Monoclonal antibody against human her2 and use thereof

Abstract

The application belongs to the technical field of biotechnology and the field of antibody engineering pharmacy, and particularly relates to an anti-human HER2 monoclonal antibody and application thereof, and specifically discloses an anti-human HER2 monoclonal antibody or an antigen binding fragment thereof, wherein the HER2 epitope recognized by the anti-human HER2 monoclonal antibody or the antigen binding fragment thereof is the third domain of HER2, and the anti-human HER2 monoclonal antibody has higher affinity.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and antibody engineering pharmaceuticals; specifically, it relates to a monoclonal antibody against human HER2 and its applications. Background Technology

[0002] The epidermal growth factor (EGF) receptor family includes four proteins: HER1 (ERBB1, EGFR), HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB4). All four contain extracellular domains, transmembrane domains, and intracellular tyrosine kinase domains, and play a role in regulating signaling pathways that control cell proliferation and survival. Unlike the other three members of the EGF receptor family, human epidermal growth factor receptor 2 (HER2) is an orphan receptor, lacking a ligand for binding. HER2 can form heterodimers with other members, with the EGFR / HER2 and HER2 / HER3 heterodimers being closely associated with tumorigenesis.

[0003] The extracellular regions of the four proteins in the EGF receptor family each consist of four domains, with domain II containing a dimerization arm. HER2, lacking a ligand, remains in an extended state. The other three members, without ligand binding, are in a compressed state, with the dimerization arm hidden within a pocket formed by domains II and IV. Upon ligand binding, the protein extends, exposing the dimerization arm, which then binds to a pocket formed by domains I and III of another protein. The dimerization leads to phosphorylation of the intracellular tyrosine kinase domain, thereby activating signaling pathways such as phosphatidylinositol triphosphate kinase (PI3K) and mitogen-activated protein kinase (MAPK), resulting in cell cycle progression, cell differentiation, and cell proliferation.

[0004] HER2 is encoded by the ERBB2 proto-oncogene on chromosome 17 of breast epithelial cells. When the ERBB2 gene amplifies and the HER2 protein is overexpressed, downstream tyrosine kinase signaling is activated in a cascade, triggering cell survival, proliferation, migration, and invasion. HER2 is a tumor-associated antigen (TAA), overexpressed in 15% of invasive breast cancer, 54-100% of colorectal cancer, 25% of cervical cancer, 17-82% of pancreatic cancer, and 34% of prostate cancer. HER2 overexpression is closely associated with poor prognosis. HER2-targeting drugs have demonstrated excellent clinical efficacy over more than two decades of development, achieving milestone after milestone in the field of cancer treatment. HER2-targeting therapeutic strategies include monoclonal antibodies (mAbs), small molecule tyrosine kinase inhibitors (TKIs), antibody-drug conjugates (ADCs), bispecific antibodies, and cell therapy.

[0005] Trastuzumab was the first monoclonal antibody drug used to treat HER2-positive breast cancer and the first monoclonal antibody drug used to treat solid tumors. Trastuzumab binds to the fourth domain of the extracellular region of HER2, inhibiting HER2 heterodimerization, suppressing the intracellular HER2 signaling pathway, leading to cell cycle arrest, and exerting an ADCC effect. Although Trastuzumab revolutionized the treatment of HER2-positive breast cancer, many patients developed resistance and relapse within a year of treatment, possibly because Trastuzumab cannot effectively inhibit the formation of heterodimers between HER2 and other members of the EGF receptor family.

[0006] Pertuzumab is the second humanized IgG1 monoclonal antibody that binds to the second extracellular domain of HER2, inhibiting HER2 heterodimerization and blocking downstream signaling pathways. Combining antibodies targeting different epitopes of the HER2 protein can produce synergistic anti-tumor effects; the combination of trastuzumab and pertuzumab exhibits a stronger inhibitory effect on downstream HER2 signaling pathways. Pertuzumab in combination with trastuzumab and docetaxel chemotherapy helps improve the complete response rate in patients with HER2-positive breast cancer. Summary of the Invention

[0007] In a first aspect, the present invention provides a monoclonal antibody against human HER2 or an antigen-binding fragment thereof, wherein the monoclonal antibody against human HER2 comprises a heavy chain variable region (VH) and a light chain variable region (VL) complementarity-determining region (CDR1, CDR2, and CDR3, respectively); the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.1; wherein the amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 are shown in SEQ ID NO.2-4; the nucleic acid sequence of the light chain variable region (VL) is shown in SEQ ID NO.4; wherein VL-CDR1, VL-CDR2, and VL-CDR3 are shown in SEQ ID NO.6-8.

[0008] Furthermore, the HER2 epitope recognized by the anti-human HER2 monoclonal antibody or its antigen-binding fragment is the third domain of HER2.

[0009] Furthermore, the sequences of the complementarity-determining regions of the heavy chain variable region (VH) and light chain variable region (VL) of the anti-human HER2 monoclonal antibody also include their mutant sequences, which have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity.

[0010] Furthermore, the anti-human HER2 monoclonal antibody or its antigen-binding fragment may also include its derivatives.

[0011] In one embodiment, the anti-human HER2 monoclonal antibody is a rabbit monoclonal antibody against human HER2.

[0012] In a second aspect, the present invention provides an isolated nucleic acid molecule that encodes a monoclonal antibody against human HER2 or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0013] Furthermore, the antibody, antibody fragment, or derivative thereof encoded by the nucleic acid molecule may be DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a chimeric nucleic acid molecule resulting from the recombination of any of those nucleic acid molecules, individually or in combination.

[0014] Thirdly, the present invention provides a recombinant vector or host cell for a nucleic acid molecule comprising a monoclonal antibody against human HER2 or an antigen-binding fragment thereof.

[0015] Furthermore, the vector is selected from DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0016] Furthermore, the viral vector is selected from lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0017] Furthermore, the host cell includes prokaryotic cells or eukaryotic cells.

[0018] Furthermore, the host cells include, but are not limited to, Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0019] Furthermore, the prokaryotic cells include, but are not limited to, Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0020] Furthermore, the eukaryotic cells include, but are not limited to, Pichia pastoris, Saccharomyces cerevisiae, Schizomyces cerevisiae, Trichoderma, insect cells such as armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

[0021] Fourthly, the present invention provides a chimeric antibody comprising the anti-human HER2 monoclonal antibody or its antigen-binding fragment as described in the first aspect of the present invention.

[0022] Fifthly, the present invention provides fusions or conjugates comprising the monoclonal antibody against human HER2 as described in the present invention or an antigen-binding fragment thereof, wherein the antibody is fused or conjugated to a heterologous molecule to produce the fusion or conjugate.

[0023] Furthermore, the fusion or conjugate is a polypeptide of the anti-human HER2 monoclonal antibody or its antigen-binding fragment described in the first aspect, fused or conjugated with one or more heterologous molecules, wherein the heterologous molecules include, but are not limited to, protein polypeptides / peptides, markers, drugs, and cytotoxic agents.

[0024] In a sixth aspect, the present invention provides a composition comprising an anti-human HER2 monoclonal antibody of the present invention or an antigen-binding fragment thereof, a derivative thereof, a nucleic acid molecule, a vector or host cell, a chimeric antibody and / or a fusion or conjugate.

[0025] In a seventh aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof, derivative, nucleic acid molecule, carrier or host cell, chimeric antibody, fusion compound or conjugate and / or composition thereof in the preparation of a diagnostic, preventive and / or therapeutic drug for tumors.

[0026] Furthermore, the tumor is preferably breast cancer.

[0027] Furthermore, the breast cancer is breast cancer with high or low expression of HER2.

[0028] Furthermore, the breast cancer is preferably breast cancer with low HER2 expression.

[0029] Eighthly, the present invention provides a pharmaceutical composition for treating breast cancer, the pharmaceutical composition comprising an anti-human HER2 monoclonal antibody or an antigen-binding fragment thereof, a derivative thereof, a nucleic acid molecule, a carrier or host cell, a chimeric antibody and / or a fusion or conjugate, and at least another pharmaceutically acceptable carrier, diluent and / or excipient.

[0030] Furthermore, the pharmaceutical composition contains additional antitumor agents.

[0031] Furthermore, the antitumor agent is selected from the group consisting of antibodies, small molecules, nanoparticles, antimetabolites, alkylating agents, topoisomerase inhibitors, microtubule-targeting agents, kinase inhibitors, protein synthesis inhibitors, immunotherapeutic agents, hormones or their analogues, DNA nanobinding agents, and / or mTOR inhibitors.

[0032] Furthermore, the antitumor agent is selected from the group consisting of gefitinib, lapatinib, sunitinib, pemetrexed, bevacizumab, cetuximab, imatinib, alemtuzumab, trastuzumab, pertuzumab, rituximab, erlotinib, and bortezomib.

[0033] In a ninth aspect, the present invention provides a kit for detecting HER2 antibodies, the kit comprising instructions and detection reagents, wherein the detection reagents are anti-human HER2 monoclonal antibodies or their antigen-binding fragments, derivatives, nucleic acid molecules, vectors or host cells, chimeric antibodies and / or fusion compounds or conjugates, and the compositions described in the sixth aspect of the present invention.

[0034] Beneficial effects

[0035] The rabbit monoclonal antibody against human HER2 prepared in this invention has higher affinity; K D The value is 2.79 × 10 -10 Trastuzumab (2.83×10) -10 ) and Pertuzumab (2.3×10 -9 This antibody can recognize epitopes different from those of Trastuzumab and Pertuzumab, increasing the number of available epitopes. Human and mouse IgG have four subtypes, while rabbit IgG has only one. Rabbit IgG has fewer amino acids in the N-terminus and DE ring, and additional disulfide bonds in the variable region of the heavy chain, which helps increase antibody stability. Furthermore, the sequence homology between rabbit and human antibodies is 60%-76%, while the sequence homology between mouse and human antibodies is slightly lower, approximately 57%-72%. Therefore, rabbit monoclonal antibodies are easier to humanize. Attached Figure Description

[0036] Figure 1 Rabbit monoclonal ELISA identification. The horizontal axis represents the antibody number, and the vertical axis represents the ratio of OD450 of the HER2-coated sample wells to the OD450 of the uncoated control wells.

[0037] Figure 2 SPR assay was performed to determine the affinity of Trastuzumab (A), Pertuzumab (B), and r40 antibody (C) for HER2 protein. The curves in the figure, from highest to lowest, correspond to different concentrations from highest to lowest.

[0038] Figure 3 Flow cytometry plot of rabbit monoclonal antibody r40. The x-axis represents fluorescence intensity, and the y-axis represents cell number. The dashed peak plot represents the addition of rabbit monoclonal antibody only, while the filled peak plot represents the addition of Trastuzumab and Pertuzumab to block the binding epitope of HER2 protein before the addition of rabbit monoclonal antibody.

[0039] Figure 4 Flow cytometry plots of HER2 antibody binding to breast cancer cell lines. From top to bottom, the plots show the flow cytometry plots of three cell lines: BT474, SKBR3, and MCF7. The horizontal axis represents the average fluorescence intensity, and the vertical axis represents the cell number.

[0040] Figure 5 Effects of HER2 antibody on BT474 cell proliferation. The x-axis represents antibody concentration, and the y-axis represents the percentage of viable cells. T: Trastuzumab; P: Pertuzumab.

[0041] Figure 6 Effects of HER2 antibody on SKBR3 cell proliferation. The x-axis represents antibody concentration, and the y-axis represents the percentage of viable cells. T: Trastuzumab; P: Pertuzumab.

[0042] Figure 7 HER2 antibody inhibited the proliferation of MCF7 cells. MCF7 cells were incubated with an antibody at a concentration of 20 μg / mL for 4 days. C: Control; T: Trastuzumab; P: Pertuzumab. Error bars, SD.****, P<0.0001, T-test.

[0043] Figure 8 Effects of r40 on the PI3K / AKT signaling pathway in BT474 and SKBR3 cell lines.

[0044] A.BT474 cells were treated with antibodies for 6 h and 24 h, and then Western blotting was used to detect the levels of AKT (Ser437), total AKT, HER2, and the internal control α-Tubulin. T: Trastuzumab; P: Pertuzumab.

[0045] B.SKBR3 cells were treated with antibodies for 6 h and 24 h before being collected. Western blotting was used to detect AKT (Ser437), total AKT, HER2, and the internal control α-Tubulin levels. T: Trastuzumab; P: Pertuzumab.

[0046] Figure 9 r40 inhibits the downstream HER2 / HER3 signaling pathway. MCF7 samples were treated with 50 μg / mL antibody for 6 h and 24 h, followed by treatment with 0.5 nM NRG1 for 10 min before collection. Western blotting was used to detect Ser437 phosphorylated AKT and total AKT, Thr202 / Tyr204 phosphorylated ERK1 / 2 and total ERK1 / 2, HER2, and the internal control GAPDH level. T: Trastuzumab; P: Pertuzumab.

[0047] Figure 10 r40 inhibits the downstream signaling pathway of EGFR / HER2. BT474 cells (left) and SKBR3 cells (right) were treated with 50 μg / mL antibody, and after 24 h, 5 nM EGF was added and incubated for 10 min before harvesting. Western blotting was used to detect Ser437 phosphorylated AKT and total AKT, Thr202 / Tyr204 phosphorylated ERK1 / 2 and total ERK1 / 2, HER2, and the internal control GAPDH level. T: Trastuzumab; P: Pertuzumab.

[0048] Figure 11 Cryo-electron microscopy structure density map of HER2-r40-Trastuzumab-Pertuzumab

[0049] A. Cryo-electron microscopy structure density map: HER2 (blue), Trastuzumab Fab (pink), Pertuzumab Fab (orange), r40 Fab (green).

[0050] B. Overall structure diagram of HER2-r40-Trastuzumab-Pertuzumab: The HER2-Trastuzumab-Pertuzumab structure (PDB: 6OGE) and the r40 antibody structure predicted by AlphaFold are constructed in an electron cloud.

[0051] Figure 12 Structural analysis of HER2-r40-Trastuzumab-Pertuzumab

[0052] A. Cryo-electron microscopy structure: HER2 domain I (light blue), domain II (blue-green), domain III (cyan-green), domain IV (blue), Trastuzumab Fab (pink), Pertuzumab Fab (yellow), r40 Fab (green).

[0053] B. The constructed HER2-r40-HER3 structure. HER2 domain I (light blue), domain II (blue-green), domain III (cyan-green), domain IV (blue), r40 Fab (green), HER3 (gray). Detailed Implementation

[0054] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0055] The term "trastuzumab" as used in this article refers to the first monoclonal antibody drug used to treat HER2-positive breast cancer and the first monoclonal antibody drug used to treat solid tumors. Trastuzumab binds to the fourth domain of the extracellular region of HER2, inhibiting HER2 heterodimerization, suppressing the intracellular HER2 signaling pathway, leading to cell cycle arrest, and exerting ADCC effects. Although trastuzumab changed the history of HER2-positive breast cancer treatment, many patients developed resistance and relapse within a year of treatment, possibly because trastuzumab cannot effectively inhibit the formation of heterodimers between HER2 and other members of the EGF receptor family.

[0056] The term "pertuzumab" used in this article refers to a second-humanized IgG1 monoclonal antibody that binds to the second domain of the extracellular region of HER2, inhibiting HER2 heterodimerization and blocking downstream signaling pathways. Combining antibodies targeting different epitopes of the HER2 protein can produce synergistic anti-tumor effects; the combination of trastuzumab and pertuzumab exhibits a stronger inhibitory effect on downstream HER2 signaling pathways.

[0057] The term "PI3K / AKT signaling pathway" used in this paper refers to an intracellular signal transduction pathway that responds to extracellular signals to promote metabolism, proliferation, cell survival, growth, and angiogenesis. This process is mediated by the phosphorylation of serine or threonine residues from a series of downstream substrates. The key genes involved are phosphatidylinositol 3-kinase (PI3K) and AKT / protein kinase b, hence the pathway is named directly after these two genes. In this invention, activation of the PI3K / AKT signaling pathway leads to phosphorylation of the serine / threonine kinase AKT protein Ser473. A weakening of p-AKT (S437) is detected, indicating that the signaling pathway is downregulated. This paper uses changes in AKT protein Ser473 phosphorylation to determine the activation and downregulation of this pathway.

[0058] The term "MAPK signaling pathway" used in this article refers to the MAPK (mitogen-activated protein kinase) signaling pathway, which regulates various important physiological and pathological effects such as cell growth, differentiation, stress, and inflammatory responses. One of the main branches of the MAPK pathway is extracellular-signal-regulated protein kinase (ERK), which primarily governs cell growth and differentiation. Activation of the signaling pathway leads to ERK phosphorylation. This article uses changes in ERK protein phosphorylation to determine the activation and downregulation of this pathway.

[0059] The terminology used herein refers to “sequence identity” between two polypeptide sequences, meaning the percentage of identical amino acids across the entire length of the amino acid sequence encoded by the CDR sequence in SEQ ID NO:1 and / or SEQ ID NO:4. Preferred polypeptide sequences of the present invention have sequence identity of at least 80%, more preferably 85%, and even more preferably 90%, 93%, 95%, 96%, 97%, 98%, or 99%.

[0060] The term antibody includes a "fragment" or "derivative" having at least one antigen-binding site and / or exhibiting the same biological activity. Furthermore, the antibodies of the present invention preferably comprise at least one immunoglobulin heavy chain and at least one immunoglobulin light chain. The immunoglobulin chain comprises variable domains and optionally constant domains. The variable domains may include complementarity-determining regions (CDRs), such as CDR1, CDR2, and / or CDR3 regions, and frame regions.

[0061] The term "separated" as used herein refers to substances obtained artificially from their natural state. If a "separated" substance or component appears in nature, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. The term "separated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the substance's activity.

[0062] The term "vector" as used herein refers to a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing said genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0063] The terms “host cell,” “host cell line,” and “host cell culture” used herein are used interchangeably and may include cells in which exogenous nucleic acids have been introduced, including the progeny of these cells. Host cells include “transformants” and “transformed cells,” which include primary transformed cells and their progeny, regardless of passage number. Progeny cells may not be identical to parent cells in terms of nucleic acid content but may contain mutations. This document includes mutant progeny cells that have the same function or biological activity as cells screened or selected in the initially transformed cells.

[0064] The determination of "antibody positive" in this article is based on the ratio of the OD value of the experimental group to the OD value of the negative control well. Generally, when the ratio is ≥2.1, the antibody test result described in this article is considered positive.

[0065] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0066] Example

[0067] The extracellular domain (ECD) of the HER2 protein was expressed and purified, and used as an immunogen to immunize rabbits. The antibody sequence was obtained using monoclonal antibody development technology based on single B lymphocyte screening. After expression and purification, ELISA experiments were performed to verify the binding ability, and the KD value of antigen-antibody binding was determined by SPR. The binding epitopes of the antibody were determined by flow cytometry and cryo-electron microscopy. Functional validation was performed using breast cancer cell lines, and finally a rabbit monoclonal antibody that can specifically bind to human HER2 protein domain III and inhibit breast cancer cell proliferation was obtained.

[0068] Example 1: Preparation of Monoclonal Antibodies

[0069] 1.1 Preparation and Immunization of HER2 ECD Protein

[0070] (1) The full-length HER2 gene was purchased from Addgene. The extracellular domain (ECD) amino acids 23-652 of the HER2 protein were selected for cloning and constructing an expression plasmid. The plasmid was used to transfect suspension Expi293 cells and the HER2 ECD protein was purified.

[0071] (2) Immunize New Zealand White rabbits

[0072] Each rabbit was immunized with 400 μg of protein per dose. For the first immunization, the immunogen was mixed with an equal volume of complete Freund's adjuvant to form an emulsion, which was then injected into the footpads. Three weeks later, 400 μg of immunogen was mixed with an equal volume of incomplete Freund's adjuvant to form an emulsion, which was then injected into the footpads as a booster immunization. Two more booster immunizations were then administered every three weeks thereafter, for a total of four immunizations. The spleen was harvested three days after the final immunization.

[0073] 1.2 Spleen cell isolation

[0074] The spleen was cut into small pieces with a scalpel and then transferred to a 100 μm cell sieve for grinding. The resulting cell suspension was filtered to remove large cell clumps and tissue membranes, centrifuged at 450 g for 7 min, and the supernatant was discarded, retaining the spleen cell clumps. The spleen cell clumps were resuspended in standard PBS buffer, and red blood cells were lysed. The suspension was then centrifuged again at 450 g for 7 min, retaining the spleen cells.

[0075] 1.3B lymphocyte sorting

[0076] According to the experimental method of this patent, spleen cells are negatively screened using biomarked non-B cell lymphocyte surface marker antibodies, and non-B cells are removed using avidin magnetic beads and magnetic separation principle to increase the relative abundance of B lymphocytes in spleen cells; 7-AAD is used for sorting to remove dead cells; and flow cytometry is used for single-cell screening to obtain target lymphocytes.

[0077] 1.4 Antibody sequence synthesis, expression, and purification.

[0078] The antibody variable region sequence was obtained using a 10× Genomics single-cell library preparation kit via single-cell V(D)J sequencing. Based on the sequencing results, a certain number of paired light and heavy chains were selected, and the variable region was loaded into a rabbit IgG antibody framework. The expression plasmid was synthesized by General Biotechnology (Anhui) Co., Ltd., and transfected into suspension Expi293 cells. The expressed antibody protein was adsorbed using a Protein A column, followed by elution with 0.1 M glycine (pH 3.0) to obtain the recombinant antibody.

[0079] The amino acid sequence of the variable region of antibody r40 is as follows:

[0080] Heavy chain variable region amino acid sequence:

[0081] QQLEQSGGGAEGGLVKPGGSLELCCSASGFAFNSYWICWVRQAPGKGLEWIGCVYS GSSGATYYASWVNGRFTLSRDVDQSTGCLQLNSLTAADTAMYYCARDLYPNSLNYGFNL WGQGTLVTVSS. (SEQ ID NO.1)

[0082] The heavy chain variable region CDR1 amino acid sequence is: CSASGFA. (SEQ ID NO.2)

[0083] Heavy chain variable region CDR2 amino acid sequence: IGCVYS. (SEQ ID NO.3)

[0084] Heavy chain variable region CDR3 amino acid sequence: CARDLYPNSLNYGFNLW. (SEQ ID NO.4)

[0085] Light chain variable region amino acid sequence:

[0086] AAVLTQTPSPVSAAVGGTVTISCQSSQSVYSNNFLSWYQQKPGQPPKVLIYGASTLASGVP SRFSGSGSGTQFTLTISDVQCDDAATYYCAGGYIATSDRLAFGGGTEVVVK. (SEQ ID NO.5)

[0087] The amino acid sequence of the CDR1 variable region in the light chain is: QSSQSVYSNNFLS. (SEQ ID NO.6)

[0088] The amino acid sequence of the CDR2 variable region of the light chain is: GASTLAS. (SEQ ID NO.7)

[0089] The amino acid sequence of the CDR2 variable region of the light chain is: CAGGYIATSDRLAF. (SEQ ID NO.8)

[0090] Example 2: Screening of high-affinity antibodies

[0091] 2.1 ELISA test

[0092] (1) Coating: Dilute HER2 protein to 0.2 ng / μL with 1×PBS, add 50 μL of diluted HER2 protein to a 96-well ELISA plate, and add an equal volume of 1×PBS to the control group. Incubate overnight at 4°C.

[0093] (2) Washing: Discard the supernatant, add 1×PBST, 150μL / well, after 5min, pour out the liquid and pat dry on absorbent paper, repeat the washing 5 times.

[0094] (3) Blocking: Add 3% BSA blocking buffer (50 μL / well) and incubate the 96-well plate at 37°C for 1 h. Discard the supernatant and wash with 1×PBST.

[0095] (4) Primary antibody incubation: Dilute the primary antibody to 2 ng / μL with 3% BSA blocking buffer, add the corresponding primary antibody dilution buffer (50 μL / well) to the wells, and incubate at 37°C for 1.5 h. Then discard the supernatant and wash with 1×PBST.

[0096] (5) Secondary antibody incubation: Dilute the HRP-conjugated secondary antibody 3000 times with 3% BSA blocking buffer, add secondary antibody dilution buffer (50 μL / well) to the wells, and incubate at 37°C for 1 h. Discard the supernatant and wash with 1×PBST.

[0097] (6) Color development: Add TMB color development solution to the wells, 50 μL / well.

[0098] (7) Termination: When the color depth is suitable, add 25 μL of 1M HCl to each well to terminate the reaction. Detect OD using a microplate reader. 450 .

[0099] The results showed that the ratio of the OD value of r40 antibody to the OD value of the uncoated antigen negative control well was greater than 10, indicating a positive antibody (see...). Figure 1 ).

[0100] 2.2 SPR (Surface Plasmon Resonance)

[0101] (1) Chip fabrication:

[0102] Antibodies were printed onto a 3D photocrosslinked chip using an Arrayjet chip spotting instrument. The chip was then placed in a vacuum drying oven and dried under vacuum for at least 1 hour until the spots were dry. The dried chip was then placed in a photocrosslinking instrument for a photocrosslinking reaction for 30 minutes. The chip was then placed in a development tank and washed sequentially with dimethylformamide, ethanol, and deionized water on a shaker for 15 minutes. It was then dried with nitrogen gas, labeled, and set aside for later use.

[0103] (2) Determine the KD value of the HER2 antibody:

[0104] Install the prepared chip onto On the HT molecular interaction detector, HER2 protein of 1.0 nM to 128 nM was sequentially passed through. The dissociation buffer was 1×PBS, and there were two regeneration buffers: 10 mM Gly-HCl pH 2.0 and 1% BSA.

[0105] The results showed that the K of the r40 rabbit monoclonal antibody D The value is 2.79 × 10 -10 The affinity for HER2 was higher than that for the positive control Trastuzumab (2.83 × 10⁻⁶). -10 ) and Pertuzumab (2.3×10 -9 )(See Figure 2 ).

[0106] Example 3: Identification of Antibody-Binding Epitopes

[0107] Since Expi293 cells express endogenous HER2 protein on their surface, there is no need to transfect the full-length HER2 protein. Untransfected Expi293 cells were used for HER2 antibody verification. The experiment was divided into two groups: one group was treated with rabbit monoclonal antibody only (single staining group); the other group was treated with 10 μg / mL Trastuzumab and Pertuzumab to completely block the HER2 binding epitope, washed three times with 1×PBS, and then treated with 10 μg / mL rabbit monoclonal antibody (saturation group).

[0108] Result interpretation: For the saturation group, if the rabbit monoclonal antibody can still bind, it indicates that it is inconsistent with the epitopes of Trastuzumab and Pertuzumab; if the rabbit monoclonal antibody cannot bind, it indicates that it is consistent with or partially overlaps with the epitopes of Trastuzumab and Pertuzumab, or that there is steric hindrance.

[0109] (1) Take 1 mL of solution with a density of 2-2.5×10⁻⁶. 6 Centrifuge Expi293 cells / mL at 1000g at 4°C for 3 min, discard the supernatant, and wash with pre-cooled 1×PBS.

[0110] (2) Divide the cells into two equal parts. Add 100 μL of 10 ng / μL rabbit monoclonal antibody to one part and add 100 μL of 10 ng / μL Trastuzumab and Pertuzumab to the other part to incubate the cells for 30 min. After washing with 1×PBS, add 100 μL of 10 ng / μL rabbit monoclonal antibody.

[0111] (3) Incubate for 30 min and wash with pre-cooled 1×PBS.

[0112] (4) Add fluorescent secondary antibody at a ratio of 1:250 and incubate for 30 min, then wash with pre-cooled 1×PBS.

[0113] (5) Add DAPI diluted 1:10000 and run it on the machine.

[0114] The results showed that antibody r40 binding to cells was hardly reduced or only slightly reduced in the presence of trastuzumab and pertuzumab, suggesting that its binding epitope to the HER2 protein differs from that of trastuzumab and pertuzumab (see [link to study]). Figure 3 ).

[0115] Example 4: HER2 antibody binding experiment with breast cancer cells

[0116] To test whether the HER2 antibody can recognize the HER2 protein on the surface of breast cancer cell lines, the antibody was incubated with three breast cancer cell lines: BT474, SKBR3, and MCF7, followed by flow cytometry analysis. The experimental steps are as follows:

[0117] (1) The three breast cancer cell lines BT474, SKBR3 and MCF7 were digested with trypsin and washed with pre-cooled 1×PBS. The cells were collected by centrifugation at 1000 rpm for 3 min and 4℃.

[0118] (2) 5×10 in each experimental group 5 Cells were resuspended in 100 μL of 1×PBS. The experimental groups were treated with 10 μg / mL Trastuzumab, Pertuzumab, and r40, respectively, while the control groups were treated with equal amounts of human IgG and rabbit IgG. A blank control was also included. All cells were incubated for 30 min.

[0119] (3) Wash with pre-cooled 1×PBS, centrifuge to collect cells, and resuspend cells in 100μL 1×PBS.

[0120] (4) Add fluorescent secondary antibody at a ratio of 1:250 and incubate in cold storage for 15 min.

[0121] (5) Wash three times with pre-cooled 1×PBS, centrifuge at 1000 rpm for 3 min to collect cells.

[0122] (6) Dilute DAPI with 1×PBS at a ratio of 1:10000, take 100μL to resuspend the cells and then perform the detection.

[0123] (7) The binding of Trastuzumab, Pertuzumab, and r40 to the HER2 protein on the surface of three breast cancer cell lines, BT474, SKBR3, and MCF7, was tested by flow cytometry. The constant regions of Trastuzumab and Pertuzumab are human IgG1 antibodies, and the isotype control is a Human IgG isotype. r40 is a rabbit monoclonal antibody, and the isotype control is a Rabbit IgG isotype.

[0124] Depend on Figure 4 It is evident that r40 antibodies can all recognize the native HER2 protein on the cell surface, comparable to Trastuzumab and Pertuzumab.

[0125] Example 5 Tumor Cell Proliferation Inhibition Experiment

[0126] The effect of the antibody on breast cancer cell proliferation was determined using the Cell Counting Kit-8 (CCK-8) assay. The method is as follows:

[0127] Breast cancer cell lines BT474, SKBR3, and MCF7 were seeded into 96-well cell culture plates at doses of 3000, 9000, and 5000 cells / well, respectively, and cultured overnight at 37°C in a 5% CO2 incubator. Antibodies were serially diluted and added to the cells for 4 days of further culture. 10 μL of CCK8 reagent was added to each well, and the plates were returned to the incubator for 2 hours. OD450 was measured using a microplate reader.

[0128] To verify whether the screened HER2 antibody could inhibit the proliferation of breast cancer cells, a CCK8 assay was performed using the HER2-highly expressing breast cancer cell line BT474. Six antibody gradients were set up, with a maximum concentration of 100 μg / mL, diluted 5-fold, and a minimum concentration of 0 μg / mL. When Trastuzumab and Pertuzumab antibodies were used in combination, the maximum concentration of both was 100 μg / mL.

[0129] The results showed that the combination of Trastuzumab and Pertuzumab antibodies had the strongest inhibitory effect on BT474 cells, followed by Trastuzumab alone. The inhibitory effects of Pertuzumab and r40 were comparable. Figure 5 ).

[0130] Subsequently, the effects of trastuzumab, pertuzumab, r40, and the combination of trastuzumab and pertuzumab on cell proliferation were verified in another HER2-high expressing breast cancer cell line, SKBR3. Compared with BT474, the overall inhibitory effect of the antibodies on SKBR3 cell proliferation was weaker, and the r40 antibody inhibited the proliferation of SKBR3 cells to a certain extent. Figure 6 ).

[0131] To investigate the effect of HER2 antibody on the proliferation of breast cancer cell lines with low HER2 expression, a CCK8 assay was performed using MCF7, and the antibody's effect in the presence of the HER3 ligand NRG1 was also tested. The antibody concentration was 20 μg / mL, and both Trastuzumab and Pertuzumab were used at 20 μg / mL in combination. The concentration of neuroregulatory protein 1 (NRG1) was 2 nM, and NRG1 was added 2 hours after the antibody was added.

[0132] In the absence of NRG1, trastuzumab, pertuzumab, r40, and the combination of trastuzumab and pertuzumab all significantly inhibited MCF7 cell proliferation; in the presence of NRG1, the four antibodies and the combination of trastuzumab and pertuzumab also significantly inhibited MCF7 cell proliferation. Figure 7 ).

[0133] In summary, r40 antibody inhibits the proliferation of both HER2-overexpressing and HER2-underexpressing breast cancer cell lines.

[0134] Example 6: Western Blot Detection of Downstream Signaling Pathway Protein Levels

[0135] BT474 and SKBR3 cells were cultured to 60-70% confluence, and 50 μg / mL of antibody was added. When Trastuzumab and Pertuzumab were used in combination, both were at a concentration of 50 μg / mL. Cells were cultured at 37°C in a 5% CO2 incubator. Cells were collected at 6 h and 24 h, respectively. Total cellular protein was extracted using RIPA lysis buffer. The levels of p-AKT (S473) and total AKT, as well as the expression level of HER2 protein, were detected by SDS-PAGE gel electrophoresis and Western blotting.

[0136] (1) Soak 1.5 × 10⁶ cells in each well of a 12-well cell culture plate. 5 One cell was added to the culture vessel the next day after the cells adhered. The antibody concentration was 50 μg / mL. The cells were then cultured for another 6 hours or 24 hours.

[0137] (2) Wash the cells twice with pre-cooled 1×PBS, add 80 μL of RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor to each well, pipette and transfer to 1.5 mL EP tube, and lyse at 4 °C for 30 min.

[0138] (3) Centrifuge the cell lysis buffer at 12,000 rpm, 4°C, for 10 min.

[0139] (4) Transfer the supernatant to a new 1.5 mL EP tube, add 20 μL of 5×SDS loading, boil the sample in a metal bath for 5 min, and then perform SDS-PAGE gel electrophoresis.

[0140] (5) After transfer, the levels of downstream signaling pathway proteins were measured by Western blotting.

[0141] The results of Western Blot analysis are shown in Figure 8. In both cell types, Trastuzumab and r40 antibody reduced p-AKT(S473) and HER2 levels, while Pertuzumab had virtually no effect on p-AKT(S473) and HER2 levels.

[0142] In BT474 cells, treatment with Trastuzumab and r40 antibodies for 6 hours slightly decreased HER2 expression, while treatment for 24 hours significantly reduced it. Figure 8 A).

[0143] In SKBR3 cells, treatment with r40 antibody for 6 h and 24 h significantly reduced the levels of p-AKT (S473) and HER2 proteins. Figure 8 B).

[0144] Therefore, r40 antibody can reduce HER2 protein levels and downregulate the PI3K / AKT signaling pathway.

[0145] Subsequently, Western blotting was used to detect the levels of p-AKT (S437), total AKT, p-ERK, total ERK, and HER2 in MCF7 cells with low HER2 expression after antibody treatment. MCF7 cells were incubated with 50 μg / mL antibody for 6 h and 24 h, followed by incubation with 0.5 nM NRG1 for 10 min before harvesting. NRG1 promotes the formation of HER2 / HER3 heterodimers, activating the PI3K / AKT and MAPK signaling pathways, resulting in a significant increase in p-AKT (S437) levels in NRG1-treated cells.

[0146] Pertuzumab affects HER2 / HER3 dimerization by targeting the second domain of HER2, thus completely inhibiting the effect of NRG1. p-AKT (Ser437) and p-ERK are maintained at the levels without NRG1 stimulation.

[0147] r40 antibody and trastuzumab reduced p-AKT (S437) and HER2 levels, with a more significant effect at 24 h than at 6 h. r40 antibody and trastuzumab had a smaller effect on p-ERK at 6 h, but also significantly reduced p-ERK levels at 24 h. Figure 9 ).

[0148] Therefore, in MCF7 cells, r40 can also downregulate the PI3K / AKT and MAPK signaling pathways by reducing HER2 protein levels.

[0149] In addition to forming a heterodimer with HER3, HER2 also primarily forms a heterodimer with another member of the HER2 family, EGFR. Both the EGFR / HER2 and HER2 / HER3 complexes have important physiological functions and are closely related to tumorigenesis. Therefore, the effects of the antibody on the EGFR / HER2 heterodimer and its downstream pathways were further investigated.

[0150] BT474 and SKBR3 cells were incubated with 50 μg / mL antibody for 24 h, then incubated with 5 nM EGF for 10 min, and RIPA lysis buffer was added. Whole cell lysates were collected for Western blot analysis.

[0151] The result is Figure 10 As shown:

[0152] In both BT474 and SKBR3 cell lines, EGF activated downstream signaling pathways, and the levels of p-AKT(S437) and p-ERK were significantly increased.

[0153] In BT474 cells, both r40 antibody and Trastuzumab significantly reduced p-AKT(S437) and p-ERK.

[0154] In SKBR3 cells, r40 antibody and trastuzumab also reduced p-AKT (S437) levels, but had no effect on p-ERK levels. Figure 10 ).

[0155] In summary, r40 can reduce HER2 expression in BT474, SKBR3, and MCF7 cells, thereby downregulating the HER2 signaling pathway and exerting an anti-cancer effect.

[0156] Example 7: Analysis of the cryo-electron microscopy structure of an antigen-antibody complex protein sample.

[0157] Antibody structure studies typically focus on the antigen-binding region, the Fab region. The Fab contains the complete light chain, the variable region (VH) of the heavy chain, and the constant region (CH1). An expression vector for the Fab was constructed and expressed. The Fab was purified using affinity chromatography. Then, HER2 protein was incubated with an excess of r40 Fab antibody and passed through a molecular sieve to collect the HER2-r40 complex. Next, HER2-r40 was incubated with an excess of Trastuzumab and passed through a molecular sieve to collect the HER2-r40-Trastuzumab complex. Finally, the HER2-r40-Trastuzumab complex was incubated with an excess of Pertuzumab and passed through a molecular sieve to collect the HER2-r40-Trastuzumab-Pertuzumab complex. Through these three steps, a quaternary complex was obtained, consisting of HER2 and three antibodies: r40, Trastuzumab, and Pertuzumab. The protein concentration was increased to 1.5 mg / mL through concentration. After hydrophilization treatment, the Quantifoil porous carbon support membrane (R1.2 / 1.3Au) was transferred to a Vitrobot Mark IV cryogenic sample preparation device pre-cooled to 4°C with humidity set to 100%. 3 μL of protein solution was added to the Quantifoil porous carbon support membrane, blotted with filter paper for 1 second, and then quickly immersed in liquid ethane. The carbon support membrane was then rapidly immersed in a sample container in liquid nitrogen for preservation.

[0158] The prepared cryo-electron microscopy samples were used for data acquisition and structural analysis using a 300 kV cryo-electron microscope (Thermo Fisher Scientific TitanKrios G4300 kV). 2965 cryo-images of HER2-r40-Trastuzumab-Pertuzumab were acquired for subsequent data processing. Through data analysis software, particle selection was performed on the cryo-images, and the particles were classified in two dimensions, three dimensions, and reconstructed, ultimately yielding the desired results. The structural density of HER2-r40-Trastuzumab-Pertuzumab was determined using high resolution. Model building was performed using the obtained cryo-electron microscopy density maps, with the HER2-Trastuzumab-Pertuzumab complex model (PDB: 6OGE) as the initial model template. The HER2-Trastuzumab portion of the model was extracted and embedded (docked) into the calculated density of HER2-Trastuzumab-m66 using UCSF Chimera via Rigid-body fitting. The entire HER2-Trastuzumab-Pertuzumab model was then extracted and combined with the r40 model predicted by AlphaFold. This was then embedded (docked) into the calculated density of HER2-Trastuzumab-Pertuzumab-r40 using UCSF Chimera via Rigid-body fitting, followed by manual model building using COOT software. All density maps and models were displayed and plotted using PyMOL (https: / / pymol.org / ), UCSF Chimera, and UCSF ChimeraX.

[0159] The results are as follows Figure 11 As shown, the electron clouds of HER2, Trastuzumab, Pertuzumab, and r40 are clearly visible. Pertuzumab binds to the second domain of HER2, while Trastuzumab binds to the fourth domain of HER2, which is consistent with previous literature reports.

[0160] Antibody r40 binds to domain III of HER2, while Pertuzumab, r40, and Trastuzumab bind to domains II, III, and IV of HER2, respectively. Figure 12 The binding of these three components to HER2 is not contradictory, which is consistent with the correct assembly of the quaternary complex. The large interaction interface between the r40 antibody and HER2 is the structural basis for ensuring the high affinity of r40 to HER2 and its good inhibitory activity. Based on the reported HER2-HER3-NRG1 structure (PDB: 7MN5) and the resolved HER2-r40-Trastuzumab-Pertuzumab structure, the HER2-r40-HER3 structure was constructed using HER2 as a reference. Figure 12 B). It is evident that the r40 antibody does not affect the formation of heterodimers between HER2 and HER3.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against human HER2, comprising a heavy chain variable region (VH) and a light chain variable region (VL) complementarity determining region sequence, the heavy chain variable region and the light chain variable region complementarity determining region each comprising a CDR1, a CDR2 and a CDR3; the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO. 1; wherein the amino acid sequences of VH-CDR1, VH-CDR2 and VH-CDR3 are shown as SEQ ID NO. 2-4; the nucleic acid sequence of the light chain variable region (VL) is shown as SEQ ID NO. 5; wherein VL-CDR1, VL-CDR2 and VL-CDR3 are shown as SEQ ID NO. 6-8.

2. The monoclonal antibody or antigen-binding fragment thereof against human HER2 according to claim 1, wherein, The HER2 epitope recognized by the monoclonal antibody or antigen-binding fragment thereof against human HER2 is the third domain of HER2.

3. An isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof against human HER2 of claim 1.

4. A recombinant vector comprising the isolated nucleic acid molecule of claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the recombinant vector of claim 4.

6. A chimeric antibody comprising the monoclonal antibody or antigen-binding fragment thereof against human HER2 of any one of claims 1-2.

7. A conjugate of the monoclonal antibody or antigen-binding fragment thereof against human HER2 of any one of claims 1-2, wherein the antibody is conjugated to a heterologous molecule to produce the conjugate.

8. A composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-2, the nucleic acid molecule of claim 3, the recombinant vector of claim 4, the host cell of claim 5, the chimeric antibody of claim 6 and / or the conjugate of claim 7.

9. Use of the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-2, the nucleic acid molecule of claim 3, the recombinant vector of claim 4, the host cell of claim 5, the chimeric antibody of claim 6, the conjugate of claim 7 and / or the composition of claim 8 in the preparation of a medicament for preventing and / or treating a tumor; the tumor is breast cancer.

10. A kit for detecting a HER2 antibody, the kit comprising instructions and a detection reagent for detecting the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-2, the nucleic acid molecule of claim 3, the recombinant vector of claim 4, the host cell of claim 5, the chimeric antibody of claim 6, the conjugate of claim 7 and / or the composition of claim 8.

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