Humanized monoclonal antibody with improved stability and uses thereof

Abstract

The present application provides a humanized monoclonal antibody or antigen-binding fragment thereof specifically binding to human ACE2 with improved stability, related products thereof, and preparation methods and uses thereof. The humanized monoclonal antibody or antigen-binding fragment thereof specifically binding to human ACE2 of the present application not only can specifically bind to the human ACE2 receptor with high affinity, but also has higher stability and better drugability, and is expected to become a therapeutic antibody drug for coronavirus infection with ACE2 as the receptor.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a humanized monoclonal antibody or antigen-binding fragment thereof with improved stability, specifically binding to human ACE2, related products thereof, and a preparation method and use thereof. BACKGROUND

[0002] Coronaviruses are classified in the order Nidovirales, family Coronaviridae, genus Coronavirus. Viruses of the genus Coronavirus are enveloped, linear, single-stranded, positive-sense RNA viruses, and are a large class of viruses widely existing in nature. Coronaviruses only infect vertebrates, such as humans, mice, pigs, cats, dogs, wolves, chickens, cattle, and birds.

[0003] There are currently seven known coronaviruses that can infect humans, namely HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome), MERS-CoV (causing Middle East respiratory syndrome), and the novel coronavirus (SARS-CoV-2). Among these coronaviruses, SARS-CoV, SARS-CoV-2, and HCoV-NL63 all use angiotensin-converting enzyme 2 (ACE2) as a receptor, utilize the RBD region of the major glycosylated spike protein (S protein) on their surface to bind to the host cell surface receptor ACE2, and complete the biological process of infecting host cells, thereby infecting human host cells and humans.

[0004] Antibodies, especially blocking antibodies, can block viral infection by binding to host cell receptor proteins, thereby blocking viral infection, blocking the process of viral invasion of host cells, and achieving prevention and treatment effects. Therefore, antibodies that target the human ACE2 receptor and block its binding to the S protein RBD of SARS-CoV, SARS-CoV-2, and HCoV-NL63 coronaviruses are likely to be effective antibodies for inhibiting infection by these viruses.

[0005] The natural stability of antibody-based protein drugs is significantly lower than that of small molecule compounds. Amino acid mutations introduced by humanization modification can also affect the physicochemical properties of antibody molecules, leading to undesirable chemical modifications, fragmentation, and aggregation during production, storage, and administration, affecting the yield and activity of antibody drugs, and high-molecular-weight aggregates can also cause increased immunogenicity and other drug safety issues.

[0006] Therefore, the development of therapeutic antibody drugs against coronaviruses with high stability and good drug properties has potential clinical application value and prospects. SUMMARY

[0007] Object of the invention

[0008] The present application aims to provide a humanized monoclonal antibody or antigen-binding fragment thereof which specifically binds to human ACE2, and has high antigen binding activity, high stability, good drugability, and a related product, as well as a preparation method and use thereof.

[0009] Solution

[0010] To achieve the above-mentioned purpose, the present application provides the following technical solutions.

[0011] In a first aspect, the present application provides a humanized monoclonal antibody or antigen-binding fragment thereof which specifically binds to human ACE2, comprising a heavy chain variable region and a light chain variable region selected from the following groups:

[0012] (1) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; or

[0013] (2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 7, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0014] In a preferred embodiment (I):

[0015] The humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as follows:

[0016] The heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 8; and,

[0017] the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 10, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 10; and,

[0018] In a particular embodiment of this preferred embodiment, the humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as follows:

[0019] the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 10, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 10; and,

[0020] For this preferred embodiment (I), it is further preferred that the humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain as follows:

[0021] the heavy chain has an amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 11; and,

[0022] the light chain has an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 12.

[0023] In a further preferred particular embodiment of preferred embodiment (I), the humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain as follows:

[0024] the heavy chain has an amino acid sequence as set forth in SEQ ID NO: 11, and the light chain has an amino acid sequence as set forth in SEQ ID NO: 12.

[0025] In a preferred embodiment (II):

[0026] the humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as follows:

[0027] the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 10, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 10; and,

[0028] The heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 10, and the light chain variable region has an amino acid sequence as set forth in SEQ ID NO: 9.

[0029] In a particular embodiment of this preferred embodiment, the humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as follows:

[0030] The heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 10, and the light chain variable region has an amino acid sequence as set forth in SEQ ID NO: 9.

[0031] For this preferred embodiment (II), it is further preferred that the humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain as follows:

[0032] The heavy chain has an amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 13; and,

[0033] The light chain has an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 12.

[0034] In a further preferred particular embodiment of preferred embodiment (II), the humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain as follows:

[0035] The heavy chain has an amino acid sequence as set forth in SEQ ID NO: 13, and the light chain has an amino acid sequence as set forth in SEQ ID NO: 12.

[0036] Further, for the humanized monoclonal antibody or antigen-binding fragment thereof described above, as preferred, the antigen-binding fragment is selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabody.

[0037] In a second aspect, the present application provides a bispecific antibody comprising the humanized monoclonal antibody or antigen-binding fragment thereof according to the first aspect described above.

[0038] In a third aspect, the present application provides a polynucleotide encoding the humanized monoclonal antibody or antigen-binding fragment thereof according to the first aspect described above, or encoding the bispecific antibody according to the second aspect described above.

[0039] In particular embodiments, the polynucleotide is DNA or mRNA.

[0040] In a fourth aspect, the present application provides a nucleic acid construct comprising a polynucleotide according to the third aspect as described above, and optionally at least one expression control element operably linked to the polynucleotide.

[0041] In a fifth aspect, the present application provides an expression vector comprising a nucleic acid construct according to the fourth aspect as described above; preferably, the expression vector is a eukaryotic expression vector.

[0042] In a sixth aspect, the present application provides a transformed host cell comprising a polynucleotide according to the third aspect as described above, a nucleic acid construct according to the fourth aspect as described above, or an expression vector according to the fifth aspect as described above.

[0043] Preferably, the host cell is a eukaryotic cell, further preferably a mammalian cell.

[0044] In a seventh aspect, the present application provides a method of producing a humanized monoclonal antibody or antigen-binding fragment thereof according to the first aspect as described above, the method comprising:

[0045] 1) culturing a transformed host cell according to the sixth aspect as described above under conditions suitable for the expression of the humanized monoclonal antibody or antigen-binding fragment thereof, so that it expresses the humanized monoclonal antibody or antigen-binding fragment thereof;

[0046] 2) recovering the expressed humanized monoclonal antibody or antigen-binding fragment thereof from the host cell or its culture.

[0047] In an eighth aspect, the present application provides a drug conjugate comprising a humanized monoclonal antibody or antigen-binding fragment thereof according to the first aspect as described above or a bispecific antibody according to the second aspect as described above, and an effector molecule conjugated directly or indirectly via a spacer to the humanized monoclonal antibody or antigen-binding fragment thereof;

[0048] Preferably, the effector molecule is a detectable label, a toxin and / or a chemotherapeutic agent;

[0049] Further preferably, the detectable label is an enzyme label, a fluorescein label, an isotope label, a biotin label, a chemiluminescent group or a metal particle.

[0050] In a ninth aspect, the present application provides a pharmaceutical composition comprising the humanized monoclonal antibody or antigen-binding fragment thereof according to the first aspect described above, the bispecific antibody according to the second aspect described above, the polynucleotide according to the third aspect described above, the nucleic acid construct according to the fourth aspect described above, the expression vector according to the fifth aspect described above, the transformed host cell according to the sixth aspect described above, and / or the drug conjugate according to the eighth aspect described above, and a pharmaceutically acceptable carrier and / or excipient.

[0051] In a specific embodiment, the pharmaceutical composition can be in the form of a nasal spray, an oral preparation, a suppository, or a parenteral preparation.

[0052] Preferably, the nasal spray is selected from the group consisting of an aerosol, a spray, and a powder spray.

[0053] Preferably, the oral preparation is selected from the group consisting of a tablet, a powder, a pill, a powder, a granule, a fine granule, a soft / hard capsule, a film-coated agent, a pellet, a sublingual tablet, and a paste.

[0054] Preferably, the parenteral preparation is a transdermal agent, an ointment, a plaster, a topical liquid, an injectable or a bolus preparation.

[0055] The amount of the effective component of the pharmaceutical composition of the present application to be administered varies depending on the subject of administration, the organ of the subject, the symptoms, the method of administration, and the like, and can be determined according to the type of dosage form, the method of administration, the age and weight of the patient, the symptoms of the patient, and the like, according to the judgment of a physician.

[0056] In a tenth aspect, the present application provides use of the humanized monoclonal antibody or antigen-binding fragment thereof according to the first aspect described above, the bispecific antibody according to the second aspect described above, the polynucleotide according to the third aspect described above, the nucleic acid construct according to the fourth aspect described above, the expression vector according to the fifth aspect described above, the transformed host cell according to the sixth aspect described above, the drug conjugate according to the eighth aspect described above, and / or the pharmaceutical composition according to the ninth aspect described above, in the preparation of a medicament for preventing and / or treating a coronavirus infection with ACE2 as a receptor.

[0057] Preferably, the coronavirus with ACE2 as a receptor is selected from the group consisting of SARS-CoV and SARS-CoV-2.

[0058] Preferably, the SARS-CoV-2 can be a SARS-CoV-2 original strain and / or a SARS-CoV-2 variant strain.

[0059] Optionally, the SARS-CoV-2 variant strain is Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2) strain, Omicron (B.1.1.529 / BA.1) sublineage strain, Omicron BA.1.1 sublineage strain, Omicron BA.2 sublineage strain, Omicron BA.2.12.1 sublineage strain, Omicron BA.2.75 sublineage strain, Omicron BA.3 sublineage strain, Omicron BA.4 sublineage strain, or Omicron BA.5 sublineage strain, further preferably Delta (B.1.617.2) strain, Omicron (B.1.1.529 / BA.1) sublineage strain, Omicron BA.2 sublineage strain, Omicron BA.2.12.1 sublineage strain, Omicron BA.2.75 sublineage strain, Omicron BA.4 sublineage strain, or Omicron BA.5 sublineage strain.

[0060] In an eleventh aspect, the present application provides a method for preventing or treating a coronavirus infection using ACE2 as a receptor, comprising: administering to a subject in need thereof a prophylactically or therapeutically effective amount of the nanobody or antigen-binding fragment thereof according to the first aspect described above, the bispecific antibody according to the second aspect described above, the polynucleotide according to the third aspect described above, the nucleic acid construct according to the fourth aspect described above, the expression vector according to the fifth aspect described above, the transformed host cell according to the sixth aspect described above, the drug conjugate according to the eighth aspect described above, and / or the pharmaceutical composition according to the ninth aspect described above.

[0061] The "prophylactically or therapeutically effective amount" varies depending on the subject of administration, the organ of the subject, the symptoms, the method of administration, etc., and can be determined according to the judgment of a doctor, taking into account the type of dosage form, the method of administration, the age and weight of the patient, the symptoms of the patient, etc.

[0062] Advantages

[0063] The humanized monoclonal antibody or antigen binding fragment thereof against human ACE2 provided by the application can specifically bind to human ACE2, thereby blocking the binding of human ACE2 to the RBD region of the coronavirus S protein, so that the coronavirus with ACE2 as the receptor loses the ability to invade the host, and the effect of preventing and treating infection of these viruses is achieved; further, the humanized monoclonal antibody or antigen binding fragment thereof against human ACE2 of the application not only can specifically bind to the human ACE2 receptor with high affinity, but also has high stability and good drugability, thereby providing a new effective choice for the prevention and / or treatment of coronavirus infection with ACE2 as the receptor. DETAILED DESCRIPTION

[0064] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0065] Unless otherwise clearly indicated, in the entire specification and claims, the term "comprise" or its variants such as "comprises" or "comprising" will be understood to encompass the stated elements or components, but not exclude other elements or components.

[0066] In addition, in order to better illustrate the present application, numerous specific details are given in the following detailed description. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some embodiments, the raw materials, elements, methods, means and the like which are well known to those skilled in the art are not described in detail, so as to highlight the main idea of the present application.

[0067] Hereinafter, the present application will be described in detail.

[0068] Definitions

[0069] "Antigen binding fragment" refers to an antigen binding fragment of an antibody and an antibody analogue, which generally includes at least part of the antigen binding region or variable region of the parent antibody, for example, one or more CDRs. The antigen binding fragment retains at least some of the binding specificity of the parent antibody. The antigen binding fragment includes Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabody, CDR-containing peptide, etc.

[0070] "Fab fragment" is composed of one light chain and the CH1 and variable region of one heavy chain.

[0071] An "Fc" region comprises two heavy chain fragments including the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0072] A "Fab' fragment" contains one light chain and a portion of one heavy chain that includes the VH domain and the CH1 domain and a region between the CH1 and CH2 domains, with an interchain disulfide bond between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0073] A "F(ab')2 fragment" contains two light chains and two heavy chains that include the constant region between the CH1 and CH2 domains, with an interchain disulfide bond between the two heavy chains. Thus, a F(ab')2 fragment is composed of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0074] An "Fv region" comprises the variable regions of both heavy and light chains, but not the constant regions.

[0075] A "single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment of an antibody that comprises the VH and VL domains of that antibody, which are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0076] A "diabody" is a small antigen-binding fragment of an antibody that has two antigen-binding sites. The fragment contains a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0077] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of an antibody of a different species, such as mouse, rat, rabbit, or nonhuman primate. In some instances, residues of the Fv framework region of a human immunoglobulin are replaced by corresponding residues of a nonhuman antibody. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.

[0078] When referring to a ligand / receptor, antibody / antigen, or other binding pair, "specific" binding refers to the determination of the presence of a binding reaction between a protein and / or other biological agent, such as human ACE2, in a heterogeneous population of proteins and / or other biological agents. Thus, under the specified conditions, a particular ligand / antigen binds to a particular receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0079] The present application also provides a pharmaceutical composition containing the anti-human ACE2 antibody or antigen-binding fragment thereof of the present application. To prepare a pharmaceutical composition, the antibody or antigen-binding fragment thereof can be mixed with a pharmaceutically acceptable carrier or excipient to prepare various desired dosage forms. As the kind of dosage form of the pharmaceutical composition of the present application, for example, tablets, powders, pills, granules, granulates, soft / hard capsules, film-coated tablets, pellets, sublingual tablets, ointments, and the like can be exemplified as oral agents, and injections, suppositories, transdermal agents, ointments, plasters, external liquid agents, and the like can be exemplified as non-oral agents, and a person skilled in the art can select an appropriate dosage form according to the administration route and the administration subject, etc.

[0080] The amount of the active ingredient of the pharmaceutical composition of the present application to be administered varies depending on the administration subject, the organ of the subject, the symptoms, the administration method, etc., and can be determined according to the kind of dosage form, the administration method, the age and body weight of the patient, the symptoms of the patient, etc., according to the judgment of a physician.

[0081] The pharmaceutical composition of the present application can further contain other agents, including but not limited to a cytotoxic agent, a cell growth inhibitor, an anti-angiogenic drug or an anti-metabolic drug, a targeted tumor drug, an immune stimulant or an immune modulator, or an antibody combined with a cytotoxic agent, a cell growth inhibitor, or other toxic drug.

[0082] The present application will be further illustrated by the following examples; in the following examples, the biological and chemical materials used are commercially available products, unless otherwise specified.

[0083] Example 1: Acquisition of murine monoclonal antibody m11B11, humanization, and construction of murine and human monoclonal antibody light and heavy chain expression plasmids

[0084] In the inventor's previous studies, by immunizing BALB / C mice with human ACE2 extracellular region (hACE2-ecto) recombinant protein, fusing the spleen cells of the immunized mice with myeloma cells to prepare hybridoma cells, and then testing the hybridoma cells by ELISA using purified hACE2 recombinant protein and by testing their blocking ability for new coronavirus pseudovirus infection, the anti-hACE2 monoclonal antibody hybridoma cell strain m11B11 was screened out. Further, the m11B11 antibody coding sequence was obtained from the anti-hACE2 monoclonal antibody hybridoma cell strain m11B11 using the 5' RACE method.

[0085] The variable region amino acid sequence of the m11B11 antibody prepared and screened according to the above method is shown in Table 1 below.

[0086] Table 1: Variable region amino acid sequence of m11B11 antibody

[0087]

[0088] Using the IMGT database, the murine m11B11 antibody was humanized using computer-aided drug design technology to form the humanized monoclonal antibody H11B11 against hACE2. The variable region amino acid sequence of the humanized monoclonal antibody H11B11 is shown in Table 2 below.

[0089] Table 2: Variable region amino acid sequence of H11B11 antibody

[0090]

[0091] The coding sequence of the human IgG1 mutant constant region (SEQ ID NO: 17) was added to the 3' end of the heavy chain variable region gene of each monoclonal antibody shown in Table 1 and Table 2, and the coding sequence of the human kappa light chain constant region (SEQ ID NO: 18) was added to the 3' end of the light chain variable region gene, to form the heavy chain and light chain of each monoclonal antibody, wherein the amino acid sequence of the heavy chain of m11B11 (m11B11-HC) is shown in SEQ ID NO: 19, the amino acid sequence of the light chain of m11B11 (m11B11-LC) is shown in SEQ ID NO: 20, the amino acid sequence of the heavy chain of H11B11 (H11B11-HC) is shown in SEQ ID NO: 21, and the amino acid sequence of the light chain of H11B11 (H11B11-LC) is shown in SEQ ID NO: 12; the full gene synthesis was performed by artificial synthesis.

[0092] The synthesized antibody light and heavy chain full genes were digested with EcoRI (NEB, Cat. No. R0101S) and NotI (NEB, Cat. No. R0189S), and were respectively ligated to the expression vector pTT5 (main elements include, in order: BSPQI digestion site, CMV Promoter (promoter for expression of the target gene), AmpR (amoxicillin resistance gene), pMB1 ori (replication initiation), ori p, signal peptide (signal peptide for expression of the target gene), and target gene) to obtain the complete light and heavy chain expression plasmids of the monoclonal antibodies m11B11 and H11B11.

[0093] Example 2: Expression of murine monoclonal antibody m11B11 and humanized monoclonal antibody H11B11

[0094] The complete light and heavy chain expression plasmids of monoclonal antibodies m11B11 and H11B11 prepared in Example 1 were extracted, and the CHO-18 cells (prepared by Suzhou Junmeng from CHO-K1 cells (ATCC, CCL-61) by suspension serum-free domestication and screening to make them suitable for transient expression) were co-transfected with the light and heavy chains in equal proportions. After co-transfection, the cells were cultured for 7 days, and then the cell culture solution was centrifuged at high speed, filtered by vacuuming the microporous filter, and then loaded into a pre-packed column for monoclonal antibody purification (HiTrap MabSelectSuRe column). The buffer containing 100 mM acetic acid-sodium acetate at pH 3.6 was used as the eluent to elute the target protein in one step, and the target sample was recovered and dialyzed into PBS. The antibody protein concentration and protein amount in the eluent were determined by NanoDrop TM One / OneC Micro UV-Vis Spectrophotometer (Thermo Scientific TM Cat. No.: ND-ONE-W), and the expression level was calculated according to the transfection volume. The determination results of the expression levels of monoclonal antibodies m11B11 and H11B11 are shown in Table 3 below.

[0095] Table 3, Light and heavy chain compatibility of m11B11 and H11B11 antibodies and their expression levels

[0096]

[0097] The results in Table 3 show that the expression level of humanized antibody H11B11 is significantly higher than that of murine antibody m11B11; that is, the expression level of murine antibody m11B11 is significantly improved after humanization.

[0098] Example 3: High-temperature stability detection of murine monoclonal antibody m11B11 and humanized monoclonal antibody H11B11

[0099] The antibody sample prepared in Example 2 was replaced into a buffer system at pH 5.5 (20 mM histidine-histidine hydrochloride buffer containing 230 mM sucrose / trehalose), and the antibody sample concentration was controlled at about 10 mg / ml, and was divided into 500 μl / branch in a test tube. The test tube loaded with the antibody sample was placed in a 40°C constant temperature incubator, and the antibody stability at 0 weeks, 2 weeks and 4 weeks was investigated.

[0100] The antibody stability was evaluated by the following parameters: (a) R-CE-SDS (reducing electrophoresis method) and NR-CE-SDS (non-reducing electrophoresis method) to detect the purity of the antibody; (b) ELISA method to detect the binding activity of the antibody to ACE2.

[0101] The specific detection methods are as follows:

[0102] Detection of antibody purity by NR-CE-SDS method

[0103] According to the final volume of 100 μl, 1% SDS, 40 mM phosphate buffer (pH 6.5), 5 μl of 0.25 M NEM, sample were added into 1.5 ml EP tube in turn, mixed, and the final concentration of the sample was 1.0 mg / mL; centrifuged at 3000 rpm for 30 s at room temperature, then incubated at 70°C for 5 min; after incubation, cooled to room temperature, centrifuged at 12000 rpm for 5 min at room temperature; 75 μl of sample solution was taken from the sample tube into the sample bottle to avoid air bubbles, and a capillary electrophoresis instrument (PA800PLUS, SCIEX) was used for detection. The antibody sample purity calculation method was: antibody protein purity (%) = antibody protein peak area / total peak area.

[0104] Detection of antibody purity by R-CE-SDS method

[0105] According to the final volume of 100 μl, 1% SDS, 40 mM phosphate buffer (pH 6.5), sample, 5 μl of β mercaptoethanol were added into 1.5 ml EP tube in turn, mixed, and the final concentration of the sample was 1.0 mg / mL; centrifuged at 3000 rpm for 30 s at room temperature, then incubated at 70°C for 15 min; after incubation, cooled to room temperature, centrifuged at 12000 rpm for 5 min at room temperature; 75 μl of sample solution was taken from the sample tube into the sample bottle to avoid air bubbles, and a capillary electrophoresis instrument (PA800PLUS, SCIEX) was used for detection. The antibody sample purity calculation method was: antibody protein purity (%) = (antibody heavy chain peak area + antibody light chain peak area) / total peak area.

[0106] Detection of antigen binding activity of antibody by ELISA method

[0107] The plate was coated with 1.0 μg / mL of human ACE-2 with mFc tag (self-made by Suzhou Junmeng, batch number: 20210203) using a Thermo Scientific microplate reader, and after blocking with 2% milk (Anchor), the gradient-diluted antibody to be tested (4.0 μg / ml as the starting concentration, 4-fold gradient dilution, a total of 12 concentrations) was added; anti-human IgG (Fc-specific) -peroxidase antibody (Sigma-Aldrich, A0170) was diluted 5000 times as a detection antibody for detection, then 0.1 mg / ml TMB (Sigma, T2885) was used for color development, and finally 2M hydrochloric acid solution was used to terminate the reaction, and the plate was read at 450nm / 620nm. The activity calculation method is: using the log(agonist) vs.response--Variable slope Least squares fit nonlinear fitting model in Graphpad software, the antibody protein concentration and color development value are fitted to obtain the EC50 value. ACE2 binding activity (%) = EC50 at different time points / EC50 at starting time point.

[0108] The instruments and column / cassette information used in each detection method are shown in Table 4 below.

[0109] Table 4, instrument and column / cassette information used for detection

[0110]

[0111]

[0112] The stability detection results of m11B11 and H11B11 monoclonal antibodies placed at 40°C for 4 weeks are shown in Table 5 below.

[0113] Table 5, stability of m11B11 and H11B11 monoclonal antibodies placed at 40°C for 4 weeks

[0114]

[0115] Note: “—” represents an abnormal spectrum, and no data is reported.

[0116] As can be seen from Table 5, after the humanized monoclonal antibody H11B11 was placed at 40°C for 4 weeks, the proportion of reduced and non-reduced CE-SDS main peak samples showed a downward trend with the extension of time, accompanied by a significant decrease in biological activity; these results suggest that the humanized monoclonal antibody H11B11 may have stability problems.

[0117] Example 4: Construction and expression of H11B11 mutant antibody molecules

[0118] In order to improve the stability of the humanized monoclonal antibody H11B11, in this embodiment, a series of mutations and screening were performed on the variable region of the H11B11 antibody, and finally two mutant antibody molecules H11B11-mut1 and H11B11-mut2 were obtained; compared with the humanized monoclonal antibody H11B11 before modification, the heavy chain variable region HCDR3 of the mutant antibody molecules H11B11-mut1 and H11B11-mut2 each contains one amino acid mutation; the HCDR1, HCDR2 and HCDR3 sequences of the antibodies H11B11, H11B11-mut1 and H11B11-mut2 are shown in Table 6 below.

[0119] Table 6, sequence of HCDR1-3 of H11B11, H11B11-mut1 and H11B11-mut2

[0120]

[0121]

[0122] As can be seen from Table 6, except that the HCDR3 is different, the remaining segments of the two mutant antibody molecules are completely identical to the antibody H11B11.

[0123] The heavy chain sequence of H11B11-mut1 is shown as SEQ ID NO: 11, and the light chain sequence is shown as SEQ ID NO: 12; the heavy chain sequence of H11B11-mut2 is shown as SEQ ID NO: 13, and the light chain sequence is shown as SEQ ID NO: 12; according to these sequence information, the full gene synthesis of the light and heavy chains of the two mutant antibodies was performed; then, according to the method described in embodiment 1, the complete light and heavy chain expression plasmids of the mutant antibodies H11B11-mut1 and H11B11-mut1 were constructed, and the expression of the antibodies and the determination of their expression levels were performed according to the method described in embodiment 2; the light and heavy chain compatibility of the monoclonal antibodies H11B11, H11B11-mut1 and H11B11-mut2 and their expression levels are shown in Table 7 below.

[0124] Table 7, light and heavy chain compatibility of H11B11, H11B11-mut1 and H11B11-mut2 and their expression levels

[0125]

[0126] As can be seen from Table 7, the expression levels of the mutant antibodies H11B11-mut1 and H11B11-mut1 are comparable to those of H11B11, and they can be efficiently expressed.

[0127] Example 5: Antigen binding activity (i.e., binding ability to human ACE2) of mutant antibodies H11B11-mut1 and H11B11-mut2

[0128] In this example, the antigen binding activity (EC50 value) of antibody H11B11 and mutant antibodies H11B11-mut1 and H11B11-mut1 were detected by ELISA method.

[0129] Specifically, using a Thermo Scientific microplate reader, the plate was coated with 1.0 pg / mL human ACE2 with mFc tag (self-made in Suzhou Junmeng, batch number: 20210203), and after blocking with 2% Milk (Anchor), the gradient-diluted test antibody (4.0 pg / ml as the starting concentration, 4-fold gradient dilution, a total of 12 concentrations) was added; anti-human IgG (Fc-specific) -peroxidase antibody (Sigma-Aldrich, A0170) was diluted 5000 times as a detection antibody for detection, then 0.1 mg / ml TMB (Sigma, T2885) was used for color development, and finally 2M hydrochloric acid solution was used to terminate the reaction, and the plate was read at 450nm / 620nm.

[0130] The four-parameter logarithmic regression (4PL) model fitting was performed using the software GraphPad Prism to obtain the EC50 (ng / mL), which can reflect the antigen binding activity of each antibody; the results are shown in Table 8 below.

[0131] Table 8: Antigen binding activity (EC50 value) of H11B11, H11B11-mut1 and H11B11-mut2 antibodies

[0132] Sample name EC50 (ng / mL) H11B11 4.1 H11B11-mut1 3.5 H11B11-mut2 2.7

[0133] As can be seen from Table 8, the antigen binding activity of mutant antibodies H11B11-mut1 and H11B11-mut1 is significantly higher than that of humanized monoclonal antibody H11B11, i.e., mutant antibodies H11B11-mut1 and H11B11-mut1 have significantly higher antigen binding activity than the original antibody H11B11.

[0134] Example 6: High temperature stability of mutant antibodies H11B11-mut1 and H11B11-mut2

[0135] In this example, according to the detection method described in Example 3, the stability of mutant antibodies H11B11-mut1 and H11B11-mut1 after being placed at 40°C for 4 weeks was determined; the results are shown in Table 9 below.

[0136] Table 9, high temperature stability of H11B11-mut1 and H11B11-mut2

[0137]

[0138] Comparing the stability data of the two mutant antibodies in Table 9 with the stability data of H11B11 in the aforementioned Table 3 (both under the same test conditions), it can be seen that the high temperature stability of the mutant antibodies H11B11-mut1 and H11B11-mut1 is significantly improved compared with the original antibody H11B11, and thus the drugability thereof is significantly improved.

[0139] The above examples show that the mutant antibodies H11B11-mut1 and H11B11-mut1 can specifically bind to human ACE2 with high binding activity, and have high stability and good drugability, and thus are expected to become therapeutic antibody drugs for coronavirus (including SARS-CoV-2) infection with ACE2 as the receptor.

[0140] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

[0141] The sequences used herein are as follows:

[0142] SEQ ID NO: 1: HCDR1 of antibody H11B11-mut1 of the present invention

[0143] DYYMN;

[0144] SEQ ID NO: 2: HCDR2 of antibody H11B11-mut1 of the present invention

[0145] FIRNKANDYTTEYST;

[0146] SEQ ID NO: 3: HCDR3 of antibody H11B11-mut1 of the present invention

[0147] HMYDHGFDF;

[0148] SEQ ID NO: 4: LCDR1 of antibody H11B11-mut1 or H11B11-mut2 of the present invention RASSSVRYMH;

[0149] SEQ ID NO: 5: LCDR2 of antibody H11B11-mut1 or H11B11-mut2 of the present invention

[0150] DTSKLAS;

[0151] SEQ ID NO: 6: LCDR3 of antibody H11B11-mut1 or H11B11-mut2 of the present invention QQWSYNPLT;

[0152] SEQ ID NO: 7: HCDR3 of antibody H11B11-mut2 of the present invention

[0153] HMYDNGFDF;

[0154] SEQ ID NO: 8: Heavy chain variable region of antibody H11B11-mut1 of the present invention

[0155] EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYSTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDHGFDFWGQGTLVTVSS;

[0156] SEQ ID NO: 9: Light chain variable region of antibody H11B11-mut1 or H11B11-mut2 of the present invention

[0157] DIQMTQSPSSLSASVGDRVTITCRASSSVRYMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSYNPLTFGQGTKLEIK;

[0158] SEQ ID NO: 10: Heavy chain variable region of antibody H11B11-mut2 of the present invention

[0159] EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYSTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDNGFDFWGQGTLVTVSS;

[0160] SEQ ID NO: 11: Heavy chain of antibody H11B11-mut1 of the present invention

[0161] EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYSTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDHGFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGAPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0162] SEQ ID NO: 12: Light chain of antibody H11B11-mut1 or H11B11-mut2 of the present invention

[0163] DIQMTQSPSSLSASVGDRVTITCRASSSVRYMHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSYNPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0164] SEQ ID NO: 13: Heavy chain of antibody H11B11-mut2 of the present invention

[0165] EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYSTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDNGFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGAPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0166] SEQ ID NO: 14: Heavy chain variable region of murine monoclonal antibody m11B11

[0167] EVKLVESGGGLVQPGGSLRLSCTTSGFTFIDYYMNWVRQPPGKALEWLGFIRNKANDYTTEYSTSVKGRFTISRDNSQSILYLQLNNLRAEDSGTYYCASHMYDDGFDFWGQGTTVTVSS;

[0168] SEQ ID NO: 15: Light chain variable region of murine monoclonal antibody m11B11

[0169] DNVLTQSPAIMSAFPGEKVTMTCNASSSVRYMHWYQQKSGTSPKRWIYDTSKLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSYNPLTFGAGTKLEIK;

[0170] SEQ ID NO: 16: Heavy chain variable region of humanized monoclonal antibody H11B11

[0171] EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYSTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDDGFDFWGQGTLVTVSS;

[0172] SEQ ID NO: 17: Human IgG1 mutant antibody constant region

[0173] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPAPEAAGAPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0174] SEQ ID NO: 18: Human kappa light chain constant region

[0175] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0176] SEQ ID NO: 19: Heavy chain of murine monoclonal antibody m11B11

[0177] EVKLVESGGGLVQPGGSLRLSCTTSGFTFIDYYMNWVRQPPGKALEWLGFIRNKANDYTTEYS TS VKGRFTISRDNSQSILYLQLNNLRAEDSGTYYCASHMYDDGFDFWGQGTTVTVSSAKTT APSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLS SSSTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIF PPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVS ALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVT LTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYS CSVVHEGLHNHHTTKSFSRTPGK;

[0178] SEQ ID NO: 20: Light chain of murine monoclonal antibody m11B11

[0179] DNVLTQSPAIMSAFPGEKVTMTCNASSSVRYMHWYQQKSGTSPKRWIYDTSKLASGVPVRF SGSGSGTSYSLTISSMEAEDAATYYCQQWSYNPLTFGAGTKLEIKRADAAPTVSIFPPSSEQ LTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTK DEYERHNSYTCEATHKTSTSPIVKSFNRNEC;

[0180] SEQ ID NO: 21: Heavy chain of humanized monoclonal antibody H11B11

[0181] EVQLVESGGGLVQPGGSLRLSCAASGFTFIDYYMNWVRQAPGKGLEWVGFIRNKANDYTTEYS TS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHMYDDGFDFWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGAPSVFLFPPKPK DTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK

Claims

1. A humanized monoclonal antibody or antigen binding fragment thereof that specifically binds to human ACE2, characterized in that, The humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (1) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; or (2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 7, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

2. The humanized monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that, The humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (1) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 8; and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 9; (2) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 10; and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:

9.

3. The humanized monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein, The humanized monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain selected from the group consisting of: (1) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 11; and a light chain having the amino acid sequence set forth in SEQ ID NO: 12; (2) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 13; and a light chain having the amino acid sequence set forth in SEQ ID NO:

12.

4. The humanized monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antigen-binding fragment of the humanized monoclonal antibody is humanized.

5. The humanized monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The monoclonal antibody is an IgG1 type monoclonal antibody.

6. The humanized monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The antigen-binding fragment is selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabody.

7. A bispecific antibody comprising the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6.

8. A polynucleotide encoding the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, or encoding the bispecific antibody of claim 7.

9. A nucleic acid construct comprising the polynucleotide of claim 8, and optionally at least one expression control element operably linked to the polynucleotide.

10. An expression vector comprising the nucleic acid construct of claim 9.

11. The expression vector of claim 10, wherein, The expression vector is a eukaryotic expression vector.

12. A transformed host cell comprising the polynucleotide of claim 8, the nucleic acid construct of claim 9 or the expression vector of any one of claims 10-11.

13. The transformed host cell of claim 12, wherein, The host cell is a eukaryotic cell.

14. The transformed host cell of claim 12, wherein, The host cell is a mammalian cell.

15. A method of producing the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, the method comprising: 1) culturing the transformed host cell of any one of claims 12-14 under conditions suitable for expression of the humanized monoclonal antibody or antigen-binding fragment thereof, so that it expresses the humanized monoclonal antibody or antigen-binding fragment thereof; 2) recovering the expressed humanized monoclonal antibody or antigen-binding fragment thereof from the host cell or its culture.

16. A drug conjugate comprising the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6 or the bispecific antibody of claim 7, and an effector molecule, which is a detectable label, conjugated directly or indirectly via a spacer to the humanized monoclonal antibody or antigen-binding fragment thereof.

17. The drug conjugate of claim 16, wherein, The detectable label is an enzymatic label, a fluorescein label, an isotopic label, a biotin label, a chemiluminescent group or a metal particle.

18. A pharmaceutical composition comprising the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, the bispecific antibody of claim 7, the polynucleotide of claim 8, the nucleic acid construct of claim 9, the expression vector of claim 10 or 11, the transformed host cell of any one of claims 12-14 and / or the drug conjugate of claim 16 or 17, and a pharmaceutically acceptable carrier and / or excipient.

19. Use of the humanized monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, the bispecific antibody of claim 7, the polynucleotide of claim 8, the nucleic acid construct of claim 9, the expression vector of claim 10 or 11, the transformed host cell of any one of claims 12-14, the drug conjugate of claim 16 or 17 and / or the pharmaceutical composition of claim 18 for the manufacture of a medicament for the prevention and / or treatment of a coronavirus infection with ACE2 as a receptor.

20. Use according to claim 19, characterized in that, The coronavirus with ACE2 as a receptor is selected from the group consisting of SARS-CoV and SARS-CoV-2.

21. Use according to claim 20, characterized in that, The SARS-CoV-2 is a SARS-CoV-2 prototype strain and / or a variant strain thereof.

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