Antibodies or antigen-binding fragments that bind to thyroglobulin

Abstract

This invention relates to the field of immunoassay technology, and more specifically, to an antibody or antigen-binding fragment thereof capable of binding to thyroglobulin. The antibody or antigen-binding fragment thereof provided by this invention exhibits excellent affinity and sensitivity for thyroglobulin.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and more specifically, to an antibody or antigen-binding fragment thereof that binds to thyroglobulin. Background Technology

[0002] Thyroglobulin (TG) is a precursor protein for the synthesis and storage of thyroid hormones. It is mainly synthesized in thyroid follicular epithelial cells and is a carrier of iodotyrosine.

[0003] In clinical practice, elevated serum thyroglobulin levels can be seen in various thyroid diseases, such as goiter, thyroid tissue inflammation or damage, and hyperthyroidism. Furthermore, serum thyroglobulin is an important indicator after total thyroidectomy for diabetic thyroiditis (DTC) and... 131 After I treatment, monitor indicators of residual disease, recurrence, and metastasis, and simultaneously monitor those who have not undergone I treatment. 131 I-1 therapy also has some indicative effect on patients. Currently, immunoassay methods are the mainstream techniques for detecting thyroglobulin, such as fluorescence immunochromatography, colloidal gold immunochromatography, and chemiluminescence immunoassay, all of which require antibodies targeting thyroglobulin. Therefore, finding more antibodies targeting thyroglobulin has significant clinical value and market prospects.

[0004] The present invention aims to provide an antibody or antigen-binding fragment thereof capable of binding to thyroglobulin, providing a more efficient tool for the early diagnosis, treatment monitoring and prognostic assessment of thyroid diseases. Summary of the Invention

[0005] This invention covers the following technical solutions: According to one aspect of the present invention, there is an antibody or antigen-binding fragment thereof that binds to thyroglobulin, comprising a heavy chain CDR1 to CDR3 with the amino acid sequence shown in SEQ ID NO:1~3 in sequence; And light chain CDR1 with amino acid sequence as shown in SEQ ID NO:4, light chain CDR2 with amino acid sequence as shown in SEQ ID NO:5, and light chain CDR3 with amino acid sequence as shown in SEQ ID NO:6.

[0006] The antibody or antigen-binding fragment thereof that binds to thyroglobulin provided by this invention has excellent affinity for thyroglobulin. Detailed Implementation

[0007] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0008] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0009] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0010] The terms "and / or," "or / and," and "and / or" used in this invention encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary 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 using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this invention, the technical solution undoubtedly includes solutions connected using "logical AND," and also undoubtedly includes solutions connected using "logical OR." For example, "A and / or B" includes three parallel solutions: 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, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0011] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0012] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0013] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0014] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0015] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0016] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0017] All references to this invention are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this invention, the referenced documents are incorporated herein by reference in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the referenced technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this invention, this invention shall prevail or modifications shall be made adaptively according to the description in this invention.

[0018] In this invention, the technical term "antibody" refers to a protein that binds to a specific antigen, and broadly refers to all proteins and protein fragments containing a complementarity-determining region (CDR), especially full-length antibodies or antibody functional fragments. The term "full-length antibody" includes polyclonal antibodies and monoclonal antibodies. The term "antigen-binding fragment" refers to a substance containing part or all of the antibody's CDR, which lacks at least some amino acids present in the full-length chain but can still specifically bind to an antigen.

[0019] In this invention, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs. Commonly used CDR labeling methods in the art include the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences led to the creation of the Kabat-Man database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., US Department of Health and Human Services, NIH, 1991, and later editions). This invention uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods are also within the scope of this invention. Here, depending on the context, the terms "CDR" and "CDRs" are used to refer to regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of the antibody to the antigen or epitope it recognizes.

[0020] In this invention, the term "affinity constant" or "K" is used. D (M)” or “K” D The representation of " is K D (M) = Kd (or koff, the dissociation rate of a specific binding molecule-target molecule interaction) / Ka (or kon, the association rate of a specific binding molecule-target molecule interaction). K D The smaller the value of (M), the stronger the affinity between the antigen and antibody. D The values ​​can be determined using methods well-known in the art, such as surface plasmon resonance (SPR) measurement in a Biacore instrument or ELISA. SPR is based on optical principles; when biomolecules bind or dissociate on the sensor chip surface, they cause a change in the refractive index of the chip surface, resulting in a change in the SPR angle. Monitoring this change allows for real-time analysis of intermolecular interactions. An exemplary SPR process mainly includes the following steps: first, the antigen or antibody to be tested is immobilized on the sensor chip surface; then, different concentration gradients of the antibody or antigen to be tested are sequentially flowed through the chip as a mobile phase, and the dynamic changes in the SPR angle during molecule binding and dissociation are monitored in real time to generate binding / dissociation curves; finally, the curve data is fitted using software to calculate the binding rate constant (kon), dissociation rate constant (koff), and affinity constant (K). DThis allows for the assessment of antibody affinity. The principle of ELISA for detecting antibody affinity is that, in the presence of a small amount of antigen (or antibody), the antibody (or antigen) is serially diluted, and the concentration of the antigen-antibody complex is measured. When the complex concentration accounts for half of the total antigen concentration, the corresponding antibody concentration is the affinity constant K. D An exemplary ELISA procedure mainly includes the following steps: First, the antigen to be tested is coated onto an enzyme-labeled plate and unbound sites are blocked; then, the antibody to be tested is serially diluted and added to the wells for incubation, followed by washing away unbound components; next, enzyme-labeled secondary antibody is added for incubation and washing; after adding the chromogenic substrate and reacting, the absorbance (OD value) is measured; finally, by plotting the relationship between OD value and antibody concentration, the antibody concentration corresponding to the half-saturation point is determined, and the affinity constant is calculated to assess the affinity.

[0021] The first aspect of the present invention relates to an antibody or antigen-binding fragment thereof that binds to thyroglobulin, comprising a heavy chain CDR1 to CDR3 with the amino acid sequence shown in SEQ ID NO:1~3. And the light chain CDR1 with amino acid sequence as shown in SEQ ID NO:4, the light chain CDR2 with amino acid sequence as shown in SEQ ID NO:5, and the light chain CDR3 with amino acid sequence as shown in SEQ ID NO:6.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof that binds to thyroglobulin comprises a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:8.

[0023] To enhance or improve the binding affinity and / or other biological properties of an antibody, suitable modifications can be introduced into the nucleotide sequence encoding the antibody, or amino acid sequence variants of the antibody can be prepared via peptide synthesis. Such modifications may include, but are not limited to, deleting one or more residues from the antibody amino acid sequence, inserting one or more residues therein, and / or substituting one or more residues therein. The deletions, insertions, and substitutions can be performed in any combination, as long as the final construct possesses the desired properties, such as antigen-binding activity. Based on conventional knowledge of those skilled in the art, it will be understood that the aforementioned amino acid sequence variants also fall within the scope of protection of this invention. The antibody binding thyroglobulin of this invention, or a variant of its antigen-binding fragment, contains, compared to any polypeptide in SEQ ID NO:1 to SEQ ID NO:6, a mutation of up to 1, 2, or 3 amino acids occurring in the CDR region (if present); or, the variant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to the overall sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0024] In this field, affinity maturation of antibody / antigen binding fragments can also be performed. Current affinity maturation methods mainly include directed evolution based on display technology, rational design, and computational simulation-assisted optimization. Specific steps include: (1) using a phage display platform, constructing an antibody mutation library through random or focused mutation, and enriching high-affinity clones through multiple rounds of antigen binding screening and amplification cycles; (2) using site-directed saturation mutagenesis technology to design mutation combinations targeting key residues in the complementarity-determining region (CDR) or framework region (FR) and evaluating binding performance; (3) applying error-prone PCR to introduce random mutations during amplification, generating a diversity library and obtaining optimized variants through functional screening; (4) using computer-aided design to predict key sites affecting affinity through molecular docking, free energy perturbation (FEP), or machine learning models to guide directed mutation. In practical applications, multiple strategies are often integrated, such as first screening potential hotspots through computation, and then combining in vitro display technology for verification and optimization to achieve efficient and accurate affinity enhancement.

[0025] In the field of antibody applications, it is also necessary to produce cysteine-engineered antibodies by artificially introducing highly reactive thiol groups (-SH). These thiol groups are then used for labeling or coating with maleimide to achieve targeted coupling. Specifically, one or more residues in the antibody are selected and replaced with cysteine ​​residues. To avoid affecting the specific binding of the antibody to the antigen, the replaced residues are usually located in the constant region of the antibody. By replacing specific amino acid residues with cysteine, reactive thiol groups are generated, making specific sites on the antibody available for conjugation to other parts, such as antibody-labeled substances, antibody-solid carriers, and antibody-specific proteins.

[0026] The isoelectric point (pI) is the pH value at which a protein has zero net charge in solution. Its value is determined by the pKa values ​​of ionizable groups in the amino acid sequence (such as the carboxyl groups in ASP / D and GLU / E, and the amino groups in LYS / K and ARG / R). In antibody preparation, pI affects solubility (e.g., colloidal stability of high-concentration formulations) and purification efficiency (ion-exchange chromatography binding characteristics). In the detection field, adjusting the antibody pI to near the pH of the reaction buffer is commonly used to prevent aggregation and improve reagent stability. Strategies for regulating the antibody isoelectric point include: site-directed mutagenesis to replace surface-charged residues (e.g., LYS→GLN to lower pI), chemical modification (PEGylation to introduce a negative charge), or glycosylation engineering (adjusting the sialylation level of the Fc region). For example, replacing basic residues in the antibody CDR region with neutral residues can lower the pI from 8.5 to 7.2. In practical applications, pI needs to be predicted using computational tools (such as ExPASy ProtParam) and verified by capillary isoelectric focusing electrophoresis.

[0027] The antibody or antigen-binding fragment of the present invention that binds to thyroglobulin may possess a constant region, which may be a constant region of any one of IgG (including any one of IgG1, IgG2, IgG3, and IgG4), IgE, IgD, IgA, and IgM. The species source of the constant region may be cattle, horses, pigs, sheep, goats, rats, mice, guinea pigs, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, alpacas, or humans, etc.

[0028] The antigen-binding fragments of antibodies provided by this invention can be designed as antibody fragments with specific functions or application advantages. Through structural simplification or modification, their physicochemical properties are optimized while retaining antigen-binding ability. Common antibody fragments include: Fab fragments (composed of the heavy chain variable region VH and the first constant region CH1 connected to the complete light chain via disulfide bonds, with a molecular weight of approximately 50 kDa), Fab' fragments (retaining part of the hinge region and containing free thiol groups on the basis of Fab, which can form a multivalent structure through chemical coupling), F(ab')2 fragments (composed of two Fab' fragments connected by disulfide bonds in the hinge region to form a bivalent binding form), Fv fragments (containing only the heavy chain and light chain variable regions VH and VL, relying on non-covalent interactions to maintain structural stability), and scFv fragments (forming a single-chain structure by tandemly connecting VH and VL through a flexible polypeptide linker, enhancing stability and facilitating genetic engineering operations). Preparation methods are divided into two categories: enzymatic digestion (such as papain hydrolyzing the hinge region of IgG to generate a monovalent Fab fragment, or pepsin cleaving the C-terminus of the IgG heavy chain to generate a bivalent F(ab')2 fragment, which is then treated with reducing agents such as β-mercaptoethanol to obtain the Fab' fragment) and recombinant expression technology (directly synthesizing fragments such as scFv through E. coli, yeast or mammalian cell expression systems, combining phage display technology to screen for high-affinity variants, and improving thermodynamic stability by introducing additional disulfide bonds or optimizing linker peptides).

[0029] The functional fragments of the aforementioned antibodies can also be synthesized using commercially available automated peptide synthesizers.

[0030] A second aspect of the invention relates to antibody conjugates comprising an antibody or antigen-binding fragment thereof that binds to thyroglobulin as described above.

[0031] Antibody conjugates are complexes formed by coupling antibodies with functional molecules (such as chemiluminescent labels, fluorescent dyes, enzymes, and radioisotopes), solid phases, or proteins. This technique is widely used in biomedical research, diagnosis, and treatment. The resulting conjugates include antibody-solid phase conjugates, antibody-signaling molecule conjugates, and antibody-peptide conjugates.

[0032] For antibody-solid phase conjugates, "solid phase" refers to the solid support used to immobilize the antibody. Its function is to provide a stable solid-liquid reaction system to achieve specific capture, separation, and signal detection of the target analyte. The uses of solid phases are well-known in the fields of chemistry, biochemistry, pharmacy, and molecular biology.

[0033] Solid phases can be classified into various types based on their properties, including organic polymers such as polystyrene, polypropylene, glass, and silicone rubber; inorganic materials such as silicon-based materials, metallic materials, and alumina; nanomaterials such as nanoparticles and nanofibers; and composite materials such as magnetic microspheres and nanocomposite materials.

[0034] Immobilization on a solid phase can be achieved by modifying or activating the solid phase to include functional groups capable of covalently coupling with antibodies or antibody complexes (antibodies can form complexes with specific molecules, including carrier proteins, biotin / avidin, FITC, etc.). The functional groups are selected from one or more of carboxyl, amino, thiol, and toluenesulfonyl groups. Antibodies or antibody complexes can also be non-covalently attached to the solid phase via, for example, ionic or hydrophobic mechanisms.

[0035] In one embodiment, the solid phase can be a sheet, pre-formed disc, cylinder, fibrous, or granular, generally allowing for proper contact. The size of the solid phase suitable for the methods of the present invention can vary depending on the method chosen. The antibody may bind to only one solid phase (e.g., a porous plate) or to multiple solid phases (e.g., beads). In one embodiment, the solid phase can be fibrous or granular to allow for optimal contact. The size of the solid phase can vary and can be selected depending on the method to be performed.

[0036] In this invention, the shape of the term "particle" (sometimes referred to as "bead," "microbead," or "microsphere") can generally vary. For example, in one particular embodiment, the particle is spherical. However, it should be understood that other shapes are also conceived in this invention, such as plate-like, rod-like, disc-like, strip-like, tubular, irregular shapes, etc. Furthermore, the size of the particles can also vary. For example, the average size (e.g., diameter) of the particles can range from approximately 0.1 nm to 1 mm in diameter, such as 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, and 500 μm.

[0037] In some embodiments, the particles are magnetic spheres, preferably magnetic nanospheres, such as magnetite nanospheres. Magnetic microspheres refer to colloidal composite materials that can be uniformly dispersed in a certain base liquid, possessing characteristics such as superparamagnetism, high specific surface area, and modifiable functional groups. Surface functional groups include, but are not limited to, active groups such as carboxyl, amino, hydroxyl, or thiol groups. Antibody-conjugated magnetic beads mainly achieve directional binding through physical adsorption or chemical coupling. Physical adsorption relies on electrostatic interactions, hydrophobic interactions, and van der Waals forces, while chemical coupling utilizes the amino, carboxyl, hydroxyl, or thiol groups modified on the surface of the magnetic beads to bind to the charged groups of the antibody. This is typically done by incubation in a suitable buffer solution and blocking non-specific sites with BSA.

[0038] For antibody-signal substance conjugates, the signal substance refers to a class of substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc., which enable qualitative or quantitative detection of the corresponding target analytes.

[0039] In some embodiments, the signaling substances include, but are not limited to, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle-based markers. In practical use, those skilled in the art can select appropriate markers based on detection conditions or actual needs; regardless of the marker used, it falls within the scope of protection of this invention.

[0040] In some embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0041] In some embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0042] In some embodiments, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0043] In some embodiments, the signaling substance is preferably a chemiluminescent signaling substance, including but not limited to luminol and its derivatives, luciferin, fluorescein of crustaceans and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofen and its derivatives, and peroxazone and its derivatives. Antibodies form antibody-signaling substance conjugates through direct and indirect coupling. Direct coupling is the direct connection between the reactive groups of the antibody and the reactive groups of the luminescent agent; indirect coupling is the connection between the antibody and the luminescent agent through a "bridge", which has advantages such as introducing new active groups and reducing steric hindrance. Common coupling methods include carbodiimide, glutaraldehyde method and N-hydroxysuccinimide active ester method; for example, if the antibody is directly labeled with ABEI, the ABEI cyclic lactone reacts with the amino residue of lysine on the antibody to form an amide bond. The main process of antibody labeling ABEI is as follows: (1) Prepare labeling buffer; (2) Antibody labeling: The antibody is labeled with ABEI and stirred at 25°C; (3) Dialyze to remove small molecules after labeling.

[0044] The antibody-peptide conjugate can be a fusion protein prepared by genetic engineering techniques, containing peptides that do not affect the basic structure and function of the antibody, such as epitope tags, purification tags, or signal peptides, fused and expressed at the N-terminus or C-terminus of the antibody. The peptides include, but are not limited to, one or more of 6×His, GST, MBP, FLAG, HA, c-Myc, SUMO, Strep, etc. The antibody-peptide conjugate can also be a complex formed by chemically or biologically conjugating the antibody to other antibodies, proteins, or specific molecules. The specific molecules include, but are not limited to, one or more of carrier proteins, biotin / avidin systems, and fluorescent dyes (such as FITC). The preparation methods of the fusion protein or complex, such as genetic engineering fusion expression or chemical cross-linking, can be carried out according to conventional methods in the art, and all should retain the basic antigen-binding activity of the antibody. All the above-mentioned conjugate forms selected according to actual needs should be understood to fall within the scope of protection of this invention.

[0045] A third aspect of the invention relates to a kit comprising an antibody or antigen-binding fragment thereof that binds to thyroglobulin as described above, or an antibody conjugate as described above.

[0046] In this invention, the term "kit" may refer to any article (e.g., packaging or container) that includes at least one device and comprises the detection reagents as described in this invention. The kit may further include instructions for use, supplementary reagents, and / or components or parts used in the methods or steps described in this invention.

[0047] Depending on the requirements, the kit may also contain other components necessary for the assay, such as one or more of the following: standards, sample pretreatment reagents (e.g., sample purification and enrichment reagents, lysis buffers, etc.), buffers / diluents (e.g., phosphate buffer), substrates for the signal substance (usually chromogenic reagents; for example, if the signal substance is ABEI, the chromogenic reagents are sodium hydroxide and hydrogen peroxide), and preservatives (any one or a mixture of two or more of sodium azide, sodium nitrite, sodium benzoate, Proclin series, and potassium sorbate). These components may be packaged separately or premixed in multiples for ease of use / preservation. One or more components in the kit may be liquids or lyophilized under low pressure to form solid powders.

[0048] The kits of the present invention include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) kits, chemiluminescence immunoassay kits, radioimmunoassay kits, and fluorescence immunoassay kits.

[0049] In some specific embodiments, the kit is a chemiluminescence kit, comprising a solid-phase reagent and a labeled reagent. The solid-phase reagent comprises an antibody-solid-phase conjugate, wherein the solid phase is magnetic nanospheres, and the labeled reagent comprises a labeled solid-phase conjugate, wherein the labeled substance is ABEI or its derivatives. The detection steps of the kit are as follows: the sample to be tested is simultaneously or sequentially contacted with the solid-phase reagent and the labeled reagent to form an antigen-antibody immune complex. The amount of the target substance in the sample is determined by detecting the amount of the immune complex. The sample to be tested can be blood, urine, cerebrospinal fluid, tissue samples, or other liquids containing the analyte.

[0050] The fourth aspect of the invention relates to antibodies that bind to thyroglobulin or antigen-binding fragments thereof, or antibody conjugates as described above, in the context of total thyroidectomy for differentiated thyroid cancer (DTC) and... 131 Application in reagents or kits for monitoring residual, relapse, and metastasis after I treatment.

[0051] The fifth aspect of the invention relates to isolated nucleic acids capable of encoding antibodies or antigen-binding fragments thereof that bind to thyroglobulin as described above.

[0052] The term "isolated nucleic acid" in this invention refers to deoxyribonucleic acid or ribonucleic acid polymers existing in single-stranded or double-stranded form. The isolated nucleic acid includes RNA genomic sequences, DNA (gDNA and cDNA), or RNA sequences transcribed from DNA, and, unless specifically indicated, the polypeptide also includes natural polynucleotides, sugars, or base-modified analogs. According to one aspect of the invention, the polynucleotide is a light-chain polynucleotide.

[0053] The isolated nucleic acids include nucleotide sequences encoding the amino acid sequence of a protein complex, as well as complementary nucleotide sequences. The complementary sequences include fully complementary sequences and substantially complementary sequences, meaning sequences that can hybridize with the nucleotide sequence encoding the amino acid sequence of the protein complex under stringent conditions known in the art.

[0054] Furthermore, the nucleotide sequence encoding the amino acid sequence of the protein complex can be altered or mutated. Such alterations include addition, deletion, or non-conservative or conserved substitution. A polynucleotide encoding the amino acid sequence of the protein complex can be interpreted as including a nucleotide sequence that is substantially identical to the isolated nucleic acid. This substantial identity involves aligning 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 sequences can show greater than 80% homology, for example, greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0055] In addition, the codons of the nucleic acids corresponding to the antibodies that bind to thyroglobulin or their antigen-binding fragments can be optimized according to the species of the host cell.

[0056] The sixth aspect of the present invention relates to a vector containing the nucleic acid described above.

[0057] The term "vector" refers to a nucleic acid delivery vehicle capable of carrying and delivering genetic material into a host cell. The vector typically contains the necessary genetic elements that allow it to replicate, maintain, and / or express the carried nucleic acid within the host cell. Typical vectors include, but are not limited to, plasmids, bacteriophages, granulocytes, minichromosomes, and viruses. Specifically, viruses that can serve as vectors include retroviruses (such as lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses, poxviruses, baculoviruses, papillomaviruses, and papillomaviruses with polycystic vacuolar structures (such as SV40). Furthermore, vectors can also be artificial chromosome systems, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC).

[0058] Based on their function, vectors can be divided into cloning vectors and expression vectors. Cloning vectors are used to transfer target nucleic acids into host cells, enabling them to replicate and amplify. They typically contain a replication origin and selection markers (such as resistance genes) that match the host cell. In addition to the above basic elements, expression vectors also contain regulatory elements necessary to drive the transcription and translation of the target nucleic acid, such as promoters, enhancers, internal ribosome entry sites (IRES), transcription termination signals, and polyadenylation signals. In some embodiments, expression vectors may further contain a sequence encoding a signal peptide to achieve secretion or membrane localization of the expression product.

[0059] The nucleic acid or fragment thereof of the present invention can be inserted into a suitable vector to construct a cloning vector or expression vector containing the nucleic acid. Such newly constructed vectors also fall within the scope of protection of this invention. After being introduced into host cells (e.g., through transformation, transduction, transfection, etc.), the vector can integrate into the host genome or exist in a free form and achieve transient or stable expression. The vector generally possesses an origin of replication and selection markers to support high copy number replication, stable maintenance, and efficient expression within the host cell.

[0060] The above description of the definition, classification, composition and use of carriers is based on conventional knowledge in the field. Without departing from the essence of the present invention, those skilled in the art can select or modify appropriate carrier systems according to actual needs. All such embodiments should be understood as part of the present invention.

[0061] The seventh aspect of the invention relates to a host cell containing the nucleic acid as described above, or transformed by the vector as described above.

[0062] The choice of host cell should be based on the specific requirements of antibody expression, including but not limited to prokaryotic or eukaryotic host systems.

[0063] Eukaryotic host cells encompass filamentous fungi, yeast, and cells from multicellular organisms (invertebrates and vertebrates), such as plant cells, insect cells, or mammalian cells. In some embodiments, suitable host cells or cell lines for expressing the antigen-binding proteins of the present invention include, but are not limited to, suspension culture-adapted cell lines: such as human embryonic kidney cells HEK293, human embryonic retinal cells PER.C6, canine kidney cells MDCK, African green monkey kidney cells COS, myeloma cells such as mouse myeloma cells NSO and Sp2 / O, hamster kidney cells BHK-21, and Chinese hamster ovary cells (CHO cells) and CAP / CAP-T cell lines. Fibroblasts may also be used. Human cells may be used, thus allowing the molecules to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells, and methods for transformation, culture, amplification, screening, and product generation and purification, are known in the art.

[0064] It can be demonstrated that prokaryotic cells, such as bacterial cells, can be used as host cells, suitable for expressing the proteins of the present invention or other embodiments. However, since proteins expressed in bacterial cells tend to be in unfolded, improperly folded, or non-glycosylated forms, any proteins produced in bacterial cells must be screened to retain antigen-binding capacity. If the molecule expressed by the bacterial cell is produced in a properly folded form, the bacterial cell will be the desired host; alternatively, in an alternative embodiment, the molecule can be expressed in a bacterial host and subsequently refolded. For example, various *Escherichia coli* strains used for expression are well-known host cells in the field of biotechnology. Various strains of *Bacillus subtilis*, *Streptomyces*, and other *Bacillus* species can also be used in this method.

[0065] If necessary, yeast cell strains known to those skilled in the art, as well as insect cells, such as fruit flies and lepidopteran insects, and virus expression systems, may also be used as host cells.

[0066] In some embodiments, the nucleic acid is inserted into the cell genome and can be stably expressed. The insertion method can be a vector as described above, or the nucleic acid can be directly transferred into the cell without being linked to a vector (e.g., liposome-mediated transfection technology).

[0067] The eighth aspect of the present invention relates to a method for preparing an antibody or antigen-binding fragment thereof that binds to thyroglobulin as described above, comprising culturing host cells as described above under culture conditions suitable for antibody expression.

[0068] In addition, this method includes an optional step of isolating and recovering the expressed antibody from the host cell or its culture medium.

[0069] The antibodies provided by this invention can be prepared using recombinant technology, as specifically disclosed in the relevant method in US Patent No. 4,816,567. In one embodiment of this invention, it involves isolating and obtaining nucleic acid fragments encoding the antibody. These nucleic acid molecules contain the amino acid sequences of the antibody's light chain variable region (VL) and / or heavy chain variable region (VH). Further, the nucleic acid can be cloned into one or more vectors, particularly expression vectors, for expression within host cells.

[0070] After the antibodies of this invention are generated, they can be purified from cell culture contents according to standard procedures in the art, including ammonium sulfate precipitation, affinity column chromatography, column chromatography, gel electrophoresis, etc. These techniques are within the conventional scope of the art and do not limit the invention. Cultured host cells can also be understood as utilizing the expression of antibodies in animals (particularly transgenic animals or nude mice). This involves using an expression system with an animal casein promoter, which, when transgenically incorporated into mammals, allows female animals to produce the desired recombinant protein in their milk. Culture media secreted with antibodies can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column containing adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product must be immediately frozen, e.g., at -70°C, or lyophilized.

[0071] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0072] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0073] The main reagents and instruments used in the following examples are from the following sources: The microplate reader used for testing was purchased from BioTek, model: ELX800; The protein separation and purification system was purchased from Cytiva, model AKTA Pure. Second anti-thyroglobulin antibody for sandwich method detection of thyroglobulin and source of thyroglobulin: Thyroglobulin assay kit (chemiluminescent immunoassay, Shenzhen New Industries Biomedical Engineering Co., Ltd., catalog number: 130253012M); Example 1 Antibody preparation steps 1) Gene extraction Positive clones were collected, mRNA was extracted, and reverse transcribed into cDNA. Using universal primers for the FR1 and FR4 regions of the light and heavy chains, the target fragment was amplified by Taq polymerase PCR, followed by A-tailing and ligation into the pMD19-T vector. The ligation product was transformed into JM109 competent cells, and after colony PCR screening, 10 positive clones from each region were selected for sequencing.

[0074] 2) Antibody gene sequence analysis and alignment The gene sequences obtained from the sequencing were analyzed and compared in the Kabat antibody database to confirm the integrity of the antibody structure. Sequence reproducibility was analyzed using SnapGene software, and selected sequences were used for construction and expression.

[0075] 3) Construction of recombinant antibody expression plasmid Based on sequencing results, specific upstream and downstream primers for light chain and heavy chain genes were designed respectively. The 5' end of the primers contains a homologous arm sequence of approximately 15 bp, which is homologous to the corresponding end of the linearized expression vector. By PCR amplification, light chain (VL) and heavy chain (VH) gene fragments with anterior and posterior homologous arms at both ends were obtained, with the structure as follows: anterior homologous arm - VL / VH gene - posterior homologous arm.

[0076] The pEE 6.1 plasmid was used as the expression vector backbone. The vector was linearized by double digestion with HindIII and EcoRI, and the linearized vector fragments were recovered by electrophoresis. The light chain or heavy chain gene fragments with homologous arms were ligated to the linearized vector via homologous recombination. The ligation products were transformed into JM109 competent cells, and positive clones were screened by colony PCR after culture. Verified positive clones were expanded, and endotoxin-free plasmids were extracted to obtain the expression vector pL carrying the light chain and the expression vector pH carrying the heavy chain.

[0077] 4) Expression purification After restoring culture, Chinese hamster ovary (CHO) cells were placed in a carbon dioxide shaker (conditions: 100 rpm, 37°C, 5% CO2) and cultured until the cell density reached ≥3×10⁻⁶. 6 Cells / mL. Cells were then passaged and expanded, collected, counted, and their viability assessed. Cells were then cultured at 1 × 10⁶ cells / mL. 6 The cells were seeded into the culture plate at a density of cells / mL and cultured overnight.

[0078] The light chain expression vector pL and the heavy chain expression vector pH were mixed at a mass ratio of 1.5:1, and the mixed plasmid was transfected into the above CHO cells according to the steps provided in the Gibco liposome transfection reagent instructions.

[0079] After transfection and culturing for an appropriate time, the cell expression supernatant was collected. Subsequently, the antibody in the supernatant was separated and purified according to the operating procedures of the GE AKTA Pure protein separation and purification system, and finally the expression antibody A of this invention was obtained.

[0080] The variable region sequence of antibody A is as follows: The heavy chain variable region sequence is shown in SEQ ID NO: 7: EVQLQQSGPELVKPGASVKMSCKASGYTFSSYVIHWVKQKPGQGLEWFGFINPYNDVTKYNEKFKGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARSTGLLWTWFTYWG QGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK; The light chain variable region sequence is shown in SEQ ID NO: 8: DIVMTQSPSSLTVTAGEKVTLNCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPARFTGSGSGTDFTLTIGSVQAEDLAVYFCQSDYSYPFTFGSGTKL EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0081] Antibody A heavy chain CDR1 is shown in SEQ ID NO: 1: SYVIH; CDR2 is shown in SEQ ID NO: 2: FINPYNDVTKYNEKFKG; CDR3 is shown in SEQ ID NO: 3: STGLLWTWFTY. Light chain CDR1 is shown in SEQ ID NO: 4: KSSQSLLNSGNQKNYLT; CDR2 is shown in SEQ ID NO: 5: WASTRES; CDR3 is shown in SEQ ID NO: 6: QSDYSYPFT.

[0082] Heavy chain constant region as shown in SEQ ID NO: 9: AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK Light chain constant regions, such as SEQ ID NO: 10: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Example 2 Antibody Performance Verification 1) Affinity Instruments and reagents: SPR instrument (Biacore 8K); Chip selection: S-series CM5 / CM7 chips (covalently coupled chips); Amino-coupled reagent kit (EDC, NHS, ethanolamine); Buffer: 10×PBS-P+ (200 mM phosphate, 27 mM KCl, 1.37 M NaCl, 0.5% Tween 20), 10 mM sodium acetate (coupling dilution, pH 4.5), 10 mM Gly-HCl (chip regeneration solution, pH 3.0).

[0083] Experimental steps: (1) Antibody A fixation: Ligand fixation was performed using the direct amino-coupled method. First, EDC / NHS (freshly prepared, 1:1 mixture) was injected for 5 min to activate the carboxyl group. Then, antibody A was diluted to 10 μg / ml with sodium acetate buffer at pH 4.5 and injected into the detection channel to the target coupling level (macromolecule analyte, 200 RU). Then, ethanolamine (pH 8.5) was injected for 10 min to block unreacted sites. Finally, the sample was washed with 1×PBS-P buffer until the baseline was stable.

[0084] (2) Analyte detection: Thyroglobulin was prepared as a 10 mM stock solution, then diluted to 100 μM. Six concentration gradients were prepared sequentially using the 2-fold dilution method. Together with the 100 μM concentration and the 0 concentration (blank control), a total of eight detection concentration points were obtained. A multi-cycle kinetic detection method was adopted, with sequential injection from low to high concentration at a flow rate of 30 μL / min for 180 s. The dissociation time was 600 s, and regeneration was performed using 10 mM Gly-HCl (pH 3.0) for 60 s.

[0085] Data fitting and analysis were performed using BIAevaluation software to calculate affinity.

[0086] Experimental results showed that the KD (M) of antibody A and thyroglobulin reached 1.4 ± 0.17 x 10⁻⁶. -11 It can meet the requirements of immune detection.

[0087] 2) Activity Dilute thyroglobulin (obtained from Shenzhen New Industries Biomedical Engineering Co., Ltd. Thyroglobulin Assay Kit, catalog number: 130253012M) to 500 ng / mL with coating buffer (main component NaHCO3), 100 μL per well, and incubate overnight at 4℃. The next day, wash twice with washing buffer (main component Na2HPO4 + NaCl) and blot dry. Add blocking buffer (20% BSA + 80% PBS), 120 μL per well, incubate at 37℃ for 1 h, and blot dry. Add diluted antibody A, 100 μL / well, incubate at 37℃ for 30 min. Wash 5 times with washing buffer and blot dry. Add goat anti-mouse IgG-HRP, 100 μL per well, incubate at 37℃ for 30 min. Wash 5 times with washing buffer and blot dry. Add chromogenic solution A (50 μL / well, containing 1.05 g / L citric acid, 0.186 g / L EDTA·1, 0.45 g / L IgG·1, 0.45 ... Add TMB and 0.2 ml / L concentrated HCl), then add colorimetric solution B (50 μL / well, containing 1.05 g / L citric acid, 0.186 g / L EDTA·2Na, 0.45 g / L TMB and 0.2 ml / L concentrated HCl), and incubate for 10 min. Add stop solution (50 μL / well, containing 0.75 g / L EDTA·2Na and 10.2 ml / L concentrated H2SO4). Read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0088] Table 1 Activity Data

[0089] 3) Stability Antibody A was placed at 4℃ (refrigerator), -80℃ (refrigerator), and 37℃ (incubator) for 21 days. Samples were taken at 7, 14, and 21 days for observation of its state, and the activity of the 21-day sample was tested. The results showed that no significant changes in protein state were observed under the three storage conditions after 21 days, and the activity did not decrease with increasing storage temperature, indicating that the antibody was stable. Table 2 below shows the OD results of enzyme immunoassay of the antibody after 21 days at different temperatures.

[0090] Table 2 Stability Data

[0091] The embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the protection scope of this disclosure. Therefore, the protection scope of this patent should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to thyroglobulin, characterized in that, The heavy chain CDR1 to CDR3 contains amino acid sequences as shown in SEQ ID NO:1~3; And light chain CDR1 with amino acid sequence as shown in SEQ ID NO:4, light chain CDR2 with amino acid sequence as shown in SEQ ID NO:5, and light chain CDR3 with amino acid sequence as shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof that binds to thyroglobulin according to claim 1, characterized in that, It includes 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 antibody conjugate, characterized in that, An antibody or antigen-binding fragment thereof that binds to thyroglobulin as described in claim 1 or 2.

4. A reagent kit, characterized in that... It can detect thyroglobulin antigen components in the blood, including the antibody or antigen-binding fragment of thyroglobulin as described in claim 1 or 2, or the antibody conjugate as described in claim 3.

5. The reagent kit according to claim 4, characterized in that, The kit is a chemiluminescence kit, comprising a solid-phase reagent and a labeling reagent, wherein the solid-phase reagent or the labeling reagent comprises the antibody or antigen-binding fragment thereof that binds to thyroglobulin as described in claim 1 or 2, or the antibody conjugate as described in claim 3.

6. The antibody or antigen-binding fragment thereof that binds to thyroglobulin as described in claim 1 or 2, or the antibody conjugate as described in claim 3, after total thyroidectomy for differentiated thyroid carcinoma (DTC) and 131 Application in reagents or kits for monitoring residual, relapse, and metastasis after I treatment.

7. The isolated nucleic acid, characterized in that, It is capable of encoding the thyroglobulin-binding antibody or its antigen-binding fragment as described in claim 1 or 2.

8. A carrier, characterized in that, It contains the nucleic acid described in claim 7.

9. A host cell, characterized in that, It comprises the nucleic acid as described in claim 7, or is transformed by the vector as described in claim 8.

Images