Anti-ace2 antibodies and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an anti-ACE2 antibody and application thereof. The antibody or antigen binding fragment thereof provided by the application comprises HCDR1, HCDR2 and HCDR3 contained in VH shown in SEQ ID NO:1, and LCDR1, LCDR2 and LCDR3 contained in VL shown in SEQ ID NO:2. The antibody provided by the application can block the combination of the spike protein of all current mutant strains and ACE2 in a broad spectrum without interfering with the enzyme activity of ACE2, and has excellent anti-SARS-CoV-2 therapeutic effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically an anti-ACE2 antibody and its uses. Background Technology

[0002] COVID-19 is a highly contagious respiratory disease caused by SARS-CoV-2 infection. Since the COVID-19 outbreak in 2019, the virus has been spreading and evolving globally. The virus's recognition and infection of target cells (such as respiratory epithelial cells) mainly depends on the binding of the SARS-CoV-2 spike protein to human angiotensin-converting enzyme 2 (ACE2) on the cell surface. This process ultimately drives the fusion of the virus with the host cell membrane.

[0003] To date, research on the prevention and immunotherapy of COVID-19 has primarily focused on introducing anti-spike protein antibodies through active immunization (vaccines) or passive immunization (therapeutic antibodies). However, the continuous evolution of SARS-CoV-2 has generated spike protein variants capable of evading humoral immune responses and / or therapeutic antibodies induced by natural infection or vaccination. Therefore, there is an urgent need for therapeutic antibodies and / or vaccines that can broadly neutralize current and future potential variants.

[0004] ACE2 has been the universal receptor for the original SARS-CoV-2 strain and all current variants, and is likely to be the receptor for future emerging variants. Therefore, antibodies covering the spike protein binding site on ACE2 represent a potential broad-spectrum anti-SARS-CoV-2 strategy. ACE2 can inactivate angiotensin II and is a negative regulator of the renin-angiotensin system; its extracellular domain is crucial for the binding and catalytic activity of angiotensin II.

[0005] Studies have shown that ACE2 can protect mice from severe acute lung injury; therefore, inhibiting ACE2 enzyme activity may lead to adverse consequences such as increased lung permeability, pulmonary edema, and cardiovascular diseases such as hypertension. For these reasons, although anti-ACE2 antibodies have been shown to block SARS-CoV-2 infection in target cells, there are still concerns about the potential risks they may pose by weakening the peptidase function of ACE2 (a function associated with severe lung pathology).

[0006] Therefore, developing an anti-ACE2 antibody that can block SARS-CoV-2 infection without impairing ACE2 enzyme activity has become an urgent technical problem to be solved in this field and has important social and commercial significance. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides an anti-ACE2 antibody and its uses. Without interfering with ACE2 enzyme activity, it can broadly block the binding of the spike protein of all current variant strains to ACE2, exhibiting excellent anti-SARS-CoV-2 therapeutic effects.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0009] The present invention provides an anti-ACE2 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising: HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 1, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2.

[0010] In this invention, the amino acid sequences of the CDRs are all as defined by the IMGT definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as the Kabat definition rule based on sequence variability (see Kabat et al., Protein Sequences in Immunology, 5th Edition, National Institutes of Health, Bethesda, Maryland (1991)) and the Chothia definition rule based on the location of the structural loop region (see JMol Biol 273:927-48, 1997). In the technical solution of this invention, the Combined definition rule, which includes both the Kabat and Chothia definitions, can also be used to determine the amino acid residues in the variable domain sequence. The Combined definition rule combines the scopes of the Kabat and Chothia definitions, thereby taking a broader range. Those skilled in the art should understand that, unless otherwise specified, the terms “CDR” and “complementarity-determining region” for a given antibody or its region (e.g., the variable region) should be understood to encompass the complementarity-determining region defined by any of the known schemes described above. Although the scope of protection sought in this invention is based on the sequence defined by the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0011] This invention provides an anti-ACE2 antibody or its antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment comprises a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), wherein: HCDR1 comprises the amino acid sequence shown in positions 27-38 of SEQ ID NO: 1; HCDR2 comprises the amino acid sequence shown in positions 56-65 of SEQ ID NO: 1; HCDR3 comprises the amino acid sequence shown in positions 105-115 of SEQ ID NO: 1; LCDR1 includes the amino acid sequence shown in positions 27-38 of SEQ ID NO: 2; LCDR2 includes the amino acid sequence shown in positions 56-65 of SEQ ID NO: 2; LCDR3 includes the amino acid sequence shown in positions 105-112 of SEQ ID NO: 2.

[0012] This invention provides an anti-ACE2 antibody or its antigen-binding fragment, comprising a heavy chain variable region. The heavy chain variable region comprises an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with SEQ ID NO: 1, or is composed of an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with SEQ ID NO: 1, or comprises the amino acid sequence of SEQ ID NO: 1, or is composed of the amino acid sequence of SEQ ID NO: 1; and the HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region are as shown at positions 27-38, 56-65, and 105-115 of SEQ ID NO: 1, respectively.

[0013] And, comprising a light chain variable region, the light chain variable region comprising an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with SEQ ID NO: 2 or consisting of an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with SEQ ID NO: 2, or comprising an amino acid sequence of SEQ ID NO: 2 or consisting of an amino acid sequence of SEQ ID NO: 2; and, the LCDR1, LCDR2, and LCDR3 contained in the light chain variable region are as shown at positions 27-38, 56-65, and 105-112 of SEQ ID NO: 2, respectively.

[0014] In this invention, the constant region of the anti-ACE2 antibody or its antigen-binding fragment is the constant region of the human antibody.

[0015] The present invention provides a humanized antibody comprising HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 1, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2.

[0016] The present invention also provides an isolated nucleic acid that encodes the above-mentioned anti-ACE2 antibody or its antigen-binding fragment.

[0017] The present invention provides a recombinant expression vector comprising the isolated nucleic acid described above; preferably, the recombinant expression vector comprises a eukaryotic cell expression vector and / or a prokaryotic cell expression vector.

[0018] The present invention provides a transformant comprising the isolated nucleic acid or the recombinant expression vector described above; preferably, the host cell of the transformant is a prokaryotic cell and / or a eukaryotic cell, wherein the prokaryotic cell is preferably an E. coli cell such as TG1 or BL21, and the eukaryotic cell is preferably an HEK293 cell or a CHO cell.

[0019] The present invention provides an immunoconjugate comprising the above-mentioned anti-ACE2 antibody or its antigen-binding fragment.

[0020] The present invention provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is the above-mentioned anti-ACE2 antibody or its antigen-binding fragment, the above-mentioned nucleic acid, or the above-mentioned immunoconjugate.

[0021] This invention provides the use of the above-mentioned anti-ACE2 antibody or its antigen-binding fragment in the preparation of a kit for diagnosing SARS-CoV-2 infection or ACE2-related diseases.

[0022] This invention provides the use of the above-mentioned anti-ACE2 antibody or its antigen-binding fragment in a drug for blocking, preventing or treating SARS-CoV-2 infection.

[0023] This invention provides the application of the above-mentioned anti-ACE2 antibody or its antigen-binding fragment in a medicament for treating colorectal adenocarcinoma or non-small cell lung cancer.

[0024] Terminology Definition It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not restrictive.

[0026] As used herein, the term “and / or” means any one of the options or two or more or all of the options.

[0027] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region composed of that specific sequence.

[0028] As used herein, the term "isolated" antibody or molecule is an antibody or molecule that has been separated from components of its natural environment. In some embodiments, the antibody or molecule is purified to a purity of more than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0029] As used herein, the terms “full antibody” or “complete antibody” are used interchangeably and refer to antibody molecules that have the molecular structure of natural immunoglobulins. In the case of a conventional four-chain IgG antibody, a complete antibody consists of two heavy chains (H) and two light chains (L) linked together by disulfide bonds. In the case of a heavy chain antibody that has only heavy chains and lacks light chains, a complete antibody consists of two heavy chains (H) linked together by disulfide bonds. For a conventional four-chain IgG antibody, the heavy chain of a complete antibody typically consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region, wherein the heavy chain constant region contains at least three domains CH1, CH2, and CH3. The light chain of a complete antibody consists of a light chain variable region (abbreviated as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0030] As used herein, the term "antigen-binding fragment" in antibody refers to a molecule distinct from a full-length antibody. It comprises a portion of the full-length antibody but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibodies, single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH, bivalent antibodies, dimeric antibodies or fragments thereof, or camelid antibodies, dimeric antibodies (diabody), single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting full-length antibodies with papain. Furthermore, F(ab')2, a dimer of Fab' and a divalent antibody fragment, is produced by digesting complete antibodies with pepsin below the disulfide bonds in the hinge region. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into Fab' monomers. Fab' monomers are essentially Fab fragments with hinge regions. The Fv fragment consists of the VL and VH domains of the antibody single arm. The two domains, VL and VH, of the Fv fragment can be encoded by independent genes, but they can also be linked using a recombinant approach with synthetic linker peptides to make it function as a single unit. As used herein, the terms “complementarity-determining region” or “CDR region” or “CDR” refer to regions within the antibody variable domain that are sequence-hypervariant and form structurally defined loops (“hypervariant loops”) and / or contain antigen contact residues (“antigen contact sites”). CDRs are primarily responsible for binding to antigen epitopes. Heavy and light chain CDRs are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. Within a given amino acid sequence of a light chain or heavy chain variable region, the precise amino acid sequence boundaries of each CDR can be determined using any of many known antibody CDR assignment schemes or combinations thereof.

[0031] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0032] The reagents and raw materials used in this invention are all commercially available.

[0033] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention explores whether anti-ACE2 monoclonal antibodies can broadly block the binding of the spike protein of all current variant strains to ACE2 without interfering with ACE2 enzyme activity. An anti-ACE2 monoclonal antibody was developed, and its potential to block the binding of the latest spike protein variant strains to ACE2 was tested in a spike protein pseudovirus infection system. Simultaneously, this invention also examined the potential impact of anti-ACE2 monoclonal antibodies on ACE2 peptidase activity and revealed the application prospects of anti-ACE2 monoclonal antibodies as a potentially safe and broad-spectrum anti-SARS-CoV-2 agent. Attached Figure Description

[0034] Figure 1 Sequencing results of the variable region of the anti-ACE2 monoclonal antibody D11, including... Figure 1 Part A represents the sequencing results of the heavy chain variable region. Figure 1 Part B represents the sequencing results of the light chain variable region.

[0035] Figure 2 The results show the experimental findings of infection of 293T and 293T-ACE2 cells with spike protein pseudoviruses of the original strain, B.1.1.529, XEC, and KP.3.1.1. Figure 2 Part A is a fluorescence microscope image; Figure 2 Part B represents the experimental statistical results of Part A.

[0036] Figure 3 For flow cytometry analysis of antibody-specific binding assays, including: Figure 3 Part A presents the experimental results of the recognition effects of PBS (blank control group), secondary antibody (fluorescent secondary antibody control group), and D11 monoclonal antibody on 293T cells, 293T-ACE2 cells, SW480 cells, and A549 cells, respectively. Figure 3 Part B represents the experimental statistical results of Part A.

[0037] Figure 4 To investigate the antibody's role in blocking SARS-CoV-2 pseudovirus invasion of target cells, the experiment included: Figure 4 Part A is a fluorescence microscope photograph; Figure 4 Part B represents the experimental statistical results of Part A.

[0038] Figure 5 The results are from the antibody titration experiment blocking the B.1.1.529 S protein pseudovirus.

[0039] Figure 6 The results show the experimental effects of antibodies on ACE2 enzyme activity. Figure 6Part A describes the effect of co-incubation of 293T-ACE2 cell lysate with antibody on ACE2 enzyme activity. Figure 6 Part B describes the effect of co-incubation of intact 293T-ACE2 cells with antibodies on ACE2 enzyme activity. Detailed Implementation

[0040] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0041] General experimental methods Methods for maintaining cell viability and normal state The L929, A549, SW480, HEK293T (293T) cells and 293T cells expressing ACE2 (293T-ACE2, constructed by transfection with a lentivirus carrying the ACE2 coding sequence) involved in the following experiments were all cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS, Serana) and 1% penicillin-streptomycin (Biosharp) at 37°C and 5% CO2.

[0042] Flow cytometry Flow cytometry analysis was performed according to the method described in the reference (Zhang, J., Wang, T., Wang, Y., Li, Y., Wang, L., Wang, J., Miao, Y., Xu, F., and Yao, Y. (2025). Bacterial pneumonia induces senescence in resident alveolar macrophages that are outcompeted by monocytes. Cell Rep 44. https: / / doi.org / 10.1016 / j.celrep.2025.115571). In short, single-cell suspensions (e.g., L929 or L929-ACE2, etc.) were prepared at 2 × 10⁶ cells per well. 6Cells were seeded in 96-well U-bottom plates and suspended in PBS. They were then stained with a monoclonal antibody (50 µL / well, diluted 1:200 with PBS containing 0.5% BSA). Subsequently, cells were stained with a fluorescently conjugated anti-mouse IgG secondary antibody (50 µL / well, diluted 1:300 with PBS containing 0.5% BSA). After washing (with PBS containing 0.5% BSA), the cells were resuspended in 200 µL of PBS containing 0.5% BSA and analyzed using LSR Fortessa.

[0043] Example 1 - Preparation and isolation of antibodies.

[0044] 1) Immunogen preparation Following the method described in the literature (Mitra, S., and Tomar, PC (2021). Hybridoma technology; advances, clinical significance, and future aspects. Preprint at SpringerScience and Business Media Deutschland GmbH, https: / / doi.org / 10.1186 / s43141-021-00264-6), L929 cells expressing anti-human ACE2 monoclonal antibodies were prepared. In short, L929 cells expressing ACE2 (L929-ACE2) were constructed by transfection with a lentivirus carrying the ACE2 coding sequence.

[0045] 2) Animal immunization L929-ACE2 cells were injected into Balb / c mice (IgG2b subtype) to stimulate the mice to produce an immune response against human ACE2.

[0046] 3) Cell fusion The spleen cells of immunized mice were then fused with SP2 / 0 myeloma cells.

[0047] 4) Hybridoma screening and cloning Hybridoma cells were screened using complete HAT medium, followed by isolation of monoclonal hybridoma cell lines using limiting dilution. The culture supernatant (containing monoclonal antibodies) of the monoclonal hybridoma cells was collected, and monoclonal cell lines capable of binding ACE2 were screened using the flow cytometry method described above.

[0048] 5) Preparation and purification of anti-ACE2 monoclonal antibodies Hybridoma cells were cultured in RPMI 1640 medium containing 10% FBS until the logarithmic growth phase, and the cell culture supernatant was collected.

[0049] To prepare ascites fluid containing anti-ACE2 antibody, 500 µL of mineral oil was injected intraperitoneally into unimmunized Balb / c mice. Seven to ten days later, each mouse was injected intraperitoneally with 1.5 × 10⁻⁶ ppm of the anti-ACE2 antibody. 6 Hybridoma cells (resuspended in 200 µL PBS). Ascites fluid was collected approximately 10 days after hybridoma cell injection, centrifuged, and the supernatant was filtered through a 0.22 µm filter membrane.

[0050] Using the Protein G Antibody Purification Kit (GenScript Biotech), following the manufacturer's instructions, the antibody in the hybridoma culture supernatant or ascites supernatant was purified by affinity chromatography to obtain high-purity anti-ACE2 monoclonal antibody D11. Detection using the Mouse Monoclonal Antibody Subtype Detection Kit (Proteintech) showed that the antibody was of the IgG2b subtype.

[0051] The following uses the IMGT scheme.

[0052] The amino acid sequence of the heavy chain variable region of the D11 antibody is shown in SEQ ID NO: 1: QVQLQQPGAELVKPGASVKLSCKASGYAFTSYYVYWVKQRPGQGLEWIGGIHPRNGGTNFNEKFKIKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTIYYDYAWFAYWGQGT. Its sequencing results are as follows: Figure 1 As shown in Part A.

[0053] The D11 heavy chain variable region includes: The amino acid sequence of the HCDR1 region is shown as positions 27-38 of SEQ ID NO: 1.

[0054] The amino acid sequence of the HCDR2 region is shown in positions 56-65 of SEQ ID NO: 1.

[0055] The amino acid sequence of the HCDR3 region is shown in positions 105-115 of SEQ ID NO: 1.

[0056] The amino acid sequence of the light chain variable region of the D11 antibody is shown in SEQ ID NO: 2: DIVLTQTTASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK. Its sequencing results are as follows: Figure 1 As shown in Part B.

[0057] The variable region of the D11 light chain includes: The amino acid sequence of the LCDR1 region is shown in positions 27-38 of SEQ ID NO: 2.

[0058] The amino acid sequence of the LCDR2 region is shown in positions 56-65 of SEQ ID NO: 2.

[0059] The amino acid sequence of the LCDR3 region is shown in positions 105-112 of SEQ ID NO: 2.

[0060] Example 2 - Preparation of SARS-CoV-2 pseudovirus and experiment on pseudovirus infection of target cells The general methods for preparing pseudoviruses and infecting target cells with pseudoviruses are as follows: S1: Preparation of SARS-CoV-2 spike protein pseudovirus Specifically, a mixture containing three transfection plasmids was prepared (tube A): psPAX2 (packaging plasmid), pCAG-SARS-CoV-2-S△C19 (enveloping plasmid containing the SARS-CoV-2 spike protein S), and pCDH-CMV-Luciferase-GFP or pHBLV-eGFP (tracer plasmid). PEI MAX (1 μg / μL, Polysciences) transfection reagent was mixed with 0.5 mL of FBS-free DMEM (tube B). After incubating tubes A and B at room temperature for 5 minutes, the two tubes were gently mixed, and incubation continued at room temperature for another 15 minutes. The transfection mixture was then added to HEK293T cell culture. After 6–12 hours, the cells were washed with complete DMEM (cDMEM) medium and cultured for another 72 hours. The cell culture supernatant containing the pseudovirus was collected, centrifuged at 1000×g for 10 minutes, and cell debris mixed in during collection was precipitated. After filtering the viral supernatant through a 0.45μm filter membrane, the supernatant was aliquoted and stored at -80℃ for later use.

[0061] S2: Cell infection experiment using SARS-CoV-2 spike protein pseudovirus Specifically, the aforementioned SARS-CoV-2 spike protein pseudovirus was mixed with polyglobulin (8-10 μg / mL; Yeasen), diluted with cDMEM, and added to 293T or 293T-ACE2 cells. After incubation at 37°C and 5% CO2 for 12 hours, the medium was replaced with fresh cDMEM and cultured for another 72 hours. In some experiments, different concentrations of D11 monoclonal antibody were added to the cell culture supernatant. GFP expression was observed using a fluorescence microscope, and infection efficiency was quantified using flow cytometry.

[0062] This invention follows the general method for preparing pseudoviruses and infecting target cells with pseudoviruses. It uses spike protein pseudoviruses of the original strain, B.1.1.529, XEC, and KP.3.1.1 to infect 293T or 293T-ACE2 cells. The experimental results are as follows: Figure 2 As shown in Part A, the experimental statistical results are as follows: Figure 2 As shown in Part B.

[0063] The results showed that pseudoviruses expressing the spike protein of the original strain and the selected variant of interest could establish infection in 293T-ACE2 cells, but not in 293T cells.

[0064] These data collectively indicate that infection by SARS-CoV-2 pseudoviruses expressing the spike protein of both the original strain and the variant strain depends on the expression of ACE2 on target cells.

[0065] Example 3 - D11 monoclonal antibody specifically binds to ACE2 on the cell surface This invention analyzes the recognition effects of PBS (blank control group), secondary antibody (positive control group), and D11 monoclonal antibody on 293T cells, 293T-ACE2 cells, SW480 cells, and A549 cells, respectively, using the flow cytometry analysis method described above. The experimental results are as follows: Figure 3 As shown in Part A, the experimental statistical results are as follows: Figure 3 As shown in Part B.

[0066] Flow cytometry analysis showed that the D11 monoclonal antibody could bind to 293T-ACE2 cells, but not to 293T cells that do not express ACE2. Furthermore, the SW480 and A549 epithelial cell lines expressing ACE2 could also be recognized by the D11 monoclonal antibody and exhibited excellent killing effects.

[0067] These data indicate that the D11 monoclonal antibody can specifically bind to ACE2.

[0068] Example 4 - D11 monoclonal antibody broadly blocks the invasion of SARS-CoV-2 pseudovirus into target cells. To investigate whether D11 monoclonal antibody can broadly block SARS-CoV-2 pseudovirus infection of target cells, this invention infected 293T-ACE2 cells with pseudoviruses expressing the spike protein S of the original strain and representative key variants (including Delta, B.1.1.529, XEC, and KP.3.1.1), and added D11 monoclonal antibody to the cell culture wells. The experimental results... Figure 4 As shown in Part A, the experimental statistical results are as follows: Figure 4 As shown in Part B.

[0069] The results showed that regardless of the spike protein variant, the addition of D11 monoclonal antibody reduced viral entry.

[0070] These data indicate that the D11 monoclonal antibody can broadly inhibit the infection of pseudoviruses of the spike protein of currently known SARS-CoV-2 variants.

[0071] This invention also used purified D11 monoclonal antibody for titration experiments to investigate its blocking effect on B.1.1.529 S protein pseudovirus using serially diluted monoclonal antibody. The experimental results are as follows: Figure 5 As shown.

[0072] The results showed that D11 monoclonal antibody at concentrations as low as 10 -5 Up to 10 -4 It can still provide effective protection at mg / mL.

[0073] Example 5 - Anti-ACE2 monoclonal antibody does not impair ACE2 enzyme activity ACE2 peptidase activity is crucial for tissue homeostasis in both healthy individuals and patients infected with SARS-CoV-2. To avoid adverse reactions due to ACE2 dysfunction, anti-ACE2 monoclonal antibodies must not interfere with ACE2 enzyme activity. This invention uses an ACE2 activity assay kit (Beyotime) to analyze ACE2 enzyme activity.

[0074] Specifically, collect lysates of 293T or 293T-ACE2 cells, centrifuge, and use the supernatant for ACE2 enzyme activity analysis. Add serially diluted D11 monoclonal antibody to the supernatant and incubate at 4°C for 30 minutes. Alternatively, incubate 293T or 293T-ACE2 cells with serially diluted D11 monoclonal antibody at 37°C for 30 minutes, then lyse the cells with H2O for enzyme activity analysis. Add substrate to the supernatant and measure fluorescence values ​​every 5 to 10 minutes at 37°C using a SynergyMx M5 microplate reader (Molecular Devices), with excitation / emission wavelengths of 325 / 393 nm.

[0075] This invention evaluated whether D11 monoclonal antibodies impair ACE2 enzyme activity using two different experimental methods.

[0076] First, 293T-ACE2 cell lysates were co-incubated with increasing concentrations of D11 monoclonal antibody, and ACE2 enzyme activity was measured. The experimental results are as follows: Figure 6 As shown in Part A.

[0077] The results showed that the ACE2 enzyme activity did not change after the addition of D11 monoclonal antibody.

[0078] Secondly, intact 293T-ACE2 cells were co-incubated with D11 monoclonal antibody, followed by cell lysis for ACE2 enzyme activity analysis. The experimental results are as follows: Figure 6 As shown in Part B.

[0079] The results consistently showed that ACE2 enzyme activity remained unchanged.

[0080] In summary, these findings indicate that the D11 monoclonal antibody does not impair the enzymatic activity of ACE2. The D11 monoclonal antibody masks the spike protein binding site but does not affect the peptidase domain of ACE2.

Claims

1. An anti-ACE2 antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment comprises: HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 1, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2; the amino acid sequences of CDRs are all in accordance with the IMGT definition rules.

2. The anti-ACE2 antibody or its antigen-binding fragment as described in claim 1, characterized in that, The amino acid sequence of HCDR1 is GYAFTSYY; The amino acid sequence of HCDR2 is IHPRNGGT; The amino acid sequence of HCDR3 is TIYYDYAWFAY; The amino acid sequence of LCDR1 is KSVSTSGYSY; The amino acid sequence of LCDR2 is LVS; The amino acid sequence of LCDR3 is QHIRELTR.

3. The anti-ACE2 antibody or its antigen-binding fragment as described in claim 2, characterized in that, It contains a heavy chain variable region, which contains an amino acid sequence having at least 90% identity with SEQ ID NO: 1 or is composed of an amino acid sequence having at least 90% identity with SEQ ID NO: 1, or contains an amino acid sequence of SEQ ID NO: 1 or is composed of an amino acid sequence of SEQ ID NO: 1; And / or, it includes a light chain variable region comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 2 or consisting of an amino acid sequence having at least 90% identity with SEQ ID NO: 2, or comprising an amino acid sequence of SEQ ID NO: 2 or consisting of an amino acid sequence of SEQ ID NO:

2.

4. An isolated nucleic acid, characterized in that, It encodes the anti-ACE2 antibody or its antigen-binding fragment as described in any one of claims 1-3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 4.

6. A host cell, characterized in that, The host cell contains the isolated nucleic acid as described in claim 4 or the recombinant expression vector as described in claim 5, wherein the host cell is a prokaryotic cell and / or a eukaryotic cell.

7. An immunoconjugate, characterized in that, It comprises an anti-ACE2 antibody or its antigen-binding fragment as described in any one of claims 1-3.

8. A pharmaceutical composition, characterized in that, It comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is an anti-ACE2 antibody or its antigen-binding fragment as described in any one of claims 1-3, a nucleic acid as described in claim 4, or an immunoconjugate as described in claim 7.

9. The use of an anti-ACE2 antibody or an antigen-binding fragment thereof as described in any one of claims 1-3 in the preparation of a medicament for blocking, preventing or treating SARS-CoV-2 infection.

10. Use of an anti-ACE2 antibody or an antigen-binding fragment thereof as described in any one of claims 1-3 in the preparation of a kit for diagnosing SARS-CoV-2 infection.

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