An antibody, antibody conjugate, kit, nucleic acid, cell against ki67, and uses thereof

Abstract

The application discloses an antibody against Ki67, an antibody conjugate, a kit, nucleic acids, cells and application thereof, and relates to the technical field of antibody preparation.The application obtains an antibody specifically combined with Ki67 protein through screening, and the antibody has good binding activity and affinity with the Ki67 protein, so that a detection product based on the Ki67 protein marker can be developed.In addition, the antibody against Ki67 or an antigen binding fragment thereof can be used for early warning, diagnosis, efficacy evaluation and prognosis of diseases with the Ki67 protein as a marker, such as tonsil cancer, and is beneficial to early warning, screening, intervention and efficacy evaluation of tumor diseases, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of antibody preparation technology, and more specifically, to an anti-Ki67 antibody, antibody-drug conjugate, kit, nucleic acid, cell, and their applications. Background Technology

[0002] Clinically, Ki67 is mainly used to label cells in the cell cycle. A high positive rate is primarily due to the fact that faster tumor growth and poorer tissue differentiation ability make them more sensitive to Ki67. Generally, such tumors have a poor prognosis and are usually difficult to cure completely. Therefore, advanced tumors, due to their rapid division and less complete cell differentiation, exhibit increased sensitivity to Ki67.

[0003] Ki-67 antigen is a cell nucleus protein related to cell division and proliferation. It is expressed in the S, G1, G2, and M phases of the cell cycle, but absent in the G0 phase. It is often used as a reliable marker of tumor cell proliferation activity.

[0004] Immunohistochemistry (IHC) is a commonly used clinical method to detect protein expression in tumor cells. The accuracy and sensitivity of IHC testing depend heavily on the quality of the monoclonal antibody that specifically binds to the protein. Therefore, developing a highly specific monoclonal antibody against the Ki67 peptide is of great significance for detecting Ki67 protein expression levels.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-Ki67 antibody, antibody-drug conjugate, kit, nucleic acid, cell, and its application to solve the above-mentioned technical problems.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides an antibody against Ki67 or an antigen-binding fragment thereof, wherein the antibody includes the following complementarity-determining region:

[0009] HCDR1 consists of the amino acid sequence shown in SEQ ID NO:1;

[0010] HCDR2 consists of the amino acid sequence shown in SEQ ID NO:2;

[0011] HCDR3 consists of the amino acid sequence shown in SEQ ID NO:3;

[0012] LCDR1, which consists of the amino acid sequence shown in SEQ ID NO:4;

[0013] LCDR2, which consists of the amino acid sequence shown in SEQ ID NO:5;

[0014] And LCDR3, which consists of the amino acid sequence shown in SEQ ID NO:6.

[0015] In a second aspect, the present invention provides an antibody conjugate comprising the antibody or its antigen-binding fragment described above.

[0016] Thirdly, the present invention provides the use of the above-described antibody or its antigen-binding fragment or the above-described antibody conjugate in the preparation of any of the following products, the products being selected from: reagents, kits or chips.

[0017] Fourthly, the present invention provides a reagent or kit for tumor risk warning, diagnosis, efficacy monitoring, prognostic assessment, Ki67 protein detection, Ki67 protein enrichment or Ki67 protein purification, which contains the above-mentioned antibody or its antigen-binding fragment or the above-mentioned antibody conjugate; and the tumor uses Ki67 protein as a detection marker.

[0018] Fifthly, the present invention provides a nucleic acid that encodes the aforementioned antibody or its antigen-binding fragment.

[0019] In a sixth aspect, the present invention provides a carrier comprising the above-described nucleic acid.

[0020] In a seventh aspect, the present invention provides a recombinant cell comprising the aforementioned vector.

[0021] The present invention has the following beneficial effects:

[0022] This invention has obtained antibodies that specifically bind to the Ki67 protein through screening. Testing showed that these antibodies exhibit good binding activity and affinity to the Ki67 protein, thus enabling the development of detection products based on Ki67 protein biomarkers, such as detection reagents, kits, or chips. Using the anti-Ki67 antibody or its antigen-binding fragment of this invention to detect Ki67 can improve the sensitivity and specificity of detection.

[0023] Furthermore, antibodies against Ki67 or their antigen-binding fragments can be used for early warning, diagnosis, efficacy evaluation, and prognosis of diseases using Ki67 protein as a biomarker, such as tonsillar cancer, which is beneficial for early warning, screening, intervention, and efficacy evaluation of tumor diseases. Therefore, this invention provides more protein options for the detection of Ki67 protein and for the early warning, diagnosis, efficacy evaluation, and prognosis of epithelial tumors. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a diagram showing the results of an immunohistochemical experiment on tonsil tissue. Detailed Implementation

[0026] 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.

[0027] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0028] Definition of noun

[0029] The term "antigen-binding fragment" broadly refers to all proteins / protein fragments containing a CDR region, particularly antibodies or antibody functional fragments. "Antigen-binding fragment" includes antigen-binding fragments of the aforementioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies, and the smallest antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments. Antibody types can include IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin."

[0030] The term “antibody” as used in this article is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired biological activity, such as specific binding to the Ki67 antigen or fragments thereof.

[0031] In this invention, the terms "complementarity-determining region" or "CDR" refer to the highly variable regions of the heavy and light chains of an immunoglobulin, specifically the regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0032] In this invention, the heavy chain complementarity-determining region (CDR) is represented by HCDR, which includes HCDR1, HCDR2, and HCDR3; the light chain complementarity-determining region (LCDR) is represented by LCDR, which includes LCDR1, LCDR2, and LCDR3. 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 database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. 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.

[0033] Typically, the variable region VH of the antibody heavy chain can be obtained by linking the following CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0034] The variable region (VL) of the antibody light chain can be obtained by linking the following numbered CDRs with FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0035] In a first aspect, the present invention provides an antibody against Ki67 or an antigen-binding fragment thereof, wherein the antibody includes the following complementarity-determining region:

[0036] HCDR1 consists of the amino acid sequence shown in SEQ ID NO:1;

[0037] HCDR2 consists of the amino acid sequence shown in SEQ ID NO:2;

[0038] HCDR3 consists of the amino acid sequence shown in SEQ ID NO:3;

[0039] LCDR1, which consists of the amino acid sequence shown in SEQ ID NO:4;

[0040] LCDR2, which consists of the amino acid sequence shown in SEQ ID NO:5;

[0041] And LCDR3, which consists of the amino acid sequence shown in SEQ ID NO:6.

[0042] HCDR1: NYIFL;

[0043] HCDR2: NINPYYGSSDYNRKFKG;

[0044] HCDR3: EYGNYGAY;

[0045] LCDR1: RASKSVSTSMH;

[0046] LCDR2: LVSNLES;

[0047] And LCDR3:QQSYKWPLT.

[0048] The amino acid sequence of the complementarity-determining region described above is a novel sequence discovered and revealed for the first time in this invention, which endows the binding protein with the ability to specifically bind to the Ki67 antigen. Based on the good binding activity and affinity of this binding protein, it can be used to develop detection products for Ki67 protein, such as detection reagents and kits. Using the binding protein of this invention to detect Ki67 peptides can improve the sensitivity and specificity of detection. Furthermore, this binding protein can be used to diagnose diseases that use Ki67 peptides as biomarkers, facilitating early detection and intervention. This invention provides more protein options for the detection of Ki67 peptides and the diagnosis of diseases using Ki67 peptides as biomarkers.

[0049] In a preferred embodiment of the present invention, the above-mentioned antibody or antigen-binding fragment binds to the Ki67 polypeptide at a K... D ≤3.448×10 9 L / mol affinity binding, K D The detection is performed in accordance with the method described in the embodiments of the present invention.

[0050] In an optional embodiment, the binding protein and the Ki67 protein have a KD ≤ 3 × 10⁻⁶. 9 L / mol, 2×10 9 L / mol, 1×10 9 L / mol, 9×10 8 L / mol, 8×10 8 L / mol, 7×10 8 L / mol, 6×10 8 L / mol, 5×10 8 L / mol, 4×10 8 L / mol, 3×10 8 L / mol or 2×10 8 Affinity in L / mol.

[0051] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0052] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes the following frame region:

[0053] The HFR1 amino acid sequence has at least 80% homology with SEQ ID NO:7;

[0054] The HFR2 amino acid sequence has at least 80% homology with the one shown in SEQ ID NO:8;

[0055] The HFR3 amino acid sequence has at least 80% homology with that shown in SEQ ID NO:9;

[0056] The HFR4 amino acid sequence has at least 80% homology with that shown in SEQ ID NO:10;

[0057] The LFR1 amino acid sequence has at least 80% homology with that shown in SEQ ID NO:11;

[0058] The LFR2 amino acid sequence has at least 80% homology with the one shown in SEQ ID NO:12;

[0059] The LFR3 amino acid sequence has at least 80% homology with that shown in SEQ ID NO:13;

[0060] The LFR4 amino acid sequence has at least 80% homology with that shown in SEQ ID NO:14.

[0061]

[0062]

[0063] It should be noted that, in other embodiments, the amino acid sequences of each framework region of the antibody or its functional fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the corresponding backbone regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14) mentioned above.

[0064] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a constant region;

[0065] In a preferred embodiment of the present invention, the constant region includes a heavy chain constant region and / or a light chain constant region;

[0066] In a preferred embodiment of the present invention, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0067] In a preferred embodiment of the present invention, the heavy chain constant region sequence is as shown in SEQ ID NO:15 or has at least 80% homology with it, and the light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% homology with it.

[0068] In other embodiments, the heavy chain constant region amino acid sequence of the antibody or its functional fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the above-mentioned SEQ ID NO:15.

[0069] In other embodiments, the light chain constant region (CL) amino acid sequence of the antibody or its functional fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the above-mentioned SEQ ID NO:16.

[0070] In a preferred embodiment of the present invention, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans;

[0071] In a preferred embodiment of the present invention, the species source of the constant region is mouse or human;

[0072] In a preferred embodiment of the present invention, the antigen-binding fragment is selected from any one of the antibody F(ab')2, Fab', Fab, Fv and scFv.

[0073] The functional fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description herein, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds.

[0074] The functional fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by, for example, automated peptide synthesizers sold by Applied BioSystems.

[0075] In a second aspect, the present invention provides an antibody conjugate comprising the antibody or its antigen-binding fragment described above.

[0076] Antibody-conjugates refer to antibodies or their antigen-binding fragments labeled with detectable markers; or, immunoconjugates in which antibodies or their antigen-binding fragments are linked to cytotoxic drugs by chemical linkers.

[0077] This antibody-drug conjugate may also be referred to as an "immunoconjugate." Those skilled in the art will understand that a "conjugate" refers to a new molecular entity formed by two or more molecules linked together by covalent bonds. In an alternative embodiment, an "immunoconjugate" refers to a monoclonal antibody linked to a cytotoxic drug (referred to as the payload) by a chemical linker. Cytotoxic drugs include, but are not limited to: cytotoxins, alkylating agents, DNA groove binders, DNA inserters, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, etc. The antibody and payload can be formed by coupling with cleavable linkers such as peptide groups or disulfide bonds.

[0078] In another alternative embodiment, the term "conjugate" as used herein refers to a compound formed by chemically linking a dye or other substance of the fluorescein precursor to an antibody. The conjugate may also include substances such as antigens, receptors, ligands, enzymes, substrates, or coenzymes.

[0079] In this invention, the term "ligand" refers to a protein molecule that can bind to a receptor. The binding of a ligand to a receptor is specific, triggering signal transduction and biochemical reactions, thereby affecting cellular behavior and physiological functions.

[0080] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment is labeled with a detectable marker. A detectable marker refers to a substance that possesses properties such as luminescence, color development, or radioactivity that can be directly observed with the naked eye or detected or probing by an instrument, thereby enabling qualitative or quantitative detection of the corresponding target analyte.

[0081] In a preferred embodiment of the present invention, the detectable markers are selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers. In actual use, those skilled in the art can select appropriate markers according to detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of the present invention.

[0082] 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, Cy5, Cy5...). .5, Cy3, etc. or 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, etc. or similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0083] In optional embodiments, the enzymes that catalyze substrate color development include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0084] In optional embodiments, radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、177 Lu、 172 Lu and 18 F.

[0085] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0086] In optional embodiments, nanoparticle-based markers include, but are not limited to, nanoparticles and colloids; nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0087] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0088] In optional embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0089] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment is coated onto a solid phase; for example, by chemical coupling, the antibody or its antigen-binding fragment is linked to the solid phase.

[0090] In a preferred embodiment of the present invention, the solid phase is selected from microspheres, plates, and membranes;

[0091] In a preferred embodiment of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0092] Thirdly, the present invention provides the use of the above-described antibody or its antigen-binding fragment or the above-described antibody conjugate in the preparation of any of the following products, the products being selected from: reagents, kits or chips.

[0093] In a preferred embodiment of the present invention, the product has at least one of the following uses: for tumor risk warning, diagnosis, efficacy monitoring, prognostic assessment, Ki67 protein detection, Ki67 protein enrichment and Ki67 protein purification; and the tumor uses Ki67 protein as a detection marker.

[0094] In a preferred embodiment of the present invention, the tumor is selected from epithelial tumors;

[0095] In a preferred embodiment of the present invention, the epithelial tumor is selected from one or more of the following: adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, low-grade glioma of the brain, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, uterine sarcoma, anal cancer, retinoblastoma, and tonsil carcinoma.

[0096] In a preferred embodiment of the present invention, Ki67 protein detection involves labeling tissue cells with antibodies or their antigen-binding fragments.

[0097] Applications of Ki67 protein enrichment and purification include, but are not limited to: coating the above-mentioned antibody or antigen-binding fragments onto a solid phase (such as setting them on an adsorption column through chemical modification), and achieving the separation, enrichment, and purification of Ki67 protein in the sample through affinity chromatography and elution.

[0098] Fourthly, the present invention provides a reagent or kit for tumor risk warning, diagnosis, efficacy monitoring, prognostic assessment, Ki67 protein detection, Ki67 protein enrichment or Ki67 protein purification, which contains the above-mentioned antibody or its antigen-binding fragment or the above-mentioned antibody conjugate; and the tumor uses Ki67 protein as a detection marker.

[0099] Such as kits for tumor risk warning, tumor diagnostic kits, tumor efficacy monitoring or recurrence monitoring kits, tumor prognosis assessment kits, Ki67 protein detection, Ki67 protein enrichment or Ki67 protein purification kits.

[0100] In a preferred embodiment of the present invention, the tumor is selected from epithelial tumors;

[0101] In a preferred embodiment of the present invention, the epithelial tumor is selected from one or more of the following: adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, low-grade glioma of the brain, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, uterine sarcoma, anal cancer, retinoblastoma, and tonsil carcinoma.

[0102] Fifthly, the present invention provides a nucleic acid that encodes the aforementioned antibody or its antigen-binding fragment. Considering the degeneracy of codons, the gene sequence encoding the aforementioned antibody can be modified in its coding region without altering the amino acid sequence to obtain a gene encoding the same antibody amino acid sequence; alternatively, the gene can be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve antibody expression efficiency.

[0103] Sixthly, the present invention provides a vector comprising the aforementioned nucleic acid. The vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct. This construct can directly or indirectly (e.g., packaged as a virus) introduce a target DNA fragment into a host cell via transformation, transfection, or transduction to express the target gene.

[0104] In a seventh aspect, the present invention provides a recombinant cell comprising the aforementioned vector.

[0105] Recombinant cells include, but are not limited to, bacterial, fungal, or mammalian cells. Mammalian cells are selected from any one of the following: 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, Vero, BHK cells, CV1 cells, HeLa cells, MDCK cells, Hep-2 cells, and Per6 cells. Among these, the 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins and are well known to those skilled in the art.

[0106] In a preferred embodiment of the present invention, the bacteria are Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli, or Bacillus subtilis.

[0107] In a preferred embodiment of the present invention, the fungus is Trichoderma reesei or yeast.

[0108] The host cells mentioned above include transformants and transformed cells, which include primary transformed cells and their offspring, regardless of passage number. Offspring may not be entirely identical to parent cells in terms of nucleic acid content, but may contain mutations.

[0109] In a preferred embodiment of the present invention, the yeast is selected from at least one of the following genera: *Dermacospermum*, *Bacillus*, *Hansenula polysacchari*, *Krypton*, *Pichia*, *Candida*, *Kluyveromyces*, *Debaryomyces*, *Kazachstania*, *Wickerhamomyces*, *Lindnera*, *Zygotorulaspora*, *Zygosaccharomyces*, *Rhodosporidium*, and *Schizosaccharomyces*.

[0110] The recombinant cells described above are obtained by transforming a recombinant expression vector into a host cell (such as a microorganism) using conventional methods in the art. The host microorganism can be any conventional host microorganism in the art, as long as it satisfies the requirement that the recombinant expression vector can stably replicate on its own and that the foreign gene it carries can be effectively expressed. The host microorganism can be bacteria or fungi.

[0111] In a preferred embodiment of the present invention, recombinant cells refer to at least one of resting cells of recombinant bacteria, live cells of recombinant bacteria, dead bacteria of recombinant bacteria, and cell fragments of recombinant bacteria.

[0112] Resting cells, also known as quiescent cells, are a special cell state. In this state, the cells do not grow or reproduce, but still contain various enzyme systems and possess oxidative and fermentative capabilities. Under suitable conditions, resting cells can resume growth. Characteristics of resting cells include: a. Retained growth potential: Although in a dormant state, these cells can re-enter the cell cycle and regain their proliferative capacity when given appropriate stimulation. b. High specificity: Resting cells exhibit high specificity in reactions, which can improve substrate conversion rates. c. Less susceptible to contamination: Due to their characteristics, resting cells can reduce the inhibition of cell growth and enzyme synthesis by products during use.

[0113] Dead bacteria in recombinant bacteria include, but are not limited to, bacteria obtained after inactivation by means of heat, pressure, radiation, etc.

[0114] Cell debris refers to cell fragments obtained by altering cell membrane permeability through methods such as ultrasound, mechanical, chemical, and biological means, resulting in the leakage of cell contents.

[0115] In a preferred embodiment of the present invention, the dead bacteria are selected from at least one of the precipitate of dead bacteria and the cell-free supernatant of dead bacteria. The cell-free supernatant of dead bacteria refers to the "exudate contents" remaining after removing the outer shell of the dead bacteria.

[0116] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0117] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0118] Example 1

[0119] This embodiment describes the preparation of a monoclonal antibody specifically targeting the Ki67 peptide.

[0120] 1. Animal (mouse) immunization

[0121] Mice were immunized with an immunogen using a standard method. The immunogen was human Ki67 polypeptide (CEDLAGFKELFQTPG), which also served as a detection antigen for serum titer and hybridoma screening. High-purity antigen increases the chance of obtaining the desired monoclonal antibody while reducing the screening workload. Five mice were immunized, each receiving 50 μg of Ki67 antigen. An antigen-protein solution was prepared using PBS. Appropriate amounts of antigen protein, PBS, and Freund's adjuvant were placed in a syringe, the syringe outlet was plugged, and the solution was emulsified thoroughly on an emulsifier to form a stable water-in-oil solution. The first tail blood serum titer was measured 7-10 days after the primary and secondary immunizations. Good titers were obtained after 2-4 booster immunizations. Mice with high serum titers were selected for final intraperitoneal immunization followed by cell fusion.

[0122] 2. Hybridoma cell fusion and screening

[0123] Preparations are required before cell fusion: (1) Culture mouse myeloma cells SP2 / 0 to the logarithmic growth phase; (2) Sacrifice a negative mouse one day before fusion, and inject mouse peritoneal trophoblast cells into the peritoneal cavity in a sterile environment using HAT medium to collect the cells and plate them in 96-well plates, 100 μL per well. These cells promote the growth of hybridoma cells. Sacrifice the immunized mouse, and collect the spleen in a sterile environment. Use PEG to chemically fuse spleen B cells and SP2 / 0 myeloma cells. Add appropriate HAT medium according to the number of cells to be plated, and finally plate the fused cells in a trophoblast cell culture plate, 100 μL per well.

[0124] After 7-10 days, the growth of surviving hybridoma cells can be observed under a microscope. Two weeks after plating, the supernatant from each well is collected, and hybridoma cells are screened using the Ki67 peptide antigen via ELISA.

[0125] The method is as follows: Coat the ELISA plate with 100 μL of PBS solution containing 2 μg / ml human Ki67 polypeptide antigen and incubate at 37°C for two hours. After washing the plate three times with PBST, add 150 μL / well of PBS solution containing 3% skim milk powder and block at 4°C overnight. Wash the plate three more times, add 80 μL / well of hybridoma supernatant, incubate at 37°C for 1 hour, and then wash three more times. Add 100 μL / well of horseradish peroxidase-labeled goat anti-mouse secondary antibody diluted 1:8000, incubate at 37°C for 45 minutes, wash three times, and blot dry. Add 100 μL / well of TMB chromogenic solution, develop at room temperature for 5-10 minutes, stop with 2M sulfuric acid solution, and measure the absorbance at 450 nm for each well. Select positive hybridoma cells.

[0126] Example 2

[0127] The ELISA-positive fusion wells from Example 1 were selected for immunohistochemical (IHC) experiments. The experimental steps are as follows:

[0128] Normal tonsil tissue sections were baked in a 60°C oven for 60 minutes. The sections were then soaked in xylene I for 15 minutes, followed by xylene II for another 15 minutes. They were then soaked in anhydrous ethanol ① for 5 minutes, anhydrous ethanol ② for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, and 75% ethanol for 5 minutes. Finally, they were soaked in ddH2O for 5 minutes and washed three times. Antigen retrieval was performed using a pressure cooker (boiling method). 10 mmol / L citrate buffer (pH 6.0) was added to the pressure cooker to submerge the sections. The mixture was heated to boiling. The sections were placed on a heat-resistant section rack and placed in the pressure cooker. The lid was closed, the pressure valve was engaged, and the mixture was heated. The pressure was maintained for 4 minutes. After the time was up, the vent valve was opened to release the pressure. Once the pressure returned to zero, the lid was opened, the inner pot was removed, and the mixture was allowed to cool to room temperature. After the solution cools to room temperature, remove the sections (approximately 40 minutes); soak in ddH2O for 5 minutes, wash twice, soak in PBST for 5 minutes, wash twice; place the sections in 20 ml of 3% H2O2-methanol solution, protect from light, and treat at room temperature for 10 minutes; soak in PBST for 5 minutes, wash three times; add one drop (approximately 25 μl) of goat serum blocking solution to each tissue group, incubate in a humidified chamber at room temperature for 45 minutes; soak in PBST for 5 minutes, wash three times.

[0129] Add the purified antibody from the ELISA-positive fusion wells in Example 1. Incubate overnight in a humidified chamber at 4°C; remove from the refrigerator at 4°C and incubate at room temperature for 60 minutes; gently rinse with PBST and soak for 5 minutes, washing 3 times; add 5 μL of HRP-labeled Long Island Biotechnology secondary antibody 2 (D-3004-0100) to each tissue group and incubate at room temperature for 45 minutes; wash; prepare DAB chromogenic solution, react in the dark for 10-15 minutes, then drop onto the sections and develop for 1-5 minutes; terminate the chromogenic reaction with distilled water; add 50 μL of hematoxylin staining solution to each tissue group and stain for 5-10 minutes, then rinse thoroughly with distilled water; decolorize the sections in 1% hydrochloric acid-ethanol for 2-3 seconds, then quickly remove and stop in distilled water, then incubate in PBST (pH 8.0) for 5-10 minutes; soak in 75% ethanol for 5 minutes; soak in 85% ethanol for 5 minutes; soak in 95% ethanol for 5 minutes; soak in anhydrous ethanol for 5 minutes. Soak in xylene for 10 minutes, then replace with xylene and soak for another 10 minutes; add neutral resin to seal the slide, then cover with a coverslip; take a microscope image.

[0130] Fusion cells that showed positive binding were selected using the ELISA assay in Example 1 and the IHC assay in Example 2. These cells were then cloned using the limiting dilution method, with each positive cell line seeded into 48 / 96-well plates and cultured further. A second round of screening was performed using ELISA to identify hybridomas that specifically recognized the Ki67 peptide and could block Ki67 binding. These hybridomas were then subcloned using the limiting dilution method to obtain a single-clone cell line, 9A11.

[0131] The monoclonal cell line was expanded, and approximately 1 × 10⁻⁶ cells were collected. 6 One cell was injected into selected mice (the mice needed to be injected with paraffin oil into their peritoneum one week in advance), and after a waiting period of 7-10 days, the mice produced ascites. The ascites was collected for antibody purification. After purification, a mouse monoclonal antibody specifically against Ki67 peptide was obtained.

[0132] Figure 1 This is an image showing the immunohistochemical results of adding Ki67-9A11 antibody to the slide. Figure 1 The specimen tested was tonsil tissue. Figure 1 The Ki67-9A11 antibody prepared in Example 1 was derived from... Figure 1 The staining results showed no significant difference in staining sites and intensity between Ki67-9A11 antibody and Maixin antibody. This confirms that the Ki67 provided by this invention has promising applications in preparing kits.

[0133] Example 3

[0134] DNA cloning and sequencing were performed, including sequencing of the variable region gene of the anti-human Ki67 monoclonal antibody.

[0135] Total RNA was extracted from mouse monoclonal cell lines using Trizol reagent. Cells cultured in 9cm dishes were transferred to 1.5ml centrifuge tubes, and the supernatant was aspirated. 1ml of Trizol reagent was added, and the cells were lysed by pipetting. The lysed sample or homogenate was incubated at room temperature for 5-10 minutes to allow complete separation of nucleoproteins and nucleic acids. 0.2ml of chloroform was added, and the mixture was vigorously vortexed for 15 seconds, then incubated at room temperature for 3 minutes. The mixture was centrifuged at 12000 rpm at 4°C for 10 minutes. The upper aqueous phase was transferred to a clean centrifuge tube, and an equal volume of isopropanol was added. The mixture was incubated at room temperature for 20 minutes. The mixture was centrifuged at 12000 rpm at 4°C for 10 minutes, and the supernatant was discarded. The precipitate was washed with 1ml of 75% ethanol. The mixture was centrifuged at 12000 rpm at 4°C for 3 minutes, and the supernatant was discarded. The mixture was dried at room temperature for 5-10 minutes. 30-50ul of RNase-free ddH2O was added. The resulting RNA solution was stored at -70°C or used for subsequent experiments.

[0136] Total RNA was reverse transcribed into cDNA using the AMV first-strand cDNA synthesis kit. The experimental configuration was as follows: 6 μL total RNA + 1 μL Oligo dT + 4 μL RNase-free water (total 11 μL). After gentle mixing, centrifuge for 3-5 seconds. The reaction mixture was pre-denatured at 65°C for 5 minutes, then incubated on ice for 30 seconds, centrifuged for 3-5 seconds, and then incubated on ice for 2 minutes. While in the ice bath, 4 μL of 5X buffer + 1 μL of dNTP mixture + 1 μL of RNase inhibitor + 1 μL of reverse transcriptase (total 20 μL) was added. After gentle mixing, centrifuge for 3-5 seconds, and then incubated on a PCR instrument at 42°C for 50 minutes and 85°C for 5 minutes to complete cDNA synthesis. Random primers are suitable for the synthesis of short-strand cDNAs under 500 bp. The transcribed RNA template does not require a poly(A) tail and can transcribe the 5' end region.

[0137] PCR amplification of the light and heavy chains. For amplifying the variable region sequence of the antibody light chain, the PCR reaction system was prepared as follows: 25 μL 2x Taq enzyme buffer + 1 μL FP-VL + 1 μL RP-VL + 2 μL cDNA + 21 μL ddH2O. For amplifying the variable region sequence of the antibody heavy chain, the PCR reaction system was prepared as follows: 25 μL 2x Taq enzyme buffer + 1 μL FP-VH + 1 μL RP-VH + 2 μL cDNA + 21 μL ddH2O. The temperature cycling for PCR amplification of the variable regions of the heavy and light chains was as follows (steps 2 to 4 were repeated 35 times):

[0138] Step 1 - Pre-denaturation: 94℃, 4 min;

[0139] Step 2 - Denaturation at 94°C for 30 seconds;

[0140] Step 3 - Annealing at 55°C for 45 seconds;

[0141] Step 4 - Extend at 72°C for 60 seconds;

[0142] Step 5: 72℃, 10 min;

[0143] Step 6 - Store at 4℃.

[0144] PCR products were analyzed by 1% agarose gel electrophoresis, and DNA bands of corresponding sizes were excised (approximately 375 bp for VH and approximately 325 bp for VL). DNA extraction was performed using the SanPrep DNA Gel Extraction Kit. The procedure is briefly described as follows: A gel block containing the target fragment was excised from the agarose gel and weighed; 3-6 times the weight of the gel block was added to buffer B2, and the gel was incubated at 50°C for 5-10 minutes to dissolve; the solution was transferred to an adsorption column and centrifuged at 8000g for 30 seconds; the liquid in the collection tube was discarded; 500 μL of wash solution was added to the column, and the column was centrifuged at 9000g for 30 seconds, and the liquid in the collection tube was discarded; the wash solution was added again, and the liquid was discarded; the adsorption column was centrifuged at 9000g for 1 minute; the adsorption column was placed in a clean 1.5 ml centrifuge tube, and 15-40 μL of Elution Buffer was added to the center of the adsorption membrane. After standing at room temperature for 1 minute, the column was centrifuged for 1 minute. The prepared DNA solution was obtained, and the PCR product was purified and sequenced to obtain the variable region sequence of the antibody.

[0145] Experimental Example 1

[0146] This experimental example tests the affinity and sensitivity of the antibody (Ki67-9A11 purified antibody) prepared in Example 2 above.

[0147] 1. Recombinant human Ki67 peptide (CEDLAGFKELFQTPG) was plated at concentrations of 3 mg / L, 1.5 mg / L, 0.75 mg / L, and 0.375 mg / L, respectively.

[0148] 2. Adjust the antibody concentration to 10. -7 mol / L level (1*10) -7 Up to 5*10 -7 (Mol / L is acceptable). Then serially dilute 1:2 to 1:256 and add to wells with different antigen coating amounts.

[0149] 3. Add secondary antibody (Sangon Biotech secondary antibody D110065), and perform TMB colorimetric assay. Measure the absorbance at 450 nm; the data are shown in Table 1.

[0150] 4. Based on the antigen-antibody binding S-curve, determine the antibody concentration with the half-maximum absorbance at different antigen concentrations. This will result in four antibody concentrations (mol / L).

[0151] 5. Substitute the values ​​into the formula K = (N-1) / (N*AB'-AB) to calculate the affinity constant. AB' and AB are the antibody concentrations that produce the half-maximum absorbance at the corresponding antigen concentrations AG (3 mg / L, 1.5 ml / L, 0.75 mg / L, 0.375 mg / L). N = AG / AG' (AG > AG').

[0152] 6. When N=2, we get three K values: 1.148, 6.410, and 3.484. When N=4, we get two K values: 2.534 and 4.110. When N=8, we get one K value: 3.002. The average of the six K values ​​is 3.448 × 10⁻⁶. 9 L / mol.

[0153] Table 1. Statistical Table of Absorbance Values ​​at 450nm

[0154]

[0155] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody against Ki67 or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment includes the following complementarity-determining region: HCDR1 consists of the amino acid sequence shown in SEQ ID NO:1; HCDR2 consists of the amino acid sequence shown in SEQ ID NO:2; HCDR3 consists of the amino acid sequence shown in SEQ ID NO:3; LCDR1, which consists of the amino acid sequence shown in SEQ ID NO:4; LCDR2, which consists of the amino acid sequence shown in SEQ ID NO:5; And LCDR3, which consists of the amino acid sequence shown in SEQ ID NO:

6.

2. The antibody against Ki67 or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes the following framework region: HFR1 has an amino acid sequence that is at least 80% homologous to SEQ ID NO:7; HFR2 has an amino acid sequence that is at least 80% homologous to that shown in SEQ ID NO:8; HFR3 has an amino acid sequence that is at least 80% homologous to that shown in SEQ ID NO:9; HFR4 has an amino acid sequence that is at least 80% homologous to that shown in SEQ ID NO:10; LFR1 has an amino acid sequence that is at least 80% homologous to that shown in SEQ ID NO:11; LFR2 has an amino acid sequence that is at least 80% homologous to that shown in SEQ ID NO:12; LFR3 has an amino acid sequence that is at least 80% homologous to that shown in SEQ ID NO:13; LFR4 has an amino acid sequence that is at least 80% homologous to that shown in SEQ ID NO:

14.

3. The anti-Ki67 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment also includes a constant region.

4. The anti-Ki67 antibody or its antigen-binding fragment according to claim 3, characterized in that, The constant region includes the heavy chain constant region and the light chain constant region.

5. The anti-Ki67 antibody or its antigen-binding fragment according to claim 4, characterized in that, The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

6. The antibody against Ki67 or its antigen-binding fragment according to claim 5, characterized in that, The heavy chain constant region sequence has at least 80% homology with the sequence shown in SEQ ID NO:15, and the light chain constant region sequence has at least 80% homology with the sequence shown in SEQ ID NO:

16.

7. The antibody against Ki67 according to claim 6, or its antigen-binding fragment, is characterized in that, The heavy chain constant region sequence is shown in SEQ ID NO:15, and the light chain constant region sequence is shown in SEQ ID NO:

16.

8. The antibody against Ki67 or its antigen-binding fragment according to claim 3, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

9. The antibody against Ki67 according to claim 8, or its antigen-binding fragment, is characterized in that, The species source of the constant region is mice or humans.

10. The antibody against Ki67 according to any one of claims 1-9, or the antigen-binding fragment thereof, characterized in that, The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

11. An antibody conjugate, characterized in that, The antibody-drug conjugate includes the antibody or antigen-binding fragment thereof as described in any one of claims 1-10, wherein the antibody-drug conjugate refers to an antibody or antigen-binding fragment thereof labeled with a detectable marker.

12. The antibody conjugate according to claim 11, characterized in that, The detectable markers are selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers; the antibody or its antigen-binding fragment is coated on a solid phase.

13. The antibody conjugate according to claim 12, characterized in that, The solid phase is selected from microspheres, plates, and membranes.

14. The antibody conjugate according to claim 12, characterized in that, The solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

15. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-10, or the antibody-drug conjugate as described in any one of claims 11-14, in the preparation of any one of the following products, characterized in that, The products are selected from: reagents, reagent kits, or chips; The product has at least one of the following uses: Ki67 protein detection, Ki67 protein enrichment, and Ki67 protein purification.

16. The application according to claim 15, characterized in that, The Ki67 protein detection involves labeling tissue cells with the antibody or its antigen-binding fragment.

17. A reagent or kit for detecting, enriching, or purifying Ki67 protein, characterized in that, It contains the antibody or antigen-binding fragment thereof as described in any one of claims 1-10 or the antibody conjugate as described in any one of claims 11-14.

18. A nucleic acid, characterized in that, It encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-10.

19. A carrier, characterized in that, It includes the nucleic acid as described in claim 18.

20. A recombinant cell, characterized in that, It includes the carrier as described in claim 19.

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