Broad-spectrum coronavirus-neutralizing antibody and use thereof

Abstract

Provided are a broad-spectrum coronavirus-neutralizing antibody and a use thereof. The antibody CYFN1006-1 comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises CDRH1 to CDRH3 having amino acid sequences as shown in SEQ ID NOs: 1-3; and the VL comprises CDRL1 to CDRL3 having amino acid sequences as shown in SEQ ID NOs: 4-6. CYFN1006-1 is a broad-spectrum highly-efficient coronavirus-neutralizing antibody, can efficiently neutralize all currently circulating SARS-CoV-2 variants, shows medium efficacy on SARS-CoV, and provides a new choice for solving the problems of viral escape and drug resistance encountered in the application of monoclonal antibody passive immunotherapy against novel coronavirus infection.

Description

Antibodies neutralizing a broad spectrum of coronaviruses and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of medical virology immunology, in particular to antibodies neutralizing a broad spectrum of coronaviruses and uses thereof. BACKGROUND

[0002] Coronaviruses are a group of enveloped RNA viruses belonging to the Coronaviridae family of the Coronavirinae genus. They are named for the crown-like projections (spikes) on their surface. Coronavirus infections occur not only in humans but also in a variety of animals. According to the International Committee on Taxonomy of Viruses (ICTV), coronaviruses are divided into four genera: alpha-, beta-, gamma-, and delta-coronaviruses.

[0003] SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) and SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2, also known as the novel coronavirus) are members of the beta-coronavirus family. Both viruses can cause severe respiratory illness. The pandemic of SARS-CoV-2 has led to massive infection and caused a large number of illness and death worldwide, and the continued spread of SARS-CoV-2 has brought serious challenges to the economy, social life and health care system. The clinical symptoms of SARS-CoV-2 infection range from asymptomatic or mild respiratory illness to severe pneumonia, acute respiratory distress syndrome, multiple organ failure and death. Certain groups of people, such as the elderly and those with underlying health problems, are at higher risk of serious consequences. At the same time, the problem of long-term sequelae (long COVID) after infection is also increasing, and more effective prevention and treatment measures are urgently needed. SARS-CoV-2 and SARS-CoV share about 79.6% genetic sequence homology. The virus mainly encodes four key proteins, of which the spike glycoprotein (S) is a key protein on the surface of the viral particle and is essential for viral infectivity. Passive immunotherapy with neutralizing antibodies against the S protein of SARS-CoV-2 is an important option for prevention and treatment, especially for individuals with low immune function, who cannot be vaccinated or have other risk factors for severe illness. During the process of viral infection, antibodies exert their therapeutic effects through a variety of mechanisms, including blocking receptor binding, cross-linking viral proteins, inhibiting fusion with host cells, neutralizing viral particles, and promoting immune clearance of infected cells and virus-containing pathogens. Therefore, several early developed neutralizing antibodies and antibody combination cocktail therapies have been granted Emergency Use Authorization (EUA) by the FDA, and have shown good therapeutic and preventive effects in clinical practice.

[0004] With the continuous evolution of the SARS-CoV-2 virus, there have been many circulating mutant strains carrying multiple mutations that have severely escaped the marketed neutralizing antibody drugs. All previously FDA-authorized emergency use of anti-SARS-CoV-2 neutralizing antibodies and antibody combination cocktail therapies have been withdrawn due to the prevalence of immune escape mutant strains. Antibodies and their combinations developed early in the SARS-CoV-2 pandemic, such as the Eli Lilly Bamlanivimab / Etesevimab antibody combination, the Regeneron Casirivimab / imdevimab (REGN-COV2) antibody combination, and the AstraZeneca Tixagevimab / Cilgavimab (Evusheld) antibody combination, have all lost their neutralizing activity against the circulating SARS-CoV-2 mutant strains. Bebtelovimab (LY-CoV1404) can efficiently neutralize many circulating SARS-CoV-2 mutant strains, including the early Omicron subvariant, but it cannot neutralize the subsequently emerging SARS-CoV-2 mutant strains BQ.1.1 and XBB lineages. Another mAb Sotrovimab (S309) isolated from convalescent individuals infected with SARS-CoV-2 retains neutralizing activity against most emerging SARS-CoV-2 variants due to recognition of a conserved epitope, however, the neutralizing activity is generally about 1 pg / mL for 50% neutralization activity (IC50) values for many SARS-CoV-2 variants, which is not sufficient to be effective.

[0005] In addition, some neutralizing antibodies in the field that have some cross-neutralizing activity against SARS-CoV-2 and its mutant strains, such as SA55, S3H3, and S309, were isolated from individuals who had received multiple vaccinations, experienced multiple infections, or both, and it is not clear whether a single infection with the SARS-CoV-2 prototype strain can also produce a broad-spectrum neutralizing antibody that is also highly effective against SARS-CoV-2 mutant viruses that the donor has not previously been exposed to.

[0006] Screening of broad-spectrum and highly efficient neutralizing antibodies targeting the conserved epitope of S protein is a strategy to cope with the emerging new variant escape. Currently, human monoclonal antibodies are mainly obtained by several strategies: (1) immunized transgenic mice. This transgenic mouse has human antibody heavy chain gene or antibody heavy chain and light chain gene transferred into it, and can produce antibodies derived from human antibody sequences after immunization with antigens, i.e. human antibodies. However, this method requires preparation of immunization antigens, and the antibodies are produced in the immune selection environment of mice, which cannot completely simulate the production of antibodies in the human body itself immune selection environment. (2) Human antibody library technology. Single-chain antibody variable region fragment (scFv) library from immune or non-immune human antibody genes is screened by this technology. Because it is a non-natural antibody gene pairing, and the antibody has not undergone in vivo immune selection and affinity maturation process, it is difficult to obtain high-affinity antibodies without safety concerns. (3) Direct cloning of antibodies from human memory B cells. With the development of antibody technology, people can directly obtain antibodies from single human B cells. Most of them use the strategy of sorting single antigen-specific memory B cells from human PBMCs by flow cytometry to clone antibodies. Although this method can quickly obtain antibodies, it has antigen selection bias, cannot directly screen antibodies based on antibody functional activity, and is easy to lose conformation-dependent antibodies with important functions produced by the body.

[0007] In summary, the problems of immune escape, potency and broad spectrum of coronavirus antibody drugs, and the uncertainty of antibody response caused by vaccines and infection need to be solved. SUMMARY

[0008] In view of the lack of broad-spectrum and highly efficient neutralizing mAb against new coronavirus variants in the prior art, a monoclonal antibody (named CYFN1006-1) which can broad-spectrum and highly efficiently neutralize all new coronavirus epidemic variants so far is isolated from a recovered coronavirus infection. The present application discloses the CYFN1006-1 antibody and its application.

[0009] The present application provides an antibody for broad-spectrum neutralization of coronavirus, which comprises a heavy chain variable region VH and a light chain variable region VL.

[0010] The VH comprises CDRH1 with an amino acid sequence as shown in SEQ ID NO. 1, CDRH2 with an amino acid sequence as shown in SEQ ID NO. 2, and CDRH3 with an amino acid sequence as shown in SEQ ID NO. 3; and the VL comprises CDRL1 with an amino acid sequence as shown in SEQ ID NO. 4, CDRL2 with an amino acid sequence as shown in SEQ ID NO. 5, and CDRL3 with an amino acid sequence as shown in SEQ ID NO. 6.

[0011] A "light chain variable region" (VL) or "heavy chain variable region" (VH) is composed of "framework" regions separated by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs (antigen binding determinants) that are primarily responsible for specific antigenic epitope binding. The CDRs include the amino acid residues in an antibody that are primarily responsible for antigen binding. Both the VL and VH domains comprise, from amino-terminus to carboxy-terminus, the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as CDRL1, CDRL2, and CDRL3, respectively; and the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as CDRH1, CDRH2, and CDRH3, respectively.

[0012] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO. 7, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO. 8.

[0013] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO. 7, and the VL comprises an amino acid sequence as set forth in SEQ ID NO. 8.

[0014] In some embodiments, the antibody is a fully human antibody, a humanized antibody, or a chimeric antibody; preferably, the antibody is of any one of the following: (i) an isotype of IgG, IgA, IgM, IgE, or IgD; (ii) a subtype of IgGl, IgG2, IgG3, or IgG4.

[0015] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody comprises a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO. 7, and a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO. 8.

[0016] The antibody can further comprise amino acid sequence modifications, including but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acids.

[0017] The present invention also provides a nucleic acid comprising a nucleotide sequence encoding the antibody described above. The nucleic acid includes, but is not limited to, DNA, RNA (including mRNA), and synthetic nucleic acids.

[0018] The present invention also provides a carrier comprising the nucleic acid.

[0019] The present invention also provides a host cell comprising the aforementioned vector.

[0020] The present invention also provides an agent for detecting, preventing, or treating coronavirus infection, comprising the aforementioned antibody, nucleic acid, vector, or host cell. In some embodiments, the agent further includes a pharmaceutically acceptable carrier or excipient.

[0021] In some embodiments, the formulation further comprises other therapeutic agents.

[0022] In some embodiments, the other therapeutic agents comprise one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, other antibodies, and vaccines.

[0023] The present invention also provides a conjugate comprising the antibody disclosed herein, and a chemical moiety conjugated thereto. In some embodiments, the chemical moiety may be selected from therapeutic agents, detectable moieties, and immunostimulatory molecules.

[0024] The present invention also provides a method for treating a subject, comprising administering to the subject an effective amount of the antibody disclosed in the present invention, the formulation disclosed in the present invention, or the conjugate disclosed in the present invention.

[0025] The antibody, composition, or conjugate described in this invention can be administered to mammals in a single dose or in a series of sub-dose over a suitable period of time, for example, daily, bi-weekly, weekly, bi-weekly, bi-weekly, bi-monthly, semi-annually, or annually as needed. The dosage unit containing an effective amount of the antibody, composition, or conjugate can be administered as a single daily dose, or the total daily dose can be administered as needed in two, three, four, or more daily sub-dose units.

[0026] In some implementation methods, the dose administered to the subject may vary depending on the implementation method, the drug used, the method of administration, and the site of treatment and the subject. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine the effective amount to administer to a person or other subject to treat a medical condition. The precise amount required for effective treatment may depend on many factors, such as antibody activity and the route of administration.

[0027] This invention also provides applications of the antibody, wherein the application is any one of the following:

[0028] (1) Used to prepare diagnostic reagents for coronaviruses and their variants;

[0029] (2) For use in the preparation of agents to prevent infection with coronaviruses and their variants;

[0030] (3) For use in the preparation of formulations for treating coronavirus and its variant infections;

[0031] (4) For the preparation of preparations for the treatment of sequelae of coronavirus and its variants or long-term coronavirus infection.

[0032] In some implementations, the coronaviruses include, but are not limited to, SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus), SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus Type 2), RaTG13, GD-Pangolin CoV, WIV1, and SHC014.

[0033] In some implementations, SARS-CoV-2 variants include, but are not limited to, Alpha variants, Beta variants, Gamma variants, Delta variants, Omicron variants, Lambda variants, JN.1 variants, and KP.2 variants.

[0034] The antibodies of this invention can be used alone or in combination with cocktail drugs for the prevention and treatment of coronaviruses. They can be used as passive immunoprophylaxis agents for high-risk groups of SARS-CoV-2 infection or as passive immunotherapy agents for patients with novel coronavirus infection (including long-term COVID-19).

[0035] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0036] 1. This invention uses unbiased B cell culture supernatant combined with SARS / COVID-19 cross-conserved antigens, along with a unique antibody cloning technology screening strategy, to directly screen for conformation-dependent anti-cross-conserved epitopes that exist in vivo but are difficult to imitate in vitro. This antibody isolation strategy does not require pre-labeling memory B cells with antigens, and is therefore not limited by labeled antigens, and can simultaneously screen for antibodies that bind to different target proteins.

[0037] 2. The antibody of the present invention can target the conserved region of the spike protein RBD on the surface of SARS-CoV-2. The pairing of the heavy chain and light chain of the antibody is naturally generated and is safe and has high affinity, making it suitable for practical applications.

[0038] 3. The CYFN1006-1 antibody of the present invention exhibits strong neutralizing activity against all SARS-CoV-2 mutant strains, with no impact on efficacy. The IC50 value is 1-5 ng / mL. It shows moderate neutralizing efficacy against SARS-CoV and is a broad-spectrum and highly effective neutralizing antibody. Its broad spectrum is comparable to or even better than that of SA55, and its neutralizing activity against most mutant strains is stronger than that of SA55.

[0039] 4. The natural human antibody CYFN1006-1 of this invention is a very broad-spectrum and highly effective coronavirus neutralizing antibody to date. It can effectively neutralize all currently circulating clinical mutant strains of SARS-CoV-2, providing a new option for solving the problems of viral escape and drug resistance faced by the application of monoclonal antibody passive immunization in SARS-CoV-2 infection. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 shows the antigen-binding characteristics of B cell culture supernatant derived from CYFN1006-1 in Example 2 of the present invention.

[0042] Figure 2 shows the antigen binding curve results of CYFN1006-1 and SA55 antibodies in Example 4 of the present invention;

[0043] Figure 3 shows the affinity analysis of CYFN1006-1 and SA55 antibodies binding to SARS-CoV-2 RBD in Example 4 of the present invention;

[0044] Figure 4 shows the neutralization curves of CYFN1006-1 and control antibodies against SARS-CoV-2 variants and other related sarbecovirus pseudoviruses in Example 5 of this invention.

[0045] Figure 5 shows the neutralizing IC50 values ​​of CYFN1006-1 and the control antibody of Example 5 of the present invention against SARS-CoV-2 variants and other related sarbecovirus pseudoviruses, in nanograms per milliliter (ng / mL). Detailed Implementation

[0046] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0047] This invention utilizes an unbiased human antibody screening method from B cells of recovered patients who have been infected with the prototype strain of SARS-CoV-2. Through unbiased in vitro memory B cell culture, screening of cross-binding antibodies against SARS-CoV-2 and SARS-CoV in the culture supernatant, gene cloning of cross-binding antibodies, and antibody characteristic analysis, a novel broad-spectrum and highly effective human monoclonal antibody, CYFN1006-1, was successfully obtained.

[0048] The CYFN1006-1 antibody binds with high affinity to the shared conserved site of the RBD of the S protein of SARS-CoV-2 and SARS-CoV. It is formed by the variable region VH of the heavy chain and the variable region VL of the light chain. CYFN1006-1 exhibits strong neutralizing activity against all currently circulating clinically diagnosed SARS-CoV mutant strains, including the latest JN.1 and KP.2 mutants, with an IC50 value of 1–5 ng / mL. It also shows cross-neutralizing activity against SARS-CoV and related sarbecovirus strains (such as WIV1, SHC014, RaTG13, and GD-pangolin). The broad-spectrum and highly effective neutralizing activity of the CYFN1006-1 antibody could provide a new generation of monoclonal antibody and antibody combination therapies for the diagnosis, prevention, and treatment of SARS-CoV-2 infection, including the treatment of chronic SARS-CoV-2.

[0049] The present invention also discovered that individuals who have been infected with the prototype strain of the novel coronavirus once can also produce broad-spectrum and highly effective neutralizing antibodies against novel mutant strains of the novel coronavirus that they have not been exposed to before, thus expanding the sources for inventing new broad-spectrum and highly effective neutralizing antibodies.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0051] Instruments and reagents:

[0052] Flow cytometer: Beckman Coulter MoFlo Astrios EQ ultra-high-speed flow cytometry cell sorting system, purchased from Shanghai Zequan Instrument Equipment Co., Ltd.;

[0053] Fluorescently labeled antibodies, including IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, IgM-PB and CD45-KO fluorescent antibodies, were purchased from Beckman Coulter.

[0054] Reverse transcription kit: SuperScript III First Strand Synthesis System, invitrogen, #18080051;

[0055] High-fidelity DNA polymerase: Phusion High-Fidelity PCR Master Mix with GC Buffer, NEB, #M0532s;

[0056] Sources of various coronavirus pseudovirus strains: laboratory preparation (preparation method refers to Wang, P. et al. Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 593, 130-135 (2021)).

[0057] Example 1: Identification and sorting of memory B cells

[0058] 1) Isolation of peripheral blood mononuclear cells (PBMCs):

[0059] Peripheral blood mononuclear cells were obtained by EDTA-anticoagulated peripheral vein blood from patients in the recovery period of COVID-19 infection and separated using density gradient centrifugation.

[0060] 2) Fluorescently labeled antibody staining:

[0061] The fluorescent antibodies used for cell staining were: IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, IgM-PB, and CD45-KO.

[0062] Specific steps: Peripheral blood mononuclear cells were washed three times with PBS buffer, then stained with antibodies and incubated at room temperature in the dark for 15 min. After washing with PBS buffer, the cells were resuspended in 400 μL of PBS buffer (phosphate buffer) and added to the flow cytometer. Fluorescently labeled antibody staining: Nine analysis tubes were used. Tubes 1-7 were filled with the corresponding single fluorescently labeled antibody or 7-AAD. Tube 9 was filled with a mixture of seven fluorescently labeled antibodies. Tube 8 was a blank tube containing only cells. The sample tubes were stained in the same way as tube 9.

[0063] 3) Sorting of memory B cells:

[0064] 7-AAD is used to distinguish between live and dead cells, CD45 is a marker for white blood cells, and CD38 is commonly used as one of the markers for B cell subsets. low It was used to mark a more mature subset of B cells that had undergone a certain degree of differentiation.

[0065] The sample tubes were analyzed, and live CD45-positive leukocytes were circled based on 7-AAD and CD45 levels. B cells were circled based on CD19 levels. Cells negative for both IgM and IgD were then circled from the CD19-positive B cell population and defined as IgD cells. - IgM - CD27 + CD38 low The population consisted of memory B cells. Memory B cells were sorted into 96-well cell culture plates containing cell culture medium, 50 cells per well, and cultured for 10 days (refer to PCT / CN2021 / 135785).

[0066] Example 2: Culture of memory B cells and screening of antibodies in culture supernatant

[0067] In Example 1, CpG2006, IL21, IL2, radiation-irradiated healthy human PBMCs, and B95-8 cell culture supernatant containing EBV were added to a 96-well cell culture plate containing memory B cells and cultured for 7–10 days (refer to PCT / CN2021 / 135785).

[0068] The presence of antibodies against the S protein of SARS-CoV-2 (NC_045512.2) and SARS-CoV (NC_004718.3) in B cell culture supernatant was screened using the capture ELISA method. This means that the culture supernatant can simultaneously bind to the SARS-CoV-2 and SARS-CoV S protein antigens, and positive B cells can be screened.

[0069] The procedure is as follows: A 96-well ELISA plate is coated with D7-tagged antibody to capture SARS-CoV-2 or SARS-CoV S, S1 or RBD antigens with D7 tags, reacts with B cell culture supernatant samples, detects the specific binding antibody signal in each well of the ELISA plate, and identifies and screens out positive B cell wells.

[0070] The results are shown in Figure 1. Figure 1 shows the antibody ELISA binding signal characteristics of the culture supernatant of B cells in a 96-well cell culture plate that screened for CYFN1006-1 antibody against SARS-CoV-2 and SARS-CoV S protein. It can be seen that the culture supernatant of the screened B cells contains cross-antibodies that can bind to the conserved RBD regions of SARS-CoV-2 and SARS-CoV simultaneously.

[0071] Example 3: Cloning of Antibodies

[0072] 1) cDNA synthesis: For positive B cells with cross-antibodies against the conserved regions of SARS-CoV-2S and SARS-CoV proteins in the supernatant selected by screening, RNA was first extracted and cDNA was synthesized using a reverse transcription kit.

[0073] 2) Nested PCR amplification of the heavy and light chain variable regions of the antibody: PCR primer sequences reference: Tiller, T., Meffre, E., Yurasov, S., Tsuiji, M., Nussenzweig, MC & Wardemann, H. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J. Immunol. Methods 329, 112-124 (2008). The reaction was performed using Phusion High-Fidelity DNA polymerase (Phusion High-Fidelity PCR Master Mix with GC Buffer, NEB, #M0532s).

[0074] The PCR products of the variable regions of the heavy and light chains of the antibodies were digested with enzymes (VH using AgeI / SalI, VK using AgeI / Xhol, and VL using AgeI / BsiWI) and cloned into antibody heavy and light chain expression vectors containing the human IgG1 constant region. The monoclonal antibody sequence information obtained by IMGT / V-Quest analysis of the gene sequences of the variable regions of the heavy and light chains is shown in Tables 1 and 2 below. V genes (Variable genes) refer to genes encoding the variable regions of the variable regions, J genes (Joining genes) refer to genes encoding the joining regions of the variable regions, and D genes (Diversity genes) refer to genes encoding the diversity regions of the variable regions.

[0075] Table 1. Sequence characteristics of monoclonal antibody CYFN1006-1

[0076] Table 2 Sequence information of monoclonal antibody CYFN1006-1

[0077] Example 4: Characterization of Antibodies

[0078] 1) Antibody production:

[0079] The heavy and light chain vectors of the constructed antibody with the defined sequence were co-transfected into 293F cells, and the culture supernatant containing the antibody was harvested after 5-7 days.

[0080] 2) Antibody purification:

[0081] The culture supernatant containing antibodies was purified using a protein A affinity column, and the IgG antibody protein content was determined.

[0082] 3) The binding reaction between antibody and S protein:

[0083] The S protein was captured using anti-tag antibodies, and a capture ELISA method was established to analyze the binding of the antibody to the S protein.

[0084] The procedure is as follows: A 96-well ELISA plate is coated with D7-tagged antibody to capture the S, S1, S2, NTD, or RBD antigens of SARS-CoV-2 or SARS-CoV with the D7 tag. The plate is then reacted with antibody samples of different serial dilution concentrations to detect the antibody signal that specifically binds to each antigen in the ELISA plate.

[0085] Biological membrane optical interferometry (BLI) was used to analyze the binding and dissociation of antibodies with the receptor binding domain (RBD). Specifically, an SA (streptavidin) sensor was used to sequentially capture antibodies and RBDs to detect the affinity of antibodies for SARS-CoV-2 on the RBD.

[0086] Experimental results:

[0087] Figure 2 shows the ELISA binding curves of monoclonal antibodies CYFN1006-1 and SA55 to the viral S protein and the truncated S subunit protein. The results show that monoclonal antibody CYFN1006-1 is an RBD antibody, binding to the S, S1, and RBD of SARS-CoV-2 with EC50 values ​​of 0.0177 μg / mL, 0.0188 μg / mL, and 0.0165 μg / mL, respectively, but not to S2 or NTD. CYFN1006-1 binds to the S and RBD of SARS-CoV with EC50 values ​​of 0.338 μg / mL and 0.0391 μg / mL, respectively. CYFN1006-1 and the control broad-spectrum neutralizing antibody SA55 have similar binding characteristics; both are cross-linked antibodies capable of simultaneously binding to the conserved RBD regions of SARS-CoV-2 and SARS-CoV, consistent with the results of B cell culture supernatant derived from CYFN1006-1 in Figure 1.

[0088] Figure 3 shows the BLI affinity analysis results of monoclonal antibodies CYFN1006-1 and SA55 with the viral RBD protein. The results show that monoclonal antibody CYFN1006-1 has a strong affinity for SARS-CoV-2 RBD, with KD(M) < 1.0 × 10⁻⁶. -12 The affinity (KD(M)) of the control broad-spectrum neutralizing antibody SA55 for SARS-CoV-2 RBD was 3.07 × 10⁻⁶. -10 It is inferior to the CYFN1006-1 antibody in this application.

[0089] Example 5: Pseudovirus Neutralization Test

[0090] Preparation of the spike protein S pseudovirus system: HEK-293T cells were transfected with an expression plasmid containing the spike protein S gene of a specific SARS-CoV-2 or SARS-CoV strain using PEI. Cells were cultured overnight at 37°C and 5% CO2 in medium containing 10% fetal bovine serum (FBS). Cells were infected with VSV-G pseudovirus ΔG variant-luciferase (G*ΔG-luciferase, Kerafast), washed three times with PBS (containing 2% FBS), and cultured in DMEM medium containing 2% FBS. After 24 h, the supernatant was collected and centrifuged at 4000 rpm for 10 min to clarify. Each viral culture was then incubated with 20% hybridoma (anti-VSV-G, CRL-2700, ATCC) supernatant at 37°C for 1 h to neutralize the contaminating VSV-G pseudotype ΔG-luciferase virus. The pseudoviruses were then titrated and stored at -80°C.

[0091] Forty-two different pseudoviruses of spike protein S were prepared using the method described above, including SARS-CoV-2 related lineages (RaTG13, GD-pangolin), SARS-CoV related lineages (WIV1, SHC014), all representative SARS-CoV-2 variants, SARS-CoV, bat or pangolin coronaviruses, MERS-CoV, and previously prevalent SARS-CoV-2 variants of concern (VOCs) B.1.1.7 and B. 1.351, P.1, B.1.617.2, BA.1; variants of interest (VOIs) B.1.525, B.1.621, C.37; previously prevalent variants BA.2, BA.2.12.1, BA.2.75, BA.4 / 5, BQ.1.1, CH.1.1; various XBB subvariants; and recently discovered and currently prevalent variants BA.2.86, BA.2.87.1, JN.1, JN.1.16, and KP.2. Expression plasmids for the spike protein S gene of all the above strains were prepared using synthetic genes (see references below).

[0092] Subsequently, a pseudovirus neutralization test was conducted, referencing the literature Wang, X. et al. Neutralization of distinct Omicron sublineages by longitudinal vaccination sera. J. Med. Virol. 94, 5090-5092 (2022). The experimental procedure is as follows:

[0093] Vero-E6 cells were planted at a density of 2 × 10⁶ cells per well. 4 Cells were seeded at a concentration of [number] cells per well in 96-well plates. The next day, 100 TCID50 of each pseudovirus was co-incubated with serially diluted concentrations of monoclonal antibodies (CYFN1006-1, SA55, S309, S3H3, or LY-CoV1404) at 37°C for 30 min, with three replicates for each concentration. The mixture was then added to the cultured cells and incubated for another 24 h. Subsequently, luminescence was measured using a luciferase assay system (RG062M, Beyotime). Control antibodies SA55, S309, S3H3, and LY-CoV1404 were prepared in the laboratory using a synthetic antibody gene.

[0094] IC50 (the antibody concentration required to neutralize 50% of the virus) is defined as the antibody concentration that, after subtracting the background of the control wells containing only cells, represents a 50% reduction in relative luminescent units compared to the virus control wells (virus and cells). IC50 values ​​were calculated using GraphPad Prism nonlinear regression.

[0095] The results are shown in Figures 4 and 5. LY-CoV1404 exhibits strong neutralizing activity against most variants, with an IC50 of approximately 1 ng / mL, but completely loses activity against BQ, CH, XBB, and JN.1 and KP.2 variant progeny. Antibody S3H3 shows moderate activity, with an IC50 of 20–50 ng / mL, possibly due to the E554K mutation in its antibody epitope. S3H3 completely inactivates variants such as BA.2.86 and JN.1. Broad-spectrum neutralizing antibody S309 shows moderate activity, with an IC50 of approximately 20 ng / mL. Although it retains activity against most Omicron progeny variants, the IC50 decreases to 100–1000 ng / mL, and it completely loses activity against BN.1 and KP.2 variants, possibly due to the combined effect of the R346T and K356T mutations. In contrast, the CYFN1006-1 antibody consistently exhibited strong neutralizing activity against all tested SARS-CoV-2 variants, with unaffected efficacy and IC50 values ​​of approximately 1–5 ng / mL, comparable to or even superior to SA55 in most cases. CYFN1006-1 also efficiently neutralized SARS-CoV-2-related animal coronaviruses (RaTG13, GD-Pangolin), and also showed neutralizing efficacy against SARS-CoV and SARS-CoV-related lineages (WIV1, SHC014), but none of the aforementioned monoclonal antibodies neutralized MERS-CoV. Therefore, the CYFN1006-1 antibody demonstrated strong neutralizing activity against all currently circulating clinical SARS-CoV-2 mutant strains, including the latest JN.1 and KP.2 mutants, with neutralizing IC50 values ​​ranging from 1 to 5 ng / mL. It also showed cross-neutralizing activity against SARS-CoV and related sarbecovirus strains such as WIV1, SHC014, RaTG13, and GD-pangolin.

[0096] In summary, the CYFN1006-1 antibody of the present invention exhibits strong neutralizing activity against all currently circulating clinical SARS-CoV mutant strains, including the latest JN.1 and KP.2 mutant strains, and also shows cross-neutralizing activity against SARS-CoV and related sarbecovirus strains (such as WIV1, SHC014, RaTG13, and GD-pangolin).

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antibody that broadly neutralizes coronaviruses, characterized in that, The antibody comprises a heavy chain variable region VH and a light chain variable region VL; The VH comprises a CDRH1 of an amino acid sequence as shown in SEQ ID NO. 1, a CDRH2 of an amino acid sequence as shown in SEQ ID NO. 2, and a CDRH3 of an amino acid sequence as shown in SEQ ID NO. 3; and the VL comprises a CDRL1 of an amino acid sequence as shown in SEQ ID NO. 4, a CDRL2 of an amino acid sequence as shown in SEQ ID NO. 5, and a CDRL3 of an amino acid sequence as shown in SEQ ID NO.

6. The VH comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 7, and the VL comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO.

8.

2. The antibody of claim 1, wherein The VH comprises an amino acid sequence as shown in SEQ ID NO. 7, and the VL comprises an amino acid sequence as shown in SEQ ID NO.

8.

3. The antibody of claim 2, wherein The antibody is a fully human antibody, a humanized antibody, or a chimeric antibody.

4. The antibody according to any one of claims 1 to 3, characterized in that, Preferably, the antibody belongs to any one of the following: (i) an isotype of IgG, IgA, IgM, IgE, or IgD; (ii) a subtype of IgG1, IgG2, IgG3, or IgG4; More preferably, the antibody is a monoclonal antibody. The nucleic acid comprises a nucleotide sequence encoding the antibody of any one of claims 1-3.

5. A nucleic acid, characterized in that, The vector comprises the nucleic acid of claim 5.

6. A vector, characterized in that, The host cell comprises the vector of claim 6.

7. A host cell, characterized in that, The preparation further comprises other therapeutic agents.

8. An agent for detecting, preventing, or treating a coronavirus infection, characterized by, Preferably, the other therapeutic agents comprise one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, other antibodies, and vaccines.

9. The formulation of claim 8, wherein, The application is any one of the following: (1) for preparing a diagnostic preparation for coronavirus and its variants; 10. Use of an antibody according to any one of claims 1 to 3, characterized in that, (2) for preparing a preparation for preventing infection of coronavirus and its variants; (3) for preparing a preparation for treating infection of coronavirus and its variants; (4) for preparing a preparation for treating sequelae or long COVID after infection of coronavirus and its variants. ​ ​

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