CN115703837A

CN115703837A - Recombinant antibody of anti-long-stimulation expression gene 2 protein

Info

Publication number: CN115703837A
Application number: CN202110944249.8A
Authority: CN
Inventors: 孟媛; 钟冬梅; 黄玉玲
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2023-02-17
Anticipated expiration: 2041-08-17
Also published as: CN115703837B

Abstract

The invention relates to an isolated binding protein containing a growth-stimulating expression gene 2 protein antigen binding domain, and researches on the aspects of preparation, application and the like of the binding protein. The binding protein has strong activity and high affinity with the growth stimulation expression gene 2 protein, and can be widely applied to the field of detection of the growth stimulation expression gene 2 protein.

Description

Recombinant antibody of anti-long-stimulation expression gene 2 protein

Technical Field

The invention relates to the technical field of immunity, in particular to a recombinant antibody of an anti-long-term stimulation expression gene 2.

Background

Growth-stimulating expression of gene 2 protein (ST 2) is one of the members of the interleukin 1 (IL-1) receptor superfamily, ST2 was first discovered in mouse fibroblasts by Tominaga et al in 1987; richard Lee, a female hospital affiliated with Harvard university Hospital, 2002, reported that myocardial stress and myocardial injury cause ST2 expression; in 2005, researchers such as Schmitz found that the ST2 gene not only participates in inflammatory reaction and pathological process of immune diseases, but also found that the specific functional ligand IL-33 of the ST2 gene; the us ACC/AHA heart failure guideline of 2013 and the heart failure diagnostic and therapeutic guideline of 2014 china introduce ST2 (soluble ST 2) into recommendations for myocardial fibrosis, a biomarker of heart failure, according to ST2 clinical study data accumulation.

Growth-stimulating expression of the gene 2 protein (ST 2), also known as the T1, IL1RL1 or Fit1 gene, and located on human chromosome 2q 12. About 40KD, the transcript of the ST2 gene has 4 subtypes, of which two are most important because of being regulated by different promoters, the first is transmembrane ST2 (ST 2L), which is composed of 3 extracellular immunoglobulin G domains, one transmembrane domain and one intracellular domain, and is a member of the membrane receptor of the interleukin-1 receptor family; the other is soluble ST2 (sST 2), sST2, due to its lack of transmembrane and intracellular domains, is free to flow in the blood, making it detectable in serum.

Research on growth stimulation expression gene 2 protein (ST 2) shows that the protein is related to cardiac dysfunction, ST2L receptor has heart protection effect, and after being combined with a specific ligand IL-33, the protein can reduce fibrosis and hypertrophic burden structure of cardiac myocardial cells, reduce myocardial damage degree after myocardial infarction, myocardial apoptosis, inflammatory myocardial activation, myocardial remodeling and other pathological symptoms. Kakkar et al found that mechanical stretching of living cells could enhance IL-33 release from cytoplasmic vesicles, could induce cardiac remodeling of IL-33 due to volume or pressure overload, vasopressin II could cause damage to the heart by producing multiple Reactive Oxygen Species (ROS), IL-33 exerted anti-myocardial hypertrophy effects by blocking the effects of vasopressin II or phenylephrine on the myocardium, and ST2L combined with IL33 activated nuclear transcription factor NF-kB and mitogen activated protein kinase MAPK pathway, resulting in the release of Th2 cytokines IL-4, IL-5 from target cells, which could exert anti-myocardial hypertrophy myocardial fibrosis effects, thereby initiating repair protection. It has also been found that in vitro administration of IL-33 enhances the activation of ST2L and its own intracellular biochemical pathways, as well as reduces the ROS production induced by angiotensin II, thereby preventing or at least reducing the worsening of the clinical and structural state of the heart;

sST2 is used as a decoy receptor of IL-33, is a marker of cardiovascular diseases, blocks a 1L-33/ST2L signal channel after being combined with IL-33, thereby reducing the effectiveness of IL-33 and a myomembrane receptor interaction, and in cardiac muscle cells with heavy mechanical load, sST2 is obviously induced, so that the occurrence of heart failure or the worsening of the previous chronic heart failure state and myocardial infarction-induced scars can not well stretch the cardiac muscle, and the conditions can be detected by measuring the blood sST2 level. The Weinberg et al study showed that myocardial infarction caused an increase in the plasma sST2 levels. Thus, an increase in sST2 can reduce the protective effect of IL-33 on cardiac myocytes and can have a negative impact on the overall cardiovascular risk profile.

Cardiovascular diseases (CVD) are the leading causes of death worldwide, brain Natriuretic Peptide (BNP) and brain natriuretic peptide precursor NT-proBNP are the best known markers of Heart Failure (HF), and troponin markers improve the diagnosis of acute and chronic coronary artery diseases, however, a single biomarker not only reflects one side of the pathological mechanism of heart failure, but also is affected by pulmonary hypertension caused by kidney function, age, BMI, etc., and the accuracy of these biomarkers is reduced. With the increased awareness of the effects of sST2 and ST2L on the cardiovascular system, the assessment of plasma sST2 levels is beginning to be considered as a new marker of cardiovascular events, in particular indices and clinical conditions associated with heart failure and ischemic heart disease, and sST2 is not affected by indices of renal function, age, body weight, etc. sST2 is also considered as a possible biomarker for acute heart failure and accompanying asthmatic patients. Studies also confirm the role of sST2 in predicting mortality, sST2 can be combined with other biomarkers as a prognostic biomarker, and sST2 can be used for monitoring the pharmacological response of heart failure, has a relevant relation with recommended treatment drugs of heart failure, and can provide reference for clinical selection of treatment drugs and schemes.

At present, the main methods for detecting ST2 include enzyme-linked immunosorbent assay (ELISA), magnetic particle chemiluminescence assay, chromatography and the like; different detection methods all require a monoclonal antibody specific for ST 2. The monoclonal antibodies currently used for the detection of ST2 have drawbacks in sensitivity or specificity.

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

Disclosure of Invention

The invention relates to a novel separated binding protein containing an ST2 antigen binding structural domain, and researches on the aspects of preparation, application and the like of the binding protein.

Wherein the antigen binding domain comprises at least one complementarity determining region selected from the group consisting of amino acid sequences set forth in SEQ ID NO:

a complementarity determining region CDR-VH1 comprising SEQ ID NO:1 or consists thereof;

a complementarity determining region CDR-VH2 comprising SEQ ID NO:2 or consists of the amino acid sequence shown in the specification;

a complementarity determining region CDR-VH3 comprising SEQ ID NO:3 or consists thereof;

a complementarity determining region CDR-VL1 comprising SEQ ID NO:4 or consists of the amino acid sequence shown in the formula (I);

a complementarity determining region CDR-VL2 comprising SEQ ID NO:5 or consists thereof;

a complementarity determining region CDR-VL3 comprising SEQ ID NO:6 or consists thereof.

The beneficial technical effects of the invention include that the binding protein has strong activity and high affinity with ST2, and is suitable for detecting the content of ST2, especially the content of human ST 2. Meanwhile, the binding protein obtained by the recombination method has small individual difference, small batch difference and stable quality, and is more beneficial to quality control and detection stability.

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 embodiments or the prior art descriptions 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 ST2 in example 1 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 otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those 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" denotes a naturally occurring or non-naturally occurring fusidic alpha-amino acid. The term "amino acid" as used in this application may include both naturally occurring amino acids and non-naturally occurring amino acids. Naturally occurring amino acids include 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 (G1 u, 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 (Va 1, V). Non-naturally occurring amino acids include, but are not limited to, alpha-aminoadipic acid, aminobutyric acid, citrulline, homocitrulline, homoleucine, homoarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.

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 natural 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, as well as antigenic compound-binding fragments of such antibodies, including Fab, F (ab') ₂ Fd, fv, scFv, diabodies and antibody minimal recognition units, 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, and IgD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric antibodiesHomozygous (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, and 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 (VL or VH) is composed of framework regions interrupted by three hypervariable regions, termed "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest), E.Kabat et al, U.S. department of Health and Human Services (U.S.. Department of Health and Human Services), (1983), and Chothia. 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.

Although the 2 domains of the Fv fragment (VL and VH) are encoded by separate genes, they can be joined using recombinant methods by synthetic linkers that enable them to be made into a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv), "single chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, and in some embodiments, the Fv polypeptide additionally comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.

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, "framework" or "FR" regions mean regions of an antibody variable domain that are exclusive of those defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions (FR 1, FR2, FR3 and FR 4) separated by CDRs.

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-FR4.

CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT, chothia and Lesk numbering scheme and the new standardized numbering system introduced by Lefranc et al 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al was the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the Kabat database, and the Kabat numbering scheme is generally considered to be a widely adopted standard for numbering antibody residues. The invention adopts Kabat annotation standard to mark CDR area, but other methods to mark CDR area also belong to the protection scope of the invention.

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 term "affinity" as used herein denotes the equilibrium constant for reversible binding of 2 agents and is expressed as KD. The affinity of a binding protein for a ligand, such as the affinity of an antibody for an epitope, can be, for example, about 100 nanomolar (nM) to about 0.1nM, about 100nM to about 1 picomolar (pM), or about 100nM to about 1 femtomolar (fM). The term "avidity" as used herein means the resistance of a complex of 2 or more agents to dissociation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA) or any other technique familiar to those skilled in the art.

The term "homology" or "identity" or "similarity" as used herein refers to sequence similarity between two peptides or between two nucleic acid molecules. Each of homology and identity can be determined by comparing the position in each sequence, which are aligned for comparison purposes. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when equivalent positions are occupied by the same or similar amino acid residue (e.g., similar in steric and/or electronic properties), then the molecules can be said to be homologous (similar) at that position. The expression percent homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Deletion of residues (amino acids or nucleic acids) or the presence of additional residues in comparing two sequences also reduces identity and homology/similarity.

The present invention relates to 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 recited in seq id nos:

a complementarity determining region CDR-VH1 comprising SEQ ID NO:1 or consists of the amino acid sequence shown in the formula (I);

In some embodiments, the binding protein comprises at least 3 CDRs (e.g., 3 light chain CDRs or 3 heavy chain CDRs).

In some embodiments, 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 an antibody, F (ab') ₂ Fab', fab, fv, scFv, diabody and antibody minimal recognition unit.

In some embodiments, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 having a sequence as set forth in SEQ ID NOS: 11-14 or having at least 90% homology thereto in order, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 having a sequence as set forth in SEQ ID NOS: 7-10 or having at least 90% homology thereto in order.

In alternative embodiments, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 having a sequence as set forth in SEQ ID NOS 11-14 or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 having a sequence as set forth in SEQ ID NOS 7-10 or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

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 IgG1, igG2, igG3, igG4, igA, igM, igE, igD.

In some embodiments, the species source of the constant region is a cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, 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:16 is shown in the figure;

the heavy chain constant region sequence is shown as SEQ ID NO: shown at 15.

In another aspect, the invention also provides an isolated nucleic acid molecule which is DNA or RNA encoding a binding protein as 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.

According to one aspect of the invention, the invention also provides a vector comprising a nucleic acid molecule 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 presence or absence of said genetic information may be selected) or an expression vector (i.e. a vector comprising the necessary genetic elements to allow expression of the genetic information of said vector in a cell). Thus, a cloning vector may comprise a selectable marker, and an origin of replication compatible with the cell type to which the cloning vector is directed, while an expression vector comprises the regulatory elements necessary to effect expression in a given 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 present invention are capable of autonomous replication and therefore are capable of providing 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 may or may not be integrated into the genome of the host cell when introduced into the host cell. Vectors typically carry a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells.

In another aspect, the present invention also provides a host cell transformed with the vector as described above.

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 subtilis, 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.

According to one aspect of the invention, the invention also provides a method of producing the above binding protein, comprising the steps of:

the host cells described above are cultured under suitable culture conditions and the produced binding protein is recovered from the culture 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 control 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.

According to an aspect of the invention, the invention also provides the use of a binding protein as described above for the preparation of a diagnostic agent or kit for the diagnosis of heart failure and for the assessment of heart function.

According to an aspect of the present invention, there is also provided a method of detecting ST2 in a test sample, comprising:

a) Contacting the ST2 antigen in the test sample with a binding protein as described above under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and

b) Detecting the presence of the immune complex.

In the above detection method, the presence of the complex in step b) is indicative of the presence of the ST2 in the test sample.

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 some embodiments, in step a), a second antibody is further included in the immune complex, which second antibody binds to the ST2;

in this embodiment, the binding protein is in the form of a primary antibody that forms a partner antibody with the secondary antibody for binding to a different epitope of ST 2.

In some embodiments, the binding protein can be labeled with an indicator that indicates the strength of the signal, such that the complex is readily detected.

In some embodiments, in step a), a second antibody is further included in the immune complex, which second antibody binds to the ST2 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 comprises Alexa 350, alexa 405, alexa 430, alexa488, alexa 555, alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY650/665, 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-nitrobenzo-2-oxa-1,3-diazole), oregon Green 488, oregon Green 500, oregon Green514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl Blue, brilliant cresol Blue, p-aminobenzoic acid, erythrosine, phthalocyanine, and the like azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyldiamine europium, europium cryptate or chelate, diamine, bispyanine glycoside, la Jolla Blue dye, allophycocyanin, allocyannin 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 comprises ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ⁹⁴ mTc、 ⁹⁴ Tc、 ⁹⁹ mTc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154-158 Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ⁵² mMn、 ⁵⁵ Co、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ⁸² mRb and ⁸³ sr.

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.

According to one aspect of the invention, the invention also relates to a detection reagent or kit comprising a binding protein as described above.

In some embodiments, the reagent or kit further comprises one or more of a pharmaceutically acceptable excipient, buffer, stabilizer, diluent, or carrier.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Example 1

This example provides an exemplary method for the preparation of recombinant antibodies against ST 2.

1. Construction of expression plasmids

Restriction enzyme, prime Star DNA polymerase, was purchased from Takara in this example. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART ^TM 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.

1.1 preparation of Anti-ST2 antibody Gene

mRNA is extracted from a hybridoma cell strain secreting Anti-ST2 monoclonal antibody, a DNA product is obtained by RT-PCR method, the product is added with A by rTaq DNA polymerase for reaction and then inserted into a pMD-18T vector, the product is transformed into DH5 alpha competent cells, after colonies are grown out, 4 clones of the Heavy Chain and Light Chain gene clones are respectively taken and sent to a gene sequencing company for sequencing.

1.2 sequence analysis of Anti-ST 2F 9 antibody variable region genes

Putting the gene sequence obtained by sequencing in a Kabat 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 a gene fragment amplified by Light Chain, the VL gene sequence is 321bp, belongs to VkII gene family, and a leader peptide sequence with the front part of 57bp is arranged; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 345bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

1.3 construction of recombinant antibody expression plasmid

pcDNA ^TM 3.4

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 pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody variable region gene in the pMD-18T, VL and VH gene specific primers of the Anti-ST211F9 antibody are designed, two ends of the primers are respectively provided with HindIII and EcoRI enzyme cutting sites and protective bases, and a 0.73KB Light Chain gene fragment and a 1.4KB Heavy Chain gene fragment are amplified by a PCR amplification method.

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. 2. Stable cell line selection

2.1 transient transfection of recombinant antibody expression plasmids into CHO cells and determination of expression plasmid Activity

Plasmid is diluted to 40ug/100ul with ultrapure water, and CHO cells are regulated at 1.43X 10 ⁷ cells/ml are put into a centrifuge tube, 100ul of plasmid is mixed with 700ul of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, sampling and counting are carried out on days 3, 5 and 7, and sampling and detecting are carried out on day 7.

Coating liquid (main component NaHCO) ₃ ) Diluting goat anti-mouse IgG 1ug/ml for microplate coating, 100uL per well, and standing overnight at 4 ℃; the next day, washing liquid (main component Na) ₂ HPO ₄ NaCl) for 2 times, patting dry; add blocking solution (20% BSA +80% PBS), 120uL per well, 37 deg.C, 1h, pat dry; adding diluted cell supernatant at 100 uL/hole at 37 deg.C for 60min; throwing off liquid in the plate, patting dry, adding 20% of mouse negative blood, sealing each hole at 120ul,37 ℃ for 1h; throwing off the liquid in the plate, patting dry, adding diluted ST2 antigen, 100uL per hole, 37 ℃,40min; washing with washing solution for 5 times, and drying; adding intoAn ST2 monoclonal antibody marked with HRP, wherein each hole is 100uL,37 ℃ and 30min; adding a developing solution A (50 uL/hole), adding a developing solution B (50 uL/hole), and carrying out 10min; adding stop solution at 50 uL/hole; OD readings were taken at 450nm (reference 630 nm) on the microplate reader. The results showed that the OD of the reaction after the cell supernatant was diluted 1000 times was still greater than 1.0, and the OD of the reaction without the cell supernatant was less than 0.1, indicating that the antibody produced after the plasmid transient transformation was active against ST2 antigen.

Remarking: liquid A (main components of citric acid, sodium acetate, acetanilide and carbamide peroxide); liquid B (main component citric acid + EDTA & 2Na + TMB + concentrated HCl); stop solution (EDTA.2 Na + concentrated H) ₂ SO ₄ )

2.2 linearization of recombinant antibody expression plasmids

The following reagents were prepared: 50ul Buffer, 100 ug/tube DNA, 10ul PvuI enzyme and sterile water are supplemented to 500ul, and the mixture is subjected to enzyme digestion in water bath at 37 ℃ overnight; extraction was performed sequentially with equal volumes of phenol/chloroform/isoamyl alcohol (lower layer) 25, followed by chloroform (aqueous phase); precipitating with 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume 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.

2.3 Stable transfection of recombinant antibody expression plasmids, pressurized selection of Stable cell lines

Plasmid is diluted to 40ug/100ul with ultrapure water, and CHO cells are regulated at 1.43X 10 ⁷ Putting cells/ml in a centrifuge tube, mixing 100ul plasmid and 700ul cells, transferring into an electric rotating cup, electrically rotating, and counting the next day; 25umol/L MSX 96-well pressure culture for about 25 days.

Observing the marked clone holes with the cells under a microscope, and recording the confluence degree; taking culture supernatant, and carrying out sample 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 10 ⁶ cells/ml,2.2ml, cell density 0.3X 10 ⁶ cell/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.

3. Recombinant antibody production

3.1 cell expansion culture

After the cells were recovered, they were cultured in 125ml size shake flasks, inoculated with 30ml Dynamis medium at a culture medium volume of 100%, and placed in a shaker at a rotation speed of 120r/min and a temperature of 37 ℃ with 8% carbon dioxide. Culturing for 72h, inoculating and expanding at an inoculation density of 50 ten thousand cells/ml, the expanding volume being calculated according to the production requirements, the medium being 100% Dynamis 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.

3.2 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 manner: daily feeding was started at 72h in the flask, 3% of the initial culture volume was fed daily by HyCloneTM Cell BoostTM Feed 7a, one thousandth of the initial culture volume was fed daily by Feed 7b, and was continued 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. Mu.g of the purified antibody was subjected to reducing SDS-PAGE, and 4. Mu.g of an external control antibody was used as a control, and the electrophorogram is shown in the figure. Two bands are shown after reducing SDS-PAGE, 1 Mr is 50KD (heavy chain), and the sequence is shown as SEQ ID NO: 18; the other Mr is 28KD (light chain), and the sequence is shown in SEQ ID NO: 20.

Example 2

Antibody affinity analysis and activity identification

The resulting antibody Anti-ST 2F 11F9 of example 1 (having the heavy chain variable region and the light chain variable region as shown in SEQ ID NO:17 and SEQ ID NO: 19) was analyzed, and the complementarity determining regions of the heavy chain:

CDR-VH1, SEQ ID NO: 1;

CDR-VH2, SEQ ID NO: 2;

CDR-VH3, SEQ ID NO: 3;

complementarity determining regions of the light chain:

CDR-VL1, SEQ ID NO: 4;

CDR-VL2, SEQ ID NO: 5;

CDR-VL3, being SEQ ID NO: 6.

Light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4, the sequences are shown as SEQ ID NO 11-14 in sequence;

heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4, the sequences are shown as SEQ ID NO 7-10 in sequence.

1. Affinity assay

Using AMC sensor, the purified antibody is diluted to 10ug/ml with PBST, and ST2 antigen is diluted with PBST in gradient;

the operation flow is as follows: equilibration for 60s in buffer 1 (PBST), immobilized antibody for 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3 (PBST), and data output. (KD represents the equilibrium dissociation constant, i.e., affinity; kon represents the association rate; kdis represents the dissociation rate.) Table 1 shows the resulting affinity assay data.

Table 1 affinity assay data

Sample name	KD(M)	kon(1/Ms)	kdis(1/s)
				Control of	7.30E-09	9.90E+05	7.23E-03
Anti-ST2 11F9	7.61E-10	9.79E+05	7.45E-04

2. Activity identification

Coating liquid (main component NaHCO) ₃ ) Diluting goat anti-mouse IgG 1ug/ml for microplate coating, 100uL per well, and standing overnight at 4 ℃; the next day, washing liquid (main component Na) ₂ HPO ₄ NaCl) for 2 times, patting dry; add blocking solution (20% BSA +80% PBS), 120uL per well, 37 deg.C, 1h, pat dry; adding diluted purified antibody at 100 uL/hole, 37 deg.C for 60min; throwing off liquid in the plate, patting dry, adding 20% of mouse negative blood, sealing each hole at 120ul,37 ℃ for 1h; throwing off the liquid in the plate, patting dry, adding diluted ST2 antigen, 100uL per hole, 37 ℃,40min; washing with washing solution for 5 times, and drying; adding HRP-labeled ST2 monoclonal antibody (capable of pairing with purified antibody and obtained from Fipeng organism) at 100uL/well, 37 deg.C for 30min; adding a developing solution A (50 uL/hole), adding a developing solution B (50 uL/hole), and carrying out 10min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 uL/hole; OD reading is carried out at 450nm (reference 630 nm) on a microplate reader, and the obtained antibody activity analysis data is shown in Table 2.

TABLE 2 antibody Activity assay data

Sample concentration (ng/ml)	31.250	15.625	7.813	3.906	1.953	0.000
							Control	1.509	0.665	0.370	0.260	0.163	0.046
Anti-ST2 11F9	1.883	1.495	0.984	0.489	0.205	0.061

3. Stability analysis

And (3) naked antibody stability assessment:

placing the self-produced antibody in a temperature range of 4 ℃ (refrigerator), -80 ℃ (refrigerator) and a temperature range of 37 ℃ (thermostat) for 21 days, taking samples in 7 days, 14 days and 21 days for state observation, and performing activity detection on the samples in 21 days, wherein the result shows that under three examination conditions, no obvious protein state change is seen in 21 days of placing the antibody, and the activity does not show a descending trend along with the rise of the examination temperature, which indicates that the self-produced antibody is stable. Table 3 shows the results of the enzyme immunity activity test OD for 21 days.

TABLE 3 antibody stability assay data

Sample concentration (ng/ml)	31.25	15.625	0
				Samples at 4 ℃ for 21 days	1.829	1.358	0.094
21 day samples at-80 deg.C	1.835	1.331	0.083
				21 day samples at 37 deg.C	1.833	1.345	0.067

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> recombinant antibody of anti-long-term stimulation expression gene 2 protein

<130> P2021030CN01

<160> 20

<170> PatentIn version 3.5

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

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Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val

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Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

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Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

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Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

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

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Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

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

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Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

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Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

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Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu Asp Ile Thr Val

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Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

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Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

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Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

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Phe Gln Ile Asn Asn Leu Arg Asn Glu Asp Thr Ala Thr Tyr Phe Cys

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Ala Arg Asp Asp Gly Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

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Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro

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Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val

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Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser

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

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Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser

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Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val

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Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys

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Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys

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Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val

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

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Asp Val Glu Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln Phe

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Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp

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Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe

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Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys

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Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys

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Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu Asp

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Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys

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Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser

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Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr

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Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Asp

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Glu Asp Met Ala Thr Tyr Phe Cys Leu Gln His Ser Tyr Leu Pro Leu

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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly

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Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

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Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

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Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

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Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

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Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

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Phe Asn Arg Asn Glu Cys

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Claims

1. An isolated binding protein comprising an ST2 antigen binding domain, wherein said antigen binding domain comprises at least one complementarity determining region selected from the group consisting of amino acid sequences recited in seq id nos:

2. The isolated binding protein comprising an antigen binding domain according to claim 1, wherein at least 3 CDRs are included in the binding protein; alternatively, the binding protein comprises at least 6 CDRs;

preferably, the binding protein is one of an antibody, F (ab ') 2, fab', fab, fv, scFv, diabody, and antibody minimal recognition unit;

preferably, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 having the sequences shown in SEQ ID NO 11-14 or at least 90% homologous thereto in sequence, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 having the sequences shown in SEQ ID NO 7-10 or at least 90% homologous thereto in sequence.

3. The isolated binding protein comprising an antigen binding domain according to claim 1 or 2, wherein the binding protein further comprises an antibody constant region sequence;

preferably, the constant region sequence is selected from the group consisting of sequences of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, igD constant regions;

preferably, the species of the constant region is from cattle, horses, cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese or humans;

preferably, the constant region is derived from a mouse;

the heavy chain constant region sequence is shown as SEQ ID NO: shown at 15.

4. An isolated nucleic acid molecule which is DNA or RNA encoding the binding protein of any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell transformed with the vector of claim 5;

preferably, the host cell is a mammalian cell;

preferably, the host cell is a Chinese Hamster Ovary (CHO) cell, a HeLa cell, a young hamster kidney cell, an NS0 mouse myeloma cell, or a variety of other cells.

7. A method of producing a binding protein according to any one of claims 1 to 3, comprising the steps of: culturing the host cell of claim 6 under suitable culture conditions and recovering the produced binding protein from the culture medium or from the cultured host cell.

8. Use of a binding protein according to any one of claims 1 to 3 for the preparation of a diagnostic agent for the diagnosis of heart failure and for the assessment of heart function.

9. A method of detecting ST2 in a test sample, comprising:

a) Contacting the ST2 antigen in the test sample with the binding protein of claim 3 under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and

b) Detecting the presence of said immune complex, the presence of said complex indicating the presence of said ST2 in said test sample;

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

preferably, in step a), a second antibody is further included in the immune complex, said second antibody binding to the ST 2.

10. A reagent or kit comprising a binding protein according to any one of claims 1 to 3;

preferably, the reagent or kit further comprises one or more of a pharmaceutically acceptable excipient, buffer, stabilizer, diluent or carrier.