CN111018978A

CN111018978A - Anti-human cardiac troponin I antibody and application thereof

Info

Publication number: CN111018978A
Application number: CN201811181363.4A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 钟冬梅; 游辉; 范凌云
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2018-10-10
Filing date: 2018-10-10
Publication date: 2020-04-17
Anticipated expiration: 2038-10-10
Also published as: CN111018978B

Abstract

The present invention relates to a novel isolated binding protein comprising a cTnI antigen binding domain and studies on the preparation, use, etc. of the binding protein. The binding protein has strong activity and high affinity with human cTnI protein, and can be widely applied to the field of detection of the cTnI protein.

Description

Anti-human cardiac troponin I antibody and application thereof

Technical Field

The invention relates to the technical field of biotechnology and medicine, in particular to an anti-human cardiac troponin I antibody and application thereof.

Background

Before the 80's of the 20 th century, the World Health Organization (WHO) has used the activity of the myocardial zymogram as one of the diagnostic criteria for Acute Myocardial Infarction (AMI). At the end of the 80's 20 th century, researchers found that troponin (Tn) had higher sensitivity and specificity than biomarkers such as phosphocreatine kinase (CK), phosphocreatine kinase isoenzyme (CK-MB), lactate dehydrogenase, and aspartate aminotransferase. The cardiac troponin (cTnI) is only present in cardiac muscle, is a marker of cardiac muscle cells, can affect the contraction and relaxation functions of the heart by abnormal change, can be used for diagnosing myocardial necrosis, judging cardiac muscle injury and the like, becomes one of the markers with the strongest damage sensitivity and specificity of the cardiac muscle cells, and is a main biochemical marker for well-known rapid diagnosis of AMI and Acute Coronary Syndrome (ACS) and assistance of risk stratification of ACS and reflecting the prognosis of ACS.

The cTnI content in normal human blood is generally lower than 0.3 mu g/L. When the integrity of the cell membrane of the cardiomyocytes is damaged by ischemia or hypoxia, the free cTnI can rapidly penetrate the cell membrane and enter the blood stream. Therefore, the rapid, sensitive and accurate determination of cTnI and its change trend in human blood at the early stage of onset has important clinical significance for the diagnosis of acute myocardial infarction, risk stratification of acute coronary syndrome, monitoring of myocardial damage caused by various factors, and the like. The clinical methods for detecting the cTnI level include enzyme-linked immunosorbent assay (ELISA), chemiluminescence, colloidal gold and the like, and different methods have respective advantages and disadvantages, but all require specific monoclonal antibodies aiming at the cTnI.

The existing cTnI antibody has low activity and poor affinity, and cannot be well applied to the detection of cTnI protein, so that the field has strong demand for an antibody which can effectively and specifically bind and detect cTnI.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The present invention relates to a novel isolated binding protein comprising a cTnI antigen binding domain and studies on the preparation, use, etc. of the binding protein.

The antigen binding domain comprises at least one complementarity determining region selected from the group consisting of the amino acid sequences described below, or has at least 80% sequence identity with the complementarity determining region of the amino acid sequences described below and has K with cTnI_D≤9.97×10^-8Affinity of mol/L;

CDR-VH1 is G-X1-I-F-X2-G-X3-T-M-N, wherein,

x1 is F or Y, X2 is S or T, X3 is N or Q;

CDR-VH2 is L-X1-N-P-S-D-X2-T-T-Y-N-X3-K-F, wherein,

x1 is L, I or V, X2 is N or GG, X3 is Q or N;

CDR-VH3 is S-X1-X2-G-S-W-X3-Q, wherein,

x1 is S or T, X2 is F, Y or W, X3 is G or A;

the CDR-VL1 is A-S-X1-S-X2-D-Y-X3-G-D-S-Y, wherein,

x1 is N or Q, X2 is I, L or V, X3 is E or D;

the complementarity determining region CDR-VL2 is V-A-S-X1-X2-D-S, wherein,

x1 is I or L, X2 is L, V or I;

the complementarity determining region CDR-VL3 is Q-Q-X1-Y-E-X2-P-X3-T, wherein,

x1 is T or S, X2 is A or G, and X3 is Y, F or W.

An important advantage is that the binding protein is highly active and has a high affinity for human cTnI.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an electrophoretogram of a monoclonal antibody against a recombinant antibody against human cardiac troponin I of the present invention.

Detailed Description

The present invention may be understood more readily by reference to the following description of certain embodiments of the invention and the detailed description of the examples included therein.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such embodiments are necessarily varied. It is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

In order that the invention may be more readily understood, selected terms are defined below.

The term "amino acid" refers to naturally occurring or non-naturally occurring fusidic α -amino acids the term "amino acid" as used herein may include naturally occurring amino acids and non-naturally occurring amino acids including alanine (three letter code: A1a, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, c), glutamine (G1N, Q), glutamic acid (G1u, E), glycine (G1Y, G), histidine (His, H), isoleucine (I1E, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Va1, V). non-naturally occurring amino acids include, but are not limited to α -amino acids, citrulline, homoserine (Tyr, histidine, etc.

The term "isolated binding protein" is a protein that, due to its derivative origin or source, does not bind to the naturally associated component with which it is associated in its native state; substantially free of other proteins from the same species; expressed by cells from different species; or do not occur in nature. Thus, a protein that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from the components with which it is naturally associated. Proteins can also be rendered substantially free of naturally associated components by separation, using protein purification techniques well known in the art.

The term "isolated binding protein comprising an antigen binding domain" broadly refers to all proteins/protein fragments that comprise a CDR region. The term "antibody" includes polyclonal and monoclonal antibodies and antigenic compound-binding fragments of these antibodies, including Fab, F (ab') 2, Fd, Fv, scFv, diabodies and minimal recognition units of antibodies, as well as single chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site. The light or heavy chain variable region is made up of framework regions interrupted by three hypervariable regions, termed "complementarity determining regions" or "CDRs". The framework regions of the antibody, which constitute the combination of the essential light and heavy chains, serve to locate and align the CDRs, which are primarily responsible for binding to the antigen.

As used herein, the "framework" or "FR" regions mean the regions of the antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3 and FR 4).

Typically, the variable domains VL/VH of the heavy and light chains are obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

As used herein, the term "purified" or "isolated" in relation to a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its native medium or native form. Thus, the term "isolated" includes a polypeptide or nucleic acid that is removed from its original environment, e.g., from its natural environment if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally bound to or normally mixed with it or in solution. Isolated polypeptides include the naturally-produced polypeptide contained in a cell lysate, the polypeptide in purified or partially purified form, recombinant polypeptides, the polypeptide expressed or secreted by a cell, and the polypeptide in a heterologous host cell or culture. In connection with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

The present invention provides an isolated binding protein comprising an antigen binding domain comprising at least one complementarity determining region selected from the group consisting of amino acid sequences set forth below, or having at least 80% sequence identity with a complementarity determining region of an amino acid sequence set forth below and having K with cTnI_D≤9.97×10^-8Affinity of mol/L;

CDR-VH1 is G-X1-I-F-X2-G-X3-T-M-N, wherein,

x1 is F or Y, X2 is S or T, X3 is N or Q;

CDR-VH2 is L-X1-N-P-S-D-X2-T-T-Y-N-X3-K-F, wherein,

x1 is L, I or V, X2 is N or GG, X3 is Q or N;

CDR-VH3 is S-X1-X2-G-S-W-X3-Q, wherein,

x1 is S or T, X2 is F, Y or W, X3 is G or A;

the CDR-VL1 is A-S-X1-S-X2-D-Y-X3-G-D-S-Y, wherein,

x1 is N or Q, X2 is I, L or V, X3 is E or D;

the complementarity determining region CDR-VL2 is V-A-S-X1-X2-D-S, wherein,

x1 is I or L, X2 is L, V or I;

x1 is T or S, X2 is A or G, and X3 is Y, F or W.

It is well known in the art that both the binding specificity and avidity of an antibody are determined primarily by the CDR sequences, and that variants with similar biological activity can be obtained by readily altering the amino acid sequence of the non-CDR regions according to well-established, well-known techniques of the art. Thus, the invention also includes "functional derivatives" of the binding proteins. "functional derivative" refers to a variant of an amino acid substitution, one functional derivative retaining detectable binding protein activity, preferably the activity of an antibody capable of binding cTnI. "functional derivatives" may include "variants" and "fragments" which have the exact same CDR sequences as the binding proteins of the invention and therefore have similar biological activities.

In some embodiments, the antigen binding domain has at least 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to the complementarity determining region of the amino acid sequence having a KD of ≦ 9.97 × 10 for cardiac troponin I^-8mol/L, KD value can also be selected to be 7.14 multiplied by 10^- ⁸mol/L、5.16×10^-8mol/L、1.34×10^-8mol/L、1.30×10^-9mol/L、4.50×10^-9mol/L、5.26×10^- ⁹mol/L、7.55×10^-9mol/L、9.68×10^-9mol/L、1.38×10^-10mol/L、4.02×10^-10mol/L、7.28×10^-10mol/L、8.78×10^-10mol/L, or 1.38X 10^-10mol/L≤KD≤9.97×10^-8mol/L, or 1.38X 10^-10mol/L≤KD≤9.68×10^-9mol/L；

Wherein the affinity is determined according to the method of the present specification.

In some embodiments of the present invention, the substrate is,

in the complementarity determining region CDR-VH1, X1 is Y;

in the complementarity determining region CDR-VH2, X3 is Q;

in the complementarity determining region CDR-VH3, X3 is G;

in the complementarity determining region CDR-VL1, X1 is Q;

in the complementarity determining region CDR-VL2, X1 is I;

in the complementarity determining region CDR-VL3, X1 is S.

In some embodiments, in the complementarity determining region CDR-VH1, X2 is S and X3 is N.

In some embodiments, in the complementarity determining region CDR-VH1, X2 is S and X3 is Q. In some cases

In embodiments, in the CDR-VH1, X2 is T and X3 is N.

In some embodiments, in the complementarity determining region CDR-VH1, X2 is T and X3 is Q.

In some embodiments, in the complementarity determining region CDR-VH2, X1 is L and X2 is N. In some cases

In an embodiment, in the CDR-VH2, X1 is L and X2 is GG.

In some embodiments, in the complementarity determining region CDR-VH2, X1 is I and X2 is N.

In some embodiments, in the complementarity determining region CDR-VH2, X1 is I and X2 is GG.

In some embodiments, in the complementarity determining region CDR-VH2, X1 is V and X2 is N.

In some embodiments, in the complementarity determining region CDR-VH2, X1 is V and X2 is GG.

In some embodiments, in the complementarity determining region CDR-VH3, X1 is S and X2 is F.

In some embodiments, in the complementarity determining region CDR-VH3, X1 is S and X2 is Y.

In some embodiments, in the complementarity determining region CDR-VH3, X1 is S and X2 is W.

In some embodiments, in the complementarity determining region CDR-VH3, X1 is T and X2 is F.

In some embodiments, in the complementarity determining region CDR-VH3, X1 is T and X2 is Y.

In some embodiments, in the complementarity determining region CDR-VH3, X1 is T and X2 is W.

In some embodiments, in the complementarity determining region CDR-VL1, X2 is I and X3 is E.

In some embodiments, in the complementarity determining region CDR-VL1, X2 is I and X3 is D.

In some embodiments, in the complementarity determining region CDR-VL1, X2 is L and X3 is E.

In some embodiments, in the complementarity determining region CDR-VL1, X2 is L and X3 is D.

In some embodiments, in the complementarity determining region CDR-VL1, X2 is V and X3 is E.

In some embodiments, in the complementarity determining region CDR-VL1, X2 is V and X3 is D.

In some embodiments, in the complementarity determining region CDR-VL2, X2 is L.

In some embodiments, in the complementarity determining region CDR-VL2, X2 is V.

In some embodiments, in the complementarity determining region CDR-VL2, X2 is I.

In some embodiments, the complementarity determining region CDR-VL3, X2 is a and X3 is Y.

In some embodiments, the complementarity determining region CDR-VL3, X2 is a and X3 is F.

In some embodiments, the complementarity determining region CDR-VL3, X2 is a and X3 is W.

In some embodiments, in the complementarity determining region CDR-VL3, X2 is G and X3 is Y.

In some embodiments, in the complementarity determining region CDR-VL3, X2 is G and X3 is F.

In some embodiments, in the complementarity determining region CDR-VL3, X2 is G and X3 is W.

In some embodiments, the mutation site of each complementarity determining region is selected from any one of the following combinations of mutations:

in some embodiments, the binding protein includes at least 3 CDRs therein; alternatively, the binding protein comprises at least 6 CDRs.

In some embodiments, the binding protein is a whole antibody comprising a variable region and a constant region.

In some embodiments, the binding protein is one of a nanobody, a F (ab ') 2, a Fab', a Fab, a Fv, a scFv, a diabody, and an antibody minimal recognition unit.

In some embodiments, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 in the sequence shown in SEQ ID NOS: 1-4, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 in the sequence shown in SEQ ID NOS: 5-8.

In some embodiments, the binding protein further comprises an antibody constant region sequence.

In some embodiments, the constant region sequence is selected from the group consisting of sequences of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.

In some embodiments, the species of the constant region is derived from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fighting, or human.

In some embodiments, the constant region is derived from a mouse;

the light chain constant region sequence is shown as SEQ ID NO. 9;

the heavy chain constant region sequence is shown in SEQ ID NO 10.

The invention also provides an isolated nucleic acid encoding the binding protein described above.

Herein, a nucleic acid comprises conservatively substituted variants thereof (e.g., substitution of degenerate codons) and complementary sequences. The terms "nucleic acid" and "polynucleotide" are synonymous and encompass genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.

The invention also provides a vector comprising the nucleic acid as described above.

Wherein the nucleic acid sequence is operably linked to at least one regulatory sequence. "operably linked" means that the coding sequence is linked to the regulatory sequences in a manner that allows for expression of the coding sequence. Regulatory sequences are selected to direct the expression of the protein of interest in a suitable host cell and include promoters, enhancers and other expression control elements.

Herein, a vector may refer to a molecule or agent comprising a nucleic acid of the invention or a fragment thereof, capable of carrying genetic information and capable of delivering the genetic information into a cell. Typical vectors include plasmids, viruses, bacteriophages, cosmids and minichromosomes. The vector may be a cloning vector (i.e., a vector for transferring genetic information into a cell, which may be propagated and in which the genetic information may be present or absent) or an expression vector (i.e., a vector which comprises the necessary genetic elements to permit expression of the genetic information of the vector in a cell). Thus, a cloning vector may contain a selectable marker, as well as an origin of replication compatible with the cell type specified by the cloning vector, while an expression vector contains the regulatory elements necessary to effect expression in a specified target cell.

The nucleic acid of the invention or fragments thereof may be inserted into a suitable vector to form a cloning or expression vector carrying the nucleic acid fragment of the invention. Such novel vectors are also part of the present invention. The vector may comprise a plasmid, phage, cosmid, minichromosome, or virus, as well as naked DNA that is transiently expressed only in a particular cell. The cloning and expression vectors of the invention are capable of autonomous replication and thus provide high copy numbers for high level expression or high level replication purposes for subsequent cloning. The expression vector may comprise a promoter for driving expression of the nucleic acid fragment of the invention, optionally a nucleic acid sequence encoding a signal peptide for secretion or integration of the peptide expression product into a membrane, a nucleic acid fragment of the invention, and optionally a nucleic acid sequence encoding a terminator. When the expression vector is manipulated in a production strain or cell line, the vector, when introduced into a host cell, may or may not be integrated into the genome of the host cell. Vectors typically carry a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells.

The expression vectors of the invention are useful for transforming host cells. Such transformed cells are also part of the invention and may be cultured cells or cell lines for propagation of the nucleic acid fragments and vectors of the invention, or for recombinant production of the polypeptides of the invention. The transformed cells of the present invention include microorganisms such as bacteria (e.g., Escherichia coli, Bacillus spp., etc.). Host cells also include cells from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably from mammals, e.g., CHO cells. The transformed cells are capable of replicating the nucleic acid fragments of the invention. When the peptide combination of the present invention is recombinantly produced, the expression product may be exported into the culture medium or carried on the surface of the transformed cell.

The invention also provides a method for producing the binding protein, which comprises the following steps:

the above-mentioned host cells are cultured in a medium, and the produced binding protein is recovered from the medium or from the cultured host cells.

The method can be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cell under suitable conditions such that the binding protein is expressed. The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of the binding protein. The bound protein may be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis.

Construction of suitable vectors containing the coding and regulatory sequences of interest can be carried out using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailed and religated as desired. Any method may be used to introduce mutations into the coding sequence to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions or the like.

The invention also provides antibodies, reactive with an epitope of cTnI, including monoclonal and polyclonal antibodies. The antibody may comprise the entire binding protein, or a fragment or derivative thereof. Preferred antibodies contain all or part of a binding protein.

The invention also provides application of the binding protein in preparing a diagnostic agent or a kit for diagnosing acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina and myocardial injury.

The invention also provides a kit characterized in that the kit comprises one or more of the above-described binding protein, the above-described isolated nucleic acid, or the above-described vector.

Preferably, the kit further comprises a label for labeling the binding protein.

According to one aspect of the invention, the invention also relates to a method for detecting troponin I antigen in a test sample, comprising:

a) contacting a troponin I antigen in the test sample with a binding protein as defined above to form an immune complex under conditions sufficient for an antibody/antigen binding reaction to occur; and

b) detecting the presence of said immune complex, the presence of said complex being indicative of the presence of said troponin I antigen in said test sample;

in this embodiment, the binding protein may be labeled with an indicator that indicates the strength of the signal, so that the complex is readily detected.

In some embodiments, in step a), a second antibody is further included in the immune complex, the second antibody binding to the binding protein;

in this embodiment, the binding protein forms a partner antibody with the second antibody in the form of a first antibody for binding to a different epitope of cTnI;

the second antibody may be labeled with an indicator showing the intensity of the signal so that the complex is easily detected.

In some embodiments, in step a), a second antibody is further included in the immune complex, which second antibody binds to the troponin I antigen;

in this embodiment, the binding protein serves as an antigen for the second antibody, which may be labeled with an indicator of signal intensity to allow the complex to be readily detected.

In some embodiments, the indicator that shows signal intensity comprises any one of a fluorescent substance, a quantum dot, a digoxigenin-labeled probe, biotin, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron-dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, colloidal gold, or an enzyme.

In some embodiments, the fluorescent species include Alexa 350, Alexa 405, Alexa 430, Alexa488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxyfluorescein, 5-carboxy-2 ', 4', 5', 7' -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1, 3-diazole), Any one of Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol Blue violet, brilliant cresol Blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, europium tripyridyldiamine, europium cryptate or chelate, diamine, bispyanine, La Jolla Blue dye, allophycocyanin, allocyanonin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas red.

In some embodiments, the radioisotope comprises¹¹⁰In、¹¹¹In、¹⁷⁷Lu、¹⁸F、⁵²Fe、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁹⁰Y、⁸⁹Zr、⁹⁴mTc、⁹⁴Tc、⁹⁹mTc、¹²⁰I、¹²³I、¹²⁴I、¹²⁵I、¹³¹I、^154-158Gd、³²P、¹¹C、¹³N、¹⁵O、¹⁸⁶Re、¹⁸⁸Re、⁵¹Mn、⁵²mMn、⁵⁵Co、⁷²As、⁷⁵Br、⁷⁶Br、⁸²mRb and⁸³sr.

In some embodiments, the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.

In some embodiments, the fluorescent microspheres are: the polystyrene fluorescent microsphere is internally wrapped with rare earth fluorescent ion europium.

As in some embodiments, the present invention provides kits for determining the presence of troponin I in a subject, e.g. a cardiomyocyte infection, comprising at least one binding protein provided herein, an associated buffer, reagents required for reacting a liquid sample with said binding protein, and reagents for determining the presence of a positive or negative binding reaction between troponin I and the binding protein. For determining the presence of troponin I, the kit may for example utilize a binding protein as an antibody carrying a label, wherein the label may be any suitable label, such as a colloidal gold label.

The following examples are provided to illustrate the present invention, but not to limit the scope of the present invention.

Example 1

Restriction enzyme, Prime Star DNA polymerase, was purchased from Takara in this example. MagExtractor-RNA extraction kit was purchased from TOYOBO. The SMARTERTM RACE cDNA amplification kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were performed by Invitrogen corporation. The hybridoma cell strain secreting Anti-cTnI 6E3 monoclonal antibody is the existing hybridoma cell strain of the applicant, and is recovered for later use.

1. Primer and method for producing the same

Amplification of Heavy Chain and Light Chain 5' RACE primers:

SMARTER II A Oligonucleotide：

5’-AAGCAGTGGTATCAACGCAGAGTACXXXXX-3’；

5'-RACE CDS Primer(5'-CDS)：5'-(T)₂₅VN-3’(N＝A,C,G,orT；V＝A,G,orC)；

Universal Primer A Mix(UPM)：5’-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’；

Nested Universal Primer A(NUP)：5’-AAGCAGTGGTATCAACGCAGAGT-3’；

mIg-KR：5’-CTAACACTCATTCCTGTTGAAGCTCTTGACAAT-3’；

mIg-HR：5’-TCATTTACCAGGAGAGTGGGAGAGGC-3’。

2. antibody variable region gene cloning and sequencing

Extracting RNA from hybridoma cell line secreting Anti-cTnI 6E3 monoclonal antibody, and extracting with SMARTER^TMThe method comprises the steps of synthesizing first strand cDNA by SMARTER II A Oligonucleotide and 5' -CDS Primer in RACE cDNA Amplification Kit and Kit, using the obtained first strand cDNA product as PCR Amplification template, amplifying Light Chain gene by Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mIg-KR Primer, amplifying Heavy Chain gene by Universal Primer A Mix (UPM), Nested Universal Primer A (NUP) and mIg-HR Primer, amplifying target band about 0.7KB by Light Chain Primer pair, amplifying target band about 1.4KB by Heavy Chain Primer pair, purifying and recovering by agarose gel electrophoresis, inserting the product into pMD-18T vector after A reaction by rTaq DNA polymerase, transforming into 5 α competent cell, cloning by the Changotide and cloning by the Hevy gene, and cloning by Invitrogen after cloning.

3. Sequence analysis of Anti-cTnI 6E3 antibody variable region Gene

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 321bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the HeavyChain primer pair, the VH gene sequence is 357bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

4. Construction of recombinant antibody expression plasmid

pcDNA^TM3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, BamHI, EcoRI and the like are introduced into the expression vector and named as a pcDNA 3.4A expression vector, and the expression vector is called as a 3.4A expression vector for short in the following; according to the sequencing result of the antibody gene in the pMD-18T, designing the specificity primers of the Heavy Chain and Light Chain of the Anti-cTnI 6E3 antibody, wherein the two ends of the primers are respectively provided with HindIII and EcoRI restriction sites and protective bases, and the primers are as follows:

cTnI-6E3-HF：5’-CCCAAGCTTATGGAATGCAGCTGTGTCATGCTCTTCTTC-3’；

cTnI-6E3-HR：5’-CCCGAATTCTCATTTACCAGGAGAGTGGGAGAGGC-3’；

cTnI-6E3-LF：5’-CCCAAGCTTATGAAGTTGCCTGTTAGGCTGTTGG-3’；

cTnI-6E3-LR：5’-CCCGAATTCCTAACACTCATTCCTGTTGAAGCTCTTGACAA-3’。

a0.72 KB Light Chain gene fragment and a 1.4KB Heavy Chain gene fragment were amplified by PCR amplification. The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the Heavy Chain gene and the Light Chain gene are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

5. Screening for Stable cell lines

5.1 plasmid diluted to 400ng/ml with ultrapure water, CHO cells were conditioned 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, the sampling counting is carried out on the 3 rd, 5 th and 7 th days, and the sampling detection is carried out on the 7 th day。

The coating solution dilutes the corresponding antigen to the designated concentration, each well is 100 mu L, and the temperature is kept overnight at 4 ℃; the next day, washing with the washing solution for 2 times, and patting dry; adding blocking solution (20% BSA + 80% PBS), and drying at 37 deg.C for 1 hr in each well; adding diluted cell supernatant at 100 μ L/well, 37 deg.C for 30min (partial supernatant for 1 h); washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu L per well at 37 ℃ for 30 min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 muL/hole), adding a developing solution B (50 muL/hole), and keeping for 10 min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 mu L/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader. And (4) calculating the content of the antibody in the cell supernatant by taking the concentration of the standard substance and the OD value as a standard curve.

5.2 linearization of recombinant antibody expression plasmids

The following reagents were prepared: 50 mul Buffer, 100 mul DNA/tube, 10 mul Puv I enzyme, and sterile water to 500 mul, water bath enzyme digestion overnight at 37 ℃; sequentially extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1 and then chloroform (water phase); precipitating with 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol on ice, rinsing with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water after ethanol is completely volatilized, and finally measuring concentration.

Stably transfecting recombinant antibody expression plasmid, and screening stable cell strains under pressure:

plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10⁷cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25 u mol/L MSX 96 hole pressure culture about 25 days.

Observing the marked clone holes with cells under a microscope, and recording the confluence degree; taking culture supernatant, and sending the culture supernatant to a sample for detection; selecting cell strains with high antibody concentration and relative concentration, transferring the cell strains into 24 holes, and transferring the cell strains into 6 holes after 3 days; after 3 days, the seeds were kept and cultured in batches, and the cell density was adjusted to 0.5X 10⁶cells/ml, 2.2ml, cell density 0.3X 10⁶cell/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample sending detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter to transfer TPP for seed preservation and passage.

6. Production of recombinant antibodies

6.1 cell expansion culture

After the cells are recovered, the cells are cultured in a shaking flask with the specification of 125ml, the inoculation volume is 30ml, the culture medium is 100% Dynamis culture medium, and the cells are placed in a shaking table with the rotation speed of 120r/min, the temperature of 37 ℃ and the carbon dioxide of 8%. Culturing for 72h, inoculating and expanding culture at an inoculation density of 50 ten thousand cells/ml, wherein the expanding culture volume is calculated according to production requirements, and the culture medium is 100% Dynamis culture medium. Then the culture is expanded every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.

6.2 Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing mode: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. The recombinant antibody was obtained after purification, and 4. mu.g of the purified antibody was subjected to reducing SDS-PAGE, and the electrophoretogram thereof is shown in FIG. 1. After reducing SDS-PAGE, two bands were shown, one of which was a light chain of about 25kD (SEQ ID NO: 11) and the other was a heavy chain of about 50kD (SEQ ID NO: 12).

Example 2

Although the antibody of sample 1 obtained in example 1 (having light and heavy chains having sequences shown in SEQ ID NOS: 11 and 12) had the ability to bind to cTnI protein, the affinity and the antibody activity were not satisfactory, and thus the applicant mutated the light and heavy chain CDRs of the antibody.

Upon analysis, the complementarity determining region (WT) of the heavy chain:

CDR-VH1 is G-F (X1) -I-F-S (X2) -G-Q (X3) -T-M-N;

CDR-VH2 is L-L (X1) -N-P-S-D-N (X2) -T-T-Y-N-N (X3) -K-F;

CDR-VH3 is S-S (X1) -F (X2) -G-S-W-A (X3) -Q;

complementarity determining regions of the light chain:

CDR-VL1 is A-S-N (X1) -S-I (X2) -D-Y-E (X3) -G-D-S-Y;

CDR-VL2 is V-A-S-L (X1) -V (X2) -D-S;

CDR-VL3 is Q-Q-T (X1) -Y-E-G (X2) -P-W (X3) -T;

wherein, X1, X2 and X3 are all mutation sites.

TABLE 1 mutant sites associated with antibody Activity

Detecting the activity of the antibody after mutation, diluting the cTnI protein to the specified concentration by using coating solution, wherein each well is 100 mu L, and the temperature is kept overnight at 4 ℃; the next day, washing with the washing solution for 2 times, and patting dry; adding diluted cTnI monoclonal antibody, 100 mu L/hole, 37 ℃,30 min; washing with washing solution for 5 times, and drying; adding goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) with the concentration of 100 mu L per well at 37 ℃ for 30 min; washing with washing solution for 5 times, and drying; adding a developing solution A (50 muL/hole), adding a developing solution B (50 muL/hole), and keeping for 10 min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 mu L/hole; OD readings were taken at 450nm (reference 630nm) on the microplate reader.

Some of the results are as follows:

TABLE 2 antibody Activity assay data

Concentration (ng/ml)	WT	Mutation 1	Mutation 2	Mutation 3	Mutation 4	Mutation 5
							1000.000	2.314	2.435	2.399	1.567	1.178	1.206
333.333	2.117	2.377	2.408	1.279	0.527	0.532
							111.111	2.009	2.410	2.374	0.894	0.221	0.217
37.037	1.956	2.367	2.353	0.514	-	-
							12.346	1.697	2.038	2.156	0.201	-	-
4.115	0.675	1.030	1.410	0.009	-	-
							1.372	0.174	0.380	0.646	-	-	-
0.000	0.017	0.001	0.032	-	-	-

"-" indicates no activity.

Affinity assay

Using AMC sensors, the antibodies with PBST diluted to 10 u g/ml, cTnI quality control recombinant protein (company self-produced recombinant antigen) with PBST gradient dilution: 769.2nmol/ml, 384.6nmol/ml, 192.3nmol/ml, 96.2nmol/ml, 48.1nmol/ml, 24nmol/ml, 12nmol/ml, 0 nmol/ml.

The operation flow is as follows: equilibrating in buffer 1(PBST) for 60s, immobilizing antibody in antibody solution for 300s, incubating in buffer 2(PBST) for 180s, binding in antigen solution for 420s, dissociating in buffer 2 for 1200s, regenerating the sensor with 10mM GLY solution pH 1.69 and buffer 3, and outputting the data. KD represents the equilibrium solvophilic constant, i.e. affinity; kon denotes the binding rate; kdis denotes the off-rate.

Table 3 affinity assay data

Different mutations	KD(M)	Kon(1/Ms)	Kdis(1/S)
				WT	6.55E-08	4.25E+04	2.78E-03
Mutation 1	7.30E-09	3.05E+04	2.23E-04
				Mutation 2	6.89E-09	4.14E+04	2.85E-04
Mutation 3	4.32E-07	4.23E+03	1.83E-03
				Mutation 4	-	-	-
Mutation 5	-	-	-

"-" indicates no detection.

As can be seen from tables 2 and 3, the activity effect and affinity of mutation 1 are the best, so that mutation sites with better potency are screened by using mutation 1 as a framework sequence (ensuring that the activity of the antibody obtained by screening is similar to that of mutation 1, and the antibody activity is +/-10%), and partial results are as follows.

TABLE 4 mutation sites related to antibody affinity

Affinity analysis, methods as above, results are shown in table 5.

Table 5 affinity assay data

As can be seen from table 5, the mutation sites listed in table 4 have little effect on the affinity of the antibody.

To verify the above results, the above experiment was repeated using WT as a backbone sequence, and affinity verification of the mutation site was performed, and some results are as follows.

TABLE 6 mutations with WT as backbone

Table 7 affinity assay data

From the analyses of tables 6 and 7, the affinity of the antibody was not greatly affected by the mutation sites listed in table 6.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Dongguan City of Pengzhi Biotech Co., Ltd

<120> anti-human cardiac troponin I antibody and application thereof

<130>2010

<160>12

<170>PatentIn version 3.3

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Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser

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Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val

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Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly

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340 345 350

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370 375 380

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435

Claims

1. An isolated binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises at least one complementarity determining region selected from the group consisting of amino acid sequences having, or having at least 80% sequence identity with a complementarity determining region of an amino acid sequence having a K with cTnI_D≤9.97×10^-8Affinity of mol/L;

CDR-VH1 is G-X1-I-F-X2-G-X3-T-M-N, wherein,

x1 is F or Y, X2 is S or T, X3 is N or Q;

CDR-VH2 is L-X1-N-P-S-D-X2-T-T-Y-N-X3-K-F, wherein,

x1 is L, I or V, X2 is N or GG, X3 is Q or N;

CDR-VH3 is S-X1-X2-G-S-W-X3-Q, wherein,

x1 is S or T, X2 is F, Y or W, X3 is G or A;

the CDR-VL1 is A-S-X1-S-X2-D-Y-X3-G-D-S-Y, wherein,

x1 is N or Q, X2 is I, L or V, X3 is E or D;

the complementarity determining region CDR-VL2 is V-A-S-X1-X2-D-S, wherein,

x1 is I or L, X2 is L, V or I;

x1 is T or S, X2 is A or G, and X3 is Y, F or W.

2. The binding protein of claim 1,

in the complementarity determining region CDR-VH1, X1 is Y;

in the complementarity determining region CDR-VH2, X3 is Q;

in the complementarity determining region CDR-VH3, X3 is G;

in the complementarity determining region CDR-VL1, X1 is Q;

in the complementarity determining region CDR-VL2, X1 is I;

in the complementarity determining region CDR-VL3, X1 is S;

further, in the complementarity determining region CDR-VH1, X2 is S, X3 is N;

further, in the complementarity determining region CDR-VH1, X2 is S, X3 is Q;

further, in the complementarity determining region CDR-VH1, X2 is T, X3 is N;

further, in the complementarity determining region CDR-VH1, X2 is T, X3 is Q;

further, in the complementarity determining region CDR-VH2, X1 is L, X2 is N;

further, in the complementarity determining region CDR-VH2, X1 is L, and X2 is GG;

further, in the complementarity determining region CDR-VH2, X1 is I, X2 is N;

further, in the complementarity determining region CDR-VH2, X1 is I, X2 is GG;

further, in the complementarity determining region CDR-VH2, X1 is V, X2 is N;

further, in the complementarity determining region CDR-VH2, X1 is V, and X2 is GG;

further, in the complementarity determining region CDR-VH3, X1 is S, X2 is F;

further, in the complementarity determining region CDR-VH3, X1 is S, X2 is Y;

further, in the complementarity determining region CDR-VH3, X1 is S, X2 is W;

further, in the complementarity determining region CDR-VH3, X1 is T, X2 is F;

further, in the complementarity determining region CDR-VH3, X1 is T, X2 is Y;

further, in the complementarity determining region CDR-VH3, X1 is T, X2 is W;

further, in the complementarity determining region CDR-VL1, X2 is I, X3 is E;

further, in the complementarity determining region CDR-VL1, X2 is I, X3 is D;

further, in the complementarity determining region CDR-VL1, X2 is L, and X3 is E;

further, in the complementarity determining region CDR-VL1, X2 is L, and X3 is D;

further, in the complementarity determining region CDR-VL1, X2 is V, X3 is E;

further, in the complementarity determining region CDR-VL1, X2 is V, X3 is D;

further, in the complementarity determining region CDR-VL2, X2 is L;

further, in the complementarity determining region CDR-VL2, X2 is V;

further, in the complementarity determining region CDR-VL2, X2 is I;

further, in the complementarity determining region CDR-VL3, X2 is a, X3 is Y;

further, in the complementarity determining region CDR-VL3, X2 is a, X3 is F;

further, in the complementarity determining region CDR-VL3, X2 is a, X3 is W;

further, in the complementarity determining region CDR-VL3, X2 is G, X3 is Y;

further, in the complementarity determining region CDR-VL3, X2 is G, X3 is F;

further, in the complementarity determining region CDR-VL3, X2 is G, X3 is W;

preferably, the mutation site of each complementarity determining region is selected from any one of the following combinations of mutations:

3. the binding protein according to any one of claims 1-2, wherein at least 3 CDRs are included in the binding protein; alternatively, the binding protein comprises at least 6 CDRs;

further, the binding protein is one of nanobody, F (ab ') 2, Fab', Fab, Fv, scFv, diabody, and antibody minimal recognition unit.

4. The binding protein according to any one of claims 1-2, wherein said binding protein comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 in the sequence given in SEQ ID NOs 1-4, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 in the sequence given in SEQ ID NOs 5-8;

further, the binding protein further comprises an antibody constant region sequence;

further, the constant region sequence is selected from the sequences of any one of constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD;

further, the species source of the constant region is a cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fighting or human;

further, the constant region is derived from a mouse;

the light chain constant region sequence is shown as SEQ ID NO. 9;

the heavy chain constant region sequence is shown in SEQ ID NO 10.

5. An isolated nucleic acid encoding the binding protein of any one of claims 1-4.

6. A vector comprising the nucleic acid of claim 5.

7. A host cell comprising the nucleic acid of claim 5 or the vector of claim 6.

8. A method of producing the binding protein of any one of claims 1 to 4, comprising the steps of:

culturing the host cell of claim 7 in a culture medium and recovering the produced binding protein from the culture medium or from the cultured host cell.

9. Use of a binding protein according to any one of claims 1 to 4 for the preparation of a diagnostic agent or kit for the diagnosis of acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina pectoris, myocardial injury.

10. A kit comprising one or more of the binding protein of any one of claims 1-4, the isolated nucleic acid of claim 5, or the vector of claim 6;

preferably, the kit further comprises a label for labeling the binding protein.