Human epidermal growth factor receptor 2 monoclonal antibodies and uses thereof

Abstract

The application discloses a human epidermal growth factor receptor 2 monoclonal antibody and a use thereof, belongs to the technical field of biotechnology, and provides a kind of monoclonal antibody or antigen binding fragment thereof for resisting human epidermal growth factor receptor 2, and its core structure is heavy chain variable region CDR1, 2, 3 region and light chain variable region CDR1, 2, 3 region, respectively as shown in SEQ ID NO:1, 2, 3, 8, 9, 10, and the application also provides corresponding application, method and polynucleotide, recombination carrier, cell, pharmaceutical composition, complex and other products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a human epidermal growth factor receptor 2 monoclonal antibody and use thereof. BACKGROUND

[0002] HER2, also known as Human Epidermal Growth Factor Receptor 2, is a transmembrane receptor-like protein with tyrosine kinase activity, which plays an important role in cell signal transduction. 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, lung cancer, etc., the expression of HER2 may be significantly increased, which is closely related to the invasiveness, recurrence and metastasis risk of tumors. For HER2-positive tumor patients, especially breast cancer patients, targeted therapy has become an important treatment method, such as trastuzumab and other anti-HER2 drugs, which can specifically act on HER2-positive cells to inhibit the growth and spread of tumors, thereby improving the treatment effect. SUMMARY

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

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

[0005] Further, the division of the CDR includes but is not limited to Kabat, IMGT, Chothia, AbM or Contact numbering system.

[0006] The term "CDR" and "complementarity determining region" used in the present application can be used interchangeably, and refers to a part of the variable chain of an antibody that participates in binding to an antigen. Therefore, the CDR is part of an "antigen binding fragment" or an "antigen binding fragment". In some embodiments, the monoclonal antibody comprises three heavy chain variable region CDRs and three light chain variable region CDRs that collectively form an antigen binding fragment. "CDR" can refer to CDR defined by any method known in the art, including a combination of methods.

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

[0008] In some embodiments, the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, 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, or at least 100% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 4-7, respectively; and the sequences of the light chain variable region framework regions FR1, FR2, FR3, 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, or at least 100% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 11-14, respectively. In some more particular embodiments, sequence identity is calculated by: determining the number of positions at which 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, Gin, Cys, and Met) occurs in both sequences in a comparison window, 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 yield the percent sequence identity.

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

[0010] Further, the sequence of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; and the sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0011] Further, the sequence of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 15; and the sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof is set forth 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-specificity dsFv, disulfide stabilized diabody, single-chain antibody molecule, scFv dimer, multispecific antibody, camelized single-domain antibody, nanobody, domain antibody, or bivalent domain antibody.

[0013] Further, the monoclonal antibody or antigen-binding fragment thereof is of human or non-human mammalian origin.

[0014] Further, the monoclonal antibody or antigen-binding fragment thereof is of human origin.

[0015] Further, 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.

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

[0017] The term "human epidermal growth factor receptor 2" is used herein in reference 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 term "antibody" is used in the broadest sense and includes various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An "antibody binding site," "antibody binding fragment," "antigen binding fragment," or "antigen binding site" is a structural part of an antibody molecule that specifically binds an antigen, comprising heavy and light chain variable and hypervariable regions (CDRs). The particular properties of an antibody are related to the immunoglobulin isotype. Antibodies or fragments can also be from species of any origin including avian (e.g., chicken, turkey, duck, goose, quail, etc.) and mammalian (e.g., human, non-human primate, mouse, rat, rabbit, bovine, goat, sheep, horse, pig, dog, cat, etc.).

[0019] The term "monoclonal antibody" is used in the art and refers to an antibody obtained from a population of substantially homogeneous antibodies that bind to the same antigenic determinant (epitope). By "substantially homogeneous" is meant that the individual antibodies are identical except for possible naturally occurring mutations that can be present. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against various different antigenic determinants (epitopes). The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins including an antibody portion, and any other modified immunoglobulin molecule that comprises an antigen recognition site. "Monoclonal antibodies" and antigen-binding fragments thereof are produced in a number of ways, including but not limited to, by hybridoma, 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 be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy chain, light chain (VH and VL, respectively) form the antigen binding site.

[0021] The present application provides use of the aforesaid monoclonal antibody or antigen-binding fragment thereof in the detection of human epidermal growth factor receptor 2 or a coding fragment thereof.

[0022] The present application provides use of the aforesaid monoclonal antibody or antigen-binding fragment thereof in the preparation of a product for the detection of human epidermal growth factor receptor 2.

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

[0024] The present application provides use of the aforementioned monoclonal antibody or antigen binding fragment thereof in the preparation of a pharmaceutical composition for modulating expression level or activity of human epidermal growth factor receptor 2.

[0025] Further, the pharmaceutical composition includes a pharmaceutically acceptable auxiliary agent.

[0026] The term "pharmaceutically acceptable" describes a material that is not biologically or otherwise undesirable, i.e., that does not cause an unacceptably high level of undesirable biological effects or interact in a deleterious manner.

[0027] The present application provides use of the aforementioned monoclonal antibody or antigen binding fragment thereof in the preparation of a pharmaceutical composition for preventing or treating a disease associated with abnormal expression of human epidermal growth factor receptor 2.

[0028] Further, the disease associated with abnormal expression of human epidermal growth factor receptor 2 is selected from any one of 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, plasmacytoma, myeloma, glioblastoma multiforme, sarcoma, osteosarcoma, and melanoma.

[0029] The term "related disease" is used herein to refer to a malignant disease, including but not limited to Adenocystic carcinoma (Adenoid cystic carcinoma), Adrenal cortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, Anal cancer, Appendix cancer, Astrocytic tumor, Basal cell carcinoma, Bile duct cancer, Bladder cancer, Bone cancer, Osteosarcoma / malignant fibrous histiocytoma, Brain stem glioma, Brain tumor, Cerebellar astrocytic tumor, Cerebellar astrocytic tumor / malignant glioma, Ependymal tumor, Medulloblastoma, Primitive neuroectodermal tumor, Visual pathway and hypothalamic glioma, Breast cancer, Bronchial adenoma / carcinoid, Burkitt lymphoma, Childhood carcinoid tumor, Gastrointestinal carcinoid tumor, Carcinoma of unknown primary, Childhood cerebellar astrocytic tumor, Childhood cerebellar astrocytic tumor / malignant glioma, Cervical cancer, Childhood cancers, Chronic lymphocytic leukemia, Colon cancer, Cutaneous T-cell lymphoma (including mycosis fungoides and Sezary Syndrome), Desmoplastic small round cell tumor, Endometrial cancer, Esophageal cancer, Ewing sarcoma family of tumors, Extracranial germ cell tumor in children, Extragonadal germ cell tumor, Extrahepatic bile duct cancer, Intraocular melanoma, Retinoblastoma, Gallbladder cancer, Gastric cancer, Gastrointestinal stromal tumor (GIST), Germ cell tumor extracranial, gonadal, or ovarian, Gestational trophoblastic tumor, Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular cancer (liver cancer), Hodgkin lymphoma, Human papillomavirus (HPV)-related cancers, Hypopharyngeal cancer, Islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, Kidney cancer (renal cell cancer), Laryngeal cancer, Lip and oral cavity cancer, Liposarcoma, Non-small cell lung cancer, Small cell lung cancer, Lymphoma, AIDS-related, Burkitt, Hodgkin, Non-Hodgkin, Primary central nervous system, Osteosarcoma / malignant fibrous histiocytoma of bone, Medulloblastoma in children, Merkel Cell Carcinoma, Malignant mesothelioma in adults, Mesothelioma in children, Multiple endocrine neoplasia syndrome of childhood, Multiple myeloma / plasma cell neoplasm, Nasal cavity and paranasal sinus cancer, Nasopharyngeal cancer, Neuroblastoma, Oral cavity pharyngeal 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 sinus and nasal cavity cancer, Parathyroid cancer, Penile cancer, Pheochromocytoma, Pineal astrocytic tumor, Pineal germinoma, Pinealoblastoma in children, Pituitary tumor, Pleuropulmonary blastoma, 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 non-seminoma), laryngeal cancer, childhood thymoma, thymic carcinoma, thyroid cancer, childhood thyroid cancer, transitional cell cancer of the renal pelvis and ureter, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, vaginal cancer, childhood visual pathway and hypothalamic glioma, vulvar cancer, and childhood Wilms tumor (kidney cancer).

[0030] The present application 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 antigen-binding fragment thereof, and detecting the binding of human epidermal growth factor receptor 2 to the aforementioned monoclonal antibody or antigen-binding fragment thereof.

[0031] The present application provides a method for preparing a cell or tissue in vitro with high inhibition of human epidermal growth factor receptor 2, the method comprising introducing a vector carrying the aforementioned monoclonal antibody or antigen-binding fragment thereof into a cell or tissue in vitro, and inhibiting the activity of human epidermal growth factor receptor 2 by expressing the aforementioned monoclonal antibody or antigen-binding fragment thereof.

[0032] The present application provides a polynucleotide encoding the aforementioned monoclonal antibody or antigen-binding fragment thereof.

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

[0034] The present application provides a recombinant vector comprising a nucleotide sequence encoding the aforementioned monoclonal antibody or antigen-binding fragment thereof.

[0035] Further, the recombinant vector includes a plasmid vector, a lentivirus vector, an adenovirus vector, and an adeno-associated virus vector.

[0036] The term "recombinant vector" is used herein to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. Recombinant vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular), nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polyribonucleotide known in the art. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, wherein the viral-derived DNA or RNA sequence is present in a vector, which is used to package into a virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a newly infected host cell, and are thus replicated with the host genome. Furthermore, certain vectors are capable of directing expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Commonly used expression vectors in recombinant DNA technology are often in the form of plasmids.

[0037] The present application provides a cell comprising a nucleotide sequence encoding the aforementioned monoclonal antibody or antigen-binding fragment thereof, or a vector thereof.

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

[0039] Further, the cell comprises a prokaryotic cell or a eukaryotic cell, wherein the prokaryotic cell comprises bacteria, and the eukaryotic cell comprises mammalian cells, insect cells, avian cells, yeast cells.

[0040] Further, the eukaryotic cell comprises an immune cell.

[0041] Further, the immune cell comprises a T cell, a B cell, a NK cell, a NKT cell, a DNT cell, a hematopoietic cell, a pluripotent stem cell, a myeloid progenitor cell, a lymphoid progenitor cell, a tumor infiltrating lymphocyte.

[0042] Further, the T cell comprises a thymocyte, a naive T lymphocyte, an immature T lymphocyte, a mature T lymphocyte, a resting T lymphocyte, or an activated T lymphocyte.

[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, αβ T cells expressing α and β chains of T cell receptor, and / or γδ T cells expressing TCR γ and δ chains.

[0044] The term "immune cell" refers to a cell that can elicit an immune response, including but not limited to T cells, B cells, NK cells, NKT cells, DNT cells, and the like, their respective precursor cells, and progeny thereof. An immune response cell can also refer to a cell of lymphoid or myeloid lineage.

[0045] The term "immune cell" and other grammatical forms thereof can refer to an immune cell of any origin. For example, an immune cell can be from blood, such as autologous T cells, allogeneic T cells, autologous NK cells, allogeneic NK cells, or can be derived from a cell line, such as an NK cell line prepared using EBV viral infection, an NK cell induced to differentiate from an embryonic stem cell and iPSC, and an NK92 cell line, and the like. An "immune cell" can be human or non-human mammalian.

[0046] The present application uses the terms "cell", "cell line", "host cell" interchangeably and encompasses plant and animal cells, and includes invertebrate, non-mammalian vertebrate, and mammalian cells. All such names include populations of cells and progeny. Exemplary non-mammalian vertebrate cells include, for example, avian cells, reptilian cells, and amphibian cells. Exemplary invertebrate cells include, but are not limited to, insect cells, such as Spodoptera (Sf9) cells, Aedes (Ae) cells, Drosophila (Dm) cells, Schneider cells, and Bombyx (Bm) 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 in accordance with the present application. 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 application provides a pharmaceutical composition, the product comprising 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, in particular delivered by intranasal, intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, subdermal, transdermal, intracranial, mucosal, anal, vaginal, oral, buccal, or they can be inhaled.

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

[0050] The present application provides a complex comprising the monoclonal antibody or antigen binding fragment thereof as described above, and a detectable label which does not affect the function of the monoclonal antibody or antigen binding fragment thereof when bound thereto.

[0051] The present application uses the term "detectable label" to mean at least one label capable of directly or indirectly producing a detectable signal. A non-exhaustive list of labels includes enzymes that produce a detectable signal (e.g., by colorimetric, fluorescent, luminescent), such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase; chromophores, such as fluorescent, luminescent dyes; groups that produce an electron density detectable by electron microscopy or by their electrical properties such as conductivity, amperometric, voltammetry, impedance; detectable groups, e.g., whose molecules have a sufficient size to induce a modification detectable in their physical and / or chemical properties, such detection can be done by optical methods such as diffraction, surface plasmon resonance, surface changes, contact angle changes or physical methods such as atomic force spectroscopy, tunnel effect or radioactive molecules such as 32 P, 35 S or 125 I. BRIEF DESCRIPTION OF DRAWINGS

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

[0053] Figure 2 is a graph of ELISA binding experiment results for human epidermal growth factor receptor 2 antibody.

[0054] Figure 3 is a graph of killing experiment results for 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 one of ordinary skill in the art upon reading the present disclosure, each of the individual aspects described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several aspects without departing from the scope or spirit of the present disclosure.

[0056] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 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 that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0057] Screening of anti-human epidermal growth factor receptor 2 antibodies

[0058] Using natural human phage antibody display library, 3 rounds of panning were performed to obtain positive phage with strong binding to human epidermal growth factor receptor 2. The positive rate of the third round of bio-panning was significantly increased, indicating that the positive phage was effectively enriched and could be used for further screening of candidate molecules. Clones with OD value > 1 were selected for sequencing verification, and finally one sequence was obtained, named HFM or HF-M.

[0059] The specific heavy chain variable region sequence is shown in Table 1.

[0060] Table 1 Heavy chain variable region sequence

[0061]

[0062] The specific light chain variable region sequence is shown in Table 2.

[0063] Table 2 Light chain variable region sequence

[0064]

[0065] By constructing to the eukaryotic expression vector pCDNA3.1, transfecting into CHO suspension cells in logarithmic growth phase, culturing for about 7 days, harvesting the culture supernatant, and identifying, the antibody with a purity of more than 95% was obtained.

[0066] Specificity detection of antibodies

[0067] Coat ELISA plate with human epidermal growth factor receptor 2 to a final concentration of 1 μg / ml, 100 μl per well, 4°C overnight. Wash the ELISA plate 3 times with plate washer. Block with 1.5% casein, 200 μl per well, 37°C for 1 hour. Wash the ELISA plate 3 times with plate washer. Prepare biotin-labeled HERCEPTIN antibody in 1X PBS, the concentration of the antibody is 0.5 μg / ml (this concentration is the lowest saturation concentration). Dilute the naked antibody (HF-M\HF\1103-1\HERCEPTIN) to 100 μg / ml, 33.33 μg / ml, 11.11 μg / ml, 3.70 μg / ml, 1.23 μg / ml, 0.41 μg / ml, 0.14 μg / ml, 0.046 μg / ml, 0.015 μg / ml, 0.005 μg / ml, respectively, with the above solution.

[0068] Add the corresponding sample to the parallel wells, 100 μl per well, and incubate at 37°C for 1.5 hours. Wash the ELISA plate 3 times with plate washer. Add 100 μl of HRP-strepavidin (1:10000) per well, and incubate at room temperature for 45 minutes. Wash the ELISA plate 3 times with plate washer. Add 100 μl of TMB color developing solution per well, and incubate at room temperature for 1 minute. Stop the reaction with stop solution, 100 μl per well. Read at 450 nm with ELISA reader.

[0069] The results are shown in Figure 1 , 2 The results show that the competition experiment can show that the antibody HF-M can compete with Herceptin / Biotin for binding to human epidermal growth factor receptor 2 protein, indicating that it overlaps with the antigen epitope recognized by Herceptin, and has a dose-dependent effect within a certain concentration range, with a LogEC50 of 0.2012, and the trend of repeated experiments is consistent. As a control, herceptin can effectively compete with its biotin-labeled antibody, and has a dose-dependent effect within a certain concentration range, with a LogEC50 of 0.03926, and the trend of repeated experiments is consistent.

[0070] Example 3 Antibody Killing Power Detection

[0071] Principle of the experiment: use human PBMC as effector cells and SKOV3-GFP (a human ovarian cancer cell line stably transfected with green fluorescent protein) as target cells, and use the lactate dehydrogenase (LDH) release method to quantitatively determine the killing activity of drug-activated PBMC on tumor cells.

[0072] The results are shown in Figure 3As shown, the EC50 value of HF-M antibody is 0.01263, the EC50 value of Herceptin is 0.003381, the EC50 value of HF is 0.01467, and the killing ability of HF-M antibody is stronger than that of positive control Herceptin and HF in targeting killing.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against human epidermal growth factor receptor 2, comprising a heavy chain variable region CDR1 as set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 as set forth in SEQ ID NO: 2, a heavy chain variable region CDR3 as set forth in SEQ ID NO: 3, and a light chain variable region CDR1 as set forth in SEQ ID NO: 8, a light chain variable region CDR2 as set forth in SEQ ID NO: 9, a light chain variable region CDR3 as set forth in SEQ ID NO:

10.

2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 4-7, respectively; and the sequences of the light chain variable region framework regions FR1, FR2, FR3, FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 11-14, respectively.

3. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, FR4 of the monoclonal antibody or antigen-binding fragment thereof are as set forth in SEQ ID NOs: 4-7, respectively; and the sequences of the light chain variable region framework regions FR1, FR2, FR3, FR4 of the monoclonal antibody or antigen-binding fragment thereof are as set forth in SEQ ID NOs: 11-14, respectively.

4. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the sequence of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; and the sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

16.

5. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the sequence of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 15; and the sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO:

16.

6. The monoclonal antibody or antigen-binding fragment thereof of claim 1, which is a Fab, Fab', F(ab')2, Fv fragment, disulfide stabilized Fv fragment, (dsFv)2, dual specific dsFv, disulfide stabilized diabody, single chain antibody molecule, scFv dimer, multi-specific antibody, nanobody, domain antibody, or bivalent domain antibody.

7. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof is of human or non-human mammalian origin.

8. The monoclonal antibody or antigen-binding fragment thereof of claim 7, wherein the monoclonal antibody or antigen-binding fragment thereof is of human origin.

9. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 for the manufacture of a product for detecting human epidermal growth factor receptor 2.

10. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 for the manufacture of a pharmaceutical composition for preventing or treating a disease associated with abnormal expression of human epidermal growth factor receptor 2; the disease associated with abnormal expression of human epidermal growth factor receptor 2 is selected from the group consisting of 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 cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, endometrial cancer, renal cancer, vulvar cancer, thyroid cancer, penile cancer, glioblastoma multiforme, sarcoma, and melanoma.

11. A method for preparing a cell or tissue in vitro with high inhibition of human epidermal growth factor receptor 2, the method comprising introducing a vector carrying the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 into a cell or tissue 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 8.

12. Any one of the following, comprising: 1) a polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; 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 8; 3) a cell comprising a nucleotide sequence encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a vector thereof; the cell does not include an individual of an animal or plant; 4) a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, 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 8, and a detectable label that does not affect the function of the monoclonal antibody or antigen-binding fragment thereof itself when bound to the monoclonal antibody or antigen-binding fragment thereof.

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