A broad-spectrum monoclonal antibody against novel coronavirus RBD, preparation method and application thereof

Abstract

The present invention relates to a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 RBD domain, and related products, preparation methods and uses thereof. The monoclonal antibody or the antigen-binding fragment thereof of the present invention can bind to the SARS-CoV-2 RBD antigen with high affinity, and can neutralize the SARS-CoV-2 prototype strain and a series of variant strains thereof with high neutralizing activity, thereby inhibiting their infection. Therefore, it has great potential application value in the clinical treatment, prevention and / or detection of infection by the SARS-CoV-2 prototype strain and its variant strains.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 RBD domain, a preparation method thereof, related products, and use thereof in the preparation of drugs for preventing, treating and / or detecting novel coronavirus infection. Background Art

[0002] The new coronavirus infection (COVID-19) is currently the largest public health event in the world.

[0003] Since there is no effective specific medicine at present, the main way to prevent and treat the new coronavirus infection is vaccination. When facing a virus attack, memory T cells and memory B cells provide the main protection, and the more important one is neutralizing antibodies. For people who have been infected, the FDA has approved the use of the antiviral drug Veklury (remdesivir) or the immunomodulator Olumiant (baricitinib) for treatment or antibody treatment.

[0004] At the beginning of the outbreak of the new coronavirus, the FDA approved the use of high-titer anti-SARS-CoV-2 convalescent serum for treatment. As the research continued, the experimenters continued to isolate effective neutralizing monoclonal antibodies. In addition to using them alone, two antibodies can also be used in combination to form antibody cocktail therapy. Currently, FDA-approved antibody cocktail therapies include REGEN-COV (Casirivimab and Imdevimab), LY-CoV016+LY-CoV555 (Bamlanivilab+Etesevimab), Evusheld (tixagevimab+cilgavimab) and monoclonal antibodies S309 (Sotrovimab), LY-CoV1404 (Bebtelovimab); these antibodies all target the receptor binding region (RBD) of the S protein on the surface of the SARS-CoV-2 virus, which is the region that directly binds to the receptor and is also one of the important targets of neutralizing antibodies.

[0005] As the virus continues to mutate, the existing therapeutic antibodies have shown a reduced ability to neutralize new mutants and epidemic strains, and their ability to prevent and treat has deteriorated. According to Xie Xiaoliang's research, the variants of Omicron can escape most of the neutralizing antibodies currently in emergency use on the market, such as REGEN-COV, LY-CoV016+LY-CoV555; although there are still antibodies such as S309 and LY-CoV1404 that can neutralize, most of them have also shown a significant reduction in neutralizing ability. Therefore, the development of new therapeutic antibodies for the new coronavirus is imminent. Summary of the invention

[0006] Purpose of the Invention

[0007] The object of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 RBD domain, a preparation method thereof, related products, and their use in the preparation of drugs for preventing, treating and / or detecting novel coronavirus infection.

[0008] Solution

[0009] Zhifei (ZF2001) is the first recombinant protein vaccine designed based on the SARS-CoV-2 RBD region in China, with good immunogenicity and safety. In addition to the SARS-CoV-2 prototype strain, ZF2001 can also provide good protection against other variants. China approved its emergency use in 2020, and the vaccine has completed phase III clinical trials in four countries.

[0010] According to the current FDA-approved high- and medium-level neutralizing therapeutic antibodies that all target RBD, isolating high- and medium-level neutralizing antibodies from volunteers immunized with the SARS-CoV-2 RBD regional protein vaccine is undoubtedly a faster way to develop new therapeutic antibodies. Therefore, the inventors of this application attempted to isolate antibodies that can broadly neutralize the most noteworthy mutant strains (VOI strains) from volunteers immunized with ZF2001. Through antibody functional screening, the inventors obtained multiple strains of high-neutralizing, high-affinity antibodies, which can be used as therapeutic antibodies or immunodetection reagents for the prototype strain of the new coronavirus and its series of variants.

[0011] Specifically, the present invention provides the following technical solutions:

[0012] In a first aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 RBD domain, which comprises a heavy chain variable region and a light chain variable region, wherein:

[0013] The heavy chain variable region comprises:

[0014] HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively;

[0015] The light chain variable region comprises:

[0016] The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.

[0017] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0018] The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:7;

[0019] The light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0020] In a further preferred embodiment, the antibody or antigen-binding fragment thereof comprises:

[0021] A heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO:7; and,

[0022] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0023] In the above-mentioned embodiments of the monoclonal antibody or antigen-binding fragment thereof of the present invention, the antibody or antigen-binding fragment thereof further comprises a constant region;

[0024] Preferably, the constant region is any one selected from the following: the constant region of IgG, IgA or IgM antibody;

[0025] More preferably, the constant region is the constant region of an IgG antibody.

[0026] In a further preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0027] a heavy chain comprising, or consisting of, the amino acid sequence of SEQ ID NO:9, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:9; and,

[0028] A light chain comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10.

[0029] As an example of a further preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0030] A heavy chain, the amino acid sequence of which is shown in SEQ ID NO:9; and

[0031] The light chain has an amino acid sequence as shown in SEQ ID NO:10.

[0032] For all the above embodiments, preferably, the antigen binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, Fd, dAb, a complementarity determining region fragment, a human antibody, a chimeric antibody or a bispecific or multispecific antibody.

[0033] In a second aspect, the present invention provides a polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above.

[0034] In a preferred embodiment, the polynucleotide is a polynucleotide set, the polynucleotide set comprising:

[0035] 1) a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 11, 12, 13 or a corresponding mRNA molecule thereof; and,

[0036] 2) A DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 14, 15, 16 or its corresponding mRNA molecule.

[0037] In a further preferred embodiment, the polynucleotide set comprises:

[0038] I) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO: 17 or its corresponding mRNA molecule; and, II) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO: 18 or its corresponding mRNA molecule.

[0039] In a further preferred embodiment, the polynucleotide set comprises:

[0040] (I) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 19 or its corresponding mRNA molecule; and,

[0041] (II) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 20 or its corresponding mRNA molecule.

[0042] In a third aspect, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above; optionally, it further comprises at least one expression control element operably linked to the polynucleotide, for example, a histidine tag, a stop codon, etc.

[0043] In a fourth aspect, the present invention provides an expression vector comprising the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above.

[0044] Preferably, the expression vector is a eukaryotic expression vector.

[0045] In a fifth aspect, the present invention provides a host cell comprising the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the expression vector as described in the fourth aspect.

[0046] Preferably, the host cell is a eukaryotic cell;

[0047] More preferably, the host cell is a mammalian cell.

[0048] In a sixth aspect, the present invention provides a method for preparing the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, the method comprising: allowing the host cell as described in the fifth aspect above to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for the expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from the culture of the host cell.

[0049] In the seventh aspect, the present invention provides a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the expression vector as described in the fourth aspect or the host cell as described in the fifth aspect, and a pharmaceutically acceptable carrier.

[0050] Preferably, the pharmaceutical composition is in the form of a nasal spray, an oral formulation, a suppository or a parenteral formulation;

[0051] Further preferably, the nasal spray is selected from aerosol, spray and powder spray;

[0052] Further preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments;

[0053] Further preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid or an injectable preparation.

[0054] The dosage of the active ingredient of the pharmaceutical composition of the present invention varies depending on the subject of administration, the subject organ, symptoms, the method of administration, etc., and can be determined based on the doctor's judgment in consideration of the type of dosage form, the method of administration, the age and weight of the patient, the patient's symptoms, etc.

[0055] In an eighth aspect, the present invention provides a kit comprising the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect and / or the pharmaceutical composition as described in the seventh aspect.

[0056] In some embodiments, the kit is a SARS-CoV-2 antigen detection kit, comprising the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above.

[0057] In the ninth aspect, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect, the pharmaceutical composition as described in the seventh aspect and / or the kit as described in the eighth aspect in the preparation of drugs for preventing, treating and / or detecting novel coronavirus infection.

[0058] Preferably, the novel coronavirus is a SARS-CoV-2 prototype strain and / or a SARS-CoV-2 variant strain.

[0059] Further preferably, the SARS-CoV-2 variant strain is selected from: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2) variants of SARS-CoV-2 and Omicron (B.1.1.529) and its sub-variants.

[0060] In the tenth aspect, the present invention provides a method for preventing and / or treating novel coronavirus infection, comprising: administering to a subject in need thereof a preventive and / or therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the host cell as described in the fifth aspect above, the pharmaceutical composition as described in the seventh aspect above, and / or the kit as described in the eighth aspect above.

[0061] Preferably, the novel coronavirus is a SARS-CoV-2 prototype strain and / or a SARS-CoV-2 variant strain.

[0062] Further preferably, the SARS-CoV-2 variant strain is selected from: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2) variants of SARS-CoV-2 and Omicron (B.1.1.529) and its sub-variants.

[0063] In a possible embodiment, the subject is a mammal, such as a human.

[0064] The monoclonal antibodies or antigen-binding fragments thereof of the invention or the pharmaceutical compositions of the invention can be administered to a subject by any appropriate route of administration, including, but not limited to, oral, buccal, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.

[0065] The "preventively and / or therapeutically effective amount" may vary depending on the subject of administration, the subject organ, symptoms, the method of administration, etc., and can be determined based on the doctor's judgment, taking into account the type of dosage form, the method of administration, the patient's age and weight, the patient's symptoms, etc.

[0066] In the eleventh aspect, the present invention provides a method for detecting novel coronavirus infection, which comprises using the monoclonal antibody or its antigen-binding fragment as described in the first aspect above.

[0067] Preferably, the novel coronavirus is a SARS-CoV-2 prototype strain and / or a SARS-CoV-2 variant strain.

[0068] Further preferably, the SARS-CoV-2 variant strain is selected from: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2) variants of SARS-CoV-2 and Omicron (B.1.1.529) and its sub-variants.

[0069] definition

[0070] Unless specifically defined otherwise herein, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0071] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0072] The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the amino acid sequences (and introducing gaps, if necessary) to obtain the maximum percentage sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine percentage amino acid sequence identity.

[0073] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analog, which generally includes at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Specifically, the antigen-binding fragment can be selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabodies, etc.

[0074] The "Fab" fragment consists of a light chain and the CH1 and variable regions of a heavy chain.

[0075] A "Fab'" fragment contains a light chain and a portion of a heavy chain including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0076] The "F(ab')2" fragment contains two light chains and portions of two heavy chains including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0077] The "Fv" region contains the variable regions from both the heavy and light chains, but lacks the constant regions.

[0078] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, which domains are contained in a single polypeptide chain. Generally speaking, the scFv polypeptide comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0079] "Diabodies" are small antigen-binding fragments with two antigen-binding sites. The fragments contain 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 on the same chain, the domains are allowed to pair with the complementary domains of another chain and form two antigen-binding sites.

[0080] Beneficial Effects

[0081] The present invention develops neutralizing antibody drugs for the new coronavirus. The monoclonal antibody of the present invention is obtained by isolating high-neutralizing antibodies from volunteers immunized with the SARS-CoV-2 RBD protein vaccine ZF2001 and screening based on antibody function. The monoclonal antibody or its antigen-binding fragment of the present invention can specifically bind to the RBD domain of the SARS-CoV-2 prototype strain or its variant strain with very high affinity, and therefore has extremely high potential to be developed as an antibody used in a new coronavirus antigen detection kit, and may be able to provide a lower detection limit and improve the sensitivity of the kit. Moreover, the monoclonal antibody or its antigen-binding fragment of the present invention exhibits high neutralizing activity against both the SARS-CoV-2 prototype strain and various variant strains, and therefore has great potential to be developed into a broad-spectrum, therapeutic antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0083] Figure 1 These are the binding kinetic curves of the two monoclonal antibodies L4.65 of the present invention recorded in Example 3 of the present invention to the RBD proteins of the prototype strain of the new coronavirus, the Omicron variant BA.2 subtype, and the Omicron variant BA.4 subtype. DETAILED DESCRIPTION

[0084] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0085] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0086] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0087] Example 1: Screening of antibodies

[0088] With the informed consent signed by the volunteers, the inventors screened three broad-spectrum neutralizing monoclonal antibodies that can neutralize the prototype strain (prototype), Beta variant, Delta variant, Omicron variant and their variant strains of the new coronavirus from the plasma of volunteers injected with Zhifei's new coronavirus recombinant protein vaccine (ZF2001).

[0089] Specifically, the PBMCs of volunteers injected with Zhifei's new coronavirus recombinant protein vaccine ZF2001 were negatively selected for B cells, and IgD was obtained by flow cytometry. - IgM - IgG + antigen + A single B cell (Note: “antigen + "covers RBD monomer, RBD dimer or S protein binding positive); then, based on the Novozymes platform, the B cells obtained by the above sorting were lysed, mRNA was extracted, and after reverse transcription into cDNA, expression constructs of light and heavy chains containing signal peptides, antibody heavy or light chain variable regions and IgG1 backbones were constructed through PCR amplification, bridging, etc., and the expression constructs were transfected into 293F cells in a 96-well deep-well plate using Polyplus's transfection reagent. After transfection, the antibodies were secreted and expressed in the cell supernatant. Three days later, the characteristics of the antibodies secreted in the cell supernatant were detected to help us select the target antibodies.

[0090] In the neutralizing antibody screening of cell supernatants, we diluted the cell supernatants 5-fold, and selected prototype (Novozyme DD1402), Beta (Novozyme DD1441), Delta (Novozyme DD1754), and Omicron (Novozyme DD1768) pseudoviruses for antibody neutralization experiments, and screened out a broad-spectrum neutralizing monoclonal antibody L4.65, which was named according to the volunteer number of the antibody source and its corresponding well number. The supernatant neutralization results are shown in Table 1 below:

[0091] Table 1. Neutralization efficiency of the screened monoclonal antibody strain L4.65 against pseudoviruses of various novel coronavirus strains

[0092] Antibody ID Prototype Beta Delta Omicron(BA.1) L4.65 100％ 99.9％ 99.8％ 97.5％

[0093] The values ​​shown in the table are the neutralization efficiency of the antibody strain against the corresponding new coronavirus pseudovirus, and the calculation method is as follows:

[0094] Neutralization efficiency = (1-fluorescence reading of corresponding well / fluorescence reading of negative well) * 100%;

[0095] In this example, 60% antibody neutralization efficiency was used as the positive screening standard, and the cDNA of a single B cell expressing the above broad-spectrum neutralizing monoclonal antibody L4.307 was sequenced to identify the nucleotide sequences of the heavy and light chain variable regions of the monoclonal antibody. The results are shown in Table 2 below:

[0096] Table 2. Nucleic acid coding sequences of the heavy and light chain variable regions of the screened monoclonal antibody strain L4.65

[0097]

[0098]

[0099] In the above Table 2, the underlined parts of each sequence are the nucleic acid coding sequences of CDR1, CDR2, and CDR3 of VH or VL, respectively.

[0100] According to the genetic code compilation rules, the nucleic acid coding sequences of the heavy chain and light chain variable regions of the antibody strains shown in Table 2 were compiled into corresponding amino acid sequences, as shown in the following Table 3:

[0101] Table 3. Amino acid sequences of the heavy and light chain variable regions of the monoclonal antibody strain L4.65

[0102]

[0103] In the above Table 3, the underlined parts of each sequence are the amino acid sequences of CDR1, CDR2, and CDR3 of VH or VL, respectively.

[0104] Example 2: Expression and purification of antibodies

[0105] The nucleotide sequences of the heavy and light chain variable regions of the monoclonal antibody strain L4.65 screened in Example 1 were obtained by artificial synthesis; then, according to the construction method in Example 1, the nucleotide sequences of the heavy and light chain variable regions were respectively constructed on the human IgG1 antibody backbone to obtain heavy and light chain expression constructs containing a signal peptide, a heavy or light chain variable region and an IgG1 backbone. Then, the expression constructs were transfected into 293F cells in a 96-well deep well plate using a Polyplus transfection reagent. After transfection, the antibodies were secreted and expressed in the cell supernatant. Three days later, the cell supernatant was collected and the antibodies in the cell supernatant were purified using Protein A magnetic beads (GenScript).

[0106] The heavy chain and light chain amino acid sequences of the monoclonal antibody L4.65 expressed above are shown in SEQ ID NOs: 9 and 10, respectively, and the encoding nucleic acid sequences thereof are shown in SEQ ID NOs: 19 and 20, respectively.

[0107] Example 3: Broad-spectrum neutralization effect of antibodies

[0108] In this example, the purified monoclonal antibody strain L4.65 obtained in Example 2 was tested for its neutralizing effect against the SARS-CoV-2 pseudovirus.

[0109] The preparation method of pseudoviruses of the prototype strain of the new coronavirus and its variants Beta, Delta, OmicronBA.1, OmicronBA.2, Omicron BA.2.12.1, Omicron BA.3, and Omicron BA.4 / 5 is based on Zhao X, Zheng A, Li D, Zhang R, Sun H, Wang Q, Gao GF, Han P, Dai L: Neutralisation of ZF2001-elicited antisera to SARS-CoV-2 variants. Lancet Microbe 2021, 2(10): e494.

[0110] The method for detecting the neutralizing effect of antibodies on the new coronavirus pseudovirus is as follows:

[0111] Except for the first well, add 60 μl of DMEM medium containing 10% FBS to each well of the 96-well round-bottom plate, dilute the antibody to be tested to a certain concentration (4 μg / ml), pipette 120 μl of antibody diluent and add it to the first well, and then perform a 2-fold dilution (i.e., pipette 60 μl of antibody diluent from the previous well and add it to the next well, and so on). After diluting the virus to an appropriate multiple (pre-test, the negative well reading after dilution is about 500-1500 fluorescent points as the appropriate dilution multiple), add 60 μl of virus diluent to each well, pat gently to mix, and place in a 37°C incubator for incubation for 1 hour. After the incubation is completed, take out the VeroE6 cells with good growth status that were plated in advance, discard the original culture supernatant, and add 100 μl of antibody-virus mixture to infect the cells. After 16-18 hours of infection, take out the cells, take photos and count the points on the CQ1 instrument, and calculate the neutralization ratio. The calculation method is:

[0112] Neutralization ratio = (1-number of fluorescence readings / number of negative well readings)*100%.

[0113] The IC50 values ​​were calculated using Graphpad and the results are shown in Table 4 below.

[0114] Table 4. IC50 values ​​of monoclonal antibody strain L4.65 against pseudoviruses of various novel coronavirus strains

[0115]

[0116] As shown in Table 4, the monoclonal antibody strain L4.65 showed a very high neutralization level against the pseudoviruses of the prototype strain and variant strains Beta, Delta, and Omicron of SARS-CoV-2, indicating that it can effectively block the invasion of these viruses. Such a neutralization level is comparable to the neutralization level of the prototype strain pseudovirus currently put into emergency use by antibodies, but most of the approved antibodies have greatly reduced neutralizing activity against the Omicron sub-strain or even no neutralizing ability, and the broad-spectrum neutralizing monoclonal antibody of the present invention can produce high neutralization efficiency against all VOC viruses, thereby supplementing the current antibody library that can neutralize new mutant strains, and has great clinical application value.

[0117] Example 4: Antibody affinity determination

[0118] In this example, the affinity of the purified monoclonal antibody strain L4.65 obtained in Example 2 was measured. Specifically, the affinity of the RBD protein of the most prevalent strain OmicronBA.2 and the strain OmicronBA.4 that may produce high antibody escape was measured, and the measurement method was as follows:

[0119] Using the OctetRed 96 instrument, 10 μg / ml of the test antibody was bound to the IHC sensor and combined with RBD proteins diluted to different concentrations for 120 seconds and dissociation for 240 seconds; the plate distribution is shown in Table 5 below.

[0120] Table 5. Panel distribution

[0121]

[0122] Then, the antibody-antigen binding was analyzed using Data Analysis 9.0 software, and the affinity values ​​and binding kinetic curves of antibody L4.65 for the RBD protein of the novel coronavirus prototype strain, Omicron variant BA.2 subtype, and Omicron variant BA.4 subtype were obtained, as shown in Table 6 and Attachment 6, respectively. Figure 1 shown.

[0123] Table 6. KD (i.e., dissociation constant) values ​​of antibody L4.65 against RBD proteins of the prototype strain (PT), Omicron variant strains BA.2 and BA.4 subtypes

[0124] KD(M) PT Omicron BA.2 Omicron BA.4 L4.65 4.35E-11 <E-12 5.47E-12

[0125] From the affinity data in Table 6 and Figure 1 It can be seen from the binding kinetic curve that the monoclonal antibody of the present invention has a very high affinity for the RBD protein of the prototype strain of the new coronavirus and the currently popular Omicron variant BA.2 subtype and Omicron variant BA.4 subtype, and has a comparable or even stronger affinity than the antibodies currently approved for marketing (for example, REGN-COV2, S309, etc.). This shows that the monoclonal antibody of the present invention has extremely high potential to be developed as an antibody used in the new coronavirus antigen detection kit, and may be able to provide a lower detection limit and improve the detection sensitivity of the kit.

[0126] Example 5: Identification and classification of antibody epitopes

[0127] In this example, the epitopes of the purified monoclonal antibody strain L4.65 obtained in Example 2 were identified and classified.

[0128] Specifically, the seven-category classification method of Hastie KM, et al (see Hastie KM, et al: Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study. Science 2021, 374(6566): 472-478.) was adopted to determine the recognition epitope by conducting a competition experiment with seven known antibodies targeting seven RBD antigen epitopes; the approximate locations of the seven RBD antigen epitopes RBD-1 to RBD-7 can be found in Hastie KM, et al. In the figure of al, the seven known antibodies against the seven antigen epitopes of RBD-1 to RBD-7 are: CV30 (PDB number: 6XE1), REGN10933 (PDB number: 6XDG), ADI-56046 (reference DOI: 10.1126 / science.abc7424), CV07-270 (PDB number: 6XKP), C110 (PDB number: 7K8V), S309 (PDB number: 7R6X), COVA1-16 (PDB number: 7JMW), CR3022 (PDB number: 6W41); for detailed information about the above seven known antibodies, please refer to Huang M, et al: Atlas of currently available human neutralizing antibodies against SARS-CoV-2 and escape by Omicron sub-variants BA.1 / BA.1.1 / BA.2 / BA.3.Immunity 2022,55(8):1501-1514e1503.

[0129] The specific identification methods are as follows:

[0130] Using the OctetRed 96 instrument, the biotin-labeled Prototype-RBD protein was bound to the SA sensor, and the response value was about 0.8nm; the whole antibody of the test antibody was used as the first antibody, and seven representative antibodies were used as the second antibody for competition experiments, that is, the sensor bound to RBD was first placed in the test antibody for 120s and then placed in the known epitope antibody 1 for 180s, and the binding response value of the second antibody in the presence of the first antibody was detected; after a round of experiments, the sensor can be washed with 0.1M glycine solution, which only dissociates the antigen-antibody binding and does not affect the binding of RBD and Sensor, and the competition between the test antibody and the known epitope antibody 2 can be repeatedly detected. The layout of the experiment is shown in Table 7 below.

[0131] Table 7

[0132] 1 2 4 5 6 A Buffer L5.34 Buffer Known antibody 1-7 0.1M glycine pH1.5 B Buffer L4.307 Buffer Known antibody 1-7 0.1M glycine pH1.5 C Buffer Buffer Buffer Known antibody 1-7 0.1M glycine pH1.5

[0133] The competition experiment results were analyzed using Data Analysis HT 9.0 software. The epitope was determined based on the competition with the seven known antibodies and the epitope region, with 70% inhibition ratio as the positive setting. The binding response values ​​(nm) and epitope analysis results detected above are shown in Table 8 below.

[0134] Table 8

[0135]

[0136] In Table 8, Site 5 represents the RBD-5 antigen epitope.

[0137] As can be seen from Table 8, antibody L4.65 occupies epitope 5.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0139] Sequences involved in this application:

[0140] SEQ ID NO: 1 (HCDR1 of L4.65)

[0141] GFSLSTSGVG

[0142] SEQ ID NO:2 (HCDR2 of L4.65)

[0143] YW

[0144] SEQ ID NO:3 (HCDR3 of L4.65)

[0145] AHFFSHYDSSNYYYGSWFDP

[0146] SEQ ID NO:4 (LCDR1 of L4.65)

[0147] QSFDSRY

[0148] SEQ ID NO:5 (LCDR2 of L4.65)

[0149] GAS

[0150] SEQ ID NO:6 (LCDR3 of L4.65)

[0151] QQFGDSPFT

[0152] SEQ ID NO:7 (VH of L4.65)

[0153] QITLKESGPTLVKPTQTLTLTCTFS GFSLSTSGVG VAWIRQPPGKALEWLAL YW RSSPSLNNRLTITKDTSKNQVVLTMTNMDPEDTATYYC AHFFSHYDSSNYYYGSWFDP WGQGTLVTVSS

[0154] SEQ ID NO:8 (VL of L4.65)

[0155] EIVLTQSPGTLSLSPGERATLSCRAS QSFDSRY LGWYQQKSGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQFGDSPFT FGQGTKLEIK

[0156] SEQ ID NO:9 (Heavy chain sequence of L4.65)

[0157] METDTLLLWVLLLWVPGSTGDQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVAWIRQPPGKALEWLALIYWDNDKRSSPSLNNRLTITKDTSKNQVVLTMTNMDPEDTAT

[0158] YYCAHFFSHYDSSNYYYGSWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA

[0159] ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI

[0160] CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE

[0161] VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW

[0162] LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

[0163] FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

[0164] HEALHNHYTQKSLSLSPGK

[0165] SEQ ID NO: 10 (light chain sequence of L4.65)

[0166] METDTLLLWVLLLWVPGSTGDEIVLTQSPGTLSLSPGERATLSCRASQSFDSRYLGWY

[0167] QQKSGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFGDSPF

[0168] TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL

[0169] QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG

[0170] ECS

[0171] SEQ ID NO: 11 (nucleotide sequence of HCDR1 of L4.65)

[0172] gggttctcactcagcactagtggagtgggt

[0173] SEQ ID NO: 12 (nucleotide sequence of HCDR2 of L4.65)

[0174] atttattgggataatgataag

[0175] SEQ ID NO: 13 (nucleotide sequence of HCDR3 of L4.65)

[0176] gcacacttcttcagtcactatgatagtagtaattattactacggctcctggttcgacccg

[0177] SEQ ID NO: 14 (nucleotide sequence of LCDR1 of L4.65)

[0178] cagagttttgacagcaggtac

[0179] SEQ ID NO: 15 (nucleotide sequence of LCDR2 of L4.65)

[0180] ggagcatcc

[0181] SEQ ID NO: 16 (nucleotide sequence of LCDR3 of L4.65)

[0182] cagcagtttggtgactcaccgttcact

[0183] SEQ ID NO: 17 (nucleotide sequence of VH of L4.65)

[0184] cagatcaccttgaaggagtctggtcctacgctggtgaaacccacacagaccctcacgctgacctgcaccttctctgggttctcactcagc

[0185] actagtggagtgggtgtggcctggatccgtcagcccccagggaaggccctggagtggcttgcactcatttattgggataatgataagcg

[0186] ctccagcccatctctgaacaacaggctcaccatcaccaaggacacctccaaaaaccaggtggtccttacaatgaccaacatggaccct

[0187] gaggacacagccacatattactgtgcacacttcttcagtcactatgatagtagtaattattactacggctcctggttcgacccgtggggcc

[0188] agggaaccctggtcaccgtctcctca

[0189] SEQ ID NO: 18 (nucleotide sequence of VL of L4.65)

[0190] gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagttttgac

[0191] agcaggtacttaggctggtaccagcagaaatctggccaggctcccaggctcctcatttatggagcatccagcagggccactggcatcc

[0192] cagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtc

[0193] agcagtttggtgactcaccgttcacttttggccaggggaccaagctggagatcaaa

[0194] SEQ ID NO:19 (Nucleotide sequence of the heavy chain of L4.65)

[0195] atggagacggatacgctgctcctgtgggttttgctgctgtgggttccaggttccactggtgaccagatcaccttgaaggagtctggtccta

[0196] cgctggtgaaacccacacagaccctcacgctgacctgcaccttctctgggttctcactcagcactagtggagtgggtgtggcctggatc

[0197] cgtcagcccccagggaaggccctggagtggcttgcactcatttattgggataatgataagcgctccagcccatctctgaacaacaggct

[0198] caccatcaccaaggacacctccaaaaaccaggtggtccttacaatgaccaacatggaccctgaggacacagccacatattactgtgca

[0199] cacttcttcagtcactatgatagtagtaattattactacggctcctggttcgacccgtggggccagggaaccctggtcaccgtctcctcag

[0200] cgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctgg

[0201] tgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgca

[0202] gagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaacc

[0203] ataaaccgagcaacaccaaagtggataaacgcgtggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctg

[0204] aactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg

[0205] gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagcc

[0206] gcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtaca

[0207] agtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggt

[0208] gtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatc

[0209] gccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctc

[0210] tacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccac

[0211] tacacgcagaagagcctctccctgtctccgggtaaa

[0212] SEQ ID NO:20 (Nucleotide sequence of the light chain of L4.65)

[0213] atggagacggatacgctgctcctgtgggttttgctgctgtgggttccaggttccactggtgacgaaattgtgttgacgcagtctccaggca

[0214] ccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagttttgacagcaggtacttaggctggtaccagcag

[0215] aaatctggccaggctcccaggctcctcatttatggagcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctg

[0216] ggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtttggtgactcaccgttcactttt

[0217] ggccaggggaccaagctggagatcaagcgcaccgtggctgcccccagcgtgtttatcttccctcccagcgacgagcagctgaagag

[0218] cggcaccgccagcgtggtctgtctcctgaacaacttctatcccagggaggccaaggtccagtggaaagtggacaacgccctgcaaag

[0219] cggcaatagccaggagtccgtcacagagcaggacagcaaggacagcacctacagcctgtccagcaccctgaccctcagcaaggcc

[0220] gactacgagaagcacaaggtgtacgcttgcgaggtgacccatcagggcctgtccagccccgtgaccaagtccttcaacaggggcga

[0221] atgcagc

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 RBD domain, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; The light chain variable region comprises: The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively; the amino acid sequence of SEQ ID NO: 5 is GAS.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region comprises or consists of an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO: 7; The light chain variable region comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

8.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein: The antibody or antigen-binding fragment thereof comprises: A heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 7; and, The light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:

8.

4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The antibody or antigen-binding fragment thereof also includes a constant region.

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein The constant region is any one selected from the following: a constant region of an IgG, IgA or IgM antibody.

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 5, wherein: The constant region is the constant region of an IgG antibody.

7. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The antibody or antigen-binding fragment thereof comprises: a heavy chain comprising, or consisting of, the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and, A light chain comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO:

10.

8. The monoclonal antibody or antigen-binding fragment thereof according to claim 7, wherein The antibody or antigen-binding fragment thereof comprises: A heavy chain, the amino acid sequence of which is shown in SEQ ID NO: 9; and, The light chain has an amino acid sequence as shown in SEQ ID NO:

10.

9. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, human antibody or chimeric antibody.

10. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. The polynucleotide according to claim 10, wherein The polynucleotide is a polynucleotide group, and the polynucleotide group includes: 1) a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 11, 12, 13 or a corresponding mRNA molecule thereof; and, 2) A DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 14, 15, or 16 or its corresponding mRNA molecule; the nucleotide sequence of SEQ ID NO: 15 is ggagcatcc.

12. The polynucleotide according to claim 10, wherein The polynucleotide set comprises: I) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO: 17 or its corresponding mRNA molecule; and, II) A DNA molecule comprising the nucleotide sequence shown in SEQ ID NO: 18 or its corresponding mRNA molecule.

13. The polynucleotide according to claim 10, wherein The polynucleotide set comprises: (I) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 19 or its corresponding mRNA molecule; and, (II) A DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 20 or its corresponding mRNA molecule.

14. A nucleic acid construct comprising the polynucleotide according to any one of claims 10 to 13.

15. The nucleic acid construct according to claim 14, wherein The nucleic acid construct further includes at least one expression control element operably linked to the polynucleotide.

16. An expression vector comprising the polynucleotide according to any one of claims 10 to 13, or the nucleic acid construct according to claim 14 or 15.

17. The expression vector according to claim 16, characterized in that The expression vector is a eukaryotic expression vector.

18. A host cell comprising the polynucleotide according to any one of claims 10 to 13, the nucleic acid construct according to claim 14 or 15, or the expression vector according to claim 16 or 17.

19. The host cell according to claim 18, characterized in that The host cell is a eukaryotic cell.

20. The host cell according to claim 19, characterized in that The host cell is a mammalian cell.

21. A method for preparing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the method comprising: The host cell according to any one of claims 18 to 20 is allowed to express the monoclonal antibody or the antigen-binding fragment thereof under conditions suitable for the expression of the monoclonal antibody or the antigen-binding fragment thereof, and the expressed monoclonal antibody or the antigen-binding fragment thereof is recovered from the culture of the host cell.

22. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the polynucleotide according to any one of claims 10 to 13, the nucleic acid construct according to claim 14 or 15, the expression vector according to claim 16 or 17, or the host cell according to any one of claims 18 to 20, and a pharmaceutically acceptable carrier.

23. The pharmaceutical composition of claim 22, wherein The pharmaceutical composition is in the form of a nasal spray, an oral preparation, a suppository or a parenteral preparation.

24. The pharmaceutical composition according to claim 23, characterized in that The nasal spray is selected from aerosols, sprays and powder sprays; and / or, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments; And / or, the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid or an injectable preparation.

25. A kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the polynucleotide according to any one of claims 10 to 13, the nucleic acid construct according to claim 14 or 15, the expression vector according to claim 16 or 17, the host cell according to any one of claims 18 to 20 and / or the pharmaceutical composition according to any one of claims 22 to 24.

26. The kit according to claim 25, characterized in that The kit is a SARS-CoV-2 antigen detection kit, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

27. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the polynucleotide according to any one of claims 10 to 13, the nucleic acid construct according to claim 14 or 15, the expression vector according to claim 16 or 17, the host cell according to any one of claims 18 to 20, the pharmaceutical composition according to any one of claims 22 to 24 and / or the kit according to claim 25 or 26 in the preparation of a medicament for preventing and / or treating SARS-CoV-2 infection and / or a detection reagent for detecting SARS-CoV-2 infection.

28. The use according to claim 27, characterized in that The SARS-CoV-2 is the prototype strain of SARS-CoV-2.

29. The use according to claim 27, characterized in that The SARS-CoV-2 is a variant strain of SARS-CoV-2, and the variant strain of SARS-CoV-2 is selected from: Beta, Delta variants and Omicron variant subtypes BA.1, BA.2, BA.2.12.1, BA.3, and BA.4 / 5 of SARS-CoV-2.

Images