 4 > HCDR2 H50 - H65 H50 - H58 H51 - H57 H52 - H56 HCDR3 H95 - H102 H95 - H102 H93 - H102 H95 - H102 LCDR1 L24 - L34 L24 - L34 L27 - L32 L24 - L34 LCDR2 L50 - L56 L50 - L56 L50 - L51 L50 - L56 LCDR3 L89 - L97 L89 - L97 L89 - L97 L89 - L97

[0046] 1The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below). The amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number".

[0047] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDR defined by the "AbM" antibody modeling software of Oxford Molecular.

[0048] 3 If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if both H35A and H35B are present, then CDR-H1 ends at position 35B.

[0049] 4 If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if both H35A and H35B are present, then CDR-H1 ends at position 34.

[0050] 5 If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35.

[0051] Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody. A person of ordinary skill in the art can clearly map the Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, "Kabat numbering" refers to numbering using the numbering system described in "Kabat et al., U.S. Dept. of Health and Human Services, 'Sequence of Proteins of Immunological Interest' (1983)". The HCDRs and LCDRs of the antibodies in this application are numbered using the above numbering system. For the specific numbering results, see Table 1. It should be noted that the polypeptide sequences of the present invention are not numbered according to the Kabat numbering system. However, a person of ordinary skill in the art can fully convert the sequence numbers in the sequence listing into Kabat numbers.

[0052] As used herein, the term "framework region" or "FR region" includes the heavy-chain framework region and the light-chain framework region, and refers to the regions in the variable region of the heavy chain of an antibody (which may be denoted as VH) and the variable region of the light chain (which may be denoted as VL) other than the CDRs; wherein, the heavy-chain framework region is denoted as "HFR" and can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light-chain framework region is denoted as "LFR" and can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0053] As used herein, the variable region of the heavy chain is obtained by connecting the CDRs numbered below and the FRs in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the variable region of the light chain is obtained by connecting the CDRs numbered below and the FRs in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0054] In this text, when the terms "identity", "homology", or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods. For example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.)). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997)); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, 8th edition, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0055] Without substantially affecting the antibody activity (retaining at least 90% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the antibody sequences. They are all considered to be included within the scope of protection of the present invention. Amino acids with similar properties are substituted in the variable region. The variant sequences of the present invention may have at least 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequences. The sequence identity described in the present invention can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in the N-terminal to C-terminal manner.

[0056] It should be noted that the positions or numbers of the mutation sites defined in the description and claims of the present invention also need to be adjusted according to the number and positions of the added and / or deleted amino acids.

[0057] In this article, the term "at least 80% homology" means at least 80% with each reference sequence, and can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology. The term "at least 90% homology" means at least 90% with each reference sequence, and can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology.

[0058] In this article, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule containing one or more nucleotide or amino acid mutations.

[0059] In this article, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating after insertion into a suitable host, which transfers the inserted nucleic acid molecule into cells or hosts and / or between cells or hosts. The vector can include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and expression vectors mainly used for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell or host. Generally, by culturing a suitable cell or host containing the vector, the vector can produce the desired expression product.

[0060] In this text, the term "cell" generally refers to a cell that has been genetically modified or recombined by genetic engineering techniques or cell fusion techniques to modify the genetic material of a host cell, and has obtained unique traits with stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell that can introduce a recombinant vector. The term "transformed" or "transfected" as used herein refers to the introduction of nucleic acid (such as a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0061] The present invention provides an anti-cTnI antibody, a nucleic acid molecule, a vector, a cell or a host, a method for preparing an antibody, a conjugate, a reagent or a kit and their uses, a method for detecting cTnI, a method for screening an anti-cTnI antibody, and a mutant library, which will be described in detail below.

[0062] Antibody

[0063] In the first aspect of the present invention, the present invention provides an anti-cTnI antibody. According to an embodiment of the present invention, the antibody includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are as follows:

[0064] HCDR 1: DYNLH;

[0065] HCDR 2: YIYPYNGX1TGYNQKFKS, where X1 is I or V;

[0066] HCDR 3: DAYX2YDYLTD, where X2 is D or W;

[0067] LCDR 1: RTX3KNX4GTNIX5, where X3 is S or R, X4 is V or M, and X5 is H, F or Y;

[0068] LCDR 2: YASERLP;

[0069] LCDR 3: QQSNNWPYT.

[0070] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0071] As can be seen from the aforementioned CDR definition principles, when different systems are used for definition, even for the same heavy chain variable region or the same light chain variable region, the obtained CDRs are different. "HCDR 1: DYNLH" in the present application means that the amino acid sequence of HCDR 1 includes or is DYNLH, and it can also be adjusted according to different systems to obtain an HCDR 1 with a sequence shorter or longer than DYNLH.

[0072] In some alternative embodiments of the present invention, X1 is I;

[0073] In some alternative embodiments of the present invention, X1 is V;

[0074] In some alternative embodiments of the present invention, X2 is D;

[0075] In some alternative embodiments of the present invention, X2 is W;

[0076] In some alternative embodiments of the present invention, X3 is S;

[0077] In some alternative embodiments of the present invention, X3 is R;

[0078] In some alternative embodiments of the present invention, X4 is V;

[0079] In some alternative embodiments of the present invention, X4 is M;

[0080] In some alternative embodiments of the present invention, X5 is H;

[0081] In some alternative embodiments of the present invention, X5 is F;

[0082] In some alternative embodiments of the present invention, X5 is Y;

[0083] In some alternative embodiments of the present invention, it is not simultaneously true that X1 is I, X2 is D, X3 is S, X4 is V, and X5 is H;

[0084] In some alternative embodiments of the present invention, X1, X2, X3, X4, and X5 are selected from any one of the following combinations:

[0085] Combination <![CDATA[X1]]> <![CDATA[X2]]> <![CDATA[X3]]> <![CDATA[X4]]> <![CDATA[X5]]> 1 I D R V H 2 I D S M H 3 I D S V F 4 I D S V Y 5 V D S V H 6 I W S V H 7 I D R V F 8 V D R V Y 9 I D R M Y 10 V W R M Y 11 I D R M F 12 V D S M Y 13 I D S M Y 14 V W S M Y 15 I W R V Y 16 I W R V F 17 V D R M Y 18 I W R M Y 19 I W R M F 20 V D R M F 21 I D R V Y 22 I D S M F 23 I W S M Y 24 V W R V Y 。

[0086] The antibody described in the first aspect of the present invention has excellent activity, affinity, stability, or specificity.

[0087] The antibody described in the first aspect of the present invention has improved activity, affinity, stability, or specificity.

[0088] In a second aspect of the present invention, the present invention provides an anti-cTnI antibody. According to an embodiment of the present invention, the antibody or functional fragment thereof comprises: a heavy chain variable region and / or a light chain variable region; the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or a variant thereof, and compared with the amino acid sequence shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 comprises a mutation at at least one of the following sites: I57V, D102W; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18 or a variant thereof, and compared with the amino acid sequence shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 comprises a mutation at at least one of the following sites: S26R, V29M, H34Y / F.

[0089] It should be noted that the numbering of the above-mentioned heavy chain variable region sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO: 17 from the N-terminus to the C-terminus. For example, the 57th position refers to the 57th position of the amino acid sequence shown in SEQ ID NO: 17 starting from the N-terminus; the "I57V" means that the isoleucine at the 57th position of the amino acid sequence shown in SEQ ID NO: 17 is replaced by valine.

[0090] The numbering of the above-mentioned light chain variable region sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO: 18 from the N-terminus to the C-terminus. For example, the 26th position refers to the 26th position of the amino acid sequence shown in SEQ ID NO: 18 starting from the N-terminus; the "S26R" means that the serine at the 26th position of the amino acid sequence shown in SEQ ID NO: 18 is replaced by arginine; the "H34Y / F" means that the histidine at the 34th position of the amino acid sequence shown in SEQ ID NO: 18 can be replaced by tyrosine or phenylalanine.

[0091] According to an embodiment of the present invention, the above-mentioned antibody may further comprise at least one of the following additional technical features:

[0092] In some alternative embodiments of the present invention, the mutation at the 57th position is I57V;

[0093] In some alternative embodiments of the present invention, the mutation at the 102nd position is D102W;

[0094] In some alternative embodiments of the present invention, the mutation at the 26th position is S26R;

[0095] In some alternative embodiments of the present invention, the mutation at the 29th position is V29M;

[0096] In some alternative embodiments of the present invention, the mutation at the 34th position is H34Y;

[0097] In some alternative embodiments of the present invention, the mutation at the 34th position is H34F;

[0098] In some alternative embodiments of the present invention, the heavy chain variable region and the light chain variable region are selected from any one of the following combinations:

[0099]

[0100]

[0101] It should be noted that the "," in the above table represents "and", that is, combined mutations. For example, "S26R, V29M, H34Y" represents a combined mutation of three mutations: S26R, V29M, and H34Y.

[0102] In the third aspect of the present invention, the present invention provides an anti-cTnI antibody. According to the embodiments of the present invention, the antibody includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3. The HCDR1, HCDR2, HCDR3 are the HCDR1, HCDR2, HCDR3 of the heavy chain variable region defined by the antibody in the second aspect; the LCDR1, LCDR2, LCDR3 are the LCDR1, LCDR2, LCDR3 of the light chain variable region defined by the antibody in the second aspect.

[0103] It should be noted that the HCDR1, HCDR2, and HCDR3 in the antibody described in the third aspect are the HCDR1, HCDR2, HCDR3 of the same heavy chain variable region defined by the antibody in the second aspect, and the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, LCDR3 of the same light chain variable region defined by the antibody in the second aspect.

[0104] For example, when the heavy chain variable region defined by the antibody in the second aspect has no mutation compared to SEQ ID NO:17, the HCDR1, HCDR2, HCDR3 of this heavy chain variable region are respectively HCDR 1: DYNLH; HCDR 2: YIYPYNGITGYNQKFKS; HCDR 3: DAYDYDYLTD; then the HCDR1, HCDR2, HCDR3 contained in the antibody described in the third aspect are also respectively HCDR 1: DYNLH; HCDR 2: YIYPYNGITGYNQKFKS; HCDR 3: VWYSGNGFDFGYFDYDAYDYDYLTD.

[0105] For example, when there is only a mutation at the S26R site in the light chain variable region defined in the antibody described in the second aspect compared to SEQ ID NO: 18, the LCDR1, LCDR2, and LCDR3 of this light chain variable region are respectively LCDR 1: RTRKNVGTNIH; LCDR2: YASERLP; LCDR 3: QQSNNWPYT; then the LCDR1, LCDR2, and LCDR3 contained in the antibody described in the third aspect are also respectively LCDR 1: RTSKNVGTNIH; LCDR 2: YASERLP; LCDR 3: QQSNNWPYT.

[0106] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0107] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM, or Contact.

[0108] According to an embodiment of the present invention, the antibody described in the first aspect or the third aspect further includes at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0109] In an alternative embodiment of the present invention, at least a part of at least one of the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting chicken antibody, or a mutant thereof.

[0110] According to an embodiment of the present invention, the HFR1 comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80% homology thereto; the HFR2 comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 80% homology thereto; the HFR3 comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 80% homology thereto; the HFR4 comprises the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 80% homology thereto; the LFR1 comprises the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 80% homology thereto; the LFR2 comprises the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 80% homology thereto; the LFR3 comprises the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 80% homology thereto; the LFR4 comprises the amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 80% homology thereto.

[0111] According to an embodiment of the present invention, the antibody described in the first aspect, the second aspect or the third aspect above may further comprise at least one of the following technical features:

[0112] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -9 M.

[0113] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -10 M.

[0114] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -11 M.

[0115] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -12 M.

[0116] According to an embodiment of the present invention, the affinity KD of the antibody < 10 -13 M.

[0117] According to an embodiment of the present invention, the antibody further comprises a constant region; wherein, the constant region comprises at least one of a heavy chain constant region and a light chain constant region.

[0118] According to an embodiment of the present invention, at least a part of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a pig antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting chicken antibody or a mutant thereof.

[0119] According to an embodiment of the present invention, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

[0120] In an alternative embodiment of the present invention, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0121] In an alternative embodiment of the present invention, the IgG is selected from IgG1, IgG2, IgG3, IgG4.

[0122] According to an embodiment of the present invention, the light chain constant region includes a κ-type or λ-type light chain constant region.

[0123] In an alternative embodiment of the present invention, the heavy chain constant region includes or is the heavy chain constant region shown in SEQ ID NO:15 or an amino acid sequence having at least 80% identity thereto; or the light chain constant region includes or is the light chain constant region shown in SEQ ID NO:16 or an amino acid sequence having at least 80% identity thereto.

[0124] According to an embodiment of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0125] In this context, the division of variable region and constant region sequences refers to the IMGT division method, see Lefranc, the international ImMunoGeneTics database. Nucl.Acids Res., 29(1):207-209(2001). DOI:10.1093 / nar / 29.1.207. PMID:11125093. and Martinez-Jean C. and Bosc N. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: housemouse (Mus musculus) IGHC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGLC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020.. There will be some amino acid differences between the variable regions divided by different methods and the C-terminus of the variable regions or the N-terminus of the constant regions divided by IMGT. The variable regions or constant regions divided by other methods well-known in the art are also within the protection scope of the present invention.

[0126] Those skilled in the art can understand that the features and advantages described for the antibodies in the first aspect also apply to the antibodies in the second aspect or the third aspect, and will not be elaborated herein.

[0127] Nucleic acid molecule, vector, cell or host, method for preparing antibody

[0128] In the process of preparing or obtaining the antibodies described in the first aspect, the second aspect or the third aspect, nucleic acid molecules expressing these antibodies can be used, connected to different vectors, and then expressed in different cells to obtain the corresponding antibodies.

[0129] In the fourth aspect of the present invention, the present invention provides a nucleic acid molecule. According to the embodiments of the present invention, the nucleic acid molecule encodes the antibodies described in the first aspect, the second aspect or the third aspect. The nucleic acid molecule according to the embodiments of the present invention can encode and obtain the above-mentioned antibodies.

[0130] According to the embodiments of the present invention, the nucleic acid molecule includes DNA or RNA.

[0131] It should be noted that for the nucleic acid molecules mentioned in this article, those skilled in the art should understand that it actually includes either any one of the complementary double strands or both. For convenience, in this article, although only one strand is given in most cases, the other complementary strand is actually also disclosed. In addition, the molecular sequences in the present invention include DNA or RNA forms. Disclosing one of them means that the other is also disclosed.

[0132] In the fifth aspect of the present invention, the present invention provides a vector. According to an embodiment of the present invention, the vector includes the nucleic acid molecule described in the fourth aspect. When connecting the above-mentioned nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can come directly from the vector itself or be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control element are operably linked. As used herein, "operably linked" means that an exogenous gene is linked to a vector such that the control elements within the vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, phages, etc. After the vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, under the mediation of the regulatory system, the expression of the aforementioned antibody can be effectively achieved, and thus a large amount of the antibody can be obtained in vitro.

[0133] In some specific embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a phage.

[0134] In an alternative embodiment of the present invention, the expression vector is a plasmid expression vector.

[0135] In the sixth aspect of the present invention, the present invention provides a cell or a host. According to an embodiment of the present invention, the cell or host includes: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expresses the antibody described in the first aspect, the second aspect or the third aspect. Using this cell under suitable conditions, the aforementioned antibody can be effectively expressed intracellularly.

[0136] According to an embodiment of the present invention, the cell is obtained by introducing the vector described in the fifth aspect into the cell.

[0137] It should be noted that the cells of the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or phages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, etc., insect cells such as Spodoptera frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.

[0138] In an alternative embodiment of the present invention, the cell is a mammalian cell, including BHK cells, CHO cells, NSO cells or COS cells, and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0139] It should be noted that the "suitable conditions" in the present invention refer to the conditions suitable for the expression of the antibody of the present invention. It is easily understood by those skilled in the art that the conditions suitable for the expression of the antibody include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell densities, suitable cell culture environments, and suitable cell culture times. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibody according to the specific environment of the laboratory.

[0140] In the seventh aspect of the present invention, the present invention provides a method for preparing the antibody described in the first aspect, the second aspect or the third aspect. According to an embodiment of the present invention, the method includes culturing the cell or host described in the sixth aspect. The methods according to some specific embodiments of the present invention can effectively obtain a large amount of the antibody.

[0141] Based on the amino acid sequence of the antibody of the present disclosure, it is easily conceivable by those skilled in the art to prepare the antibody by using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, isolating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing the antibody described in any one of the above. This is easily achievable for those skilled in the art. Based on this, no matter what technique is used to prepare the antibody of the present disclosure, it falls within the protection scope of the present disclosure.

[0142] Those skilled in the art can understand that the characteristics and advantages described above for the antibody described in the first aspect, the second aspect or the third aspect also apply to the nucleic acid molecule, the vector, the cell or the host, and the method for preparing the antibody, and will not be repeated here.

[0143] Conjugates, reagents or kits and their uses

[0144] In the eighth aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate includes: the antibody described in the first aspect, the second aspect or the third aspect, and a conjugate moiety conjugated thereto. The conjugate according to the embodiment of the present invention can specifically bind to cTnI and can be used for qualitative or quantitative detection of cTnI, or for indicating cTnI-related diseases.

[0145] According to an embodiment of the present invention, the above conjugate may further include at least one of the following additional technical features:

[0146] According to an embodiment of the present invention, the conjugate moiety includes at least one of a purification tag, an affinity substance, a marker, and a solid-phase carrier.

[0147] According to an embodiment of the present invention, the purification tag includes at least one of a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag.

[0148] In this context, the affinity substance may be, for example, biotin, a biotin derivative, or streptavidin, or one of nucleic acids of a sense strand and an antisense strand that are complementary to each other.

[0149] In some alternative embodiments of the present invention, the affinity substance includes at least one selected from biotin, a biotin derivative, or streptavidin.

[0150] In this context, a "marker" refers to a class of substances having characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and through which qualitative or quantitative detection of a corresponding target can be achieved.

[0151] According to an embodiment of the present invention, the marker includes at least one selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based markers.

[0152] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the protection scope of the present invention.

[0153] According to an embodiment of the present invention, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0154] According to an embodiment of the present invention, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0155] According to an embodiment of the present invention, the radioisotopes include, but are not limited to 212 Bi 131 I 111 In 90 Y 186 Re 211 At 125 I 188 Re 153 Sm 213 Bi 32 P 94 mTc 99 mTc 203 Pb 67 Ga 68 Ga 43 Sc 47 Sc 110 mIn 97 Ru 62 Cu 64 Cu 67 Cu 68 Cu 86 Y 88 Y 121 Sn 161 Tb 166 Ho 105 Rh177 Lu, 172 Lu and 18 F.

[0156] According to an embodiment of the present invention, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rosalic acid and its derivatives, and peroxyoxalate and its derivatives.

[0157] According to an embodiment of the present invention, the nanoparticle-based label includes, but is not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0158] According to an embodiment of the present invention, the colloid includes, but is not limited to, colloidal metal, dispersed dyes, dye-labeled microspheres, and latex.

[0159] According to an embodiment of the present invention, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0160] In this article, the solid-phase carrier can be a substance that can be suspended or dispersed in a liquid phase (for example, solid-phase carriers such as particles, magnetic beads, etc.), or a solid phase that can accommodate or carry a liquid phase (for example, supports such as plates, membranes, test tubes, etc., and containers such as microtiter plates, microfluidic channels, glass capillaries, nano-columns, monolithic columns, etc.).

[0161] According to an embodiment of the present invention, the solid-phase carrier includes at least one selected from microspheres, plates, and membranes.

[0162] In a ninth aspect of the present invention, the present invention provides a reagent or a kit. According to an embodiment of the present invention, the reagent or the kit includes: the antibody according to the first aspect, the second aspect, or the third aspect, or the conjugate according to the eighth aspect. As described above, the antibodies in some specific embodiments or examples of the present invention can bind to cTnI. Therefore, the reagent or the kit containing the antibody can effectively perform qualitative or quantitative detection of cTnI. By applying the reagent or the kit provided by the present invention, for example, it can be used for detections such as immunoblotting and immunoprecipitation that involve the specific binding performance of cTnI and its antibody. As described above, the mutant antibodies of the present invention have improved cTnI binding activity, affinity, stability, or specificity. Therefore, the reagent or the kit containing the antibody has improved detection sensitivity or specificity.

[0163] The above-mentioned kit may include any one or more of the following: treatment solution, anti-cTnI antibody, cTnI quality control product, anti-IgG antibody, instruction manual or literature, etc. The anti-cTnI antibody can be used in different types of diagnostic tests. For example, it can detect the presence of various diseases, drugs, or other proteins in vitro or in vivo. For example, it can be used to test related diseases by detecting the serum or blood of a subject.

[0164] In the tenth aspect of the present invention, the present invention provides the use of the antibody according to the first, second, or third aspect, the conjugate according to the eighth aspect, the reagent or kit according to the ninth aspect in detecting cTnI, indicating cTnI-related diseases, preparing a product for detecting cTnI, or preparing a product for indicating cTnI-related diseases.

[0165] According to an embodiment of the present invention, the "product" includes but is not limited to reagents, test strips, reagent plates, or kits.

[0166] According to an embodiment of the present invention, the "indicating" includes but is not limited to prediction, diagnosis, prognosis, or medication guidance.

[0167] According to an embodiment of the present invention, the "cTnI-related disease" refers to a disease with cTnI as a biomarker.

[0168] In one or more embodiments of the present disclosure, cTnI-related diseases include but are not limited to acute myocardial infarction, unstable angina, acute coronary syndrome, or perioperative myocardial injury.

[0169] In the eleventh aspect of the present invention, the present invention provides a method for detecting cTnI. According to an embodiment of the present invention, the method includes: contacting a sample to be detected with the antibody according to the first, second, or third aspect, the conjugate according to the eighth aspect, or the reagent or kit according to the ninth aspect to form an immune complex.

[0170] According to an embodiment of the present invention, based on the signal of the immune complex, it is determined whether the sample to be detected contains cTnI or the content of cTnI.

[0171] According to an embodiment of the present invention, the immune complex further includes a second antibody that binds to the antibody.

[0172] According to an embodiment of the present invention, the signal includes a fluorescence signal.

[0173] Method, mutant library

[0174] In the twelfth aspect of the present invention, a method for screening cTnI antibodies is proposed. According to an embodiment of the present invention, the method includes: a) designing primers for amino acid substitution at 1, 2, 3, 4, or 5 sites among X1, X2, X3, X4, and X5 defined in the antibody of the first aspect, or at the mutation sites defined in the antibody of the second aspect; b) constructing a mutant library with the primers described in a) using the nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, or the cell or host of the sixth aspect as a template;

[0175] c) screening cTnI antibodies from the mutant library.

[0176] According to an embodiment of the present invention, the mutant library is a single-site saturation mutant library.

[0177] According to an embodiment of the present invention, the cTnI antibody includes or is the antibody described in the first aspect, the second aspect, or the third aspect.

[0178] In the thirteenth aspect of the present invention, a mutant library is proposed, and the mutant library includes the antibody described in the first aspect, the second aspect, or the third aspect.

[0179] According to an embodiment of the present invention, the mutant library is obtained by the method described in the twelfth aspect.

[0180] In the fourteenth aspect of the present invention, a method for indicating cTnI-related diseases is proposed. According to an embodiment of the present invention, the method includes: contacting a sample to be tested from a subject with the antibody described in the first aspect, the second aspect, or the third aspect, the conjugate of the eighth aspect, or the reagent or kit of the ninth aspect to form an immune complex.

[0181] According to an embodiment of the present invention, based on the signal of the immune complex, the status of cTnI-related diseases in the subject is indicated.

[0182] According to an embodiment of the present invention, the method described in the above fourteenth aspect may further include at least one of the following technical features:

[0183] According to an embodiment of the present invention, the immune complex further includes a second antibody that binds to the antibody.

[0184] According to an embodiment of the present invention, the signal includes a fluorescence signal.

[0185] As used herein, the term "subject" refers to a vertebrate, preferably a mammal, and most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, game animals, and pets. Also included are tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro.

[0186] The amino acid sequences involved in this article are shown in Table 2:

[0187] Table 2: Amino Acid Sequences

[0188]

[0189]

[0190]

[0191] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specified as to the manufacturer, they are all conventional products that can be obtained commercially.

[0192] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise noted, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0193] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (ed. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (ed. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (ed. Mullis et al., 1994); and "Current Protocols in Immunology" (ed. J.E. Coligan et al., 2011), each of which is hereby expressly incorporated by reference.

[0194] In this example, the restriction endonuclease, T4 DNA ligase, and DNA polymerase were purchased from New England Biolabs, the Taq DNA polymerase was purchased from TaKaRa, the gel extraction kit and plasmid extraction kit were commercially available, and primer synthesis and gene sequencing were completed by an outsourcing company. The sequence of the cTnI monoclonal antibody (hereinafter referred to as the WT antibody) was derived from the sequencing of mouse hybridoma cells.

[0195] Example 1: Construction and Screening of a Mutation Library

[0196] 1. Construction of the wild-type (WT) cTnI antibody (abbreviated as WT antibody) template plasmid

[0197] (1) Synthesis of WT antibody gene:

[0198] The nucleotide sequences of VH and VL of the WT antibody were optimized for E. coli codons, and then the antibody gene sequences were synthesized. The amino acid sequences of VH and VL of the WT antibody are shown in SEQ ID NO:17 and SEQ ID NO:18 respectively, and the amino acid sequences of the heavy chain and light chain are shown in SEQ ID NO:19 and SEQ ID NO:20 respectively.

[0199] (2) Amplification of WT antibody gene fragments:

[0200] The nucleotide sequences of the synthesized antibody VH and VL in step (1) were amplified by PCR using DNA polymerase, and then the antibody bands were separated by agarose gel electrophoresis. Subsequently, the antibody gene fragments were purified using a gel extraction kit.

[0201] (3) Enzymatic digestion and ligation of WT antibody gene fragments:

[0202] The antibody gene fragments obtained in step (2) and the V01 vector plasmid (including the nucleotide sequence of the constant region) were simultaneously double-digested with restriction endonucleases, and then the antibody gene fragments and the V01 vector with sticky ends were purified using a gel extraction kit. Subsequently, the antibody gene fragments and the V01 vector were ligated with T4 DNA ligase at 22 °C for 4 hours. Then, the ligation reaction product was recovered and purified, and the DNA concentration was measured. Finally, 100 ng of the plasmid was transformed into 100 μl of TG1 E. coli competent cells to obtain a bacterial solution. Then, the entire bacterial solution was spread on a plate containing ampicillin resistance and cultured overnight at 37 °C.

[0203] (4) Extraction and sequencing verification of the WT template plasmid

[0204] Ten monoclonal colonies from the overnight culture in step (3) were selected, and colony PCR and gel electrophoresis were performed using Taq DNA polymerase. The bacteria with the correct insertion of the antibody gene sequence were selected for culture and amplification. The WT template plasmid was obtained using a plasmid extraction kit and sent to a sequencing company for gene sequencing verification.

[0205] 2. Construction of the single-point mutation library

[0206] In this part of the experiment, single-point saturation mutagenesis was performed on the entire CDR regions of VH and VL of the WT antibody obtained in step 1 to construct a single-point mutation library.

[0207] (1) Primer design and synthesis

[0208] Using degenerate base codons, upstream and downstream primer pairs for single-point saturation mutagenesis of amino acid sites in the entire CDR regions of VH and VL were designed and handed over to an outsourcing company for primer synthesis.

[0209] (2) Single-site saturation mutation plasmid PCR amplification

[0210] The primers obtained in step (1) were used to perform PCR amplification on the single-point saturation mutation plasmid using the PCR method. The reaction system was configured according to Table 3, and then the PCR reaction conditions in Table 4 were used to amplify and prepare the single-point saturation mutation library plasmid. Finally, the WT template plasmid obtained in step 1 was digested with a restriction endonuclease at 37° C. for 1 hour to obtain the plasmid of the mutation library of the amino acid sites in the entire CDR region.

[0211] Table 3: PCR amplification reaction system

[0212] WT template plasmid 50 ng DNA polymerase 1 μl DNA polymerase buffer 10 μl dNTP (2.5 mM) 4 μl Forward primer (10 μM) 1 μl Reverse primer (10 μM) 1 μl ddH2O Volume made up to 50 μl

[0213] Table 4: PCR reaction conditions

[0214] Step1 Step2 Step3 Step4 Step5 Step6 Temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ Time 5 min 30s 30s 2 min 5 min ∞

[0215] Note: Step 2 to Step 4 are repeated 22 times.

[0216] (3) Single-site saturation mutation plasmid transformation:

[0217] Take the plasmid of the mutation library of amino acid sites in the entire CDR region obtained in step (2), take 10 μl of the reaction product and transform it into 100 μl of TG1 Escherichia coli competent cells to obtain bacterial solution, then spread the entire bacterial solution on a plate containing ampicillin resistance and culture at 37°C overnight.

[0218] 3. Screening of single point mutation library

[0219] (1) Mutation library antibody expression

[0220] 500 μl of culture medium was added to a 96-well culture plate in advance. For each single-point mutation library, a monoclonal colony transformed with the single-point saturation mutation plasmid cultured overnight in step 2-(3) was selected, and WT, negative control (i.e., colonies without VH\VL genes inserted), and blank control (i.e., only culture medium without colonies) colonies were set. After culturing at 37°C for 5-6 hours, the bacterial solution was transferred to a new 96-well culture plate, and then cultured at 37°C for 1-2 hours. Finally, an induction medium was added, and the antibodies were expressed at 37°C overnight to obtain the antibody expression supernatant of the mutation library of the entire CDR region amino acid site.

[0221] (2) Mutation library screening and sequencing

[0222] The commercially available cTnI antigen was added to an ELISA plate at a concentration of 0.004 μg / ml and a volume of 100 μl / well, and incubated overnight at 4°C for coating. The next day, it was blocked with 1-2% skim milk powder. After diluting the antibody expression supernatant of the mutant library of the amino acid sites in the full CDR region obtained in step (1), it was added to the ELISA plate wells at a volume of 100 μl / well, and WT, negative control (i.e., colonies without inserted VH\VL genes) and blank control (i.e., only medium without colonies) were set. Incubate at room temperature for 2 hours, and then perform subsequent plate washing, color development, and reading using the detection method of conventional ELISA; finally, organize and analyze the data results. The results showed that the activity of the mutants was better than that of WT. The clones with improved activity were sent for sequencing, and finally, the sequencing results were analyzed to select the mutant sites of 6 unique mutant candidate clones (see Table 5) for the construction of a combinatorial mutant library. (The Ratio value in Table 5 represents the degree of affinity improvement. When the Ratio value is equal to 1, it means that the affinity of the mutant clone is the same as that of WT).

[0223] Table 5: Screening results and mutant sites of candidate clones

[0224]

[0225] Note: In this article, WT always represents no mutation relative to the wild-type sequence (the same as in Table 6 and Table 7); the positions of the mutant amino acids in this article were obtained by sequentially numbering the amino acid sequences of VH or VL of WT from the N-terminus to the C-terminus in order.

[0226] 4. Construction of combinatorial mutant library

[0227] (1) Design and synthesis of library primers:

[0228] According to the mutant sites on VH and VL of the cTnI antibody obtained in step 3-(2), amplification primers for the combinatorial mutant library were designed and primers were synthesized.

[0229] (2) Fragment amplification and ligation

[0230] According to the PCR system in Table 3 and the PCR reaction conditions in Table 4, the antibody mutant fragments were amplified, and then the antibody mutant fragments were recovered by gel extraction. Using the method of Overlap PCR, the antibody mutant fragments were spliced into complete antibody fragments (heavy chain variable region or light chain variable region).

[0231] Finally, the antibody fragment was inserted into the V01 vector by restriction enzyme digestion and ligation to form a complete antibody expression plasmid (for the specific steps, see the steps of "Restriction Enzyme Digestion and Ligation of WT Antibody Gene Fragment" in Step 1-(3)). 100 ng of the plasmid was transformed into 100 μl of TG1 Escherichia coli competent cells, and the bacterial solution was spread evenly on a plate containing ampicillin resistance and cultured overnight at 37°C.

[0232] 5. Screening of the combinatorial mutant library

[0233] Eighteen monoclonal bacteria randomly selected from the above Step 4-(2) were used for the expression of combinatorial mutant antibody supernatant, ELISA screening and detection, and positive clone sequencing analysis. The mutant site information and Ratio value determination are shown in Table 6. The results show that the binding activities of mutants 1-18 are significantly better than that of WT.

[0234] Table 6: Information of combinatorial mutant candidate clones

[0235]

[0236]

[0237] Example 2: Expression of mutant cTnI antibody

[0238] In this example, the mutant cTnI antibody screened in Example 1 was expressed. The specific experimental operations are as follows:

[0239] 1. Construction of eukaryotic recombinant expression plasmid

[0240] pcDNA TM The pcDNA vector is the constructed recombinant antibody eukaryotic expression vector. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI have been introduced into this expression vector, which is named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short. According to the variable region gene sequences of the 24 candidate clones screened in Example 1, specific amplification primers for the VL and VH genes of the corresponding antibody sequences and constant region overlap primers were designed. The two ends of the primers respectively carried HindIII and EcoRI enzyme digestion sites and protective bases, and a 0.73 KB light chain gene fragment and a 1.40 kb heavy chain gene fragment were amplified by PCR.

[0241] The heavy chain and light chain gene fragments were digested with HindIII / EcoRI double enzymes, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. The digested antibody light and heavy chain gene fragments and the vector were purified and recovered. Then, the gene fragments encoding the antibody light and heavy chains were respectively ligated into the 3.4A expression vector and transformed into DH5α Escherichia coli competent cells. After colonies grew, single colonies were picked respectively for PCR identification of positive clones, and the positive clones were picked for sequencing to determine the correctness of the sequence. The clones with correct sequencing were selected for plasmid extraction for standby.

[0242] 2. Preparation of recombinant antibody samples

[0243] Resuscitate HEK293 cells in advance, passage and culture them to a 200 ml system to make the cell density reach (3 - 5)×10 6 cells / ml, and the cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml as the cell dilution. Prepare plasmid DNA and transfection reagent dilutions with the medium respectively. Add the transfection reagent dilution to the plasmid DNA dilution, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell dilution within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in a 35°C constant temperature incubator for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, collect the samples by centrifugation. Purify the antibody with a protein A affinity chromatography column.

[0244] Example 3: Affinity analysis

[0245] The affinity of some of the obtained mutant antibodies was detected and analyzed. The specific steps were as follows: Test the binding and dissociation curves of the antigen and antibody on a Biacore8K+ device, and the instrument automatically fits to obtain the affinity constant, binding rate, and dissociation rate. In the affinity detection results, KD represents the equilibrium dissociation constant, that is, the affinity constant. The smaller the KD value, the higher the affinity; ka represents the binding rate; kd represents the dissociation rate. The results showed that the affinity of the mutant antibody for cTnI was better than that of the wild-type antibody for cTnI.

[0246] Table 7: Affinity detection data

[0247]

[0248]

[0249] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0250] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An anti-cTnI antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are the amino acid sequences shown below: HCDR 1: DYNLH; HCDR 2: YIYPYNGX1TGYNQKFKS, wherein X1 is I or V; HCDR 3: DAYX2YDYLTD, where X2 is D or W; LCDR 1: RTX3KNX4GTNIX5, where X3 is S or R, X4 is V or M, and X5 is H, F or Y; LCDR 2:YASERLP; LCDR 3:QQSNNWPYT; Optionally, X1 is 1; Optionally, X1 is V; Optionally, X2 is D; Optionally, X2 is W; Optionally, X3 is S; Optionally, X3 is R; Optionally, X4 is V; Optionally, X4 is M; Optionally, X5 is H; Optionally, X5 is F; Optionally, X5 is Y; Optionally, X1 is I, X2 is D, X3 is S, X4 is V and X5 is H are not simultaneously true; Optionally, X1, X2, X3, X4 and X5 are selected from any one of the following combinations: 。 2. An anti-cTnI antibody, characterized in that: include: a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or a variant thereof, wherein compared with the amino acid sequence shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 comprises a mutation in at least one of the following sites: I57V, D102W; The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18 or a variant thereof, wherein compared with the amino acid sequence shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 comprises a mutation in at least one of the following sites: S26R, V29M, and H34Y / F; Optionally, the mutation at position 57 is I57V; Optionally, the mutation at position 102 is D102W; Optionally, the mutation at position 26 is S26R; Optionally, the mutation at position 29 is V29M; Optionally, the mutation at position 34 is H34Y; Optionally, the mutation at position 34 is H34F; Optionally, the heavy chain variable region and the light chain variable region are selected from any one of the following combinations:

3. An anti-cTnI antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region defined by the antibody according to claim 2; the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region defined by the antibody according to claim 2; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems; Optionally, the antibody comprises at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; Optionally, at least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 is derived from at least one of mouse antibody, human antibody, primate antibody, bovine antibody, horse antibody, dairy cow antibody, porcine antibody, sheep antibody, goat antibody, dog antibody, cat antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting cock antibody or mutants thereof; Optionally, the HFR1 comprises an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% homology thereto; The HFR2 comprises an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% homology thereto; The HFR3 comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% homology thereto; The HFR4 comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto; The LFR1 comprises an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto; The LFR2 comprises an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto; The LFR3 comprises an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% homology thereto; The LFR4 comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto.

4. The antibody according to any one of claims 1 to 3, characterized in that The antibody further comprises a constant region; Wherein, the constant region includes at least one of a heavy chain constant region and a light chain constant region; Optionally, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the light chain constant region comprises a light chain constant region selected from a κ type or a λ type; Optionally, the heavy chain constant region comprises or is the heavy chain constant region shown in SEQ ID NO: 15 or an amino acid sequence having at least 80% identity thereto; or The light chain constant region comprises or is the light chain constant region shown in SEQ ID NO: 16 or an amino acid sequence having at least 80% identity thereto.

5. A nucleic acid, a vector, a cell or a method for preparing the antibody according to any one of claims 1 to 4, characterized in that: The nucleic acid encodes the antibody according to any one of claims 1 to 4; the vector contains the above-mentioned nucleic acid; the cell contains the above-mentioned nucleic acid or vector; and the method comprises culturing the above-mentioned cell.

6. A conjugate, characterized in that: include: The antibody according to any one of claims 1 to 4 and the conjugated part thereof; Optionally, the coupling part includes at least one of a purification tag, an affinity substance, a marker and a solid phase carrier; Optionally, the purification tag includes at least one of a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag; Optionally, the solid support comprises at least one selected from microspheres, plates and membranes; Optionally, the label comprises at least one selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the affinity substance includes at least one selected from biotin, a biotin derivative or streptavidin.

7. A reagent or a kit, characterized in that: include: The antibody according to any one of claims 1 to 4 or the conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1 to 4, the conjugate according to claim 6, the reagent or the kit according to claim 7 in detecting cTnI, preparing a product for detecting cTnI, or preparing a product for indicating a cTnI-related disease; Optionally, the cTnI-related disease includes at least one of acute myocardial infarction, unstable angina, acute coronary syndrome or perioperative myocardial injury.

9. A method for detecting cTnI, characterized in that: include: The antibody according to any one of claims 1 to 4, the conjugate according to claim 6, or the reagent or kit according to claim 7 is contacted with a sample to be detected to form an immune complex; Optionally, based on the signal of the immune complex, it is determined whether the sample to be tested contains cTnI or the content of cTnI.

10. A method for screening cTnI antibodies, characterized in that: include: a) designing primers for performing amino acid substitution at the sites of X1, X2, X3, X4 and X5 defined in the antibody of claim 1, or at the mutation sites defined in the antibody of claim 2; b) using the nucleic acid, vector or cell of claim 5 as a template and the primers described in a) to construct a mutation library; c) screening cTnI antibodies from the mutant library; Optionally, the mutation library is a single-site saturation mutation library; Optionally, the cTnI antibody includes or is the antibody according to any one of claims 1 to 4.

11. A mutation library, characterized in that The mutant library comprises the antibody according to any one of claims 1 to 4; Optionally, the mutant library is obtained by the method of claim 10.

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