Humanized monoclonal antibody efficiently neutralizing SARS-cov-2 variant strain and use thereof

Abstract

Specifically, the present invention relates to a humanized monoclonal antibody efficiently neutralizing a SARS-CoV-2 variant strain and a use thereof. Six CDR regions of the antibody are as shown in SEQ ID NOs: 1-6; a heavy chain variable region of the monoclonal antibody has a full length of an amino acid sequence as shown in SEQ ID NO: 7; and a light chain variable region of the monoclonal antibody has a full length of an amino acid sequence as shown in SEQ ID NO: 8. By using B-cell culture combined with antibody cloning technology and by means of a cross-reactive antibody screening strategy, a human monoclonal antibody 2F8 with efficient neutralization activity is obtained from a patient having recovered from original SARS-CoV-2 infection. The antibody can effectively resist neutralization escape by the SARS-CoV-2 variant strain and can be used for passive prevention in a population at a high risk of SARS-CoV-2 infection and the treatment of COVID-19.

Description

A Highly Effective Human Monoclonal Antibody for Neutralizing SARS-CoV-2 Variant and Its Application Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a highly efficient human monoclonal antibody that neutralizes SARS-CoV-2 variants and its applications. Background Technology

[0002] In December 2019, a novel coronavirus—Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)—triggered a large-scale epidemic. According to the World Health Organization (WHO), as of February 25, 2024, the number of COVID-19 cases worldwide (cumulative total) was 774,771,942, of which China had 99,334,752 confirmed cases (cumulative total) and 121,993 deaths.

[0003] SARS-CoV-2 is an enveloped single-stranded RNA virus that shares approximately 79.6% genetic sequence homology with SARS coronavirus (SARS-CoV). The virus primarily encodes four key proteins, among which the spike glycoprotein (S) is a crucial protein on the surface of the viral particle and is essential for viral infectivity. It not only facilitates the binding of the virus to host cells but also participates in the process of viral entry into host cells.

[0004] SARS-CoV-2 continues to mutate during its transmission. Under the selective pressure of active immunity and passive vaccine immunity in the human body, the virus has evolved from the initial wild-type strain (WT) into multiple lineages of variants (Alpha, Beta, Gamma, Delta, and Omicron, etc.), accumulating more than 30 mutation sites on the S protein. Recently, JN.1 and KP.2 variants have emerged.

[0005] The emergence of novel variants poses challenges to the development of vaccines and antibody drugs. Newly emerging variants can render corresponding antibodies incapable of neutralizing the virus, and even cause two combined antibodies to lose their activity (Reference: Cell Rep Med. 2021 Apr20; 2(4):100255.). Currently, most early-developed neutralizing antibodies have escaped before clinical trials are completed, resulting in reduced or lost protective efficacy. Most of the commercially available neutralizing antibodies previously marketed domestically and internationally have now been largely phased out (Reference: Nature. 2022 Aug; 608(7923):593-602.). Screening for and obtaining highly efficient, broad-spectrum neutralizing antibodies targeting more conserved epitopes of the S protein is a strategy to address the escape of constantly emerging new variants (Reference: Cell Rep. 2022 Dec 20; 41(12):111845.).

[0006] Currently, there is a lack of human monoclonal antibodies that can effectively neutralize variants of the novel coronavirus. Isolating fully human monoclonal antibodies that can neutralize the SARS-CoV-2 virus from recovered COVID-19 patients can effectively overcome the above problems and is one of the main directions for the development of anti-COVID-19 drugs.

[0007] Summary of the Invention

[0008] This invention aims to provide a highly effective human monoclonal antibody that neutralizes SARS-CoV-2 variants and its applications, specifically involving a novel, highly effective, broad-spectrum human monoclonal antibody that neutralizes SARS-CoV-2. This invention isolates a highly effective human monoclonal antibody from recovered COVID-19 patients that neutralizes SARS-CoV-2 variants. This antibody targets the receptor-binding domain (RBD) of the viral spike (S) protein and effectively resists neutralization escape by SARS-CoV-2 variants. The highly effective, broad-spectrum human monoclonal antibody that neutralizes SARS-CoV-2 provided by this invention can be used for the diagnosis, prevention, and treatment of COVID-19.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The first objective of this invention is to provide a highly efficient human monoclonal antibody that neutralizes SARS-CoV-2 variants, wherein the six CDR regions of the antibody are:

[0011] The CDR1 sequence of the heavy chain variable region is shown in SEQ ID NO.1, the CDR2 sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the CDR3 sequence of the heavy chain variable region is shown in SEQ ID NO.3.

[0012] The CDR1 sequence of the light chain variable region is shown in SEQ ID NO.4, the CDR2 sequence of the light chain variable region is shown in SEQ ID NO.5, and the CDR3 sequence of the light chain variable region is shown in SEQ ID NO.6.

[0013] The CDR1 sequence of the heavy chain variable region: GYTFSYYW (SEQ ID NO.1);

[0014] The CDR2 sequence of the heavy chain variable region: IYPGDSDT (SEQ ID NO.2);

[0015] The CDR3 sequence of the heavy chain variable region: ARQGDLGDWILLGY (SEQ ID NO.3);

[0016] The CDR1 sequence of the light chain variable region: SSDVGGYNY (SEQ ID NO.4);

[0017] The CDR2 sequence of the light chain variable region: EVN (SEQ ID NO.5);

[0018] The CDR3 sequence of the light chain variable region: CSYALSRVV (SEQ ID NO.6).

[0019] Preferably, the full-length heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.7; the full-length light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.8.

[0020] Preferably, the full-length heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.9; and the full-length light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.10.

[0021] Preferably, the monoclonal antibody is a human IgG antibody.

[0022] Preferably, the antigen bound by the monoclonal antibody is the spike protein S1 of the SARS-CoV-2 virus.

[0023] Preferably, the antigen structural region bound by the monoclonal antibody is the RBD domain in the spike protein S1 of the SARS-CoV-2 virus.

[0024] A second objective of this invention is to provide a nucleic acid fragment encoding the aforementioned monoclonal antibody.

[0025] Another object of the present invention is to provide the application of the above-described monoclonal antibody in the preparation of reagents for detecting SARS-CoV-2 virus.

[0026] Another object of the present invention is to provide the use of the above-described monoclonal antibody in the preparation of reagents for inhibiting SARS-CoV-2 virus.

[0027] Another object of the present invention is to provide the use of the above-described monoclonal antibody in the preparation of a medicament for the prevention and / or treatment of diseases caused by SARS-CoV-2 virus infection.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) This invention uses B cell culture combined with antibody cloning technology and cross-antibody screening strategy to obtain a human monoclonal antibody 2F8 with high neutralizing activity from the original COVID-19 infected recovered patients.

[0030] (2) The human monoclonal antibody 2F8 provided by the present invention can effectively resist the neutralization and escape of the novel coronavirus variant JN.1, etc., and provides a new candidate drug for the passive prevention of SARS-CoV-2 infection in high-risk groups and the treatment of novel coronavirus pneumonia. Attached Figure Description

[0031] Figure 1 shows the sequence characteristics of the newly obtained antibody;

[0032] Figure 2 shows the binding results of the new antibodies to the viral S protein and the truncated S protein.

[0033] Figure 3 shows the affinity analysis between the newly obtained antibody and the viral S protein;

[0034] Figure 4 shows the IC50 results of the neutralization experiments of the new antibody against various mutant pseudoviruses.

[0035] Figure 5 shows the neutralization curves of the new antibodies against various mutant pseudoviruses. Detailed Implementation

[0036] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments.

[0037] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0038] The flow cytometer is an ultra-high-speed flow cytometer cell sorting system, purchased from Beckman Coulter, model MoFloAstrios EQ;

[0039] The fluorescently labeled antibodies IgD-FITC, CD19-ECD, 7-AAD, CD27-PC7, CD38-APCA750, IgM-PB, and CD45-KO were purchased from Beckman Coulter, model B38673.

[0040] The EBV-containing B95.8 cells were purchased from Guangzhou Shengchuang Company, model ATCC VR-1492;

[0041] The cDNA synthesis kit, SuperScript III First Strand Synthesis System, was purchased from Invitrogen, model 18080051.

[0042] Example 1: Identification and sorting of memory B cells

[0043] 1.1 Isolation of Peripheral Blood Mononuclear Cells: Patients who had recovered from SARS-CoV-2 infection and were discharged from the hospital were followed up at the outpatient clinic of the Eighth Affiliated Hospital of Guangzhou Medical University. With informed consent, intravenous EDTA-anticoagulated blood was collected, and peripheral blood mononuclear cells were isolated using density gradient centrifugation and aliquoted into 5×10⁶ cells / mL containers. 6 / tube, placed in liquid nitrogen for cryopreservation;

[0044] 1.2 Fluorescent Antibody Staining: Peripheral blood mononuclear cells were thawed in a 37°C water bath, washed three times with PBS buffer, and then stained with antibodies according to the information in Table 1 below. The cells were incubated at room temperature in the dark for 15 min, washed again with PBS buffer, and then resuspended in 400 μl of PBS buffer before being fed into a flow cytometer. The flow cytometer used was a Beckman Coulter MoFlo Astrios EQ ultra-high-speed flow cytometry cell sorting system. Fluorescent antibody staining was performed in nine analysis tubes. Tubes 1-7 contained the corresponding single fluorescently labeled antibody, tube 9 contained a mixture of seven fluorescently labeled antibodies, and tube 8 was a blank tube containing only cells. The sample tubes were stained in the same manner as tube 9.

[0045] Table 1. Information on fluorescently labeled antibody combinations in each tube for memory B cell sorting.

[0046] 1.3 Sorting of Memory B Cells: Sample tubes were analyzed, and live CD45-positive leukocytes were circled based on 7-AAD and CD45 levels. B cells were circled based on CD19 levels. IgD was defined within the IgM and IgD-negative B cell population. - IgM - CD27 + CD38 lowThe population consisted of memory B cells. The memory B cells were sorted into 96-well cell culture plates containing cell culture medium, at a density of 50–100 cells per well.

[0047] Example 2: Culture of memory B cells and screening of antibodies from culture supernatant

[0048] CpG, IL21, IL2, radiation-irradiated healthy human PBMCs, and B95.8 cell culture supernatant containing EBV were added to 96-well cell culture plates containing memory B cells and cultured for 10 days. The presence of antibodies against SARS-CoV-2 and SARS-CoVS proteins in the B cell culture supernatant was screened using a capture ELISA.

[0049] Example 3: Cloning of Antibodies

[0050] 3.1 cDNA Synthesis: For B cells that were screened and found to contain antibodies against the SARS-CoV-2 S protein in their supernatant, RNA was extracted and then reverse transcribed into cDNA. The cDNA synthesis kit was used (SuperScript III First Strand Synthesis System, Invitrogen, #18080051).

[0051] 3.2 Nested PCR amplification of the heavy and light chain variable regions of the antibody: PCR primer sequences are referenced (J Immunol Methods. 2008 Jan 1; 329(1-2):112-24.), and the reaction was performed using Phusion high-fidelity DNA polymerase (Phusion High-Fidelity PCR Master Mix with GC Buffer, NEB, #M0532s).

[0052] 3.3 The PCR products of the antibody heavy and light chain variable regions were digested with enzymes (VH, AgeI / SalI, VK, AgeI / Xhol, VL, AgeI / BsiWI) and cloned into the corresponding expression vectors (IgG1, IgK, or IgL, reference: J Immunol Methods (2008) 329(1–2):112–24.). The obtained heavy and light chain variable region gene sequences were analyzed by IMGT / V-Quest.

[0053] Example 4: Characterization of Antibodies

[0054] 4.1 Antibody production: The heavy chain and light chain vectors of the constructed antibody with defined sequences were co-transfected into 293T cells, and the culture supernatant containing the antibody was harvested 5 days later.

[0055] 4.2 Antibody purification: The culture supernatant containing antibodies was purified using a Protein A affinity column (purchased from GE Healthcare, model 17-5138-03), and the protein content was determined.

[0056] 4.3 Antibody binding reaction with S protein:

[0057] The S protein in the supernatant was captured by anti-tag antibodies and the S protein was truncated. An ELISA method was established, and the binding of the antibody and the protein was analyzed by the capture ELISA method.

[0058] Biological membrane optical interferometry (BLI) was used to analyze the binding and dissociation of antibodies and RBD. Specifically, an SA sensor (Sartorius, 18-5019) was used to sequentially detect the antibody and RBD protein to detect the affinity between the antibody and RBD.

[0059] Experimental Results: Figure 1 shows the obtained monoclonal antibody 2F8 and its sequence characteristics; Figure 2 shows the binding of monoclonal antibody 2F8 to the viral S protein and truncated S protein. The results show that monoclonal antibody 2F8 is an RBD antibody, binding to SARS-CoV-2 S, S1, and RBD with EC50 values ​​of 0.0295 μg / mL, 0.0372 μg / mL, and 0.025 μg / mL, respectively. It does not bind to S2 and NTD. 2F8 shows weak binding to SARS-CoV S and RBD. Figure 3 shows the affinity analysis of monoclonal antibody 2F8 to the viral S protein, indicating that 2F8 has a high affinity for SARS-CoV-2 RBD (Kd < 1.0 x 10⁻⁶). -12 (1 / s).

[0060] 4.4 The neutralizing activity of antibodies against SARS-CoV-2 pseudoviruses:

[0061] Vero-E6 cells were planted at a density of 2 × 10⁶ cells per well. 4 The pseudovirus (VSV-G, Kerafast) was seeded at a concentration of [number] units into 96-well plates. The next day, three copies of the serially diluted monoclonal antibody were incubated at 37°C for 30 minutes. The mixture was then added to cultured cells and incubated for another 24 hours. Fluorescence values ​​were subsequently measured using a luciferase detection system (RG062M, Beyotime).

[0062] IC50 is defined as the dilution of a virus control well (virus and cells) by 50% relative to light units after subtracting the background of the cell-only control well. IC50 values ​​are calculated using a non-linear regression method in GraphPad Prism.

[0063] Experimental results: As shown in Figures 4 and 5, it can effectively neutralize currently prevalent viral variants, including the recently emerged JN.1, with IC50 values ​​all below 10 ng / mL. It also maintains good neutralizing activity against KP.2 (IC50 of 70 ng / mL).

[0064] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A highly effective human monoclonal antibody that neutralizes SARS-CoV-2 variants, characterized in that, The six CDR regions of the antibody are: The CDR1 sequence of the heavy chain variable region is shown in SEQ ID NO.1, the CDR2 sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the CDR3 sequence of the heavy chain variable region is shown in SEQ ID NO.

3. The CDR1 sequence of the light chain variable region is shown in SEQ ID NO.4, the CDR2 sequence of the light chain variable region is shown in SEQ ID NO.5, and the CDR3 sequence of the light chain variable region is shown in SEQ ID NO.

6.

2. The monoclonal antibody according to claim 1, characterized in that, The full-length heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.7; the full-length light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.

8.

3. The monoclonal antibody according to claim 1 or 2, characterized in that, The monoclonal antibody is a human IgG type antibody.

4. The monoclonal antibody according to claim 1 or 2, characterized in that, The monoclonal antibody binds to the spike protein S1 of the SARS-CoV-2 virus.

5. The monoclonal antibody according to claim 4, characterized in that, The antigen structural region bound by the monoclonal antibody is the RBD domain in the spike protein S1 of the SARS-CoV-2 virus.

6. A nucleic acid fragment encoding the monoclonal antibody according to any one of claims 1 to 5.

7. The use of the monoclonal antibody according to any one of claims 1 to 5 in the preparation of reagents for detecting SARS-CoV-2 virus.

8. The use of the monoclonal antibody according to any one of claims 1 to 5 in the preparation of reagents for inhibiting SARS-CoV-2 virus.

9. The use of the monoclonal antibody according to any one of claims 1 to 5 in the preparation of a drug, characterized in that, The drug is a drug for the prevention and / or treatment of diseases caused by SARS-CoV-2 virus infection.

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