CN114957455B

CN114957455B - Novel coronavirus monoclonal antibody and application thereof

Info

Publication number: CN114957455B
Application number: CN202210489241.1A
Authority: CN
Inventors: 周兵; 张政; 刘磊; 鞠斌; 范晴; 戚甫荣
Original assignee: Shenzhen National Clinical Research Center For Infectious Diseases; Third Peoples Hospital of Shenzhen
Current assignee: Shenzhen National Clinical Research Center For Infectious Diseases; Third Peoples Hospital of Shenzhen
Priority date: 2022-05-06
Filing date: 2022-05-06
Publication date: 2023-05-16
Anticipated expiration: 2042-05-06
Also published as: CN114957455A

Abstract

The application discloses a novel coronavirus monoclonal antibody and application thereof. The novel coronavirus monoclonal antibody is STS165, and consists of a heavy chain and a light chain matched with the heavy chain; the sequences of CDR1, CDR2 and CDR3 of the heavy chain of STS165 are in sequence "GFTFDDYG", "inwngst" and "ARVGYCGGDCYSLSLAGFPDY", and the sequences of CDR1, CDR2 and CDR3 of the light chain are in sequence "QSVSYN", "DAS" and "QQYNNWPPYT". The novel coronavirus monoclonal antibody has cross neutralization and binding activities on 13 novel coronaviruses including omacron variants, and provides a novel choice for the prevention and treatment of the novel coronaviruses.

Description

Novel coronavirus monoclonal antibody and application thereof

Technical Field

The application relates to the technical field of novel coronavirus treatment, in particular to a novel coronavirus monoclonal antibody and application thereof.

Background

2019 coronavirus disease (covd-19) caused by infection with different varieties of SARS-CoV-2 is still a global endeavor, bringing great loss to the life health of people and economic development of society in various regions of the world, and thus effective therapeutic intervention is still needed.

The omacron variant of SARS-CoV-2 is a new mutant found in month 11 of 2021, which rapidly spreads around the world. One of the reasons for the popularity of Omicron variants is that the Spike protein on the surface of the virion has more than 30 mutations, which makes it more infectious and immune escaping.

During viral infection, the trimeric Spike protein binds to the receptor angiotensin converting enzyme 2 (ACE 2) on the host target cell and is cleaved by the host protease into S1 and S2 subunits. The S1 subunit comprises an N-terminal domain (NTD) and a Receptor Binding Domain (RBD), which bind directly to host receptors. The S2 subunit is responsible for membrane fusion between the virus and the host cell. Binding of S1 to the receptor and protease digestion triggers conformational changes in the trimer Spike and exposes the fusion peptide of S2, allowing the virus to enter the host cell. Like the original SARS-CoV-2, the Omacron variant still utilizes the receptor ACE2 on the target cell to infect the host. Blocking RBD-ACE2 interactions prevents virus from infecting cells, making them key candidate targets for antiviral drugs and neutralizing antibodies (neutralizing antibody, nAb). Whereas mutations that occur in RBD often affect viral infection and transmission, even resulting in viral escape, natural infection or vaccination-induced antibody neutralization.

The nAbs against SARS-CoV-2 is a promising clinical intervention. At present, monoclonal antibodies targeting SARS-CoV-2 have been extensively studied and reported; however, many monoclonal antibodies, including some of the antibody drugs currently used for emergency authorization, have reduced, or even completely lost neutralizing activity against the newly emerging omacron variant. Thus, to solve the problem of omacron variant abuse worldwide, the development of new novel coronavirus antibodies that address the potential risk of continuous evolution and recombination of SARS-CoV-2 and neutralizing antibody escape remains a major and difficult point of research in the art.

Disclosure of Invention

The object of the present application is to provide a novel coronavirus monoclonal antibody and its use.

In order to achieve the above purpose, the present application adopts the following technical scheme:

in a first aspect the present application discloses a novel coronavirus monoclonal antibody, which is STS165; the monoclonal antibodies are composed of heavy chains and light chains paired with the heavy chains; the sequences of CDR1, CDR2 and CDR3 of the heavy chain of STS165 are in sequence "GFTFDDYG", "inwngst" and "ARVGYCGGDCYSLSLAGFPDY", and the sequences of CDR1, CDR2 and CDR3 of the light chain are in sequence "QSVSYN", "DAS" and "QQYNNWPPYT".

It should be noted that, the key point of the present application is that a fully human monoclonal antibody, namely STS165, is isolated and identified from peripheral blood specific single memory B cells of convalescence individuals, and the monoclonal antibody can perform neutralization on 13 SARS-CoV-2 variant pseudoviruses including novel coronavirus omacron variants, specifically including: WT (wild type), alpha, beta, gamma, delta, lambda, mu, kappa, eta, epsilon, iota, c.1.2, omicron.

In one implementation of the present application, the heavy chain variable region sequence of the novel coronavirus monoclonal antibody STS165 is the sequence shown by Seq ID No.1, and the light chain variable region sequence is the sequence shown by Seq ID No. 2.

It should be noted that the heavy chain variable region and the light chain variable region of the above specific sequences are only monoclonal antibody sequences specifically employed in one implementation of the present application; it will be appreciated that for monoclonal antibodies, the regions that affect their exact complementarity to an epitope are complementarity determining regions (abbreviated CDRs), specifically, CDR1, CDR2 and CDR3 of the heavy chain, and CDR1, CDR2 and CDR3 of the light chain; therefore, the functions and actions of the novel coronavirus monoclonal antibody can be basically realized as long as the CDR1, CDR2 and CDR3 sequences of the heavy chain and the light chain are ensured to be unchanged. That is, the specific sequences of the novel coronavirus monoclonal antibodies of the present application are not limited to the heavy chain variable region and the light chain variable region of the above specific sequences.

In a second aspect, the present application discloses a nucleic acid fragment encoding a novel coronavirus monoclonal antibody of the present application, which encodes the heavy and light chains of STS165.

In one embodiment of the present application, the nucleic acid fragment comprises a nucleic acid fragment encoding the heavy chain variable region shown in Seq ID No.1, and the sequence thereof is the sequence shown in Seq ID No. 3; a nucleic acid fragment encoding the light chain variable region shown in Seq ID No.2, which has the sequence shown in Seq ID No. 4.

It is to be noted that the above specific nucleic acid sequences are only those specifically employed in one implementation of the present application, and it is understood that there may be a plurality of codons for one amino acid; thus, depending on the degeneracy of the codons, there may be several nucleic acid sequences encoding the same heavy or light chain, in addition to the nucleic acid sequences defined above, while ensuring that the coding sequence is unchanged, all falling within the scope of the present application.

In a third aspect the present application discloses a recombinant plasmid comprising a nucleic acid fragment of the present application.

It should be noted that, the recombinant plasmid of the present application is to be able to effectively express the nucleic acid fragment of the present application, thereby obtaining the corresponding heavy chain, light chain or novel coronavirus monoclonal antibody; thus, in principle, vectors which are capable of transfecting nucleic acid fragments into host cells for expression of nucleic acids may be used in the present application.

In a fourth aspect, a recombinant cell comprising a nucleic acid fragment of the present application or a recombinant plasmid of the present application is disclosed.

The recombinant cell of the present application refers to a host cell transfected with the nucleic acid fragment or recombinant plasmid of the present application; heavy, light or novel coronavirus monoclonal antibodies of the present application can generally be obtained by direct culture of such host cells.

The fifth aspect of the application discloses a preparation method of the novel coronavirus monoclonal antibody, which comprises the steps of adopting recombinant cells of the application to carry out protein expression on recombinant plasmids of the application, extracting and purifying the expressed protein, and obtaining the novel coronavirus monoclonal antibody of the application.

The sixth aspect of the application discloses the application of the novel coronavirus monoclonal antibody, the nucleic acid fragment, the recombinant plasmid or the recombinant cell in the application in preparing novel coronavirus vaccine, novel coronavirus prevention and treatment drug or novel coronavirus detection reagent.

It should be noted that the novel coronavirus monoclonal antibodies of the present application can have better cross-neutralizing and binding activities for different variants, especially for Omicron variants; therefore, the method can be used for preparing corresponding novel coronavirus vaccines or other medicaments with similar functions for preventing and treating novel coronaviruses. Similarly, the cross-neutralizing and binding activities of the monoclonal antibodies of the present application to different variants can also be used to detect corresponding novel coronavirus variants. As for the nucleic acid fragments, recombinant plasmids and recombinant cells, these can be used as raw materials for preparing novel coronavirus monoclonal antibodies, thereby being used for preparing novel coronavirus vaccines, novel coronavirus control drugs or novel coronavirus detection reagents.

A seventh aspect of the present application discloses a novel coronavirus vaccine comprising the novel coronavirus monoclonal antibodies of the present application, or a substance capable of inducing the production or in vivo expression of the novel coronavirus monoclonal antibodies of the present application.

In an eighth aspect of the present application, a novel coronavirus detection reagent is disclosed, which comprises the novel coronavirus monoclonal antibody of the present application, or an antigen capable of specifically binding to the novel coronavirus monoclonal antibody of the present application.

Due to the adoption of the technical scheme, the beneficial effects of the application are that:

the novel coronavirus monoclonal antibody has cross neutralization and binding activities on 13 novel coronaviruses including omacron variants, and provides a novel choice for the prevention and treatment of the novel coronaviruses.

Drawings

FIG. 1 shows the results of a pseudo-virus neutralization assay of 13 SARS-CoV-2 variants and SARS-CoV by monoclonal antibody STS165 of the examples of the present application;

FIG. 2 shows SPR test results of monoclonal antibody STS165 on RBD proteins of different SARS-CoV-2 variant strains in the examples of the present application;

FIG. 3 shows epitope identification results of monoclonal antibody STS165 in the examples of the present application;

FIG. 4 shows the results of a neutralization assay of monoclonal antibody STS165 in combination with approved clinically used non-competing antibodies in the examples of this application.

Detailed Description

The constant advent and spread throughout the world of SARS-CoV-2 variants has resulted in the inactivation of a large number of monoclonal antibodies, including antibody drugs for clinical emergency authorization. Notably, omicon reduces the neutralizing activity of other emergency-authorized antibodies besides S309. Thus, there is an urgent need to investigate the broad-spectrum neutralizing antibodies of new coronavirus variants while saving these nAbs from losing neutralizing effect on Omicron and on-going variants.

The application uses a novel coronavirus RBD protein as a bait, separates specific single memory B cells from peripheral blood of individual volunteers in a convalescence period through flow cytometry, and clones and identifies the specific single memory B cells from the peripheral blood by using a Smart-seq-2 technology to obtain a fully human monoclonal antibody, namely STS165. It has an IC50 of 0.128. Mu.g/mL against the novel coronavirus WT strain (wild-type) pseudovirus.

The present application further predicts the binding epitope of STS165 by competition ELISA with ACE2 and 4 classes of representative neutralizing antibodies (class 1: P2C-1F11, REGN10933, CB6; class 2: BD-368-2, C144, P2B-2F6; class 3: S309, REGN10987, C110, C135; class 4: EY6A, H014, S304, S2A 4). The results show that STS165 shows high intensity competition with ACE2 receptor and 4 class 4 antibodies, indicating that STS165 belongs to class 4 antibodies whose recognition sites are distant from the Receptor Binding Motif (RBM).

Next, the present application tested STS165 monoclonal neutralizing antibodies for cross-neutralizing, binding activity against novel coronaviruses WT, alpha, beta, gamma, delta, lambda, mu, kappa, eta, epsilon, iota, c.1.2 and omacron series variants. The results show that STS165 neutralizes 13 SARS-CoV-2 variant pseudoviruses; in addition, STS165 is also effective in neutralizing SARS-CoV pseudovirus. STS165 has higher affinity with wild-type and mutant SARS-CoV-2 and SARS-CoV RBD proteins (RBD-WT, RBD-Omicron, RBD-Lambda, RBD-Mu, RBD-C.1.2 and RBD-SARS-CoV), and shows the same pattern as the neutralization activity, indicating a strong correlation between binding affinity and neutralization, resulting in STS165 having broad-spectrum cross-neutralization activity.

Finally, as many different SARS-CoV-2 varieties appear and spread throughout the world. These variants are resistant to many specific monoclonal antibodies. In particular, omacron has reduced neutralization of most emergency authorization antibodies except S309. Therefore, there is an urgent need to rescue the neutralizing activity of these nAbs against omacron and like variants. Cocktail therapy, i.e., the combination of antibodies, is not only an effective treatment, but also prevents antibody resistance caused by viral escape mutations. STS165 can neutralize 13 varieties including Beta, delta, and omacron, an ideal partner for therapeutic antibody cocktail therapy. The present application evaluates the neutralizing effect of STS165 on 11 pseudoviruses in combination with 3 other non-competing antibodies (REGN 10933, REGN10987, and S309) that have been urgently approved for clinical use. The results showed that REGN10933 showed a significant decrease in the neutralizing IC50 for Beta, while REGN10933 and REGN10987 showed less than 50% inhibition of both Omicron and SARS-CoV pseudoviruses at the highest concentrations (50. Mu.g/mL). When REGN10933 and REGN10987 were used in combination with STS165, respectively, the neutralization activity against Omacron and SARS-CoV pseudoviruses was restored to some extent. At the same time, the neutralizing activity against variant pseudoviruses was also maintained for other variants using REGN10933, REGN10987 and S309 antibodies, respectively, with STS165 cocktail therapy. Taken together, these results indicate that STS165 is not only a broad-spectrum neutralizing antibody with good potential for use, but also can form cocktails with different emergency-approved antibodies for use against the emerging SARS-CoV-2 variety.

The novel coronavirus monoclonal antibody has cross neutralization and binding activities on novel coronaviruses WT, alpha, beta, gamma, delta, lambda, mu, kappa, eta, epsilon, iota, C.1.2 and Omicron mutant, and can effectively neutralize SARS-CoV. The neutralizing effect is wide, can be used for passively preventing various SARS-CoV-2 variants, and provides a new candidate therapeutic antibody for novel coronavirus prevention and treatment.

The invention will be described in further detail below with reference to the drawings by means of specific embodiments. The following examples are merely illustrative of the present application and should not be construed as limiting the present application. Unless otherwise specified, the instruments and materials used in the examples below are those conventionally used in laboratories.

Examples

1. Materials and methods

1. Study approval and biological samples

The study was approved by the ethical committee of the third people hospital in Shenzhen City (approval number: 2020-084). The participants provided written informed consent for sample collection and subsequent analysis. The recovery phase individual test time was about 2 weeks after virus negative, and Peripheral Blood Mononuclear Cell (PBMC) samples were collected and stored in liquid nitrogen.

In this example, 1 sample is specifically collected, and the sample collection method is as follows:

peripheral blood mononuclear cell isolation: 10mL of venous blood is collected by an anticoagulation tube, transferred into a 50mL centrifuge tube, diluted by 10mL of PBS solution and gently mixed; two 15mL centrifuge tubes were taken and 5mL Ficoll separate solution was added to each tube. Then adding 10mL of diluted blood into the upper layer of the ficoll separating liquid; centrifuging at 2000rpm for 20 minutes; sucking the leukocyte layer into a clean 15mL centrifuge tube; PBS was added to 10mL, centrifuged at 1500rpm for 10 min, the supernatant was removed, resuspended in 3mL of cell cryopreservation solution, 1mL of cells were each taken into 3 cryopreservation tubes, placed in a cryopreservation cassette and placed in a-80℃refrigerator overnight, and the cells were transferred into liquid nitrogen the next day for long-term storage.

2. Isolation of monoclonal antibodies from B cells of convalescent individuals

The frozen peripheral blood mononuclear cells were resuscitated and washed 2 times with 10mL of PBS. CD19-PE-Cy7, CD3-Pacific Blue, CD8-Pacific Blue, CD14-Pacific Blue, CD27-APC/Cy7, igG-FITC (all from BD Biosciences) and His-tagged novel coronavirus WT strain RBD (Sino Biological) probe mixtures were added to 100. Mu.L of staining buffer (PBS+2% fetal bovine serum), peripheral blood mononuclear cells were resuspended, and stained at 4℃for 30 minutes. After washing 2 times with PBS, APC and PE-labeled anti-His tag secondary antibody (Abcam) was added to 100. Mu.L of staining buffer (PBS+2% fetal bovine serum), and cells were stained at 4℃for 30 minutes. After 2 washes in PBS, novel coronavirus WT strain RBD-specific igg+b cells were sorted by BD FACS Aria II-sorting flow cytometer.

Taking out the cell suspension, adding Oligo (dT) primer to reverse transcribe RNA (mainly mRNA) with polyA tail; two strands of cDNA were synthesized using TSO (template-switching oligo) primers, thereby replacing RNA complementary to one strand of cDNA; next, the cDNA was amplified to ng-scale (Takara) by PCR amplification enrichment; breaking DNA by using the modified high-activity Tn5 transposase, adding a linker to two ends of cDNA, and marking the DNA fragment which is usually 200-600bp; amplifying the library labeled in the previous step by adding N/S5xx and N7xx index primers (Vazyme); finally, sequencing was performed using an Illumina sequencer (Smart-seq 2 transcriptome sequencing, beverand biotechnology limited, beijing).

The sequence of the antibody variable region obtained by sequencing was synthesized by the company of the Kirschner Biotechnology Co., ltd, and the variable regions of the heavy and light chains of the antibody were cloned into the full-length IgG1 heavy and light chain expression vector pcDNA3.4 (the company of the Kirschner Biotechnology Co., ltd.) respectively, and plasmids of the heavy and light chains of the antibody were prepared in large quantities. After obtaining plasmids prepared by aurey corporation, 293F cells (500 mL, for example) were co-transfected with pairs of heavy and light chain expression plasmids using PEI transfection reagent to express monoclonal antibodies from the culture supernatant using a protein A adsorption column. The method comprises the following specific steps:

293F cells were cultured in 8% CO ₂ The 293F cell concentration was adjusted to 1.2X10 in an incubator at 37 ℃ ⁶ Culturing for 2 hours at a concentration of one mL/mL; preparing solution A: to 12.5mL opti-MEM (31985070, gibco)Adding 250 μg of antibody heavy chain plasmid and 250 μg of antibody light chain plasmid; preparing a solution B: to 12.5mL of opti-MEM was added 2.5mL of 1mg/mL PEI transfection reagent (24885-2, polyscices) and allowed to stand for 5 minutes; mixing the solution A and the solution B, and standing for 20 minutes to obtain an AB mixed solution; 25mL of the AB mixed solution is added into 500mL of 293F cells dropwise, and the mixture is uniformly shaken while being added dropwise; culturing the cells for 5 days; then, the 293F cells were centrifuged at 3000g for 20 minutes, and the supernatant was collected and filtered with a 0.45 μm filter; opening a cover in the Protein A gravity column, completely flowing out 20% ethanol solution in the column by utilizing gravity, and balancing the Protein A gravity column by using 10mM PBS solution with 5 times of column volume; adding the filtered cell supernatant into a Protein A gravity column, and flowing out by virtue of gravity; the Protein a gravity column was washed with 3 column volumes of PBS solution and eluted with 5 volumes of 0.1M glycine-hydrochloric acid solution (ph=3.0); placing the eluent into an ultrafiltration concentration tube with 30KD, supplementing with PBS, centrifuging at 3500rpm for 40 min at 4deg.C, discarding the waste liquid in the collection tube, adding 20mL of PBS solution, centrifuging at 3500rpm for 40 min at 4deg.C, absorbing the concentrated and displaced antibody solution, and determining the concentration of antibody protein.

3. Competitive ELISA method for detecting antibody epitope

ACE2 protein and STS165 antibody were labeled with HRP labeling kit (ab 102890, abcam). The method comprises the following specific steps:

100. Mu.g of the protein or antibody to be labeled was diluted to 100. Mu.L with PBS, 10. Mu.L of Modifier reagent was added, and gently swirled and mixed. The cap of the HRP-conjugated mixture was opened, and the antibody sample (with Modifier reagent added) was pipetted with a pipette tip, directly onto the lyophilized powder material, and gently resuspended. The bottle cap is covered, and the bottle is placed for 3 hours at room temperature (20 ℃ to 25 ℃) in a dark place. After 3 hours (or longer) incubation, 1 μl of the quantiser reagent was added to each 10 μl of antibody in the reaction and gently mixed. After 30 minutes the conjugated antibody was used, 100. Mu.L glycerol was added and the mixture was mixed and stored at-20℃to give a solution (about 0.5. Mu.g/mL).

SARS-CoV-2RBD protein (40592-V08B, sino Biological) was coated overnight at 4℃at 2. Mu.g/mL in 96-well ELISA plates, 100. Mu.L per well. PBST was washed 5 times (PBS solution containing 0.5% Tween-20); sealing with sealing liquid at normal temperature for 1 hr, each timeWells 150 μl, followed by 5 washes with PBST, blocking solution formulation: 5% skim milk+2% BSA (PBS formulation), all following antibody dilutions were formulated as blocking solution; the antibodies to be detected were serially diluted 5-fold from the highest concentration of 20. Mu.g/mL, 8 dilutions total, mixed with equal volumes of HRP-labeled ACE2 or STS165 antibodies, added to 96-well plates, and incubated at 37℃for 1 hour at 100. Mu.L per well. Washing with PBST for 5 times; mixing the color development solution A and solution B (in the process) according to a ratio of 1:1, and reacting for 20 minutes in a dark place at normal temperature with 100 mu L of each hole; then 50 mu L of 2M H ₂ SO ₄ The reaction was terminated. Optical density was measured at 450nm (OD) using a Varioskan LUX multimode microplate reader (Thermo Scientific).

4. Surface Plasmon Resonance (SPR) binding analysis

Binding assays for wild-type and mutant SARS-CoV-2 and SARS-CoV RBD protein (Sino Biological) to STS165 antibodies were performed using the Biacore 8K system (GE Healthcare). The method comprises the following steps:

one flow cell of the CM5 sensor chip was covalently coated with 10mM sodium acetate buffer (pH 5.0) to obtain about 250 final Response Units (RU), while the other flow cell remained uncoated and was blocked as a control. All assays were performed in HBS-EP buffer (10 mM HEPES pH 7.4, 150mM NaCl, 3mM EDTA and 0.05% Tween-20) at a flow rate of 30. Mu.L/min. Serial dilutions of antibody were injected for 60 seconds, respectively, and the resulting data were fitted in a 1:1 binding model using Biacore assessment software (GE Healthcare). Each measurement was performed twice and a single value was used to generate the average affinity constant.

5. Detection of neutralizing Capacity of antibodies

HEK-293T cells and HEK-293T-hACE2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin at 37℃with 5% CO ₂ Culturing in an incubator. The novel coronavirus pseudovirus was generated by co-transfecting HEK-293T cells with 10. Mu.g of novel coronavirus spike protein expression plasmid and 20. Mu.g of env-deficient HIV-1 backbone vector plasmid (pNL 4-3.Luc. R-E-). Wherein the novel coronavirus spike protein expression plasmid is prepared by synthesis by the company of Kirsrui Biotechnology.

False diseaseThe specific experimental steps for toxin preparation are as follows: when the confluence of HEK-293T cells in a T75 cell culture flask reaches about 80%, sucking and removing the culture medium, digesting with pancreatin, adding culture medium to resuspend cells, centrifuging at 1000rpm for 5min, removing the supernatant, adding 10mL of culture medium to resuspend cells, counting, and taking 6×10 ⁶ Cells were cultured overnight in new T75 cell flasks; taking 2 1.5mL centrifuge tubes, adding 400 mu L of serum-free culture medium into each centrifuge tube, respectively adding 120 mu L of PEI transfection reagent and 20 mu g of pNL 4-3.Luc.R-E-plus 10 mu g of SARS-CoV-2 spike protein expression plasmid, mixing uniformly, standing at room temperature for 5 minutes, mixing the two, and standing at room temperature for 20 minutes; adding the transfection reagent into a HEK-293T 75 cell bottle, uniformly mixing, culturing in an incubator for about 7 hours, sucking off the culture medium, adding a fresh culture medium, and continuing culturing for 48 hours; the cell culture medium containing the pseudovirus is sucked, transferred into a 50mL centrifuge tube, centrifuged at 3000rpm for 10 minutes, and the supernatant is filtered by a 0.45 mu m filter, split-packed and frozen in a low-temperature refrigerator at minus 80 ℃ for standby.

To determine the neutralizing activity of the antibodies, the monoclonal antibodies were diluted 9 times at a 3-fold ratio of 100 μg/mL starting in 96-well plates, added with an equal volume of diluted pseudovirus and incubated for 1 hour at 37 ℃. Subsequently, 100. Mu.L of HEK-293T-hACE2 cells, i.e.2.5 ten thousand cells, were added per well. At 37℃5% CO ₂ After 48 hours of incubation in the incubator, the cell culture medium was removed and 100 μl of Bright Lite luciferase reagent (Vazyme Biotech) was added to the cell wells. After incubation for 2 minutes at room temperature, 90 μl of cell lysate was transferred to 96 Kong Baiban and chemiluminescent signals were detected using a multifunctional microplate reader. The 50% inhibitory concentration (IC 50) was calculated by a logarithmic (inhibitor) and normalized response-variable slope (four parameter) model using GraphPad prism8.0 software. Wherein, in the evaluation of neutralizing activity of the antibody combination, 2 monoclonal antibodies were diluted 9 times at a 3-fold ratio of 50. Mu.g/mL, respectively, and the remaining steps were the same as those of the single antibody neutralizing activity evaluation.

2. Results and analysis

1. Isolation of monoclonal antibodies

Specific individual memory B cells were isolated from convalescent individuals peripheral blood using SARS-CoV-2RBD protein as a bait. Single cell library building, transcriptome sequencing and BCR sequence assembling are performed by using Smart-seq2 technology, and finally the fully human monoclonal antibody, namely STS165, is obtained.

The heavy chain variable region and light chain variable region sequences of the monoclonal antibody STS165 obtained by the isolation of this example, and the sequencing results are shown in tables 1 and 2. Wherein, table 1 is monoclonal antibody and its sequence, table 2 is monoclonal antibody heavy chain variable region and light chain variable region single cell library-building sequencing result.

Table 1 monoclonal antibody sequences and nucleic acid sequences thereof

TABLE 2 monoclonal antibody nucleic acid sequencing results

The analysis showed that the sequences of CDR1, CDR2 and CDR3 of the heavy chain of STS165 were "GFTFDDYG", "inwngst" and "ARVGYCGGDCYSLSLAGFPDY" in sequence, and the sequences of CDR1, CDR2 and CDR3 of the light chain were "QSVSYN", "DAS" and "QQYNNWPPYT" in sequence.

Pseudo-virus neutralization assay results of STS165 monoclonal antibody

The neutralizing activity of STS165 antibodies against novel coronavirus variants was examined by a pseudovirus neutralization assay. The present example produced a pseudovirus of the WT, alpha, beta, gamma, delta, lambda, mu, kappa, eta, epsilon, iota, C.1.2 and Omicron varieties, together with a SARS-CoV pseudovirus. As shown in FIG. 1, STS165 can neutralize 13 SARS-CoV-2 variant pseudoviruses, which shows great potential use of the antibody. STS165, in particular, is also effective in neutralizing SARS-CoV, IC ₅₀ 2.15. Mu.g/mL. These data indicate that STS165 is a broad-spectrum neutralizing antibody with potential application.

3. Surface Plasmon Resonance (SPR) results

The affinity of STS165 to the novel coronaviruses and variants thereof, as well as the SARS-CoV RBD protein, was detected by surface plasmon resonance and the results are shown in table 3 and fig. 2.

TABLE 3 surface plasmon resonance results for STS165 antibodies

KD(nM)	RBD-WT	RBD-Omicron	RBD-Lambda	RBD-Mu	RBD-C.1.2	RBD-SARS-CoV
							STS165	0.1021	0.3520	0.0938	0.0733	0.0619	1.9900

The results in Table 3 and FIG. 2 show that STS165 has a higher binding affinity for both the SARS-CoV-2 variant and the RBD protein of SARS-CoV, and that it exhibits the same pattern of neutralizing activity, indicating a strong correlation between binding affinity and neutralizing activity, resulting in a broad cross-neutralizing activity for STS165.

Epitope identification of STS165 monoclonal antibody

It is well known that RBD-specific nAbs are classified into four classes based on competition with ACE2 and conformational epitopes on RBD that recognize up and down. This example uses competition ELISA to predict binding epitopes from the competition of STS165 with ACE 2. As a result, as shown in FIG. 3, STS165 showed strong competition with ACE2 receptor, similar to the class 1 representative antibody P2C-1F11 and the class 2 representative antibody BD-368-2. In order to more accurately identify the epitope recognized by STS165, we further determined the competition relationship of STS165 with other 14 monoclonal antibodies whose epitopes have been well studied by structural biology techniques. Of these 14 antibodies fall into four classes of typical antibodies that have been identified: category 1: P2C-1F11, REGN10933, CB6; category 2: BD-368-2, C144, P2B-2F6; category 3: s309, REGN10987, C110, C135; category 4: EY6A, H014, S304, S2A4. The results show that STS165 antibodies can compete with the 4 class 4 antibodies, indicating that STS165 belongs to class 4 antibodies whose recognition sites are distant from the Receptor Binding Motif (RBM).

Combination of STS165 monoclonal antibodies

STS165 is an ideal partner for the development of therapeutic antibody cocktail therapies because it neutralizes 13 variants, including Beta, delta, and Omacron. Such cocktails are not only an effective treatment, but also prevent antibody inactivation by viral escape mutants that might escape from the selective pressure of single antibody therapy. This example evaluates the neutralization of 11 pseudoviruses by STS165 in combination with 3 other non-competing antibodies (REGN 10933, REGN10987, and S309) that have been urgently approved for clinical use. As shown in FIG. 4, REGN10933 has lower neutralizing activity against Beta than WT, IC ₅₀ Significantly reduced, only 2.318 μg/mL. Furthermore, the inhibition by Omicron was less than 50% at the highest concentration of 50 μg/mL for both REGN10933 and REGN 10987. When REGN10933 and REGN10987 antibodies were used in combination with STS165, respectively, the neutralizing activity against Omicron and SARS-CoV pseudoviruses was restored to some extent. At the same time, the neutralizing activity against variant pseudoviruses was also maintained for other variants using REGN10933, REGN10987 and S309 antibodies, respectively, with STS165 cocktail therapy.

Taken together, these results indicate that STS165 is not only a broad-spectrum neutralizing antibody with good potential for use, but also can form cocktails with different emergency-approved antibodies for use against the emerging SARS-CoV-2 variety. In summary, the functional studies of this example revealed a novel broad-spectrum neutralizing antibody STS165, the findings of this example underscores the importance of antibody-based COVID-19 therapeutic intervention.

The foregoing is a further detailed description of the present application in connection with the specific embodiments, and it is not intended that the practice of the present application be limited to such descriptions. It will be apparent to those skilled in the art to which the present application pertains that several simple deductions or substitutions may be made without departing from the spirit of the present application.

SEQUENCE LISTING

<110> Shenzhen national institute of clinical medicine for infectious diseases

Shenzhen Third People's Hospital

<120> a novel coronavirus monoclonal antibody and use thereof

<130> 22I33726

<160> 4

<170> PatentIn version 3.3

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<213> STS165 heavy chain variable region

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Tyr Asp Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 3

<211> 384

<212> DNA

<213> STS165 heavy chain variable region

<400> 3

gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120

ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180

gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240

ctgcaaatga acagtctgag agccgaggac acggccttgt atcactgtgc gagagtcggg 300

tattgtggtg gtgattgcta ttctttatcg cttgctggtt ttcctgacta ctggggccag 360

ggaaccctgg tcaccgtctc ctca 384

<210> 4

<211> 324

<212> DNA

<213> STS165 light chain variable region

<400> 4

gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc tacaacttag cctggtacca gcagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccacca gggccactgg tatcccagcc 180

aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag tctgcagtct 240

gaagattttg cagtttatta ctgtcagcag tataataact ggcctccgta cacttttggc 300

caggggacca agctggagat caaa 324

Claims

1. A novel coronavirus monoclonal antibody, characterized in that: the novel coronavirus monoclonal antibody is STS165; the monoclonal antibody consists of a heavy chain and a light chain paired with the heavy chain;

the sequences of CDR1, CDR2 and CDR3 of the heavy chain of STS165 are in sequence "GFTFDDYG", "inwngst" and "ARVGYCGGDCYSLSLAGFPDY", and the sequences of CDR1, CDR2 and CDR3 of the light chain are in sequence "QSVSYN", "DAS" and "QQYNNWPPYT".

2. The novel coronavirus monoclonal antibody of claim 1, wherein: the heavy chain variable region sequence of STS165 is the sequence shown by Seq ID No.1, and the light chain variable region sequence is the sequence shown by Seq ID No. 2.

3. A nucleic acid fragment encoding the novel coronavirus monoclonal antibody of claim 1 or 2, said nucleic acid fragment encoding the heavy and light chains of STS165.

4. A nucleic acid fragment according to claim 3, wherein: a nucleic acid fragment comprising a heavy chain variable region encoding the sequence shown in Seq ID No.1, the sequence of which is the sequence shown in Seq ID No. 3; a nucleic acid fragment encoding the light chain variable region shown in Seq ID No.2, which has the sequence shown in Seq ID No. 4.

5. A recombinant plasmid comprising the nucleic acid fragment of claim 3 or 4.

6. A recombinant cell comprising the nucleic acid fragment of claim 3 or 4 or the recombinant plasmid of claim 5.

7. The method for preparing a novel coronavirus monoclonal antibody according to claim 1 or 2, wherein: comprising the steps of carrying out protein expression on the recombinant plasmid of claim 5 by adopting the recombinant cell of claim 6, extracting and purifying the expressed protein, and obtaining the novel coronavirus monoclonal antibody.

8. Use of a novel coronavirus monoclonal antibody according to claim 1 or 2 or a nucleic acid fragment according to claim 3 or 4 or a recombinant plasmid according to claim 5 or a recombinant cell according to claim 6 for the preparation of a novel coronavirus control drug or a novel coronavirus detection reagent.

9. A novel coronavirus vaccine characterized in that: the novel coronavirus vaccine comprises the novel coronavirus monoclonal antibody according to claim 1 or 2.

10. A novel coronavirus detection reagent, characterized in that: the novel coronavirus detection reagent comprises the novel coronavirus monoclonal antibody according to claim 1 or 2.