CN117362421B

CN117362421B - Broad-spectrum monoclonal antibody for novel coronavirus RBD, preparation method and application thereof

Info

Publication number: CN117362421B
Application number: CN202211538937.5A
Authority: CN
Inventors: 戴连攀; 高福; 郭舒欣; 郑宇轩; 段敏润; 徐坤; 崔庆为
Original assignee: Shanxi Institute Of Higher Innovation; Institute of Microbiology of CAS
Current assignee: Shanxi Institute Of Higher Innovation; Institute of Microbiology of CAS
Priority date: 2022-12-02
Filing date: 2022-12-02
Publication date: 2025-01-07
Anticipated expiration: 2042-12-02
Also published as: WO2024114830A1; CN117362421A

Abstract

本发明涉及特异性结合SARS‑CoV‑2RBD结构域的单克隆抗体或其抗原结合片段、其相关产品、制备方法及用途。本发明的单克隆抗体或其抗原结合片段能以较高的亲和力与SARS‑CoV‑2RBD抗原结合，且能以较高的中和活性中和SARS‑CoV‑2原型毒株及其一系列变异毒株，从而抑制其感染，因此，在临床治疗、预防和/或检测SARS‑CoV‑2原型毒株及其变异毒株感染方面具有巨大的潜在应用价值。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

Broad-spectrum monoclonal antibody for novel coronavirus RBD, preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to a monoclonal antibody or an antigen binding fragment thereof which specifically binds to a SARS-CoV-2RBD structural domain, a preparation method thereof, related products and application thereof in preparing medicines for preventing, treating and/or detecting new coronavirus infection.

Background

Novel coronavirus infection (COVID-19) is currently the greatest public health event worldwide.

Because there is no effective specific drug for a while, the current way to prevent and treat novel coronavirus infection is mainly vaccination. Memory T cells and memory B cells provide the primary protective effect when exposed to viral challenge, and of which neutralizing antibodies are of greater importance. In the face of infected people, FDA approves treatment with antiviral drugs Veklury (remdesivir) or immunomodulators Olumiant (baricitinib) or with antibodies.

In the early stages of the new coronavirus outbreak, the FDA approved the use of high titer anti-SARS-CoV-2 convalescence serum for treatment, and as research continues, laboratory personnel continued to isolate potent neutralizing monoclonal antibodies, which could be used alone or in combination to form an 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 antibody S309 (Sotrovimab), LY-CoV1404 (Bebtelovimab), all of which target the Receptor Binding Domain (RBD) of the SARS-CoV-2 viral surface S protein, which is the domain that binds directly to the receptor, as well as one of the important targets for neutralizing antibodies.

With the continuous variation of viruses, the neutralizing ability of existing therapeutic antibodies to new mutant strains and epidemic strains is reduced, and the control ability is deteriorated. According to Xie Xiaoliang, the Omacron variant was shown to escape most of the currently commercially used neutralizing antibodies, such as REGEN-COV, LY-CoV016+LY-CoV555, and most of the antibodies such as S309, LY-CoV1404 were also capable of neutralization, but the neutralizing ability was greatly reduced. Therefore, development of new therapeutic antibodies against coronaviruses is urgent.

Disclosure of Invention

Object of the Invention

The invention aims at providing a monoclonal antibody or an antigen binding fragment thereof which specifically binds to SARS-CoV-2RBD structural domain, a preparation method thereof, related products and application thereof in preparing medicines for preventing, treating and/or detecting new coronavirus infection.

Solution scheme

Zhifei (ZF 2001) is a first recombinant protein vaccine designed based on SARS-CoV-2RBD region in China, and has good immunogenicity and safety. In addition to the SARS-CoV-2 prototype strain, ZF2001 can also provide good protection against other variants. The use of the vaccine was urgently approved in 2020 in China, and three clinical trials have been completed in four countries at present.

The isolation of high neutralizing antibodies from volunteers immunized with SARS-CoV-2RBD domain protein vaccine is certainly a more rapid method of developing new therapeutic antibodies, depending on the situation where both high neutralizing therapeutic antibodies currently approved by the FDA are targeted to RBD. Accordingly, the inventors of the present application tried to isolate antibodies from volunteers immunized with ZF2001 that could neutralize the mutant strain (VOI strains) of most interest currently in a broad spectrum. Through antibody function screening, the inventor obtains a plurality of high-neutrality and high-affinity antibodies, which can be used as therapeutic antibodies of a novel coronavirus prototype strain and a series of variants thereof or an immunodetection reagent thereof.

Specifically, the invention provides the following technical scheme:

In a first aspect, the invention provides a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to a SARS-CoV-2RBD domain, comprising a heavy chain variable region and a light chain variable region, wherein,

The heavy chain variable region comprises:

the amino acid sequences are shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3 respectively;

the light chain variable region comprises:

the amino acid sequences are shown as LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively.

In a preferred embodiment, 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 set forth in SEQ ID NO. 7 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 7;

the light chain variable region comprises or consists of an amino acid sequence as set forth in SEQ ID NO. 8 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 8.

In a further preferred embodiment, the antibody or antigen binding fragment thereof comprises:

a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 7, and,

The amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

In the above-described embodiments of the monoclonal antibodies or antigen-binding fragments thereof of the invention, the antibodies or antigen-binding fragments thereof further comprise a constant region;

Preferably, the constant region is any one selected from the group consisting of IgG, igA, and IgM antibodies;

Further preferably, the constant region is a constant region of an IgG antibody.

In a still further preferred embodiment, the monoclonal antibody or antigen binding fragment thereof comprises:

A heavy chain comprising or consisting of the amino acid sequence shown as 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 shown as SEQ ID NO. 9, and,

A light chain comprising or consisting of an amino acid sequence as set forth 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 set forth in SEQ ID No. 10.

As an example of a still further preferred embodiment, the monoclonal antibody or antigen binding fragment thereof comprises:

a heavy chain having an amino acid sequence as shown in SEQ ID NO.9, and,

The amino acid sequence of the light chain is shown as SEQ ID NO. 10.

For all of the above embodiments, preferably, the antigen binding fragment is selected from the group consisting of Fab, fab '-SH, fv, scFv, F (ab') 2, fd, dAb, complementarity determining region fragment, human antibody, chimeric antibody, or bispecific or multispecific antibody.

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

In a preferred embodiment, the polynucleotide is a set of polynucleotides comprising:

1) A DNA molecule comprising the nucleotide sequences shown as SEQ ID NO. 11, 12, 13 or a corresponding mRNA molecule, and,

2) A DNA molecule comprising the nucleotide sequences shown in SEQ ID NO. 14, 15, 16 or a corresponding mRNA molecule thereof.

In a further preferred embodiment, the set of polynucleotides comprises:

I) A DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO. 17 or a corresponding mRNA molecule thereof, and II) a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO. 18 or a corresponding mRNA molecule thereof.

In a still further preferred embodiment, the set of polynucleotides comprises:

(I) A DNA molecule having a nucleotide sequence shown in SEQ ID NO. 19 or a corresponding mRNA molecule thereof, and,

(II) DNA molecule with nucleotide sequence shown as SEQ ID NO. 20 or corresponding mRNA molecule thereof.

In a third aspect, the invention provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above, optionally further comprising at least one expression control element, e.g.a histidine tag, a stop codon, etc., operably linked to said polynucleotide.

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

Preferably, the expression vector is a eukaryotic expression vector.

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

Preferably, the host cell is a eukaryotic cell;

Further preferably, the host cell is a mammalian cell.

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

In a seventh aspect, the invention provides a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof according to the first aspect, a polynucleotide according to the second aspect, a nucleic acid construct according to the third aspect, an expression vector according to the fourth aspect or a host cell according to the fifth aspect, and a pharmaceutically acceptable carrier.

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

further preferably, the nasal spray is selected from the group consisting of aerosols, sprays and powder sprays;

Further preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, granules, soft/hard capsules, film coatings and ointments;

further preferably, the parenteral formulation is a transdermal agent, an ointment, a plaster, a topical liquid or an injectable formulation.

The amount of the active ingredient to be administered of the pharmaceutical composition of the present invention varies depending on the administration subject, the organ to be administered, the symptoms, the administration method, etc., and can be determined by considering the type of the dosage form, the administration method, the age and weight of the patient, the symptoms of the patient, etc., and the judgment of the doctor.

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

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

In a ninth aspect, the present invention provides the use of a monoclonal antibody or antigen binding fragment thereof according to the first aspect, a polynucleotide according to the second aspect, a nucleic acid construct according to the third aspect, an expression vector according to the fourth aspect, a host cell according to the fifth aspect, a pharmaceutical composition according to the seventh aspect and/or a kit according to the eighth aspect for the preparation of a medicament for the prevention, treatment and/or detection of a novel coronavirus infection.

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

Further preferably, the SARS-CoV-2 variant strain is selected from the group consisting of Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1), delta (B.1.617.2) variants and Omicron (B.1.1.529) and its subvariants.

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

In a possible embodiment, the subject is a mammal, e.g., a human.

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

The "prophylactically and/or therapeutically effective amount" may vary depending on the administration subject, the organ of the subject, the symptoms, the administration method, etc., and may be determined by considering the type of dosage form, the administration method, the age and weight of the patient, the symptoms of the patient, etc., and the judgment of the doctor.

In an eleventh aspect, the present invention provides a method of detecting a novel coronavirus infection comprising the use of a monoclonal antibody or antigen binding fragment thereof as described in the first aspect above.

Definition of the definition

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

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

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 reference amino acid sequence after aligning the amino acid sequences (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percent amino acid sequence identity.

The term "antigen binding fragment" refers to antigen binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen binding or variable regions of the parent antibody, e.g., one or more CDRs. Fragments of the antibodies retain at least some of the binding specificity of the parent antibody. Specifically, the antigen binding fragment may be selected from Fab, fab '-SH, fv, scFv, F (ab') 2, diabodies, and the like.

The "Fab" fragment consists of CH1 and variable domains of one light and one heavy chain.

The "Fab '" fragment contains a light chain and a portion of a heavy chain comprising a portion of the constant region between the VH domain, the CH1 domain, and the CH1 and CH2 domains, with an inter-chain disulfide bond formed between the two heavy chains of the two Fab ' fragments to form a F (ab ') 2 molecule.

The "F (ab') 2" fragment contains two light chains and portions of two heavy chains comprising portions of the VH domain, the CH1 domain, and the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

The "Fv" region comprises variable regions from both the heavy and light chains, but lacks constant regions.

"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. In general, scFv polypeptides comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

A "diabody" is a small antigen-binding fragment having two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain. By using a linker that is so short that it is not possible to pair between two domains of the same strand, the domains pair with complementary domains of the other strand and form two antigen binding sites.

Advantageous effects

The monoclonal antibody or antigen binding fragment thereof can be specifically combined with RBD structural domain of SARS-CoV-2 prototype strain or variant strain thereof with very high affinity, thus having extremely high potential to develop the antibody used in a new coronaantigen detection kit, possibly providing lower detection line and improving the sensitivity of the kit, and the monoclonal antibody or antigen binding fragment thereof of the invention shows higher neutralization activity for SARS-CoV-2 prototype strain and various variant strains, thus having great potential to be developed into a broad-spectrum therapeutic antibody.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1 shows the binding kinetics of two monoclonal antibodies L4.65 of the invention described in example 3 of the invention to RBD proteins of the novel coronavirus prototype strain, the Omicron variant BA.2 subtype and the Omicron variant BA.4 subtype.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.

In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

Example 1 screening of antibodies

Upon obtaining informed consent signed by volunteers, the inventors screened three broad-spectrum neutralizing monoclonal antibodies capable of neutralizing the new coronavirus prototype strain (prototype), beta variant, delta variant, omicron variant and variant strains thereof from volunteer plasma injected with new coronavirus recombinant protein vaccine of Zhifei (ZF 2001).

Specifically, B cells are negatively selected by volunteer PBMC injected with a new coronavirus recombinant protein vaccine ZF2001, single B cells of IgD ^-IgM^-IgG⁺antigen⁺ are obtained through flow sorting (injection: an 'anti ⁺' covers RBD monomer, RBD dimer or S protein binding positive), then the B cells obtained through sorting are subjected to cleavage based on a Noruzan company platform, mRNA is extracted, the mRNA is reversely transcribed into cDNA, and then expression constructs of light chains and heavy chains containing signal peptides, antibody heavy chain or light chain variable regions and an IgG1 skeleton are constructed through PCR amplification, then 293F cells are transfected in a 96-well deep-hole plate by using a transfection reagent of Polyplus, after transfection, antibodies are secreted and expressed in cell supernatants, and three days later, the antibody characteristics secreted in the cell supernatants are detected, so that the target antibodies are helped to be selected.

In the neutralization antibody screening of cell supernatants, we selected prototype (Novain DD 1402), beta (Novain DD 1441), delta (Novain DD 1754), omicron (Novain DD 1768) pseudoviruses for antibody neutralization experiments after 5-fold dilution of cell supernatants, and screened to obtain a broad-spectrum neutralizing monoclonal antibody L4.65, which was named according to the volunteer number of antibody source and its corresponding well number, and the supernatant neutralization results were as shown in Table 1 below:

Table 1 neutralization efficiency of the selected monoclonal antibody strain L4.65 against pseudoviruses of each novel coronavirus strain

Antibody numbering	Prototype	Beta	Delta	Omicron(BA.1)
					L4.65	100%	99.9%	99.8%	97.5%

The values shown in the table are the neutralization efficiency of the antibody strain for the corresponding novel coronavirus strain pseudovirus, and the calculation method is as follows:

Neutralization efficiency = (1-corresponding well fluorescence reading/negative well fluorescence reading) = 100%;

In this example, the nucleotide sequences of the heavy and light chain variable regions of the monoclonal antibodies were identified by using 60% of antibody neutralization efficiency as a positive screening standard and sequencing the cdnas of single B cells expressing the above broad-spectrum neutralizing monoclonal antibodies L4.307, as shown in the following table 2:

TABLE 2 nucleic acid coding sequences of the heavy and light chain variable regions of the selected monoclonal antibody strain L4.65

In Table 2, the underlined portions of the sequences are the nucleic acid coding sequences of CDR1, CDR2 and CDR3 of VH or VL, respectively.

The nucleic acid coding sequences of the heavy and light chain variable regions of the antibody strains shown in table 2 were compiled into the corresponding amino acid sequences according to the genetic code compilation rules, as shown in table 3 below:

TABLE 3 amino acid sequence of heavy and light chain variable regions of monoclonal antibody strain L4.65

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

Example 2 expression and purification of antibodies

The nucleotide sequences of the heavy and light chain variable regions of the monoclonal antibody strain L4.65 selected in example 1 were obtained by artificial synthesis, then the nucleotide sequences of the heavy and light chain variable regions were constructed on human IgG1 antibody backbones, respectively, in the manner of construction in example 1, to obtain heavy and light chain expression constructs containing signal peptide, heavy or light chain variable region and IgG1 backbones, and then 293F cells were transfected with the expression constructs using a transfection reagent of Polyplus in 96-well deep well plates, after which the antibodies were expressed in cell supernatants by secretion, after three days, the cell supernatants were collected, and the antibodies in the cell supernatants were purified by using Protein A magnetic beads (gold srey).

The heavy chain and light chain amino acid sequences of the expressed monoclonal antibody L4.65 are shown as SEQ ID NO. 9 and SEQ ID NO. 10 respectively, and the encoding nucleic acid sequences are shown as SEQ ID NO. 19 and SEQ ID NO. 20 respectively.

Example 3 broad-Spectrum neutralization Effect of antibodies

In this example, the neutralizing effect against a novel coronavirus pseudovirus was examined for the purified monoclonal antibody strain L4.65 obtained in example 2.

Preparation of novel coronavirus prototype strain (prototype) and variants thereof Beta, delta, omicron BA.1, omicron BA.2, omicron BA.2.12.1, omicron BA.3, omicron BA.4/5 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-2variants.Lancet Microbe 2021,2(10):e494.

The method for detecting the neutralizing effect of the antibody on the novel coronavirus pseudovirus comprises the following steps:

In addition to the first well, 60. Mu.l of DMEM medium containing 10% FBS was added to each well of a 96-well round bottom plate, the antibody to be tested was diluted to a certain concentration (4. Mu.g/ml), 120. Mu.l of the antibody dilution was aspirated to the first well, and then 2-fold ratio dilution was performed (i.e., 60. Mu.l of the antibody dilution from the previous well was aspirated to the lower well, and so forth). After dilution of the virus to a suitable multiple (pre-detection, with a negative well reading of approximately 500-1500 spots at a suitable dilution), 60 μl of virus dilution was added per well, and after tapping and mixing, the wells were incubated in a 37 ℃ incubator for 1h. After the incubation, veroE6 cells with good growth state plated in advance were taken out, the original culture supernatant was discarded, and 100. Mu.l of an antibody-virus mixture was added to carry out cell infection. After 16-18h of infection, the cells were taken out, photographed several points on a CQ1 instrument, and the neutralization ratio was calculated by the following method:

Neutralization ratio= (1-fluorescence read number/negative well read number) ×100%.

IC50 values were plotted and calculated in Graphpad and the results are shown in table 4 below.

TABLE 4 neutralization of IC50 values of pseudoviruses of each novel coronavirus strain by monoclonal antibody strain L4.65

As is clear from Table 4, the monoclonal antibody strain L4.65 showed very high neutralization levels against pseudoviruses of each of the SARS-CoV-2 prototype strain and variant strains Beta, delta and Omicron, indicating that it was able to block invasion of these viruses with high efficiency. The neutralization level is equivalent to that of the existing antibodies which are put into emergency use to the pseudovirus of the prototype strain, but the neutralization activity of most approved antibodies on the market to the omacron strain is greatly reduced or even no neutralization capacity exists, and the broad-spectrum neutralization monoclonal antibody can generate high neutralization efficiency to all VOC viruses, thereby supplementing the antibody library which can neutralize the novel mutant strain at present, and having great clinical application value.

Example 4 affinity determination of antibodies

In this example, affinity measurement was performed on the purified monoclonal antibody strain L4.65 obtained in example 2. Specifically, affinity assays were performed on RBD proteins of the currently most prevalent strains omicronba.2 and omicronba.4 strains that are likely to produce high antibody escape, respectively, as follows:

Using an OctetRed 96 instrument, 10. Mu.g/ml of the antibody to be tested was bound to IHC sensor and to RBD protein diluted to different concentrations for 120s and dissociated for 240s, and the plate distribution was shown in Table 5 below.

TABLE 5 distribution of row plates

Then, the binding of the antibody to the antigen was analyzed by DATA ANALYSIS 9.0.0 software to obtain the affinity values and binding kinetics curves of the antibody L4.65 for RBD proteins of the novel coronavirus prototype strain, the Omicron variant BA.2 subtype and the Omicron variant BA.4 subtype, as shown in Table 6 and FIG. 1, respectively.

Table 6, antibody L4.65 KD (i.e., dissociation constant) values for RBD proteins of the prototype strain (PT), omacron variants BA.2 and BA.4 subtypes

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

As can be seen from the affinity data of table 6 and the binding kinetics curves of fig. 1, the monoclonal antibodies of the present invention have very high affinity for both the new coronavirus prototype strain and the currently prevalent Omicron variant ba.2 subtype and the Omicron variant ba.4 subtype RBD proteins, and have a considerably higher or even stronger affinity than the currently approved antibodies (e.g., REGN-COV2, S309, etc.), which suggests that the monoclonal antibodies of the present invention have a very high potential to be developed as antibodies for use in the new coronaantigen detection kit and may be able to provide lower detection line and increase the detection sensitivity of the kit.

Example 5 identification and Classification of antibody epitopes

In this example, epitope identification and classification were performed on the purified monoclonal antibody strain L4.65 obtained in example 2.

Specifically, seven classes of classification methods of Hastin KM, et al (see Hastie KM,et al:Defining variant-resistant epitopes targeted by SARS-CoV-2antibodies:A global consortium study.Science 2021,374(6566):472-478.), for identifying epitopes by competition experiments with seven known antibodies directed against seven RBD epitopes, the approximate positions of the seven RBD epitopes RBD-1 to RBD-7 can be seen in Hastin KM, et al, the seven known antibodies directed against seven RBD-1 to RBD-7 are CV30 (PDB number: 6XE 1), REGN10933 (PDB number: 6 XDG), ADI-56046 (reference DOI:10.1126/science. Abc7424), CV07-270 (PDB number: 6 XKP), C110 (PDB number: 7K 8V), S309 (PDB number: 7R 6X), COVA1-16 (PDB number: 7 JMW), CR3022 (PDB number: 6W), respectively), and specific information on the seven known antibodies can be seen Huang M,et al:Atlas of currently available human neutralizing antibodies against SARS-CoV-2and escape by Omicron sub-variants BA.1/BA.1.1/BA.2/BA.3.Immunity 2022,55(8):1501-1514e1503.

The specific identification method comprises the following steps:

The method comprises the steps of using an OctetRed 96 instrument to combine Prototype-RBD protein marked with biotin on an SA Sensor, wherein the response value is about 0.8nm, using the whole antibody to be detected as a first antibody and using seven classes of representative antibodies as a second antibody to perform competition experiments, namely, placing a Sensor combined with RBD in the antibody to be detected for 120s, then placing the Sensor in the antibody 1 with a known epitope for 180s, detecting the binding response value of the second antibody under the condition that the first antibody exists, and after the experiment is finished, washing the Sensor by using a glycine solution with the concentration of 0.1M, wherein the glycine solution only dissociates the antigen-antibody binding, does not influence the RBD to the Sensor binding, and repeatedly detecting competition of the antibody to be detected and the antibody 2 with the known epitope again. The row plate distribution for this experiment is shown in table 7 below.

TABLE 7

	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

The results of the competition experiments were analyzed by DATA ANALYSIS HT 9.0.0 software, and epitope determination was performed based on the competition with the seven known antibodies, binding to the epitope region, with a positive 70% inhibition ratio, and the detected binding response values (nm) and epitope analysis results are shown in table 8 below.

TABLE 8

In Table 8, site 5 represents the RBD-5 epitope.

As can be seen from table 8, antibody L4.65 occupies epitope No. 5.

It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention.

The sequence involved in the application:

SEQ ID NO. 1 (HCDR 1 of L4.65)

GFSLSTSGVG

SEQ ID NO. 2 (HCDR 2 of L4.65)

IYWDNDK

SEQ ID NO. 3 (HCDR 3 of L4.65)

AHFFSHYDSSNYYYGSWFDP

SEQ ID NO. 4 (LCDR 1 of L4.65)

QSFDSRY

SEQ ID NO.5 (LCDR 2 of L4.65)

GAS

SEQ ID NO.6 (LCDR 3 of L4.65)

QQFGDSPFT

SEQ ID NO. 7 (VH of L4.65)

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVAWIRQPPGKALEWLALIYWDNDKRSSPSLNNRLTITKDTSKNQVVLTMTNMDPEDTATYYCAHFFSHYDSSNYYYGSWFDPWGQGTLVTVSS

SEQ ID NO. 8 (VL of L4.65)

EIVLTQSPGTLSLSPGERATLSCRASQSFDSRYLGWYQQKSGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFGDSPFTFGQGTKLEIK

SEQ ID NO. 9 (heavy chain sequence of L4.65)

METDTLLLWVLLLWVPGSTGDQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVAWIRQPPGKALEWLALIYWDNDKRSSPSLNNRLTITKDTSKNQVVLTMTNMDPEDTAT

YYCAHFFSHYDSSNYYYGSWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI

CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW

LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

SEQ ID NO. 10 (light chain sequence of L4.65)

METDTLLLWVLLLWVPGSTGDEIVLTQSPGTLSLSPGERATLSCRASQSFDSRYLGWY

QQKSGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFGDSPF

TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL

QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG

ECS

SEQ ID NO. 11 (nucleotide sequence of HCDR1 of L4.65)

gggttctcactcagcactagtggagtgggt

SEQ ID NO. 12 (nucleotide sequence of HCDR2 of L4.65)

atttattgggataatgataag

SEQ ID NO. 13 (nucleotide sequence of HCDR3 of L4.65)

gcacacttcttcagtcactatgatagtagtaattattactacggctcctggttcgacccg

SEQ ID NO. 14 (nucleotide sequence of LCDR1 of L4.65)

cagagttttgacagcaggtac

SEQ ID NO. 15 (nucleotide sequence of LCDR2 of L4.65)

ggagcatcc

SEQ ID NO. 16 (nucleotide sequence of LCDR3 of L4.65)

cagcagtttggtgactcaccgttcact

SEQ ID NO. 17 (nucleotide sequence of VH of L4.65)

cagatcaccttgaaggagtctggtcctacgctggtgaaacccacacagaccctcacgctgacctgcaccttctctgggttctcactcagc

actagtggagtgggtgtggcctggatccgtcagcccccagggaaggccctggagtggcttgcactcatttattgggataatgataagcg

ctccagcccatctctgaacaacaggctcaccatcaccaaggacacctccaaaaaccaggtggtccttacaatgaccaacatggaccct

gaggacacagccacatattactgtgcacacttcttcagtcactatgatagtagtaattattactacggctcctggttcgacccgtggggcc

agggaaccctggtcaccgtctcctca

SEQ ID NO. 18 (nucleotide sequence of VL of L4.65)

gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagttttgac

agcaggtacttaggctggtaccagcagaaatctggccaggctcccaggctcctcatttatggagcatccagcagggccactggcatcc

cagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtc

agcagtttggtgactcaccgttcacttttggccaggggaccaagctggagatcaaa

SEQ ID NO. 19 (nucleotide sequence of heavy chain of L4.65)

atggagacggatacgctgctcctgtgggttttgctgctgtgggttccaggttccactggtgaccagatcaccttgaaggagtctggtccta

cgctggtgaaacccacacagaccctcacgctgacctgcaccttctctgggttctcactcagcactagtggagtgggtgtggcctggatc

cgtcagcccccagggaaggccctggagtggcttgcactcatttattgggataatgataagcgctccagcccatctctgaacaacaggct

caccatcaccaaggacacctccaaaaaccaggtggtccttacaatgaccaacatggaccctgaggacacagccacatattactgtgca

cacttcttcagtcactatgatagtagtaattattactacggctcctggttcgacccgtggggccagggaaccctggtcaccgtctcctcag

cgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctgg

tgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgca

gagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaacc

ataaaccgagcaacaccaaagtggataaacgcgtggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctg

aactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtg

gtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagcc

gcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtaca

agtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggt

gtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatc

gccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctc

tacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccac

tacacgcagaagagcctctccctgtctccgggtaaa

SEQ ID NO.20 (nucleotide sequence of light chain of L4.65)

atggagacggatacgctgctcctgtgggttttgctgctgtgggttccaggttccactggtgacgaaattgtgttgacgcagtctccaggca

ccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagttttgacagcaggtacttaggctggtaccagcag

aaatctggccaggctcccaggctcctcatttatggagcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctg

ggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtttggtgactcaccgttcactttt

ggccaggggaccaagctggagatcaagcgcaccgtggctgcccccagcgtgtttatcttccctcccagcgacgagcagctgaagag

cggcaccgccagcgtggtctgtctcctgaacaacttctatcccagggaggccaaggtccagtggaaagtggacaacgccctgcaaag

cggcaatagccaggagtccgtcacagagcaggacagcaaggacagcacctacagcctgtccagcaccctgaccctcagcaaggcc

gactacgagaagcacaaggtgtacgcttgcgaggtgacccatcagggcctgtccagccccgtgaccaagtccttcaacaggggcga

atgcagc

Claims

1. A monoclonal antibody or 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:

the light chain variable region comprises:

the amino acid sequences are shown as LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively, wherein the amino acid sequence of SEQ ID NO. 5 is GAS.

2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein,

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody or antigen-binding fragment thereof comprises:

4. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof further comprises 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 group consisting of IgG, igA, and IgM antibodies.

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

7. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof comprises:

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 having an amino acid sequence as shown in SEQ ID NO. 9, and,

The amino acid sequence of the light chain is shown as SEQ ID NO. 10.

9. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab '-SH, fv, scFv, F (ab') 2, human or chimeric antibodies.

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

11. The polynucleotide of claim 10, wherein the polynucleotide is a set of polynucleotides comprising:

2) A DNA molecule or a corresponding mRNA molecule comprising the nucleotide sequences shown as SEQ ID NO. 14, 15 and 16, wherein the nucleotide sequence of SEQ ID NO. 15 is ggagcatcc.

12. The polynucleotide of claim 10, wherein the set of polynucleotides comprises:

I) A DNA molecule comprising the nucleotide sequence shown as SEQ ID NO. 17 or a corresponding mRNA molecule thereof, and,

II) a DNA molecule comprising the nucleotide sequence shown as SEQ ID NO. 18 or a corresponding mRNA molecule thereof.

13. The polynucleotide of claim 10, wherein the set of polynucleotides comprises:

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

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

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

17. The expression vector of claim 16, wherein the expression vector is a eukaryotic expression vector.

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

19. The host cell of claim 18, wherein the host cell is a eukaryotic cell.

20. The host cell of claim 19, wherein the host cell is a mammalian cell.

21. A method of making the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-9, comprising allowing the host cell of any one of claims 18-20 to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from a culture of the host cell.

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

24. The pharmaceutical composition of claim 23, wherein the nasal spray is selected from the group consisting of aerosols, sprays, and powder sprays;

and/or the oral formulation is selected from the group consisting of tablets, powders, pills, granules, soft/hard capsules, film coatings and ointments;

and/or the parenteral formulation is a transdermal agent, an ointment, a plaster, a topical liquid or an injectable formulation.

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

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

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

28. The use according to claim 27, wherein the SARS-CoV-2 is a prototype strain of SARS-CoV-2.

29. The use according to claim 27, wherein the SARS-CoV-2 is a variant strain of SARS-CoV-2, and the variant strain of SARS-CoV-2 is selected from the group consisting of Beta, delta variant and Omicron variant subtypes BA.1, BA.2, BA.2.12.1, BA.3, and BA.4/5 of SARS-CoV-2.