UMtCK antibodies or antigen binding fragments thereof

Abstract

The present disclosure relates generally to the technical field of laboratory medicine, in particular to a uMtCK antibody or an antigen binding fragment thereof. The uMtCK detection kit can be specifically combined with uMtCK, is basically not subjected to cross reaction with CK-MM, CK-BB or CK-MB, has relatively high affinity, and has a relatively good application prospect in the field of uMtCK detection.

Description

Technical Field

[0001] The present disclosure generally relates to the field of laboratory medicine technology, and in particular, to a uMtCK antibody or an antigen-binding fragment thereof. Background Art

[0002] Creatine kinase (CK, EC 2.7.3.2) belongs to the phosphokinase family and plays an important role in the energy metabolism of vertebrates by catalyzing reversible phosphorylation reactions. CK has four isoenzyme forms, including muscle type (CK-MM), brain type (CK-BB), hybrid type (CK-MB) and mitochondrial type (MtCK), which are mainly present in brain tissue, skeletal muscle and myocardium. Due to the important role of creatine kinase in cellular energy cycle, it plays an important role in regulating glycolysis, phagocytosis of phagocytes, release of neurotransmitters and transmembrane transport of ions. One of the important indicators for clinical diagnosis of myocardial infarction is the content of CK in serum, and CK is also presumed to be a potential clinical biomarker and therapeutic target for many diseases. Human mitochondrial universal isoform of creatine kinase (uMtCK) plays an important role in mitochondrial oxidative phosphorylation and cell apoptosis. Recent studies have found that uMtCK has abnormal expression in many cancers.

[0003] At present, the main method for measuring uMtCK is electrophoresis, which requires enzyme activity measurement to display isoenzymes on different electrophoretic bands. However, uMtCK is sensitive to oxygen free radicals and is easily inactivated by free radicals, which may be the reason for the low detection rate of uMtCK in human serum. In addition, although immunological detection can avoid the disadvantages of electrophoresis, it is not easy to provide antibodies that can specifically recognize uMtCK due to the similar structures of the four isoenzymes of CK. Summary of the invention

[0004] This disclosure covers the following technical solutions:

[0005] (1) uMtCK antibody or antigen-binding fragment thereof, comprising a heavy chain complementary determining region whose amino acid sequence is sequentially shown in SEQ ID NOs: 1 to 3 and a light chain complementary determining region whose amino acid sequence is sequentially shown in SEQ ID NOs: 4 to 6.

[0006] (2) The uMtCK antibody or antigen-binding fragment thereof according to (1), comprising a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8.

[0007] (3) An isolated nucleic acid that can encode the uMtCK antibody or antigen-binding fragment thereof described in (1) or (2).

[0008] (4) A vector comprising the nucleic acid described in (3).

[0009] (5) A host cell comprising the nucleic acid described in (3) or transformed with the vector described in (4).

[0010] (6) A method for preparing the uMtCK antibody or antigen-binding fragment thereof described in (1) or (2), comprising culturing the host cell described in (5) under appropriate conditions, and recovering the target product from the cell culture.

[0011] (7) An antibody combination product comprising the uMtCK antibody or antigen-binding fragment thereof described in (1) or (2) and a second uMtCK antibody produced by Huamei with the product number CSB-MA149995.

[0012] (8) A kit comprising the uMtCK antibody or antigen-binding fragment thereof described in (1) or (2), or the antibody combination product described in (7).

[0013] (9) Use of the uMtCK antibody or antigen-binding fragment thereof according to (1) or (2), or the antibody combination product according to (7) in the preparation of a tumor diagnostic reagent or kit.

[0014] (10) According to the use described in (9), the tumor is selected from: breast cancer, gastric cancer, prostate cancer, cervical cancer, liver cancer, ovarian cancer, lung cancer, lymphoma, colorectal cancer, nasopharyngeal carcinoma and osteosarcoma.

[0015] Beneficial effects:

[0016] The antibodies or antigen-binding fragments thereof provided by the present disclosure can specifically bind to uMtCK, have little cross-reaction with CK-MM, CK-BB or CK-MB, and have high affinity, and have good application prospects in the field of uMtCK detection. DETAILED DESCRIPTION

[0017] References to embodiments of the present disclosure will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0018] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present disclosure is the same as that commonly understood by those of ordinary skill in the art to which the present disclosure belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present disclosure. The terms used herein in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0019] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0020] The terms "and / or", "or / and", and "and / or" used herein have a selection range that includes any one of two or more related listed items, and also includes any and all combinations of related listed items, wherein the any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present disclosure, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0021] As used in this disclosure, the terms "comprising," "including," and "including" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0022]

[0013] Numerical ranges recited in this disclosure as endpoints include all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0023] As used herein, reference to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0024] The present disclosure relates to concentration values, and the meaning includes fluctuations within a certain range. For example, fluctuations can be allowed within a corresponding precision range. For example, 2%, a fluctuation within the range of ±0.1% can be allowed. For values ​​that are large or do not require too fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, fluctuations within the range of ±1%, ±2%, ±5%, etc. can be allowed. Involving molecular weight, the meaning is allowed to include fluctuations of ±10%.

[0025] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0026] In the present disclosure, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0027] In the present disclosure, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0028] In the present disclosure, "preferred", "better", "more preferred", and "suitable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present disclosure. In the present disclosure, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0029] In the present disclosure, the technical term "antibody" refers to a protein that binds to a specific antigen, and generally refers to all proteins and protein fragments that contain a complementary determining region (CDR region), especially full-length antibodies or antibody functional fragments. The term "full-length antibody" includes polyclonal antibodies and monoclonal antibodies, and the term "antibody functional fragment" refers to a substance that contains part or all of the antibody CDR, which lacks at least some of the amino acids present in the full-length chain but is still able to specifically bind to the antigen.

[0030] In the present disclosure, the term "complementarity determining region" or "CDR" refers to the highly variable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed" US Department of Health and Human Services, NIH, 1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. Here, depending on the context, the terms "CDR" and "CDRs" are used to refer to a region comprising one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody to its recognized antigen or epitope.

[0031] As used herein, the term "signal substance" refers to any component, molecule, functional group, compound, fragment or part that can provide a signal and be detected. In some embodiments, the detection entity is provided or used alone. In some embodiments, the detection entity is provided and / or used in conjunction with (e.g., conjugated to) another agent. Examples of detection entities include, but are not limited to, various ligands, radionuclides (e.g., 3 H. 14 C. 18 F. 19 F. 32 P. 35 S. 135 I. 125 I. 123 I. 64 Cu, 187 Re, 111 In, 90 Y. 99 mTc, 177 Lu, 89 Zr, etc.), fluorescent dyes, chemiluminescent agents (such as acridinium esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (such as gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (such as dyes, colloidal gold, etc.), biotin, digoxigenin, haptens, and proteins to which antisera or monoclonal antibodies can be applied.

[0032] All documents mentioned in the present disclosure are cited as references in the present disclosure, just as each document is cited as a reference individually. Unless they conflict with the invention objectives and / or technical solutions of the present disclosure, the cited documents involved in the present disclosure are cited with all their contents and all their purposes. When the present disclosure involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present disclosure involves cited documents, the examples and preferred methods of the cited relevant technical features may also be incorporated into the present disclosure as references, but are limited to the implementation of the present disclosure. It should be understood that when the cited content conflicts with the description in the present disclosure, the present disclosure shall prevail or be modified adaptively according to the description of the present disclosure.

[0033] The first aspect of the present disclosure provides a uMtCK antibody or an antigen-binding fragment thereof, which comprises a heavy chain complementary determining region whose amino acid sequence is sequentially shown in SEQ ID NOs: 1 to 3 and a light chain complementary determining region whose amino acid sequence is sequentially shown in SEQ ID NOs: 4 to 6.

[0034] In some embodiments, the uMtCK antibody or antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:8.

[0035] According to the conventional understanding of those skilled in the art, it should be considered that variants of the above amino acid sequences are also within the scope of the present disclosure. The uMtCK antibody or antigen-binding fragment variant disclosed herein comprises mutations of up to 1, 2 or 3 amino acids occurring in the CDR region (if any) compared to any polypeptide of SEQ ID NO: 1 to SEQ ID NO: 6, respectively; or, the variant has a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity relative to the overall sequence of SEQ ID NO: 7 to SEQ ID NO: 8. The mutation may be a substitution, deletion or addition of an amino acid or any combination thereof; preferably, the mutation is a conservative substitution.

[0036] "Conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main-chain conformation and rigidity, etc.), such that changes can frequently be made without altering the biological activity of the protein.

[0037] The substitutions generally considered as conservative substitutions are substitutions between aliphatic amino acids Ala, Val, Leu and Ile, interchanges of hydroxyl residues Ser and Thr, exchanges of acidic residues Asp and Glu, substitutions between amide residues Asn and Gln, exchanges of basic residues Lys and Arg, and substitutions between aromatic residues Phe and Tyr, etc. It is known to those skilled in the art that, in general, single amino acid substitutions in non-essential regions of polypeptides do not substantially change biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, substitutions of amino acids with similar structures or functions are unlikely to destroy biological activity.

[0038] In some embodiments, the antigen-binding fragment is one of F(ab')2, Fab, scFv and bispecific antibody.

[0039] The term "F(ab')2" is obtained by digesting the entire full-length antibody with pepsin to remove most of the Fc region while leaving some of the hinge region intact. The F(ab')2 fragment has two antigen-binding Fab parts linked together by a disulfide bond, so the F(ab')2 fragment is a bivalent antibody. For example, the F(ab')2 prepared from an IgG antibody has a molecular weight of about 110 kDa.

[0040] The term "Fab" refers to the antibody structure that can still bind to the antigen, is monovalent and does not contain the Fc portion. Papain digestion of a full-length antibody yields two Fab fragments and one Fc fragment, each of which is approximately 50 kDa.

[0041] The term "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. In the present disclosure, the term "linker" may be a flexible or rigid peptide, usually flexible, which can reduce the steric hindrance between the fusion protein and the target protein, thereby being more conducive to the correct folding of the protein. For example, it is composed of a repeated GGGGS amino acid sequence or a variant thereof, and for example, a variant using 1 to 4 repetitions (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. The linker peptide is usually flexible and can reduce the steric hindrance between the fusion protein and the target protein, thereby being more conducive to the correct folding of the protein.

[0042] In some embodiments, the uMtCK antibody or antigen-binding fragment thereof has a constant region.

[0043] In some embodiments, the species of origin of the constant region is mouse, rat, guinea pig, hamster, rabbit, horse, sheep, goat, donkey, cow, chicken, duck and human.

[0044] In some embodiments, the heavy chain constant region sequence of the antibody or antigen-binding fragment thereof is selected from human IgG1.

[0045] In some embodiments, the heavy chain constant region sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 7. In some embodiments, the light chain constant region sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 8.

[0046] A second aspect of the present disclosure relates to an isolated nucleic acid capable of encoding the uMtCK antibody or antigen-binding fragment thereof as described above.

[0047] The term "isolated nucleic acid" herein refers to a deoxyribonucleic acid or ribonucleic acid polymer in single-stranded or double-stranded form. The isolated nucleic acid includes an RNA genomic sequence, a DNA (gDNA and cDNA) or an RNA sequence transcribed from a DNA, and, unless otherwise specified, the polypeptide also includes analogs of natural polynucleotides, sugars, or base changes. According to one aspect of the present disclosure, the polynucleotide is a light chain polynucleotide.

[0048] The isolated nucleic acid includes a nucleotide sequence encoding the amino acid sequence of the protein complex, and also includes a nucleotide sequence complementary thereto. The complementary sequence includes a completely complementary sequence and a substantially complementary sequence, which refers to a sequence that can hybridize with the nucleotide sequence encoding the amino acid sequence of the protein complex under stringent conditions known in the art.

[0049] Moreover, the nucleotide sequence encoding the amino acid sequence of the protein complex may be altered or mutated. The alteration includes addition, deletion, or non-conservative substitution or conservative substitution. The polynucleotide encoding the amino acid sequence of the protein complex may be interpreted as including a nucleotide sequence that has substantial identity with respect to the isolated nucleic acid. The substantial identity aligns the nucleotide sequence with another random sequence in a manner that maximizes their correspondence, and when the aligned sequences are analyzed using algorithms commonly used in the art, the sequence may show greater than 80% homology, for example, greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0050] In addition, the codons of the nucleic acid corresponding to the uMtCK antibody or its antigen-binding fragment can be optimized according to the species of the host cell.

[0051] The third aspect of the present disclosure relates to a vector comprising the nucleic acid as described above.

[0052] The term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomas (such as SV40). In some embodiments, the vector disclosed herein contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0053] In the present disclosure, the vector can be a composition, such as a mixture of multiple plasmids, and different plasmids carry a portion of the antibody or its antigen-binding fragment, for example, nucleic acids encoding antibody heavy chains and light chains are independently inserted into different vectors.

[0054] A fourth aspect of the present disclosure relates to a host cell comprising the nucleic acid as described above, or transformed with the vector as described above.

[0055] Host cells or cell lines suitable for expressing the antigen-binding proteins of the present disclosure include mammalian cells such as NS0, Sp2 / 0, CHO, COS, HEK, fibroblasts and myeloma cells. Human cells can be used, thus allowing the molecules to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines can be used. The selection of suitable mammalian host cells, as well as methods for transformation, culture, amplification, screening and product production and purification, are known in the art.

[0056] It may be demonstrated that bacterial cells may be used as host cells suitable for expressing proteins or other embodiments of the present disclosure. However, since proteins expressed in bacterial cells tend to be in an unfolded form or in an incorrectly folded form or in a non-glycosylated form, any protein produced in bacterial cells must be screened to retain antigen binding ability. If the molecule expressed by the bacterial cell is produced in a properly folded form, the bacterial cell would be the desired host, or, in an alternative embodiment, the molecule may be expressed in a bacterial host and subsequently refolded. For example, various strains of E. coli used for expression are well known host cells in the biotechnology field. Various strains of Bacillus subtilis, Streptomyces, other Bacillus species, etc. may also be used in the method.

[0057] If desired, yeast cell strains known to those skilled in the art as well as insect cells, such as Drosophila and Lepidoptera insects and viral expression systems can also be used as host cells.

[0058] In some embodiments, the nucleic acid is inserted into the cell genome and can be stably expressed. The insertion method can be selected from the above-mentioned vectors, or the nucleic acid is not connected to the vector and directly transferred into the cell (such as liposome-mediated transfection technology).

[0059] The fifth aspect of the present disclosure relates to a method for preparing the uMtCK antibody or antigen-binding fragment thereof as described above, which comprises culturing the host cell as described above under suitable conditions, and recovering the target product from the cell culture.

[0060] The culture method disclosed herein is generally a serum-free culture method, and cells are generally cultured in a serum-free suspension. Similarly, once the antibodies disclosed herein are produced, they can be purified from the cell culture contents according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc. Such techniques are within the scope of conventional techniques in this field and do not limit the present disclosure. Cultivating host cells can also be understood as expressing antibodies in animals (particularly transgenic animals or nude mice). This involves an expression system utilizing an animal casein promoter, which, when transgenically incorporated into a mammal, allows female animals to produce the desired recombinant protein in their milk. The culture fluid secreting the antibody can be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected using SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product should be immediately frozen, such as at -70°C, or freeze-dried.

[0061] The sixth aspect of the present disclosure relates to an antibody combination product, which comprises the uMtCK antibody or antigen-binding fragment thereof as described above and a second uMtCK antibody produced by Huamei with a product number of CSB-MA149995.

[0062] The above-mentioned antibody combination product is a paired antibody. The two uMtCK antibodies can bind to different epitopes of uMtCK, and then use double antibody sandwich technology to detect it and obtain higher detection specificity.

[0063] A seventh aspect of the present disclosure relates to a kit comprising the uMtCK antibody or antigen-binding fragment thereof as described above, or the antibody combination product as described above.

[0064] The term "kit" in the present disclosure may refer to any article (e.g., package or container) comprising at least one device, which includes a detection agent as described in the present disclosure. The kit may further include instructions for use, supplementary reagents and / or components or assemblies used in the methods or steps thereof described in the present disclosure.

[0065] In some embodiments, the kit comprises the antibody combination product as described above; wherein the uMtCK antibody or the antigen-binding fragment thereof is labeled with a signal substance, and the second uMtCK antibody is conjugated to a solid phase carrier.

[0066] Signal substances include any moiety that can be detected using an assay, for example due to its specific functional properties and / or chemical characteristics. Non-limiting examples of such agents include enzymes, radioactive labels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands (such as biotin). In some embodiments of the present disclosure, the signal substance is selected from luminol and its derivatives, isoluminol and its derivatives, alkaline phosphatase, and horseradish peroxidase.

[0067] As required, the kit may also include any one or more of the following components: sample pretreatment reagents (such as sample purification and enrichment reagents, lysate, etc.), cleaning solutions (such as PBS, etc.), buffers (such as phosphate buffer), colorimetric reagents for signal substances (such as horseradish peroxidase, the colorimetric reagent is ECL), preservatives (sodium azide, sodium nitrite, sodium benzoate, Proclin series, potassium sorbate, any one or a mixture of two or more). These components can be packaged separately, or two or more can be premixed for ease of use / storage.

[0068] In the present disclosure, the solid phase carrier can be a substance used to conjugate antibodies and facilitate detection. In some embodiments, the solid phase carrier is a plastic, a microparticle or a membrane carrier; the plastic can be polystyrene; the membrane carrier can be a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane. In some embodiments, the solid phase carrier is selected from a test tube, an EP tube, a multi-well plate, a chromatography column, and a micro-reaction plate concave hole.

[0069] In the present disclosure, the term "microparticles" may be spheres, nearly spheres, cubes, polyhedrons or irregular shapes. The diameter of the microspheres is preferably 10 nm to 1 mm, such as 100 nm, 500 nm, 1 μm, 10 μm, 100 μm, 500 μm; preferably 400 nm to 10 μm. The microparticles are preferably magnetic microparticles, and their components contain magnetic substances. The magnetic substance may be a metal (metallic element or alloy), a non-metal, or a composite formed by a metal and a non-metal. Metals include iron, aluminum nickel cobalt metals, etc.; non-metals include ferrite non-metals (preferably Fe2O3 or Fe3O4 magnetic nanoparticles); composites formed by metals and non-metals include neodymium iron boron rubber magnetic composite materials.

[0070] In some specific embodiments, the solid phase carrier is a membrane carrier, and is used for lateral flow detection. Lateral flow detection is also referred to as lateral flow immunoassay determination (LFIA), laminar flow, immunochromatographic determination or test strip test. LFIA test is a form of immunoassay, in which the test sample flows along a solid matrix by capillary action. After the sample is applied to the test strip, it encounters a colored reagent (generally including an antibody specific for the test target antigen) that is bonded to particulates, the reagent is mixed with the sample and passes through a substrate (for example, specific for the detection label on the detectable label on the target nucleic acid or the complementary nucleic acid of the target nucleic acid) contacted with the line or region pretreated with the antibody or with conjugated as herein described or unconjugated antibody pretreatment. According to the level of the target present in the sample, the signal substance (usually a colored substance) can be captured and combined in the test line or test zone. LFIA is essentially applicable to immunoassays operated along a single axis, to adapt to the test strip format or dipstick format. Dip test strips are extremely versatile and can be easily modified by those skilled in the art to detect a variety of antigens from fluid samples such as urine, blood, water, and / or homogenized tissue samples. Typically, a lateral flow test strip consists of: a sample pad, a conjugate pad, a detection membrane, and an optional absorbent pad.

[0071] An eighth aspect of the present disclosure relates to the use of a uMtCK antibody or an antigen-binding fragment thereof, or the antibody combination product as described above, in the preparation of a tumor diagnostic reagent or kit.

[0072] In some embodiments, the tumor is selected from the group consisting of breast cancer, gastric cancer, prostate cancer, cervical cancer, liver cancer, ovarian cancer, lung cancer, lymphoma, colorectal cancer, nasopharyngeal carcinoma, and osteosarcoma.

[0073] The embodiments of the present disclosure will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance given in the present disclosure, and can also be based on the experimental manuals or conventional conditions in the art, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0074] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0075] Example 1 Preparation and titer evaluation of antibodies

[0076] 1. Antibody Preparation

[0077] Synthetic gene: According to the amino acid sequence of SEQ ID NO:7-8, it is determined that the signal peptide sequence and the target protein sequence must be included (the heavy chain signal peptide is shown in SEQ ID NO:11, and the light chain signal peptide is shown in SEQ ID NO:12), and then Nanjing GenScript Biotech Co., Ltd. provides gene synthesis services (including codon optimization). The upstream and downstream of the antibody light and heavy chains are HindIII and EcoRI restriction sites, and the downstream of the target fragment contains a stop codon. The light and heavy chain genes are synthesized into the pUC57 vector respectively, and finally pUC57-AL and pUC57-AH are obtained.

[0078] Recombinant plasmid construction: First, the light chain plasmid pUC57-AL and the vector pCDNA3.1 (from Invitrogen) were double-digested with HindIII and EcoRI. The enzyme digestion system should be carried out according to the instructions of the Invitrogen manufacturer. After incubation at 37°C for 1 hour, the double-digestion product was subjected to 1% agarose gel electrophoresis. After identification on the gel imaging instrument, the gel block containing the target fragment was cut out and recovered with a gel recovery kit. The target fragment was set aside. T4 DNA ligase was added, and the ligation system was incubated at 22°C for 1 hour, and then transformed into E. coli Top10 competent state, coated on an ampicillin resistant plate, and cultured at 37°C for 12-16 hours.

[0079] Single colonies were selected for PCR identification. The reaction system should be carried out according to the instructions of Invitrogen manufacturer. The identification primers were selected as universal primers for upstream and downstream of the vector. PCR reaction was carried out at 95℃ for 30s, 60℃ for 30s, and 72℃ for 5s.

[0080] 60s, 30 cycles, 72℃60s program for amplification. The PCR reaction system was subjected to 1% agarose gel electrophoresis, and positive clones were determined on a gel analyzer. The identified positive clones were inoculated into LB medium, cultured at 37℃ for 12-16h, and the plasmid was extracted using an Omega general extraction kit. The plasmid was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. If the sequence was consistent with the synthesized gene sequence, it indicated that the light chain recombinant plasmid pCDNA3.1-AL was successfully constructed.

[0081] Similarly, the heavy chain recombinant plasmid pCDNA3.1-AH can be obtained.

[0082] Recombinant plasmid extraction: follow the instructions of the Omega endotoxin-free plasmid extraction kit to extract the recombinant plasmid and detect the concentration on a UV spectrophotometer.

[0083] Transfection of mammalian cells: The recombinant plasmid was transfected into mammalian CHO cells according to the liposome transfection instructions of Invitrogen, wherein the mass ratio of light chain to heavy chain recombinant plasmid was 2:1, the mass ratio of transfection reagent PEI to expression plasmid was 3:1, the subculture density was 0.5E6, and the culture medium used was Maibang MaxY medium; after culturing at 37°C, 5% CO2 in a shaker for 72 hours, feed was added at 5% of the original volume, and then added every other day until the sample was collected on the 14th day.

[0084] Protein purification: Follow the instructions of the GE AKTA Pure protein separation and purification system and purify through the A column to obtain the initially purified antibody. First, balance the column with 20mM sodium phosphate, 20mM sodium citrate, pH 7.5, and then load the supernatant of the expression sample after centrifugation onto the Protein A column (HiTrap Protein a FF, 5mL, GE Healthcare). After washing with the same buffer, use 20mM sodium phosphate, 100mM sodium chloride, 100mM glycine pH 3.0 for stepwise elution. The eluted antibody was neutralized with 0.5M sodium phosphate pH 8.0 (1:10), concentrated, and the purified antibody was obtained after changing the solution.

[0085] 2. Antibody titer assessment

[0086] uMtck-1 antibody: purchased from Wuhan Huamei Biotechnology

[0087] uMtck-2 antibody: the antibody prepared by the above method

[0088] uMtck-3 antibody: Wuhan Cloud Clone Biotechnology

[0089] uMtck Antigen: Wuhan Cloud Clone Biology

[0090] Antibodies from different manufacturers were coated on magnetic microspheres, and luminescent markers were used to label the ubiquitous mitochondrial creatine kinase antigen.

[0091] Methods for testing the affinity between antibodies from different manufacturers and uMtck antigen:

[0092] (1) Add magnetic beads coated with antibodies from different manufacturers into different wells of reaction cups.

[0093] (2) adding the ubiquitous mitochondrial creatine kinase antigen labeled with a luminescent marker to the step 1) respectively, and incubating to allow the ubiquitous mitochondrial creatine kinase antigen labeled with a luminescent marker to bind to the antibody-coated magnetic microspheres; wherein the incubation temperature is 25° C. to 37° C., and the incubation time is 30 min.

[0094] (3) Magnetic separation to obtain a conjugate of the ubiquitous mitochondrial creatine kinase antigen labeled with a luminescent marker and the magnetic microspheres coated with an antibody.

[0095] (4) adding a luminescent excitation substrate to the conjugate obtained in step (3), detecting the intensity of the light signal, and comparing the luminescent intensity; the luminescent excitation substrate includes but is not limited to sodium hydroxide and hydrogen peroxide.

[0096] The data is as follows:

[0097] Antibody uMtck-1 uMtck-2 uMtck-3 Luminous intensity mean 542340 1346430 274367

[0098] As can be seen from the above table, the uMtck-2 antibody has the highest luminescence intensity and has the strongest affinity for binding to the uMtck antigen.

[0099] Example 2 Kit Composition Information

[0100] 1. Component A: solution of coated magnetic microspheres (Huamei; catalog number CSB-MA149995), the diluent is phosphate buffer, wherein:

[0101] a) uMtck-1 antibody specifically binds to ubiquitous mitochondrial creatine kinase (monoclonal preparation).

[0102] b) The magnetic microspheres are any of the following: magnetic microspheres modified with epoxy, amino, aldehyde, sulfonyl, carboxyl, amide, thiol or hydroxyl groups on the surface.

[0103] The working concentration of magnetic microspheres is 1-5 mg / ml, and the working concentration of uMtck-1 antibody is 5-10 μg / ml;

[0104] 2. Component B: uMtck-2 antibody marked with a luminescent marker (the antibody obtained in Example 1), wherein the working concentration of the luminescent marker is 1-1.50 μg / ml, and the working concentration of the uMtck-2 antibody is 300-500 ng / ml. The diluent is phosphate buffer.

[0105] The luminescent marker is any one of the following: luminol and its derivatives, isoluminol and its derivatives, alkaline phosphatase, horseradish peroxidase.

[0106] 3. Calibrator solution: Prepare low-concentration calibrator solution (concentration 39.06 ng / ml) and high-concentration calibrator solution (concentration 1250 ng / ml) in phosphate buffer.

[0107] 4. Reaction buffer: phosphate buffer.

[0108] Preparation of phosphate buffer: Dissolve 0.2g potassium dihydrogen phosphate in 800ml purified water, add 2.9g 12-hydrated disodium hydrogen phosphate, add BSA and preservatives, and dilute to 1000mL after complete dissolution, adjust pH to 7.3 for use. The mass volume percentage concentration of BSA is 0.1%-0.5%, and the preservative is any one of sodium azide, sodium nitrite, sodium benzoate, Proclin series, potassium sorbate, or a mixture of two or more.

[0109] Example 3 Kit Performance Verification

[0110] 1. Cross-reactivity verification

[0111] Use phosphate buffer to prepare 4 ml of low-concentration calibrator solution A1 (concentration 10.0 ng / ml), medium-concentration calibrator solution A2 (concentration 30.0 ng / ml), and high-concentration calibrator solution A3 (concentration 80.0 ng / ml). The three concentration gradient calibrators were divided into 4 equal parts, of which the first sample was not treated and served as the absolute control sample; the potential cross-substance solution was added to the second and third samples to prepare the test group samples; the potential cross-substance solvent was added to the fourth sample to prepare the relative control sample. The potential cross-substance was prepared as follows:

[0112] Preparation of CK-MB cross-interference stock solution: CK-MB antigen (purchased from Saitukang Biotechnology Co., Ltd.) was prepared with phosphate buffer to a concentration of 10 μg / ml;

[0113] Preparation of CK-BB cross-interference stock solution: Take CK-BB antigen (purchased from Saitukang Biotechnology Co., Ltd.) and prepare it with phosphate buffer to a concentration of 200 μg / ml;

[0114] Preparation of CK-MM cross-interference stock solution: Take CK-MM antigen (purchased from Saitukang Biotechnology Co., Ltd.) and prepare it with phosphate buffer to a concentration of 700 μg / ml.

[0115] Preparation of phosphate buffer: dissolve 0.2g potassium dihydrogen phosphate in 800ml purified water, add 2.9g 12-hydrated disodium hydrogen phosphate, add BSA and preservatives, and after complete dissolution, make up to 1000mL, adjust pH to 7.4 for use.

[0116] Prepare cross-substance control and test group samples C0, C1, C2 and C3 according to the following table, where C0 is used as the absolute control group sample, C1 is used as the relative control group sample, and C2 and C3 are used as the test group samples. Each sample is measured 3 times, and the mean value of the test results of each sample is recorded.

[0117]

[0118] The test results are as follows:

[0119]

[0120]

[0121]

[0122] According to the above experimental results, when the concentration of cross-interference substances CK-MB is 500ng / ml, CK-BB is 10μg / ml, and CK-MM is 35μg / ml, the interference rate on the detection results of 10.0ng / ml / 30.0ng / ml / 80.0ng / ml uMtCK calibrator is within 10%, and the detection results are basically not interfered by CK-MB, CK-BB, and CK-MM.

[0123] 2. Blank limit assessment

[0124] Select blank samples and repeat the test 20 times, calculate the average (M) and standard deviation (SD) of the RLU values ​​(relative luminescence value) of the 20 test results, and the blank limit is the concentration value determined by the MFI value of M+2SD. The test results of the blank limit are detailed in the table below.

[0125]

[0126]

[0127] From the results in the table, it can be seen that the blank limit detection concentration is 1.0ng / ml, and the maximum concentration that can be detected for the blank sample is only 1ng / ml.

[0128] 3. Sensitivity Assessment

[0129] The uMtCK standard (purchased from Wuhan Cloud Clone Bio) was used to prepare a ten-point curve with phosphate buffer, and the uMtCK kit in Example 2 was used for detection. The following table shows the RLU light intensity detection data of the ten-point curve, where the 0 point is phosphate buffer, and RLU: relative light unit.

[0130] Preparation concentration ng / ml RLU 0 4449 9.75 11028 19.5 20133 39.06 45359 78.125 101610 156.25 236305 312.5 496942 625 821110 1250 1221613 2500 1584605

[0131] From the above ten-point curve results, it can be seen that at a concentration of 0-2500 ng / ml, the difference in light intensity range is 1580156, and the concentration standards at each stage can be clearly distinguished.

[0132] 4. Repeatability Assessment

[0133] Three calibrators with different concentration levels were tested, 10 parallel tests were performed, and the coefficient of variation CV was calculated to characterize the repeatability of the tumor-related index detection kit.

[0134]

[0135]

[0136] From the above results, it can be seen that in the intra-batch experiment, the coefficient of variation CV of the low, medium and high calibrators were all within 6%.

[0137] 5. Positive detection rate assessment:

[0138] The uMtCK luminescence kit was used to detect 50 clinical samples, which were composed of serum collected from confirmed cancer patients and normal human serum.

[0139] Sensitivity = positive test value / total number of cancer samples × 100%;

[0140] Specificity = number of negative samples / number of normal samples × 100%.

[0141]

[0142] As can be seen from the above table, the antibodies disclosed herein have good sensitivity and specificity for the detection of clinical tumor samples.

[0143] The above-mentioned embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. uMtCK antibody or antigen-binding fragment thereof, characterized in that It comprises a heavy chain complementary determining region whose amino acid sequence is sequentially shown in SEQ ID NOs: 1 to 3 and a light chain complementary determining region whose amino acid sequence is sequentially shown in SEQ ID NOs: 4 to 6.

2. The uMtCK antibody or antigen-binding fragment thereof according to claim 1, characterized in that: It comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

8.

3. An isolated nucleic acid, characterized in that Capable of encoding the uMtCK antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A carrier, characterized in that Containing the nucleic acid according to claim 3.

5. A host cell, characterized in that Contains the nucleic acid of claim 3, or is transformed with the vector of claim 4.

6. A method for preparing the uMtCK antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The host cell of claim 5 is cultured under appropriate conditions, and the target product is recovered from the cell culture.

7. An antibody combination product, characterized in that: The method comprises the uMtCK antibody or antigen-binding fragment thereof according to claim 1 or 2 and a second uMtCK antibody produced by Huamei with the product number CSB-MA149995.

8. A kit, characterized in that A product comprising the uMtCK antibody or antigen-binding fragment thereof according to claim 1 or 2, or the antibody combination product according to claim 7.

9. Use of the uMtCK antibody or antigen-binding fragment thereof according to claim 1 or 2, or the antibody combination product according to claim 7 in the preparation of a tumor diagnostic reagent or kit.

10. The use according to claim 9, characterized in that: The tumor is selected from the group consisting of breast cancer, gastric cancer, prostate cancer, cervical cancer, liver cancer, ovarian cancer, lung cancer, lymphoma, colorectal cancer, nasopharyngeal carcinoma and osteosarcoma.

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