Anti-human HER2 monoclonal antibody and application thereof

Abstract

The invention belongs to the fields of biotechnology and antibody engineering pharmacy. In particular to an anti-human HER2 monoclonal antibody and application thereof. In particular to an anti-human HER2 monoclonal antibody or an antigen binding fragment thereof, an HER2 epitope recognized by the anti-human HER2 monoclonal antibody or the antigen binding fragment thereof is a III structural domain of HER2, and the anti-human HER2 monoclonal antibody disclosed by the invention has higher affinity.

Description

Technical Field

[0001] The present 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 Art

[0002] The epidermal growth factor (EGF) receptor family consists of four proteins, namely HER1 (ERBB1, EGFR), HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB4), all of which contain an extracellular domain, a transmembrane domain, and an intracellular tyrosine kinase domain, and play a role in regulating signal pathways that control cell proliferation and survival. Different from the other three members of the EGF receptor family, human epidermal growth factor receptor 2 (HER2) is an orphan receptor without a ligand that binds to it. HER2 can form heterodimers with other members, among which the EGFR / HER2 and HER2 / HER3 heterodimers are closely related to tumorigenesis.

[0003] The extracellular region of each of the four proteins in the EGF receptor family is composed of four domains, and the second domain contains a dimerization arm. HER2 has no ligand and is always in an extended state. The other three members are in a compressed state when not bound to a ligand, and the dimerization arm is hidden in a pocket formed by the second and fourth domains. After ligand binding, the protein extends to expose the dimerization arm, and the dimerization arm binds to a pocket formed by the first and third domains of another protein. The dimerization leads to phosphorylation of the intracellular tyrosine kinase domain, thereby activating signal pathways such as phosphatidylinositol 3-phosphate kinase (PI3K) and mitogen-activated protein kinase (MAPK), resulting in cell cycle progression, cell differentiation, and cell proliferation, etc.

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

[0005] Trastuzumab is the first monoclonal antibody drug used to treat HER2-positive breast cancer and also the first monoclonal antibody drug used to treat solid tumors. Trastuzumab binds to the fourth domain of the extracellular region of HER2, inhibits the heterodimerization of HER2, inhibits the intracellular HER2 signaling pathway, causes cell cycle arrest, and exerts the ADCC effect. Although Trastuzumab has changed the treatment history of HER2-positive breast cancer, many patients develop drug resistance and recurrence within one year after 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, which binds to the second domain of the extracellular region of HER2, inhibits HER2 heterodimerization, and blocks the downstream signaling pathway. The combined use of antibodies targeting different epitopes of the HER2 protein can produce a synergistic anti-tumor effect. The combined use of Trastuzumab and Pertuzumab has a stronger inhibitory effect on the HER2 downstream signaling pathway. The combination of Pertuzumab, Trastuzumab, and docetaxel chemotherapy helps to improve the complete remission rate of 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. The monoclonal antibody against human HER2 comprises complementary determining region sequences of a heavy chain variable region (VH) and a light chain variable region (VL). The complementary determining regions of the heavy chain variable region and the light chain variable region respectively contain CDR1, CDR2, and CDR3. The amino acid sequence of the heavy chain variable region (VH) is as shown in SEQ ID NO.1. Among them, the amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 are as shown in SEQ ID NOs.2-4. The nucleic acid sequence of the light chain variable region (VL) is as shown in SEQ ID NO.4. Among them, VL-CDR1, VL-CDR2, and VL-CDR3 are as shown in SEQ ID NOs.6-8.

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

[0009] Further, the sequences of the complementary determining regions of the heavy chain variable region (VH) and the light chain variable region (VL) of the monoclonal antibody against human HER2 further include their mutant sequences, and the mutant sequences 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%, at least 99% sequence identity.

[0010] Further, the monoclonal antibody against human HER2 or its antigen-binding fragment further includes its derivatives.

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

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

[0013] Further, the antibody, antibody fragment, or its derivative encoded by the nucleic acid molecule can be DNA, cDNA, RNA, or synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of those nucleic acid molecules alone or in combination.

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

[0015] Further, 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 lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0017] Further, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0018] Furthermore, the host cell includes, but is not limited to, Escherichia coli, yeast cells, mammalian cells, phages, or a combination thereof.

[0019] Further, the prokaryotic cell includes, but is not limited to, Escherichia coli, Bacillus subtilis, Lactobacillus, Streptomyces, Proteus mirabilis, or a combination thereof.

[0020] Further, the eukaryotic cell includes, but is not limited to, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as Spodoptera frugiperda, plant cells such as Nicotiana tabacum, BHK cells, CHO cells, COS cells, myeloma cells, or a combination thereof.

[0021] In a fourth aspect, the present invention provides a chimeric antibody, which comprises the anti-human HER2 monoclonal antibody or an antigen-binding fragment thereof according to the first aspect of the present invention.

[0022] In a fifth aspect, the present invention provides a fusion or conjugate comprising the anti-human HER2 monoclonal antibody or an antigen-binding fragment thereof according to the present invention, wherein the antibody is fused or conjugated to a heterologous molecule to produce a fusion or conjugate.

[0023] Further, the fusion or conjugate is formed by fusing or conjugating a polypeptide of the anti-human HER2 monoclonal antibody or an antigen-binding fragment thereof according to the first aspect with one or more heterologous molecules, wherein the heterologous molecules include, but are not limited to, protein polypeptides / peptides, markers, drugs, cytotoxic agents.

[0024] In a sixth aspect, the present invention provides a composition, which comprises the anti-human HER2 monoclonal antibody or an antigen-binding fragment thereof, derivatives, nucleic acid molecules, vectors or host cells, chimeric antibodies and / or fusion or conjugates according to the present invention.

[0025] In a seventh aspect, the present invention provides the use of the antibody or an antigen-binding fragment thereof, derivatives, nucleic acid molecules, vectors or host cells, chimeric antibodies, fusion or conjugates according to the present invention and / or the composition according to the present invention in the preparation of drugs for diagnosing, preventing and / or treating tumors.

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

[0027] Furthermore, the breast cancer is HER2-highly expressed or HER2-lowly expressed breast cancer.

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

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

[0030] Further, the pharmaceutical composition contains an additional anti-tumor agent.

[0031] Further, the anti-tumor agent is selected from the group consisting of antibodies, small molecules, nanoparticles, antimetabolites, alkylating agents, topoisomerase inhibitors, agents targeting microtubules, kinase inhibitors, protein synthesis inhibitors, immunotherapeutic agents, hormones or their analogs, DNA nano-binding agents and / or mTOR inhibitors.

[0032] Further, the anti-tumor 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 including an instruction manual and a detection reagent, the detection reagent being an anti-human HER2 monoclonal antibody or an antigen-binding fragment, derivative, nucleic acid molecule, vector or host cell, chimeric antibody and / or fusion or conjugate thereof and the composition described in the sixth aspect of the present invention.

[0034] Beneficial effects

[0035] The rabbit monoclonal antibody against human HER2 prepared by the present invention has a higher affinity; the K D value is 2.79×10 -10 ; Trastuzumab (2.83×10 -10 ) and Pertuzumab (2.3×10 -9 ); meanwhile, this antibody can recognize epitopes different from those of Trastuzumab and Pertuzumab, increasing the number of available epitopes; both human and mouse IgG have four subtypes, while rabbit IgG has only one. Rabbit IgG has fewer amino acids at the N-terminus and the D-E loop, and has an additional disulfide bond in the variable region of the heavy chain, which helps to increase the stability of the antibody. At the same time, the antibody sequence homology between rabbits and humans can reach 60%-76%, and the antibody sequence homology between mice and humans is slightly lower, about 57%-72%. Therefore, rabbit monoclonal antibodies are easier to be humanized. Description of the drawings

[0036] Figure 1 Identification of rabbit monoclonal antibodies by ELISA. The abscissa represents the antibody number, and the ordinate represents the ratio of the OD450 value of the well coated with HER2 to the OD450 value of the control well without coated protein.

[0037] Figure 2 SPR detection of the affinity of Trastuzumab (A), Pertuzumab (B), and r40 antibody (C) with HER2 protein. The order of all curves from high to low in the figure corresponds to different concentrations from high to low.

[0038] Figure 3 Flow cytometry of rabbit monoclonal antibody r40. The abscissa is the fluorescence intensity, and the ordinate is the cell number. The dotted peak graph is the addition of only rabbit monoclonal antibody, and the filled peak graph is the addition of Trastuzumab and Pertuzumab to block the binding epitopes of HER2 protein first, and then the addition of rabbit monoclonal antibody.

[0039] Figure 4 Flow cytometry of the binding of HER2 antibodies to breast cancer cell lines. From top to bottom are the flow cytometry graphs of three cell lines, BT474, SKBR3, and MCF7. The abscissa is the mean fluorescence intensity, and the ordinate is the cell number.

[0040] Figure 5 Effect of HER2 antibodies on the proliferation of BT474 cells. The abscissa is the concentration of the antibody, and the ordinate is the ratio of viable cells. T: Trastuzumab; P: Pertuzumab.

[0041] Figure 6 Effect of HER2 antibodies on the proliferation of SKBR3 cells. The abscissa is the concentration of the antibody, and the ordinate is the ratio of viable cells. T: Trastuzumab; P: Pertuzumab.

[0042] Figure 7 HER2 antibodies inhibit the proliferation of MCF7 cells. MCF7 cells were incubated with antibodies 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 Effect of r40 on the PI3K / AKT signaling pathway in BT474 and SKBR3 cell lines.

[0044] After treating A.BT474 cells with antibodies for 6 h and 24 h and harvesting the samples, Western Blot was used to detect the levels of AKT (Ser437), total AKT, HER2 and the internal reference α-Tubulin. T: Trastuzumab; P: Pertuzumab.

[0045] After treating B.SKBR3 cells with antibodies for 6 h and 24 h and harvesting the samples, Western Blot was used to detect the levels of AKT (Ser437), total AKT, HER2 and the internal reference α-Tubulin. T: Trastuzumab; P: Pertuzumab.

[0046] Figure 9 r40 inhibits the HER2 / HER3 downstream signaling pathway. MCF7 cells were treated with 50 μg / mL antibodies for 6 h and 24 h, then 0.5 nM NRG1 was added and incubated for 10 min before harvesting the samples. Western Blot was used to detect the levels of phosphorylated AKT at Ser437 and total AKT, phosphorylated ERK1 / 2 at Thr202 / Tyr204 and total ERK1 / 2, HER2 and the internal reference GAPDH. T: Trastuzumab; P: Pertuzumab.

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

[0048] Figure 11 HER2-r40-Trastuzumab-Pertuzumab cryo-EM structure density map

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

[0050] B. Overall structure of HER2-r40-Trastuzumab-Pertuzumab: The HER2-Trastuzumab-Pertuzumab structure (PDB: 6OGE) and the r40 antibody structure predicted by AlphaFold were built into the electron cloud.

[0051] Figure 12 HER2-r40-Trastuzumab-Pertuzumab Structural Analysis

[0052] A. Cryo-EM Structure: Domain I of HER2 (light blue), Domain II (blue-green), Domain III (cyan), Domain IV (blue), Trastuzumab Fab (pink), Pertuzumab Fab (yellow), r40 Fab (green).

[0053] B. Constructed HER2-r40-HER3 Structure. Domain I of HER2 (light blue), Domain II (blue-green), Domain III (cyan), Domain IV (blue), r40 Fab (green), HER3 (gray). Detailed Implementation Modes

[0054] The following further describes the detailed implementation modes of the present invention. It should be noted here that the description of these implementation modes is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the following described implementation modes can be combined with each other as long as they do not conflict with each other.

[0055] The term "Trastuzumab" involved in this article: namely (Trastuzumab), is the first monoclonal antibody drug used to treat HER2-positive breast cancer and also the first monoclonal antibody drug used to treat solid tumors. Trastuzumab binds to Domain IV of the extracellular region of HER2, inhibits the heterodimerization of HER2, inhibits the intracellular HER2 signaling pathway, causes cell cycle arrest, and exerts the ADCC effect. Although Trastuzumab has changed the treatment history of HER2-positive breast cancer, many patients develop drug resistance and recurrence within one year after treatment, probably because Trastuzumab cannot effectively inhibit the formation of heterodimers between HER2 and other members of the EGF receptor family.

[0056] The term "Pertuzumab" involved in this article: namely (Pertuzumab), is the second humanized IgG1 monoclonal antibody, binds to Domain II of the extracellular region of HER2, inhibits the heterodimerization of HER2, and blocks the downstream signaling pathway. The combined use of antibodies targeting different epitopes of the HER2 protein can produce a synergistic anti-tumor effect. The combined use of Trastuzumab and Pertuzumab has a stronger inhibitory effect on the HER2 downstream signaling pathway.

[0057] The term "PI3K / AKT signaling pathway" as described herein: The PI3K / AKT signaling pathway is an intracellular signal transduction pathway that responds to extracellular signals and promotes metabolism, proliferation, cell survival, growth, and angiogenesis. This process is mediated by the phosphorylation of serine or threonine of a series of downstream substrates, and the key genes involved are phosphatidylinositol 3-kinase (PI3K) and AKT / protein kinase b, so this pathway is directly named after these two genes; in the present invention, when the PI3K / AKT signaling pathway is activated, it will lead to the phosphorylation of serine 473 of the serine / threonine kinase AKT protein, and detecting that p-AKT(S437) becomes weak indicates that this signaling pathway is downregulated. In this article, the activation and downregulation of this pathway are judged by the change of the phosphorylation of AKT protein at Ser473.

[0058] The term "MAPK signaling pathway" as described herein: The MAPK (mitogen-activated protein kinase) signaling pathway can regulate various important physiological / pathological effects such as cell growth, differentiation, stress, and inflammatory response. One of the main branch routes of the MAPK pathway is extracellular-signal-regulated protein kinase (ERK), and ERK is mainly responsible for cell growth and differentiation. When the signaling pathway is activated, it will lead to the phosphorylation of ERK. In this article, the activation and downregulation of this pathway are judged by the change of the phosphorylation of ERK protein.

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

[0060] The term antibody includes an "fragment" or "derivative" having at least one antigen-binding site of the antibody and / or showing the same biological activity. In addition, 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 a variable domain and optionally a constant domain. The variable domain may comprise complementarity-determining regions (CDRs), such as CDR1, CDR2, and / or CDR3 regions, and framework regions.

[0061] As used herein, the term "isolated" refers to being obtained by artificial means from its natural state. If a certain "isolated" substance or component occurs in nature, it may be that its natural environment has changed, or the substance has been isolated from its natural environment, or both situations have occurred. The term "isolated" does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0062] As used herein, the term "vector" refers to a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the gene or sequence in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

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

[0064] The determination of "antibody positive" as involved herein: 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 ≥ 2.1, it is determined that the antibody detection result described herein is positive.

[0065] Unless otherwise specified, the experimental methods in the following examples are all conventional methods, and unless otherwise specified, the test materials used in the following examples can all be obtained through conventional commercial channels.

[0066] Example

[0067] Express and purify the extracellular domain (ECD) of HER2 protein, immunize experimental animal rabbits with it as an immunogen, and obtain antibody sequences using monoclonal antibody development technology based on the screening of single B lymphocytes; after expression and purification, an ELISA verification experiment was carried out to prove the binding ability, the KD value of antigen-antibody binding was measured by SPR, and the binding epitope of the antibody was measured by flow cytometry and cryo-electron microscopy structure research; functional verification was carried out using breast cancer cell lines, and finally a rabbit monoclonal antibody that can specifically bind to the human HER2 protein domain III and inhibit the proliferation of breast cancer cells was obtained.

[0068] Example 1 Preparation of Monoclonal Antibody

[0069] 1.1 Preparation and Immunization of HER2 ECD Protein

[0070] (1) The full-length HER2 gene was purchased from Addgene. The 23-652 amino acids of the extracellular domain (ECD) of the HER2 protein were selected for cloning and constructing an expression plasmid. The plasmid was transfected into 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 each time. For the first immunization, the immunogen was mixed with an equal volume of complete Freund's adjuvant to make an emulsifier and injected into the footpads. Three weeks later, 400 μg of the immunogen was mixed with an equal volume of incomplete Freund's adjuvant to make an emulsifier and injected into the footpads for booster immunization. After that, booster immunization was carried out again twice every 3 weeks, for a total of 4 immunizations. Three days after the last immunization, the spleen was taken.

[0073] 1.2 Isolation of Spleen Cells

[0074] The spleen was cut into pieces with a surgical blade and then transferred to a 100 μm cell sieve for grinding. The resulting cell suspension was filtered to remove larger cell clumps and tissue envelopes, centrifuged at 450 g for 7 min, and the supernatant was removed to retain the spleen cell mass. The spleen cell mass was resuspended in a conventional PBS buffer and lysed to lyse red blood cells, and then centrifuged again at 450 g for 7 min to retain the spleen cells.

[0075] 1.3 Sorting of B Lymphocytes

[0076] According to the experimental method of this patent, the spleen cells were negatively screened using an antibody against a non-B cell lymphocyte surface marker labeled with a biological marker, and then non-B cells were removed using the principle of avidin magnetic beads and magnetic separation to increase the relative abundance of B lymphocytes in the spleen cells; 7-AAD was used for sorting to remove dead cells; a flow cytometer was used for single-cell screening to obtain the target lymphocytes.

[0077] 1.4 Antibody Sequence Synthesis, Expression and Purification.

[0078] The variable region sequences of antibodies were obtained by single-cell V(D)J sequencing using the 10× Genomics single-cell library construction kit. According to the sequencing results, a certain number of paired heavy and light chains were selected, and the variable regions were loaded into the rabbit IgG antibody framework. The expression plasmid was synthesized by General Biosystems (Anhui) Co., Ltd., transfected into suspension Expi293 cells, and the expressed antibody protein was adsorbed with a protein A column. Then, it was eluted with 0.1 M glycine (pH 3.0) to obtain the recombinant antibody.

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

[0080] Amino acid sequence of the heavy chain variable region:

[0081] QQLEQSGGGAEGGLVKPGGSLELCCSASGFAFNSYWICWVRQAPGKGLEWIGCVYSGSSGATYYASWVNGRFTLSRDVDQSTGCLQLNSLTAADTAMYYCARDLYPNSLNYGFNLWGQGTLVTVSS. (SEQ ID NO.1)

[0082] Among them; the amino acid sequence of CDR1 of the heavy chain variable region: CSASGFA. (SEQ ID NO.2)

[0083] Amino acid sequence of CDR2 of the heavy chain variable region: IGCVYS. (SEQ ID NO.3)

[0084] Amino acid sequence of CDR3 of the heavy chain variable region: CARDLYPNSLNYGFNLW. (SEQ ID NO.4)

[0085] Amino acid sequence of the light chain variable region:

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

[0087] Among them, the amino acid sequence of CDR1 of the light chain variable region: QSSQSVYSNNFLS. (SEQ ID NO.6)

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

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

[0090] Example 2 Screening of High-Affinity Antibodies

[0091] 2.1 ELISA Assay

[0092] (1) Coating: Dilute HER2 protein to 0.2 ng / μL with 1×PBS. Add the diluted HER2 protein to a 96-well ELISA plate, 50 μL / well. 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, 150 μL / well. After 5 min, pour out the liquid and pat dry on absorbent paper. Repeat washing 5 times.

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

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

[0096] (5) Incubation with Secondary Antibody: Dilute the HRP-conjugated secondary antibody 3000-fold with 3% BSA blocking solution. Add the secondary antibody dilution (50 μL / well) to the wells. 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 appropriate, add 1 M HCl to the wells, 25 μL / well, to terminate the reaction. Detect the OD value using an ELISA reader 450 .

[0099] The result shows that the ratio of the OD value of antibody r40 to the OD value of the negative control well without coated antigen is greater than 10, indicating a positive antibody (see Figure 1 ).

[0100] 2.2 SPR (Surface Plasmon Resonance)

[0101] (1) Chip Preparation:

[0102] Print the antibody on the 3D photocrosslinking chip using an Arrayjet chip spotter. Place the chip in a vacuum drying oven for vacuum drying for more than 1 h until the sample spots are dry. Place the dried chip in a photocrosslinker for a photocrosslinking reaction for 30 min. Place the chip in a developing tank and wash it successively with dimethylformamide, ethanol, and deionized water on a shaker for 15 min, dry it with nitrogen, make a mark, and reserve it for use.

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

[0104] Install the prepared chip onto the HT molecular interaction detector, and successively circulate HER2 proteins with concentrations ranging from 1.0 nM to 128 nM. The dissociation buffer is 1×PBS, and there are two regeneration buffers, namely 10 mM Gly-HCl pH 2.0 and 1% BSA.

[0105] The results show that the K D value of the r40 rabbit monoclonal antibody is 2.79×10 -10 , and its affinity for HER2 is higher than that of the positive controls Trastuzumab (2.83×10 -10 ) and Pertuzumab (2.3×10 -9 ) (see Figure 2 ).

[0106] Example 3 Identification of antibody binding epitope

[0107] Since endogenous HER2 protein is expressed on the surface of Expi293 cells, there is no need to transfect the full-length HER2 protein, and untransfected Expi293 cells are used for HER2 antibody verification. The experiment is divided into two groups. One group only adds the rabbit monoclonal antibody (single staining group); the other group first adds 10 μg / mL of Trastuzumab and Pertuzumab to completely block the binding epitopes of HER2, wash it three times with 1×PBS, and then add 10 μg / mL of the rabbit monoclonal antibody (saturation group).

[0108] Result determination: For the saturation group, if the rabbit monoclonal antibody can still bind, it indicates that its epitope is different from those of Trastuzumab and Pertuzumab; if the rabbit monoclonal antibody cannot bind, it indicates that its epitope is the same as or partially overlaps with those of Trastuzumab and Pertuzumab or there is steric hindrance.

[0109] (1) Take 1 mL of Expi293 cells with a density of 2 - 2.5×10 6 cells / mL, centrifuge at 1000 g for 3 min at 4°C, 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. For the other part, first add 100 μL of 10 ng / μL Trastuzumab and Pertuzumab, incubate the cells for 30 min, wash with 1×PBS, and then 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 the fluorescent secondary antibody at a ratio of 1:250, incubate for 30 min, and wash with pre-cooled 1×PBS.

[0113] (5) Add DAPI diluted 1:10000 and load onto the machine.

[0114] The results showed that the binding of antibody r40 to the cells was hardly reduced or only slightly reduced in the presence of Trastuzumab and Pertuzumab. It can be considered that their binding epitopes to the HER2 protein are different from those of Trastuzumab and Pertuzumab (see Figure 3 ).

[0115] Example 4 Experiment on the Binding of HER2 Antibodies to Breast Cancer Cells

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

[0117] (1) Digest the three breast cancer cell lines, BT474, SKBR3, and MCF7, with trypsin, wash with pre-cooled 1×PBS, centrifuge at 1000 rpm for 3 min at 4°C to collect the cells.

[0118] (2) For each experimental group, resuspend 5×10 5 cells in 100 μL of 1×PBS. Add 10 μg / mL Trastuzumab, Pertuzumab, and r40 to the experimental groups respectively, add equal amounts of human IgG and rabbit IgG to the control groups respectively, and set up a blank control. Incubate for 30 min.

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

[0120] (4) Add the fluorescent secondary antibody at a ratio of 1:250 and incubate in a cold storage transfection 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 100 μL to resuspend the cells and then detect them on the machine.

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

[0124] From Figure 4 It can be seen that the r40 antibody can recognize the natural HER2 protein on the cell surface, comparable to Trastuzumab and Pertuzumab.

[0125] Example 5 Tumor Cell Proliferation Inhibition Experiment

[0126] Use the Cell Counting Kit-8 (CCK-8) kit to determine the effect of the antibody on the proliferation of breast cancer cells. The method is as follows:

[0127] Seed the breast cancer cell lines BT474, SKBR3 and MCF7 into 96-well cell culture plates at 3000 cells / well, 9000 cells / well and 5000 cells / well respectively, and culture overnight in a 37 °C, 5% CO 2 incubator; dilute the antibody in gradients, add it to the cells and continue to culture for 4 days; add 10 μL of CCK8 reagent to each well, put it back into the incubator and continue to culture for 2 h; measure OD450 using an enzyme-linked immunosorbent assay reader.

[0128] To verify whether the screened HER2 antibody can inhibit the proliferation of breast cancer cells, use the breast cancer cell line BT474 with high HER2 expression for the CCK8 experiment. Set 6 antibody gradients, the highest is 100 μg / mL, diluted at a 5-fold gradient, and the lowest concentration is 0 μg / mL. The highest concentration of Trastuzumab and Pertuzumab antibodies in combination is 100 μg / mL for both.

[0129] The results show that the combination of Trastuzumab and Pertuzumab antibodies has the strongest inhibitory effect on BT474 cells, followed by Trastuzumab, and the inhibitory effects of Pertuzumab and r40 are 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 breast cancer cell line SKBR3 with high HER2 expression; compared with BT474, the inhibitory effects of the antibodies on SKBR3 proliferation were generally weaker, and the r40 antibody inhibited the proliferation of SKBR3 cells to a certain extent( Figure 6 ).

[0131] To explore the effects of HER2 antibodies on the proliferation of breast cancer cell lines with low HER2 expression, CCK8 experiments were performed on MCF7, and the effects of the antibodies in the presence of the HER3 ligand NRG1 were tested. The antibody concentration was 20 μg / mL, and the concentrations of both Trastuzumab and Pertuzumab were 20 μg / mL when used in combination. The concentration of neuregulin 1 (NRG1) was 2 nM, and NRG1 was added 2 h after the addition of the antibodies.

[0132] In the absence of NRG1, Trastuzumab, Pertuzumab, r40, and the combination of Trastuzumab and Pertuzumab could all significantly inhibit the proliferation of MCF7 cells; in the presence of NRG1, the four antibodies and the combination of Trastuzumab and Pertuzumab could also significantly inhibit the proliferation of MCF7 cells( Figure 7 ).

[0133] In summary, the r40 antibody has an inhibitory effect on the proliferation of breast cancer cell lines with high or low HER2 expression.

[0134] Example 6 Detection of the levels of downstream signaling pathway proteins by Western Blot

[0135] BT474 and SKBR3 cells were cultured to 60 - 70% confluence respectively, and 50 μg / mL of the antibody was added. When Trastuzumab and Pertuzumab were used in combination, the concentrations of both were 50 μg / mL, and the cells were cultured in an incubator at 37°C and 5% CO 2 The cells were collected at 6 h and 24 h respectively, and the total cell proteins were extracted by adding 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 Blot.

[0136] (1) Seed 1.5×10 5 cells in each well of a 12-well cell culture plate. After the cells adhered the next day, add the antibody at a concentration of 50 μg / mL and continue to culture for 6 h or 24 h.

[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 to mix, and transfer to a 1.5 mL EP tube. Lyse the cells at 4°C for 30 min.

[0138] (3) Centrifuge the cell lysate at high speed at 12,000 rpm at 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 buffer, boil the sample in a metal bath for 5 min, and then perform SDS-PAGE gel electrophoresis.

[0140] (5) After transferring the membrane, detect the protein levels of downstream signaling pathways by Western Blot.

[0141] As shown in Figure 8, the results of Western Blot detection indicate that in both types of cells, Trastuzumab and r40 antibody can reduce the levels of p-AKT (S473) and HER2, while Pertuzumab has basically no effect on the levels of p-AKT (S473) and HER2.

[0142] In BT474 cells, the expression level of HER2 in cells treated with Trastuzumab and r40 antibody for 6 h decreased slightly, and significantly decreased after 24 h ( Figure 8 A).

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

[0144] Therefore, the r40 antibody can reduce the protein level of HER2 and down-regulate the PI3K / AKT signaling pathway.

[0145] Subsequently, the levels of p-AKT (S437), total AKT, p-ERK, total ERK, and HER2 were detected by Western Blot 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, and then incubated with 0.5 nM NRG1 for 10 min before harvesting the cells. NRG1 activates the PI3K / AKT and MAPK signaling pathways by promoting the formation of heterodimers of HER2 / HER3, and the level of p-AKT (S437) in cells treated with NRG1 increased significantly.

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

[0147] The r40 antibody and Trastuzumab can reduce the levels of p-AKT(S437) and HER2, which is more significant at 24 h than at 6 h. The r40 antibody and Trastuzumab have less effect on p-ERK at 6 h and also significantly reduce the level of p-ERK at 24 h ( Figure 9 ).

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

[0149] In addition to forming heterodimers with HER3, HER2 mainly forms heterodimers with another member of the family, EGFR. Both the EGFR / HER2 and HER2 / HER3 complexes have important physiological functions and are closely related to the occurrence of tumors. Therefore, the effects of the antibody on the EGFR / HER2 heterodimer and downstream pathways were further tested.

[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 then RIPA lysis buffer was added to collect whole cell lysates for Western Blot detection.

[0151] The results are shown by Figure 10 :

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

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

[0154] In SKBR3 cells, the r40 antibody and Trastuzumab can also reduce the level of p-AKT(S437), but have no effect on the level of p-ERK ( Figure 10 ).

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

[0156] Example 7: Analyzing the Cryo-EM Structure of an Antigen-Antibody Complex Protein Sample

[0157] In general, only the antigen-binding segment, i.e., the Fab segment, is selected for antibody structure research. The Fab contains the complete variable regions VH and the constant region CH1 of the light and heavy chains. The expression vector of Fab was constructed and expressed, and Fab was purified by affinity chromatography. Then, the HER2 protein was incubated with an excess of r40 Fab antibody and passed through a molecular sieve, and the HER2-r40 complex was collected; then, HER2-r40 was incubated with an excess of Trastuzumab and passed through a molecular sieve, and the HER2-r40-Trastuzumab complex was collected; finally, the HER2-r40-Trastuzumab complex was incubated with an excess of Pertuzumab, and the HER2-r40-Trastuzumab-Pertuzumab complex was collected through a molecular sieve. Through three-step assembly, a quaternary complex formed by HER2 and three antibodies, r40, Trastuzumab, and Pertuzumab, was obtained. The protein concentration was increased to 1.5 mg / mL by concentration. After hydrophilizing the Quantifoil porous carbon support film (R1.2 / 1.3Au), it was transferred to a cryo-sampling device Vitrobot Mark IV pre-cooled to 4 °C, and the humidity was set to 100%. 3 μL of the protein solution was aspirated and added to the Quantifoil porous carbon support film, blotted with filter paper for 1 s, quickly immersed in liquid ethane, and then the carbon support film was quickly immersed in a sample box in liquid nitrogen and stored in liquid nitrogen for later use.

[0158] The prepared cryo-EM sample was used for data collection and structure analysis using a 300 kV cryo-EM (Thermo Fisher Scientific Titan Krios G4300 kV). 2965 cryo-photographs of HER2-r40-Trastuzumab-Pertuzumab were collected for subsequent data processing. Through the processing of the data analysis software, particle picking of the cryo-photographs was carried out, and the particles were subjected to two-dimensional classification, three-dimensional classification, and reconstruction. Finally, Structural density of HER2-r40-Trastuzumab-Pertuzumab at the resolution. Using the cryo-EM density map obtained above for model building, the HER2-Trastuzumab-Pertuzumab complex model (PDB: 6OGE) was used as the initial model building template. The HER2-Trastuzumab part of the model was taken, and it was embedded (Docked) into the calculated density of HER2-Trastuzumab-m66 by the method of Rigid-body fitting using UCSF Chimera. Taking the whole model of HER2-Trastuzumab-Pertuzumab and combining it with the r40 model predicted by AlphaFold, it was embedded (Docked) into the calculated density of HER2-Trastuzumab-Pertuzumab-r40 by the method of Rigid-body fitting using UCSF Chimera, and then manual building was carried out using the COOT software. All density maps and models were displayed and drawn using PyMOL (https: / / pymol.org / ), UCSF Chimera, and UCSF ChimeraX.

[0159] The results are as Figure 11 shown, and the electron clouds of HER2, Trastuzumab, Pertuzumab, and r40 can be clearly seen. Pertuzumab binds to the second domain of HER2, while Trastuzumab binds to the fourth domain of HER2, which is consistent with the reported literature.

[0160] The antibody r40 binds to the third domain of HER2. Pertuzumab, r40, and Trastuzumab bind to the second, third, and fourth domains of HER2 respectively ( Figure 12 ). The binding of the three to HER2 does not conflict at all, which is consistent with the result of the correct assembly of the quaternary complex. The interaction interface between the r40 antibody and HER2 is relatively large, which is the structural basis for ensuring the high affinity of r40 for HER2 and its good inhibitory activity. Based on the reported HER2-HER3-NRG1 structure (PDB: 7MN5) and the resolved HER2-r40-Trastuzumab-Pertuzumab structure, with HER2 as the reference, the HER2-r40-HER3 structure ( Figure 12 B) was built. It can be seen that the r40 antibody does not affect the formation of heterodimers between HER2 and HER3.

Claims

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

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

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

4. A recombinant vector or host cell comprising a nucleic acid molecule encoding the monoclonal antibody against human HER2 or an antigen-binding fragment thereof.

5. A chimeric antibody comprising the monoclonal antibody against human HER2 or an antigen-binding fragment thereof according to claim 1.

6. A fusion or conjugate of the monoclonal antibody against human HER2 or an antigen-binding fragment thereof according to claim 1, wherein the antibody is fused or conjugated to a heterologous molecule to produce a fusion or conjugate.

7. A composition comprising the monoclonal antibody against human HER2 or an antigen-binding fragment thereof, nucleic acid molecule, vector or host cell, chimeric antibody, and / or fusion or conjugate according to claims 1-6.

8. Use of the antibody or an antigen-binding fragment thereof, derivative, nucleic acid molecule, vector or host cell, chimeric antibody, fusion or conjugate according to claims 1-7 and / or the composition according to the present invention in the preparation of drugs for diagnosing, preventing, and / or treating tumors.

9. A pharmaceutical composition for treating breast cancer, the pharmaceutical composition containing the monoclonal antibody against human HER2 or an antigen-binding fragment thereof, derivative, nucleic acid molecule, vector or host cell, chimeric antibody, and / or fusion or conjugate according to claims 1-7, and at least one other pharmaceutically acceptable carrier, diluent, and / or excipient.

10. A kit for detecting HER2 antibodies, the kit comprising an instruction manual and a detection reagent, the detection reagent being the monoclonal antibody against human HER2 or an antigen-binding fragment thereof, derivative, nucleic acid molecule, vector or host cell, chimeric antibody, and / or fusion or conjugate according to the present invention and the composition according to the sixth aspect of the present invention.

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