Monoclonal antibody of human epidermal growth factor receptor 2 and application thereof

Abstract

The invention discloses a human epidermal growth factor receptor 2 monoclonal antibody and application thereof, belongs to the technical field of biology, and provides an anti-human epidermal growth factor receptor 2 monoclonal antibody or an antigen binding fragment thereof, the core structure of the monoclonal antibody is heavy chain variable regions CDR1, 2, 3 and light chain variable regions CDR1, 2, 3, 8, 9, 10, which are respectively shown as SEQ ID NO: 1, 2, 3, 8, 9, 10, the invention also provides a corresponding application, a method, polynucleotide, a recombinant vector, a cell, a pharmaceutical composition, a compound and other products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a human epidermal growth factor receptor 2 monoclonal antibody and a use thereof. Background Art

[0002] HER2, the full name of which is Human Epidermal Growth Factor Receptor 2, is a transmembrane receptor-like protein with tyrosine kinase activity that plays an important role in cell signal transduction. The HER2 gene is located on chromosome 17q12-21.32 and encodes a protein with a relative molecular mass of 185kD. In normal tissues, the expression level of HER2 is low. However, in some malignant tumors, such as breast cancer, gastric cancer, and lung cancer, the expression of HER2 may be significantly increased, which is closely related to the aggressiveness, recurrence, and metastasis risk of the tumor. Targeted therapy has become an important treatment for patients with HER2-positive tumors, especially breast cancer patients. Anti-HER2 drugs such as trastuzumab can specifically act on HER2-positive cells, inhibit the growth and spread of tumors, and thus improve the treatment effect. Summary of the invention

[0003] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0004] The present invention provides an anti-human epidermal growth factor receptor 2 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain variable region CDR1 as shown in SEQ ID NO: 1, a heavy chain variable region CDR2 as shown in SEQ ID NO: 2, a heavy chain variable region CDR3 as shown in SEQ ID NO: 3, a light chain variable region CDR1 as shown in SEQ ID NO: 8, a light chain variable region CDR2 as shown in SEQ ID NO: 9, and a light chain variable region CDR3 as shown in SEQ ID NO: 10.

[0005] Furthermore, the CDR division method includes but is not limited to Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0006] As used herein, the terms "CDR" and "complementarity determining region" are used interchangeably and refer to a portion of the variable chain of an antibody that is involved in binding to an antigen. Thus, a CDR is a portion of an "antigen binding fragment" or an "antigen binding fragment". In some embodiments, a monoclonal antibody comprises three heavy chain variable region CDRs and three light chain variable region CDRs that together form an antigen binding fragment. "CDR" may refer to a CDR defined by any method known in the art (including a combination of methods).

[0007] Furthermore, the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity with the amino acid sequences described in SEQ ID NOs: 4-7, respectively; the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity with the amino acid sequences described in SEQ ID NOs: 11-14, respectively.

[0008] In some embodiments, the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and at least 100% sequence identity to the amino acid sequences described in SEQ ID NOs: 4-7, respectively. In some embodiments, the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment have at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and at least 100% sequence identity to the amino acid sequences described in SEQ ID NOs: 11-14, respectively. In some more specific embodiments, the sequence identity is calculated by comparing the two best aligned sequences in the window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) appears in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the sequence identity percentage.

[0009] Furthermore, the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or its antigen-binding fragment are shown in SEQ ID NOs: 4-7, respectively; the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or its antigen-binding fragment are shown in SEQ ID NOs: 11-14, respectively.

[0010] Furthermore, the sequence of the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 15; the sequence of the light chain variable region of the monoclonal antibody or its antigen-binding fragment has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0011] Furthermore, the sequence of the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO:15; the sequence of the light chain variable region of the monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO:16.

[0012] Further, it is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment, (dsFv)2, dual-specific dsFv, disulfide-stabilized diabody, single-chain antibody molecule, scFv dimer, multispecific antibody, camelized single domain antibody, nanobody, domain antibody or bivalent domain antibody.

[0013] Furthermore, the monoclonal antibody or antigen-binding fragment thereof is derived from human or non-human mammals.

[0014] Furthermore, the monoclonal antibody or antigen-binding fragment thereof is derived from human.

[0015] Furthermore, the monoclonal antibody or antigen-binding fragment thereof may include one or more replacement or modification of amino acid residues that do not affect binding specificity.

[0016] In some embodiments, the monoclonal antibody or its antigen-binding fragment is a monoclonal antibody, a human, a humanized, or a chimeric antibody. In some embodiments, the monoclonal antibody or its antigen-binding fragment is an IgG antibody. In some embodiments, the monoclonal antibody or its antigen-binding fragment is a multispecific antibody. In some embodiments, the monoclonal antibody or its antigen-binding fragment is a full-length antibody. In some embodiments, the monoclonal antibody or its antigen-binding fragment includes aglycosylation site mutation. In some embodiments, the monoclonal antibody or its antigen-binding fragment includes a fucosylation site mutation.

[0017] The term "human epidermal growth factor receptor 2" used in the present invention refers to the full-length human epidermal growth factor receptor 2 protein from humans (NCBI Gene ID: 2064), which is abbreviated as HER2. "ErbB2", "HER2" and "human epidermal growth factor receptor 2" are used interchangeably herein.

[0018] The present invention uses the term "antibody" in the broadest sense and includes various antibody structures, including, but not limited to, structures known from monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen binding activity. "Antibody binding site", "antibody binding fragment", "antigen binding fragment" or "antigen binding site" is a structural portion of an antibody molecule comprising heavy and light chain variable regions and hypervariable regions (CDRs) that specifically binds to an antigen. As known in the art, the specific properties of an antibody are related to immunoglobulin isotypes. Antibodies or fragments can also be from species of any origin including birds (e.g., chickens, turkeys, ducks, geese, quails, etc.) and mammals (e.g., humans, non-human primates, mice, rats, rabbits, cattle, goats, sheep, horses, pigs, dogs, cats, etc.).

[0019] The present invention uses the term "monoclonal antibody" intended for use in the art, and refers to an antibody obtained from a substantially homologous antibody population that binds to the same antigenic determinant (epitope). "Substantially homologous" means that the individual antibodies are identical except for possible naturally occurring mutations that may be present in small amounts. This is in contrast to polyclonal antibodies, which typically include different antibodies against a variety of different antigenic determinants (epitopes). The term "monoclonal antibody" encompasses complete and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. "Monoclonal antibodies" and their antigen-binding fragments are made in many ways, including, but not limited to, by hybridomas, phage selection, recombinant expression, and transgenic animals.

[0020] In some embodiments, the VH region (heavy chain variable region) and VL region (light chain variable region) can also be subdivided into regions with high variability (called complementarity determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) in each heavy chain and light chain respectively form antigen binding sites.

[0021] The present invention provides the use of the above-mentioned monoclonal antibody or its antigen-binding fragment in detecting human epidermal growth factor receptor 2 or its coding fragment.

[0022] The present invention provides use of the above-mentioned monoclonal antibody or its antigen-binding fragment in preparing a product for detecting human epidermal growth factor receptor 2.

[0023] Furthermore, the product includes a test kit, a test paper, a nucleic acid membrane strip, a chip, a system, or a device.

[0024] The present invention provides use of the above-mentioned monoclonal antibody or antigen-binding fragment thereof in preparing a pharmaceutical composition for regulating the expression level or activity of human epidermal growth factor receptor 2.

[0025] Furthermore, the pharmaceutical composition includes a pharmaceutically acceptable adjuvant.

[0026] The term "pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, ie, does not cause unacceptable levels of undesired biological effects or interact in a deleterious manner.

[0027] The present invention provides use of the above-mentioned monoclonal antibody or its antigen-binding fragment in preparing a pharmaceutical composition for preventing or treating diseases related to abnormal expression of human epidermal growth factor receptor 2.

[0028] Furthermore, the disease associated with abnormal expression of human epidermal growth factor receptor 2 is selected from any one of the following: breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, head and neck cancer, gastroesophageal junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, bladder cancer, prostate cancer, urothelial carcinoma, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, renal cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasma cell tumor, myeloma, glioblastoma multiforme, sarcoma, osteosarcoma and melanoma.

[0029] The term "related disease" used in the present invention refers to a malignant disease, including but not limited to adenoid cystic carcinoma, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma, cerebellar astrocytoma / malignant glioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma lymphoma, childhood carcinoid tumors, gastrointestinal carcinoid tumors, cancer of unknown primary, childhood cerebellar astrocytoma, childhood cerebellar astrocytoma / glioblastoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, colon cancer, cutaneous T-cell lymphoma (including mycosis fungoides and Sezary syndrome) Syndrome), desmoplastic small round cell tumor, endometrial cancer, esophageal cancer, Ewing sarcoma in the Ewing family of tumors, extracranial germ cell tumors in children, extragonadal germ cell tumors, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor (GIST), extracranial, extragonadal, or ovarian germ cell tumors, gestational trophoblastic tumors, brain stem gliomas, juvenile cerebral astrocytomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma (liver cancer), Hodgkin lymphoma, human papillomavirus (HPV)-related cancers, hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), Kaposi sarcoma sarcoma), kidney cancer (renal cell carcinoma), laryngeal cancer, lip and oral cancer, liposarcoma, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, Burkitt lymphoma, Hodgkin lymphoma non-Hodgkin lymphoma, primary central nervous system lymphoma, malignant fibrous histiocytoma of the bone / osteosarcoma, childhood medulloblastoma, Merkel cell carcinoma Carcinoma), Malignant Mesothelioma in Adults, Mesothelioma in Children, Multiple Endocrine Neoplasm Syndrome in Children, Multiple Myeloma / Plasma Cell Neoplasms, Multiple Myeloma (Bone Marrow Cancer), Nasal and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Oropharyngeal Cancer, Ovarian Cancer, Ovarian Epithelial Cancer (Surface Epithelial-Stromal Tumor), Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Islet Cell Pancreatic Cancer, Paranasal and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pineal Astrocytoma, Pineal Germ Cell Tumor, Pineocytoma in Children, Pituitary Adenoma, Pleuropulmonary Blastomycin, Prostate Cancer, Rectal Cancer, Renal Pelvis and Ureter Cancer, Rhabdomyosarcoma in Children, Salivary Gland Cancer, Soft Tissue Sarcoma, Uterine Sarcoma, Skin Cancer (Non-Melanoma), Skin Cancer (Melanoma), Small Intestine Cancer, Squamous Cell Carcinoma,Metastatic occult primary squamous cell carcinoma of the neck, primitive neuroectodermal tumors of childhood, testicular cancer (seminoma and nonseminoma), pharyngeal cancer, thymoma of childhood, thymic carcinoma, thyroid cancer, thyroid cancer of childhood, transitional cell carcinoma of the renal pelvis and ureter, gestational trophoblastic tumor, urethral cancer, endometrial cancer, vaginal cancer, gliomas of the visual pathways and hypothalamus of childhood, vulvar cancer, and Wilms tumor (kidney cancer) of childhood.

[0030] The present invention provides a method for detecting human epidermal growth factor receptor 2 in a sample for non-diagnostic and non-therapeutic purposes, the method comprising contacting the sample to be detected with the aforementioned monoclonal antibody or its antigen-binding fragment, and detecting the binding status of human epidermal growth factor receptor 2 with the aforementioned monoclonal antibody or its antigen-binding fragment.

[0031] The present invention provides a method for preparing cells or tissues with highly inhibited human epidermal growth factor receptor 2 in vitro, the method comprising introducing a vector carrying the aforementioned monoclonal antibody or its antigen-binding fragment into cells or tissues in vitro, and inhibiting the activity of human epidermal growth factor receptor 2 by expressing the aforementioned monoclonal antibody or its antigen-binding fragment.

[0032] The present invention provides a polynucleotide encoding the above-mentioned monoclonal antibody or antigen-binding fragment thereof.

[0033] Furthermore, the polynucleotide includes DNA and mRNA.

[0034] The present invention provides a recombinant vector, which comprises a nucleotide sequence encoding the above-mentioned monoclonal antibody or antigen-binding fragment thereof.

[0035] Furthermore, the recombinant vector includes a plasmid vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0036] The present invention uses the term "recombinant vector" to refer to a nucleic acid molecule that is capable of transporting another nucleic acid molecule connected thereto. Recombinant vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, no free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and other various polynucleotides known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which a virally derived DNA or RNA sequence is present in a vector for packaging into a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) are capable of autonomous replication in the host cells into which they are introduced. Other vectors (e.g., non-episodic mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, and are thereby replicated together with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Common expression vectors used in recombinant DNA techniques are usually in the form of plasmids.

[0037] The present invention provides a cell, which comprises a nucleotide sequence encoding the above-mentioned monoclonal antibody or antigen-binding fragment thereof, or a vector thereof.

[0038] Furthermore, the cell includes any cell that can replicate, preserve, and express the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, or the aforementioned vector.

[0039] Furthermore, the cells include prokaryotic cells or eukaryotic cells, wherein prokaryotic cells include bacteria, and eukaryotic cells include mammalian cells, insect cells, bird cells, and yeast cells.

[0040] Furthermore, the eukaryotic cells include immune cells.

[0041] Furthermore, the immune cells include T cells, B cells, NK cells, NKT cells, DNT cells, hematopoietic cells, pluripotent stem cells, myeloid progenitor cells, lymphoid progenitor cells, and tumor infiltrating lymphocytes.

[0042] Furthermore, the T cells include thymocytes, initial T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes.

[0043] Further, the T cells include helper T cells, cytotoxic T cells, tumor-infiltrating cytotoxic T cells, CD4+CD8+T cells, CD4-CD8-T cells, αβT cells expressing the α and β chains of the T cell receptor and / or γδT cells expressing the TCRγ and δ chains.

[0044] The term "immune cell" refers to a cell that can initiate an immune response, including but not limited to T cells, B cells, NK cells, NKT cells, DNT cells, etc., their respective precursor cells and their progeny. Immune response cells can also refer to cells of lymphoid or myeloid lineages.

[0045] The term "immune cell" and its grammatical other forms may refer to immune cells of any origin. For example, immune cells may be derived from blood, such as autologous T cells, allogeneic T cells, autologous NK cells, allogeneic NK cells, or derived from cell lines, such as NK cell lines prepared by infection with EBV virus, NK cells induced and differentiated from embryonic stem cells and iPSCs, and NK92 cell lines, etc. "Immune cells" may be from humans or non-human mammals.

[0046] The present invention uses the terms "cell", "cell line", "host cell" interchangeably and covers plant and animal cells, and includes invertebrates, non-mammalian vertebrates and mammalian cells. All such names include cell populations and progeny. Exemplary non-mammalian vertebrate cells include, for example, avian cells, reptile cells and amphibian cells. Exemplary invertebrate cells include, but are not limited to, insect cells, such as caterpillar (Spodoptera frugiperda) cells, mosquito (Aedes aegypti) cells, fruit fly (Drosophila melanogaster) cells, Schneider cells and silkworm cells. Cells can be differentiated, partially differentiated or undifferentiated, such as stem cells, including embryonic stem cells and pluripotent stem cells. Additional tissue samples derived from organs or organ systems can be used according to the present invention. Exemplary mammalian cells include, for example, cells derived from humans, non-human primates, cats, dogs, sheep, goats, cows, horses, pigs, rabbits, rodents including mice, hamsters, rats and guinea pigs, and any derivatives and progeny thereof.

[0047] The present invention provides a pharmaceutical composition, which comprises the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned polynucleotide, the aforementioned recombinant vector, and / or the aforementioned cell.

[0048] Further, the pharmaceutical composition is administered systemically, specifically via intranasal route, intravenous route, intraperitoneal route, intramuscular route, intradermal route, subcutaneous route, subdermal route, transdermal route, intracranial route, mucosal route, anal route, vaginal route, oral route, buccal route or they can be delivered by inhalation.

[0049] The pharmaceutical composition can be used to treat a cell proliferative disorder or delay the progression of a cell proliferative disorder in a subject in need thereof.

[0050] The present invention provides a complex, which includes the monoclonal antibody or its antigen-binding fragment as described above, and a detectable marker, wherein the detectable marker does not affect the function of the monoclonal antibody or its antigen-binding fragment itself when binding to the monoclonal antibody or its antigen-binding fragment.

[0051] The term "detectable marker" as used herein refers to at least one marker that is capable of directly or indirectly generating a detectable signal. A non-exhaustive list of markers includes enzymes that generate a detectable signal (e.g., by colorimetry, fluorescence, luminescence), such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase; chromophores, such as fluorescent, luminescent dyes; groups whose electron density is detected by electron microscopy or by their electrical properties, such as conductivity, amperometry, voltammetry, impedance; detectable groups, such as molecules having a sufficient size to induce detectable modifications in their physical and / or chemical properties, such detection can be by optical methods such as diffraction, surface plasmon resonance, surface changes, contact angle changes or physical methods such as atomic force spectroscopy, tunneling or radioactive molecules such as 32 P. 35 S or 125 I to complete. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a graph showing the binding results of human epidermal growth factor receptor 2 antibody.

[0053] Figure 2 This is the result of ELISA binding experiment of human epidermal growth factor receptor 2 antibody.

[0054] Figure 3 This is a graph showing the results of a lethality experiment of human epidermal growth factor receptor 2 antibody. DETAILED DESCRIPTION

[0055] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As will be apparent to those skilled in the art after reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that can be easily separated or combined with features of any of the other several aspects without departing from the scope or essence of this disclosure.

[0056] It should also be understood that the terms used herein are only used for the purpose of describing specific aspects and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the specification and related art, and should not be interpreted as idealized or overly formal unless explicitly defined herein.

[0057] Example 1 Screening of Anti-Human Epidermal Growth Factor Receptor 2 Antibodies Using the natural human phage antibody display library, a total of 3 rounds of panning were performed to obtain positive phages that strongly bind to human epidermal growth factor receptor 2. The positive rate of the third round of bio-panning increased significantly, indicating that the positive phages were effectively enriched and can be used for further screening of candidate molecules. The clones with OD values ​​> 1 were selected for sequencing verification, and finally 1 sequence was obtained, named HFM or HF-M.

[0058] The specific heavy chain variable region sequences are shown in Table 1.

[0059] Table 1 Heavy chain variable region sequences

[0060] The specific light chain variable region sequences are shown in Table 2.

[0061] Table 2 Light chain variable region sequences

[0062] The protein was constructed into the eukaryotic expression vector pCDNA3.1 and transfected into CHO suspension cells in the logarithmic growth phase. The culture supernatant was harvested after culturing for about 7 days and the antibody with a protein purity greater than 95% was obtained after identification.

[0063] Example 2 Antibody Specificity Detection Dilute human epidermal growth factor receptor 2 with coating solution to a final concentration of 1μg / ml, coat the ELISA plate, 100μl per well, and coat overnight at 4℃. Wash the ELISA plate 3 times with a plate washer. Block with 1.5% casein, 200μl per well, and block at 37℃ for 1 hour. Wash the ELISA plate 3 times with a plate washer. Prepare biotin-labeled HERCEPTIN antibody with 1XPBS, and the antibody concentration is 0.5μg / ml (this concentration is the lowest saturation concentration). Use the above solution to dilute naked antibody (HF-M\HF\1103-1\HERCEPTIN) to 100μg / ml, 33.33μg / ml, 11.11μg / ml, 3.70μg / ml, 1.23μg / ml, 0.41μg / ml, 0.14μg / ml, 0.046μg / ml, 0.015μg / ml, and 0.005μg / ml respectively.

[0064] Add the corresponding samples to the upper and lower parallel wells, 100μl per well, and react at 37℃ for 1.5 hours. Wash the ELISA plate 3 times with a plate washer. Add 100μl HRP-avidin (strep-hrp) (1:10000) to each well and react at room temperature for 45 minutes. Wash the ELISA plate 3 times with a plate washer. Add 100μl TMB colorimetric solution to each well and react at room temperature for 1 minute. Stop the reaction with stop solution, 100μl per well. Read the enzyme-linked immunosorbent assay at 450nm.

[0065] The results are as follows Figure 1 , 2 As shown, the results show that the competition experiment shows that the antibody HF-M can compete with the binding of Herceptin / Biotin to the human epidermal growth factor receptor 2 protein, indicating that it overlaps with the antigen epitope recognized by Herceptin, and is dose-dependent within a certain concentration range. The LogEC50 is 0.2012, and the trend of repeated experiments is consistent. As a control, herceptin can effectively compete with its own biotin-labeled antibody, and is dose-dependent within a certain concentration range. The LogEC50 is: 0.03926, and the trend of repeated experiments is consistent.

[0066] Example 3 Antibody lethality detection Experimental principle: Human PBMCs were used as effector cells and SKOV3-GFP (human ovarian cancer cell line stably transfected with green fluorescent protein) as target cells. The lactate dehydrogenase (LDH) release method was used to quantitatively determine the killing activity of drug-activated PBMCs against tumor cells.

[0067] The results are as follows Figure 3As shown, the EC50 value of HF-M antibody is 0.01263, the EC50 value of Herceptin is 0.003381, and the EC50 value of HF is 0.01467. In terms of targeted killing, HF-M antibody is more lethal than the positive controls Herceptin and HF.

Claims

1. An anti-human epidermal growth factor receptor 2 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain variable region CDR1 as shown in SEQ ID NO: 1, a heavy chain variable region CDR2 as shown in SEQ ID NO: 2, a heavy chain variable region CDR3 as shown in SEQ ID NO: 3, a light chain variable region CDR1 as shown in SEQ ID NO: 8, a light chain variable region CDR2 as shown in SEQ ID NO: 9, and a light chain variable region CDR3 as shown in SEQ ID NO:

10.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity with the amino acid sequences of SEQ ID NOs: 4-7, respectively; and the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity with the amino acid sequences of SEQ ID NOs: 11-14, respectively; Preferably, the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 4-7, respectively; the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 11-14, respectively; Preferably, the sequence of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 15; the sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; Preferably, the sequence of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof is shown as SEQ ID NO:15; the sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof is shown as SEQ ID NO:

16.

3. The anti-human epidermal growth factor receptor 2 monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1, which is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment, (dsFv)2, dual-specific dsFv, disulfide-stabilized diabody, single-chain antibody molecule, scFv dimer, multi-specific antibody, camelized single domain antibody, nanobody, domain antibody or bivalent domain antibody; Preferably, the monoclonal antibody or antigen-binding fragment thereof is derived from a human or non-human mammal; Preferably, the monoclonal antibody or antigen-binding fragment thereof is derived from human; Preferably, the monoclonal antibody or antigen-binding fragment thereof further comprises one or more substitutions or modifications of amino acid residues that do not affect binding specificity.

4. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in detecting human epidermal growth factor receptor 2 or a coding fragment thereof.

5. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a product for detecting human epidermal growth factor receptor 2.

6. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a pharmaceutical composition for regulating the expression level or activity of human epidermal growth factor receptor 2.

7. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a pharmaceutical composition for preventing or treating diseases related to abnormal expression of human epidermal growth factor receptor 2; Preferably, the disease associated with abnormal expression of human epidermal growth factor receptor 2 is selected from any one of the following: breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, head and neck cancer, gastroesophageal junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, bladder cancer, prostate cancer, urothelial carcinoma, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasma cell tumor, myeloma, glioblastoma multiforme, sarcoma, osteosarcoma and melanoma.

8. A method for detecting human epidermal growth factor receptor 2 in a sample for non-diagnostic and non-therapeutic purposes, the method comprising contacting the sample with the monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 3, and detecting the binding of human epidermal growth factor receptor 2 to the aforementioned monoclonal antibody or its antigen-binding fragment.

9. A method for preparing cells or tissues that highly inhibit human epidermal growth factor receptor 2 in vitro, the method comprising introducing a vector carrying the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 into cells or tissues in vitro, and inhibiting the activity of human epidermal growth factor receptor 2 by expressing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

10. Any of the following substances, including: 1) A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; 2) A recombinant vector comprising a nucleotide sequence encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; 3) A cell comprising a nucleotide sequence encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or a vector thereof; 4) A pharmaceutical composition, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the aforementioned polynucleotide, the aforementioned recombinant vector, and / or the aforementioned cell; 5) A complex, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and a detectable marker, wherein the detectable marker does not affect the function of the monoclonal antibody or antigen-binding fragment thereof when binding to the monoclonal antibody or antigen-binding fragment thereof.

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