CN118530348A - A broad-spectrum coronavirus neutralizing antibody and its application - Google Patents

Abstract

The present invention discloses an antibody for neutralizing coronaviruses with a broad spectrum and its application. The antibody CYFN1006-1 comprises a heavy chain variable region VH and a light chain variable region VL; wherein the VH comprises CDRH1-3 with amino acid sequences as shown in SEQ ID NO.1-3; and the VL comprises CDRL1-3 with amino acid sequences as shown in SEQ ID NO.4-6. CYFN1006-1 is currently a very broad-spectrum and highly effective coronavirus neutralizing antibody, which can effectively neutralize all clinically prevalent mutant strains of SARS-CoV-2 so far, and has a moderate effect on SARS-CoV, providing a new option for solving the problems of virus escape and drug resistance faced by the application of monoclonal antibody passive immunization in novel coronavirus infection.

Description

A broad-spectrum coronavirus neutralizing antibody and its application

Technical Field

The present invention relates to the field of medical virus immunology, and in particular to an antibody capable of neutralizing a broad spectrum of coronaviruses and an application thereof.

Background Art

Coronavirus is a type of enveloped RNA virus belonging to the genus Coronavirus in the family Coronaviridae. It is named because of the crown-shaped spike protein on its surface. Coronavirus infection occurs not only in humans, but also in a variety of animals. According to the classification of the International Committee on Taxonomy of Viruses (ICTV), coronaviruses are divided into the following four genera: alpha-coronavirus, beta-coronavirus, gamma-coronavirus, and delta-coronavirus.

SARS-CoV (severe acute respiratory syndrome coronavirus) and SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2, also known as the new coronavirus) are members of the beta-coronavirus family. Both viruses can cause severe respiratory diseases. The pandemic of the new coronavirus has caused large-scale infections and caused a large number of morbidity and mortality worldwide. The continued spread of the new coronavirus poses serious challenges to the economy, social life and health care systems. The clinical symptoms of new coronavirus infection range from asymptomatic or mild respiratory diseases to severe pneumonia, acute respiratory distress syndrome, multiple organ failure and death. Certain populations, such as the elderly and those with underlying health problems, are at higher risk of serious consequences. At the same time, the long-term sequelae of infection (long new crown) are also increasing. More effective prevention and treatment methods are urgently needed. SARS-CoV-2 and SARS-CoV share about 79.6% genetic sequence homology. The virus mainly encodes four key proteins, among which the spike glycoprotein (S) is a key protein on the surface of the virus particle, which is essential for the infectivity of the virus. Passive immunotherapy with neutralizing antibodies targeting the S protein of the new coronavirus is an important option for prevention and treatment, especially for individuals who are immunocompromised, cannot be vaccinated, or have other risk factors for severe illness. During viral infection, antibodies exert their therapeutic effects through multiple 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 pathogens containing viral proteins. Therefore, several early-stage neutralizing antibodies and antibody combination cocktail therapies have obtained FDA emergency use authorization (EUA) and have shown good therapeutic and preventive effects in clinical practice.

As the novel coronavirus continues to evolve, many popular mutant strains carrying multiple mutations have emerged, seriously escaping the marketed neutralizing antibody drugs. All neutralizing antibodies and antibody combination cocktail therapies against the novel coronavirus that have previously been authorized for emergency use by the FDA have been withdrawn due to the prevalence of immune escape mutant strains. Antibodies and combinations developed in the early stages of the novel coronavirus, such as Eli Lilly's Bamlanivimab/Etesevimab antibody combination, Regeneron's Casirivimab/imdevimab (REGN-COV2) antibody combination, and AstraZeneca's Tixagevimab/Cilgavimab (Evusheld) antibody combination, have lost their neutralizing activity against popular novel coronavirus mutant strains. Although Bebtelovimab (LY-CoV1404) can effectively neutralize many popular novel coronavirus mutant strains, including the early Omicron subvariant, it cannot neutralize the subsequent novel coronavirus mutant strains BQ.1.1 and XBB lineages. Another mAb Sotrovimab (S309) isolated from individuals recovering from SARS-CoV infection retains neutralizing activity against most emerging SARS-CoV variants due to recognition of conserved epitopes. However, its neutralizing activity is general, with a 50% neutralizing activity (IC50) value of approximately 1 μg/mL for many SARS-CoV variants, which is not enough to be effective.

In addition, some neutralizing antibodies in the field that have certain cross-neutralizing activity against the new coronavirus and its mutant strains, such as SA55, S3H3 and S309, were isolated from individuals who have received multiple vaccinations, experienced multiple infections, or both vaccination and infection. It is not clear whether a single infection with the prototype strain of the new coronavirus can also produce broad-spectrum neutralizing antibodies that are highly effective against the new coronavirus mutant viruses that the donor has not previously been exposed to.

Screening for broad-spectrum and highly effective neutralizing antibodies targeting conserved epitopes of the S protein is a strategy to deal with the emergence of new variants that are constantly escaping. Currently, human monoclonal antibodies are mainly obtained by several strategies: (1) Immunization of transgenic mice. Since these transgenic mice have been transferred with human antibody heavy chain genes or antibody heavy chain and light chain genes, they can produce antibodies derived from human antibody sequences, i.e., human antibodies, after antigen immunization. However, this method requires the preparation of antigens for immunization, and the antibodies are produced in the immune selection environment of mice, which cannot completely mimic the production of antibodies in the human body's own immune selection environment. (2) Human antibody library technology. This technology is used to screen single-chain antibody variable region fragments (scFv) libraries from immune or non-immune human antibody genes. Since it is a non-natural antibody gene pairing and the antibodies have not undergone in vivo immune selection and affinity maturation processes, it is difficult to obtain high-affinity antibodies without safety concerns. (3) Directly cloning 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 adopt the strategy of sorting single antigen-specific memory B cells from human PBMCs by flow cytometry for antibody cloning. Although this method can quickly obtain antibodies, it has antigen selection bias and cannot directly perform antibody screening based on antibody functional activity. What is obtained are antibodies that can bind to recombinant antigens, and it is easy to lose conformation-dependent antibodies with important functions produced by the body.

In summary, there are urgent issues that need to be addressed, such as immune escape, efficacy and broad spectrum of coronavirus antibody drugs, as well as uncertainty in antibody responses caused by vaccines and infections.

Summary of the invention

In view of the lack of mAbs that can neutralize novel coronavirus variants with broad spectrum and high efficiency in the prior art, the present invention isolates a monoclonal antibody (named CYFN1006-1) from patients who have recovered from novel coronavirus infection, which can neutralize all novel coronavirus variants that have been prevalent so far with broad spectrum and high efficiency. The present invention discloses the CYFN1006-1 antibody and its application.

The present invention provides an antibody that broadly neutralizes coronaviruses, the antibody comprising a heavy chain variable region VH and a light chain variable region VL;

Wherein, the VH comprises CDRH1 as shown in SEQ ID NO.1, CDRH2 as shown in SEQ ID NO.2 and CDRH3 as shown in SEQ ID NO.3; the VL comprises CDRL1 as shown in SEQ ID NO.4, CDRL2 as shown in SEQ ID NO.5 and CDRL3 as shown in SEQ ID NO.6.

The "light chain variable region" (VL) or "heavy chain variable region" (VH) consists of a "framework" region separated by three "complementarity determining regions" or "CDRs". The framework region is used to align the CDRs (antigen binding determining regions) that specifically bind to antigenic epitopes. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL domain and the VH domain contain the following framework regions (FRs) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to as CDRL1, CDRL2, and CDRL3, respectively, in the present invention; the CDR1, CDR2, and CDR3 of the VH domain are also referred to as CDRH1, CDRH2, and CDRH3, respectively, in the present invention.

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.

In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO.7, and the VL comprises the amino acid sequence shown in SEQ ID NO.8.

In some embodiments, the antibody is a fully human antibody, a humanized antibody or a chimeric antibody; 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.

In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain 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 light chain 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.

The antibodies may also contain 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 acid modifications.

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

The present invention also provides a vector comprising the nucleic acid.

The present invention also provides a host cell, which comprises the vector.

The present invention also provides a preparation for detecting, preventing or treating coronavirus infection, comprising the antibody, nucleic acid, vector or host cell. In some embodiments, the preparation further comprises a pharmaceutically acceptable carrier or excipient.

In some embodiments, the formulation further comprises an additional therapeutic agent.

In some embodiments, the other therapeutic agents comprise one or more of the group consisting of hormonal agents, targeted small molecule agents, 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.

The present invention also provides a conjugate, which comprises the antibody disclosed in the present invention and a chemical moiety conjugated thereto. In some embodiments, the chemical moiety can be selected from a therapeutic agent, a detectable moiety and an immunostimulatory molecule.

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.

The dosage of the antibody, composition or conjugate of the present invention can be administered to a mammal once or in a series of sub-doses over an appropriate time period, for example, daily, semi-weekly, weekly, biweekly, semi-monthly, bimonthly, semi-annually or annually as needed. A dosage unit comprising an effective amount of an antibody, composition or conjugate can be administered in a single daily dose, or the total daily dose can be administered in two, three, four or more divided doses administered daily as needed.

In some embodiments, the dosage administered to a subject may vary with the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician can determine the effective amount to be administered 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 the activity of the antibody and the route of administration.

The present invention also provides an application of the antibody, which is any one of the following:

(1) Used in the preparation of diagnostic preparations for coronavirus and its variants;

(2) Preparations for preventing infection with coronavirus and its variants;

(3) Preparations for treating coronavirus and its variants;

(4) Used to prepare preparations for treating sequelae of infection with coronavirus and its variants or COVID-19.

In some embodiments, the coronavirus includes but is 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.

In some embodiments, variants of SARS-CoV-2 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.

The antibodies of the present invention can be used alone or in combination with cocktail drugs in the prevention and treatment of coronaviruses, and can be used as passive immunotherapy for people at high risk of SARS-CoV-2 infection or passive immunotherapy for patients infected with the new coronavirus (including long-term COVID-19).

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

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

2. The antibodies of the present invention can target the conserved region of the RBD of the SARS-CoV-2 surface spike protein. The heavy chain and light chain pairing of the antibodies is original to naturally produced antibodies, which is safe and has high affinity, and is suitable for practical applications.

3. The CYFN1006-1 antibody of the present invention exhibits a strong neutralizing effect on all SARS-CoV-2 mutants, and the efficacy is not affected. The IC50 value is 1-5 ng/mL, and it exhibits moderate efficacy in neutralizing SARS-CoV. It is a broad-spectrum and highly efficient neutralizing antibody, and its broad-spectrum is comparable to or even better than that of SA55, and its neutralizing activity against most mutants is stronger than that of SA55.

4. The natural human antibody CYFN1006-1 of the present invention is a very broad-spectrum and highly effective coronavirus neutralizing antibody so far. It can effectively neutralize all clinically prevalent mutant strains of SARS-CoV-2 so far, and provides a new option for solving the problems of virus escape and drug resistance faced by the application of monoclonal antibody passive immunization in new coronavirus infection.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

FIG1 shows the antigen binding properties of the CYFN1006-1 derived B cell culture supernatant of Example 2 of the present invention;

FIG2 is a graph showing the antigen binding curve results of CYFN1006-1 and SA55 antibodies of Example 4 of the present invention;

FIG3 is an affinity analysis of CYFN1006-1 and SA55 antibodies binding to SARS-CoV-2 RBD according to Example 4 of the present invention;

FIG4 is a graph showing the neutralization curves of CYFN1006-1 and control antibodies of Example 5 of the present invention against SARS-CoV-2 variants and other related sarbecovirus pseudoviruses;

FIG5 shows the neutralization IC50 values of CYFN1006-1 of Example 5 of the present invention and control antibodies against SARS-CoV-2 variants and other related sarbecovirus pseudoviruses, in nanograms per milliliter (ng/mL).

DETAILED DESCRIPTION

In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

The present invention uses an unbiased human antibody screening method from B cells of recovered patients who have been infected with a single novel coronavirus prototype strain, through unbiased in vitro memory B cell culture, screening of SARS-CoV-2 and SARS-CoV cross-binding antibodies in the culture supernatant, gene cloning of cross-binding antibodies and antibody characteristic analysis, and successfully obtains a new human monoclonal antibody CYFN1006-1 that is broad-spectrum and highly effective in neutralizing the novel coronavirus.

The CYFN1006-1 antibody can bind with high affinity to the shared conserved sites of the RBD of the S protein of the new coronavirus and the SARS virus. It is formed by the antibody heavy chain variable region VH and the light chain variable region VL. CYFN1006-1 has shown a strong neutralizing effect on all clinical new coronavirus mutant virus strains so far, including the latest JN.1 and KP.2 mutant strains, and its neutralizing activity IC50 value remains at 1-5ng/mL. It also has cross-neutralizing activity against SARS-CoV and related sarbecovirus strains (such as WIV1, SHC014, RaTG13 and GD-pangolin). The broad-spectrum and highly efficient neutralizing activity of the CYFN1006-1 antibody can bring a new generation of monoclonal antibodies and antibody combination specific drugs for the diagnosis, prevention and treatment of new coronavirus infection, including the treatment of long new crown.

The present invention also found that people who have been infected with the prototype strain of the new coronavirus once can also produce broad-spectrum and highly effective neutralizing antibodies against mutant coronavirus strains that they have never been exposed to before, expanding the source of the invention of new broad-spectrum and highly effective neutralizing antibodies.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

Instruments and reagents:

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

Fluorescently labeled antibodies: including IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, IgM-PB, and CD45-KO fluorescent antibodies, purchased from Beckman Coulter;

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

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

Source of each coronavirus pseudovirus strain: 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)).

Example 1 Identification and sorting of memory B cells

1) Isolation of peripheral blood mononuclear cells (PBMCs):

EDTA-anticoagulated blood was collected from peripheral veins of patients infected with the novel coronavirus during the recovery period, and peripheral blood mononuclear cells were isolated by density gradient centrifugation.

2) Fluorescently labeled antibody staining:

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

Specific steps: Peripheral blood mononuclear cells were washed 3 times with PBS buffer, then antibodies were added for staining, incubated at room temperature in the dark for 15 minutes, washed with PBS buffer, and then 400 μL PBS buffer (phosphate buffer) was added to suspend the cells and placed on a flow cytometer. Fluorescently labeled antibody staining: 9 analysis tubes were set up, 1 to 7 tubes were added with the corresponding single fluorescently labeled antibody or 7-AAD, the 9th tube was added with 7 mixed fluorescently labeled antibodies, the 8th tube was a blank tube with only cells, and the sample tube was stained in the same way as the 9th tube.

3) Isolation of memory B cells:

7-AAD is used to distinguish between living cells and dead cells, CD45 is a marker for white blood cells, CD38 is usually used as one of the markers for B cell subsets, and CD38 ^low is used to mark more mature B cell subsets that have undergone a certain degree of differentiation.

The sample tubes were analyzed on the machine, and live CD45-positive white blood cells were circled based on 7-AAD and CD45. B cells were circled based on CD19. In the CD19-positive B cell population, IgM and IgD double-negative cells were circled, and the IgD ^- IgM ^- CD27 ⁺ CD38 ^low population was defined as memory B cells. Memory B cells were sorted into a 96-well cell culture plate containing cell culture medium, 50 cells/well, and cultured for 10 days (refer to PCT/CN2021/135785).

Example 2 Memory B cell culture and screening of culture supernatant antibodies

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

The capture ELISA method was used to screen the presence of antibodies against the S protein of SARS-CoV-2 (NC_045512.2) and SARS-CoV (NC_004718.3) in the B cell culture supernatant, that is, the culture supernatant can bind to SARS-CoV-2 and SARS-CoV S protein antigens at the same time to screen positive B cells.

The operation steps are as follows: the 96-well ELISA plate is coated with the D7-tagged antibody, the S, S1 or RBD antigen of SARS-CoV-2 or SARS-CoV with the D7 tag is captured, and the antigen is reacted with the B cell culture supernatant sample, and the specific binding antibody signal of each well in the ELISA plate is detected to identify and screen the positive B cell wells.

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

Example 3 Cloning of Antibodies

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

2) Nested PCR amplification of heavy and light chain variable region genes of antibodies: PCR primer sequence reference: Tiller, T., Meffre, E., Yurasov, S., Tsuiji, M., Nussenzweig, M.C. & 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 uses Phusion high-fidelity DNA polymerase (Phusion High-Fidelity PCR Master Mix with GC Buffer, NEB, #M0532s).

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

Table 1 Sequence characteristics of monoclonal antibody CYFN1006-1

Table 2 Sequence information of monoclonal antibody CYFN1006-1

Example 4 Analysis of Antibody Characteristics

1) Antibody production:

The heavy chain and light chain vectors of the constructed antibody with determined sequences were co-transfected into 293F cells, and the culture supernatant containing the antibody was harvested after 5 to 7 days.

2) Antibody purification:

The culture supernatant containing antibodies was purified by protein A affinity column, and the IgG antibody protein content was determined.

3) Binding reaction between antibodies and S protein:

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

The operation steps are as follows: the 96-well ELISA plate is coated with the D7-tagged antibody to capture the S, S1, S2, NTD or RBD antigens of SARS-CoV-2 or SARS-CoV with the D7 tag, and react with antibody samples of different serial dilution concentrations to detect the antibody signals specifically binding to each antigen in the ELISA plate.

Biomembrane interferometry (BLI) was used to analyze the binding and dissociation of antibodies and RBD (receptor binding domain). Specifically, SA (streptavidin) sensor was used to capture antibodies and RBD in turn to detect the affinity of antibodies and SARS-CoV-2 to RBD.

Experimental results:

Figure 2 shows the ELISA binding curves of monoclonal antibodies CYFN1006-1 and SA55 with viral S protein and truncated S subunit protein. The experimental results show that monoclonal antibody CYFN1006-1 is an RBD antibody, binding to S, S1 and RBD of SARS-CoV-2, with EC50 of 0.0177μg/mL, 0.0188μg/mL and 0.0165μg/mL, respectively, and does not bind to S2 and NTD. CYFN1006-1 binds to S and RBD of SARS-CoV, with EC50 of 0.338μg/mL and 0.0391μg/mL, respectively. CYFN1006-1 and the control broad-spectrum neutralizing antibody SA55 have similar binding properties, and both are cross-antibodies that can simultaneously bind to the conserved regions of RBD of SARS-CoV-2 and SARS-CoV, which is consistent with the results of the CYFN1006-1-derived B cell culture supernatant in Figure 1.

Figure 3 shows the BLI affinity analysis results of monoclonal antibodies CYFN1006-1 and SA55 with viral RBD protein. The results show that monoclonal antibody CYFN1006-1 has a strong affinity with SARS-CoV-2 RBD, KD(M)<1.0×10 ^-12 . The control broad-spectrum neutralizing antibody SA55 has an affinity with SARS-CoV-2 RBD KD(M)=3.07×10 ^-10 , which is lower than the CYFN1006-1 antibody of the present application.

Example 5 Pseudovirus Neutralization Test

Preparation of spike protein S pseudovirus system: HEK-293T cells were transfected with expression plasmids containing the spike protein S gene of a specific SARS-CoV-2 or SARS-CoV strain using PEI. The cells were cultured overnight in a medium containing 10% fetal bovine serum (FBS) at 37°C and 5% _CO2 . The cells were infected with the VSV-G pseudotype virus ΔG variant-luciferase marker (G*ΔG-luciferase, Kerafast), and then washed three times with PBS (containing 2% FBS) and cultured in DMEM medium containing 2% fetal bovine serum. After 24 hours, the supernatant was collected and clarified by centrifugation at 4000rpm for 10 minutes. Each virus solution was then reacted with 20% hybridoma (anti-VSV-G, CRL-2700, ATCC) supernatant at 37°C for 1 hour to neutralize the contaminated VSV-G pseudotype ΔG-luciferase virus, and then the pseudovirus was titrated and stored at -80°C.

According to the above method, 42 different strains of spike protein S pseudoviruses were prepared, 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 coronavirus, MERS-CoV, and the once popular SARS-CoV-2 variants of concern (VOCs) B.1.1.7, B. 1.351, P.1, B.1.617.2, BA.1, variants of interest (VOIs) B.1.525, B.1.621, C.37, once popular 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 popular variants BA.2.86, BA.2.87.1, JN.1, JN.1.16 and KP.2. The expression plasmids of the spike protein S gene of all the above strains are synthetic genes (see the references below).

Subsequently, a pseudovirus neutralization test was performed with reference to Wang, X. et al. Neutralization of distinct Omicron sublineages by longitudinal vaccination sera. J. Med. Virol. 94, 5090-5092 (2022). The test steps are as follows:

Vero-E6 cells were seeded in 96-well plates at a concentration of 2×10 ⁴ cells per well. The next day, 100TCID50 of each pseudovirus was 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). The control antibodies SA55, S309, S3H3, and LY-CoV1404 were all prepared in the laboratory by synthesizing antibody genes.

IC50 (antibody concentration required to neutralize 50% of the virus) is defined as the antibody concentration that reduces the relative luminescence units by 50% compared to virus control wells (virus and cells) after subtracting the background in control wells containing cells only. IC50 values were calculated using GraphPad Prism nonlinear regression.

The results are shown in Figures 4 and 5. LY-CoV1404 has strong neutralizing activity against most variants, with an IC50 of about 1 ng/mL, but completely loses its activity against BQ, CH, XBB, and JN.1 and KP.2 variants. Antibody S3H3 has average activity, with an IC50 of 20 to 50 ng/mL. It may be due to the presence of the E554K mutation in its antibody epitope, and S3H3 is completely inactivated against variants such as BA.2.86 and JN.1. The broad-spectrum neutralizing antibody S309 has average activity, with an IC50 of about 20 ng/mL. Although it remains active against most post-omicron variants, the IC50 is reduced to 100 to 1000 ng/mL, and it completely loses its activity against BN.1 and KP.2 variants, probably due to the combined effects of the R346T and K356T mutations. In contrast, the CYFN1006-1 antibody showed consistent strong neutralization against all tested SARS-CoV-2 variants, with unaffected potency, and IC50 values of approximately 1 to 5 ng/mL, which is comparable to or even superior to SA55. CYFN1006-1 can also effectively neutralize SARS-CoV-2-related animal coronaviruses (RaTG13, GD-Pangolin), and also has neutralizing potency against SARS-CoV and SARS-CoV-related lineages (WIV1, SHC014), but none of the above monoclonal antibodies can neutralize MERS-CoV. Therefore, the CYFN1006-1 antibody has shown strong neutralization against all clinical novel coronavirus mutant virus strains so far, including the latest JN.1 and KP.2 mutant strains, with neutralization activity IC50 values in the range of 1 to 5 ng/mL, and also has cross-neutralizing activity against SARS-CoV and related sarbecovirus strains such as WIV1, SHC014, RaTG13, and GD-pangolin.

In summary, the CYFN1006-1 antibody of the present invention exhibits a strong neutralizing effect on all clinical novel coronavirus mutant virus strains so far, including the latest JN.1 and KP.2 mutant strains, and also has cross-neutralizing activity against SARS-CoV and related sarbecovirus strains (such as WIV1, SHC014, RaTG13 and GD-pangolin).

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 principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. An antibody for broad spectrum neutralization of coronaviruses, said antibody comprising a heavy chain variable region VH and a light chain variable region VL;

Wherein the VH comprises CDRH1 with an amino acid sequence shown as SEQ ID NO.1, CDRH2 with an amino acid sequence shown as SEQ ID NO.2 and CDRH3 with an amino acid sequence shown as SEQ ID NO. 3;

The VL comprises CDRL1 with an amino acid sequence shown as SEQ ID NO.4, CDRL2 with an amino acid sequence shown as SEQ ID NO.5 and CDRL3 with an amino acid sequence shown as SEQ ID NO. 6.

2. The antibody of claim 1, wherein 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.

3. The antibody of claim 2, wherein the VH comprises an amino acid sequence set forth in SEQ ID No.7 and the VL comprises an amino acid sequence set forth in SEQ ID No. 8.

4. The antibody according to any one of claims 1 to 3, wherein the antibody is a fully human, humanized or chimeric antibody;

Preferably, the antibody belongs to any one of the following:

(i) IgG, igA, igM, igE or an isotype of IgD;

(ii) A subtype of IgG1, igG2, igG3 or IgG 4;

more preferably, the antibody is a monoclonal antibody.

5. A nucleic acid comprising a nucleotide sequence encoding the antibody of any one of claims 1-3.

6. A vector comprising the nucleic acid of claim 5.

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

8. A formulation for detecting, preventing or treating a coronavirus infection comprising the antibody of any one of claims 1 to 3, the nucleic acid of claim 5, the vector of claim 6 or the host cell of claim 7.

9. The formulation of claim 8, further comprising an additional therapeutic agent;

Preferably, the additional therapeutic agent comprises one or more of the group consisting of hormonal agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, additional antibodies and vaccines.

10. Use of an antibody according to any one of claims 1 to 3, wherein the use is any one of the following:

(1) Is used for preparing diagnostic preparations of coronaviruses and variants thereof;

(2) For preparing a preparation for preventing coronavirus and its variant infection;

(3) For preparing a formulation for treating coronavirus and variant infection thereof;

(4) Is used for preparing the preparation for treating the infection sequelae or the long new crown of the coronavirus and the variant strain thereof.