Monoclonal antibodies against the rbd region of the sars-cov-2 glycoprotein and uses thereof

Abstract

The application discloses four monoclonal antibodies XA326.7, XA326.8, XA326.18 and XA326.49 against novel coronavirus glycoprotein, and light chains and heavy chains thereof. The light chain and heavy chain variable region sequences of the monoclonal antibody with high neutralization activity are provided, the amino acid sequence of the light chain variable region is shown in SEQ.ID.NO.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ.ID.NO.2. The monoclonal antibody against novel coronavirus glycoprotein is prepared through a conventional hybridoma technology, four hybridoma cells XA326.7, XA326.8, XA326.18 and XA326.49 capable of stably secreting the antibody with high neutralization activity are screened, and the mAb obtained by preparing an ascites can be applied to blocking infection of novel coronavirus on ACE2 positive cells. The uniqueness of the gene sequence and the corresponding protein sequence and the CDR sequence are confirmed, and technical support is provided for development and preparation of diagnostic and therapeutic reagents.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a monoclonal antibody targeting the RBD region of the SARS-CoV-2 glycoprotein and its application. Background Technology

[0002] In addition to clinical treatment and rescue measures, preventive vaccine development and epidemiological control measures, it is also necessary to strengthen the monitoring of its host adaptability, viral evolution and infectivity, pathogenicity, and especially the development of neutralizing active antibody preparations for COVID-19, which is an acute viral pneumonia caused by the novel coronavirus SARS-CoV-2. The reserve of related technical means is of great importance.

[0003] SARS-CoV-2, along with SARS-CoV (which caused the 2003 severe acute respiratory syndrome) and MERS-CoV (which caused the 2012 Middle East respiratory syndrome), belongs to the genus β of the family Coronaviridae. Its genome is a continuous linear single-stranded RNA, encoding four structural proteins: spike, envelope, membrane, and nucleoprotein. The S protein is the most important surface protein of coronaviruses, binding to host cells expressing ACE2 molecules through its RBD region, thus determining the virus's host range and infection specificity. Therefore, specific epitopes on the S protein have become core targets for the development of therapeutic neutralizing antibodies. The N protein, on the other hand, possesses strong immunogenicity, and its specific antibodies can be used in virological, pathological, and clinical diagnostic research.

[0004] Antibodies are a core component of the body's humoral immune response and the molecular basis for the protective effect of vaccines. In past medical practice, the high specificity and neutralizing properties of antibodies have been utilized to develop therapeutic agents for various infectious diseases and toxin poisonings. For example, neutralizing antibodies against tetanus toxin and snake venom have been successfully used in the treatment of clinical patients. Regarding the novel coronavirus, scientists such as Xie Xiaoliang and Chen Wei have cloned neutralizing human antibodies from the peripheral blood of convalescent patients, and Regeneron's "cocktail" neutralizing antibody formulations are already in clinical use. Monoclonal antibodies, with their standardized and controllable immunization procedures, offer higher affinity, more diverse selection, and potential for genetic engineering modification, remaining an important means of developing antibody-based biologics.

[0005] Due to the large molecular weight of the SARS-CoV-2 virus, neutralizing antibodies constitute a relatively small proportion of all prepared antibodies. International research on therapeutic monoclonal antibodies is still in its early stages, and the number of antibodies with neutralizing activity that have been prepared is limited. An antibody monomer molecule is a tetrapeptide chain structure composed of two identical heavy chains (H chains) and two identical light chains (L chains) linked by interchain disulfide bonds. The H and L chains include an amino (N) terminus and a carboxyl (C) terminus. The variable region (V region) near the N-terminus consists of a hypervariable / complementarity-determining region (HVR / CDR) and a backbone region (FR); the constant region (C region) is located near the C-terminus. The protein fold formed by the heavy chain variable region (VH) and the light chain variable region (VL) is the antigen-binding site. The CDR / HVR is the site where the antibody binds complementary to the antigenic determinant, and the C region triggers the antigen-antibody recognition reaction.

[0006] Although human antibodies derived from peripheral blood B lymphocytes of recovered COVID-19 patients already exist, antibodies from different sources vary in affinity, neutralizing activity, and binding sites, resulting in different therapeutic effects. Continued development of new antibody agents is urgently needed to combat COVID-19, a highly dangerous acute infectious disease. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a monoclonal antibody targeting the RBD region of the SARS-CoV-2 glycoprotein and its application.

[0008] To achieve the above objectives, the present invention employs the following technical solution:

[0009] This invention discloses four monoclonal antibodies targeting the RBD region of the SARS-CoV-2 glycoprotein, including:

[0010] Named XA326.8, it comprises a light chain and a heavy chain, the amino acid sequence of which is shown in SEQ.ID.NO.1 and the amino acid sequence of which is shown in SEQ.ID.NO.2.

[0011] Named XA326.7, it comprises a light chain and a heavy chain, the amino acid sequence of which is shown in SEQ.ID.NO.3 and the amino acid sequence of which is shown in SEQ.ID.NO.4.

[0012] Named XA326.18, it comprises a light chain and a heavy chain, the amino acid sequence of which is shown in SEQ.ID.NO.5 and the amino acid sequence of which is shown in SEQ.ID.NO.6.

[0013] Named XA326.49, it comprises a light chain and a heavy chain, the amino acid sequence of which is shown in SEQ.ID.NO.7 and the amino acid sequence of which is shown in SEQ.ID.NO.8.

[0014] The present invention also includes isolated nucleic acid molecules that can encode the aforementioned monoclonal antibodies.

[0015] The present invention also discloses an expression vector based on the above-mentioned nucleic acid molecule. In addition to the nucleic acid molecule described above, the expression vector also includes an expression regulatory sequence that is operatively linked to the sequence of the above-mentioned nucleic acid molecule.

[0016] An expression vector is a nucleic acid delivery tool that enables the expression of its carried genetic material elements within host cells through transformation, transduction, or transfection. Types of vectors include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0017] The present invention also discloses a host cell comprising the above-described nucleic acid molecule or the above-described expression vector. The host cell may be a prokaryotic cell (such as a bacterial cell), a lower eukaryotic cell (yeast cell), or a higher eukaryotic cell (mammalian cell).

[0018] The present invention also discloses a detection reagent or detection kit comprising the above-mentioned monoclonal antibody, nucleic acid molecule, expression vector or host cell targeting the RBD region of the SARS-CoV-2 glycoprotein.

[0019] The present invention also discloses the application of the above-mentioned monoclonal antibody, nucleic acid molecule, expression vector or host cell targeting the RBD region of the SARS-CoV-2 glycoprotein in the preparation of SARS-CoV-2 detection products.

[0020] The present invention also discloses the application of the above-mentioned monoclonal antibody, nucleic acid molecule, expression vector or host cell targeting the RBD region of the SARS-CoV-2 glycoprotein in the preparation of drugs that inhibit SARS-CoV-2.

[0021] The present invention also discloses the application of the above-mentioned monoclonal antibody, nucleic acid molecule, expression vector or host cell targeting the RBD region of the SARS-CoV-2 glycoprotein in the preparation of antibody drug formulations for the prevention or treatment of pneumonia caused by SARS-CoV-2.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Based on the pathogenic characteristics of viral infection, this study used recombinant S1 and S1-RBD as immunogens and screening antigens, and employed mature monoclonal antibody preparation technology to screen four neutralizing antibodies from a large number of positive clones. These antibodies were used for subsequent determination of neutralizing protective activity. This provides extremely convenient conditions for the development and research of genetically engineered antibodies, with specific advantages including:

[0024] 1. The four monoclonal antibodies against the RBD region of the SARS-CoV-2 glycoprotein provided by this invention are high- and medium-activity monoclonal antibodies against the SARS-CoV-2 glycoprotein: they can block the infection of ACE2-positive cells by SARS-CoV-2 pseudovirus and can be further used for in vivo experimental research.

[0025] 2. This invention clones the amino acid sequences of the light chain and heavy chain variable regions of a monoclonal antibody against the RBD region of the SARS-CoV-2 glycoprotein. Sequence analysis confirmed the uniqueness of the antibody sequence.

[0026] 3. Analyze and obtain the amino acid sequences of the light chain and heavy chain variable regions, and on this basis, provide support for constructing highly specific and high-affinity chimeric or humanized genetically engineered antibodies against the SARS-CoV-2 glycoprotein RBD region. Attached Figure Description

[0027] Figure 1 This is a diagram showing the DNA electrophoresis results of the heavy chain (H) and light chain (K) after reverse transcription and PCR of the mRNA obtained from the four monoclonal antibodies in this invention.

[0028] Figure 2 The results of the half-inhibitory concentration (IC50) of the neutralizing activity of the four antibodies in this invention are shown, along with the specific IC50 values.

[0029] Figure 3 This is a cartoon illustration simulating the structure of the antibody variable region of the present invention;

[0030] Where A is a cartoon diagram simulating the variable region structure of XA326.7;

[0031] Where B is a cartoon diagram simulating the variable region structure of XA326.8;

[0032] Where C is a cartoon diagram simulating the variable region structure of XA326.49. Detailed Implementation

[0033] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0034] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0035] This invention immunizes Babl / c mice with recombinantly expressed SARS-CoV-2 glycoprotein molecules to screen hybridoma cell lines that stably secrete monoclonal antibodies (mAbs) against the RBD region of the SARS-CoV-2 glycoprotein XA326.7, XA326.8, XA326.18, and XA326.49 against the SARS-CoV-2 glycoprotein. Highly specific anti-SARS-CoV-2 glycoprotein RBD region mAbs are obtained by preparing ascites fluid; and the uniqueness of this protein sequence is confirmed. The invention is described in detail below with reference to specific monoclonal antibody preparation methods, antibody specificity and activity detection, and sequence detection and uniqueness determination.

[0036] 1. Preparation and specificity identification of monoclonal antibodies against the RBD region of SARS-CoV-2 glycoprotein

[0037] 1.1 Preparation and purification of monoclonal antibodies

[0038] Following the monoclonal antibody preparation method (Experimental Techniques in Cellular and Molecular Immunology (First Edition) , pp. 9-17), CD226KO mice were immunized with the SARS-CoV-2 glycoprotein antigen (50 μg per mouse, for a total of 3 mice). The initial immunization used Freund's complete adjuvant, and subsequent immunizations used Freund's incomplete adjuvant, with each immunization spaced 3 weeks apart, administered via subcutaneous injection at multiple sites, for a total of 4 immunizations. Blood was collected from the tail tip 7-10 days after the final immunization to determine serum antibody titers and assess the immunization effect. The mouse with the highest serum titer was given a booster immunization via intraperitoneal injection of the antigen. Three days later, the mouse was sacrificed, and spleen cells were isolated for cell fusion.

[0039] The cell fusion procedure was as follows: Logarithmically growing mouse myeloma cells SP2 / 0 were counted, and a suspension of immunized mouse spleen cells was prepared simultaneously. Myeloma cells and spleen cells were mixed at a ratio of 1:10, and PEG was added for cell fusion. The fused cell suspension was added to a 96-well plate containing feeder cells (normal Balb / c mouse peritoneal macrophages) and incubated at 37°C in a 5% CO2 incubator. After clones appeared, the SARS-CoV-2 glycoprotein antigen was coated onto a polystyrene ELISA plate, and the cell supernatant was detected by indirect ELISA to select positive clones. Cells containing positive clones were cloned using a limiting dilution method. After four cloning processes, a total of 53 monoclonal hybridoma cell lines capable of stably secreting positive antibodies were obtained. The different clones were numbered sequentially from XA326.1 to XA326.53.

[0040] Ascites induction and antibody purification: After obtaining a hybridoma cell line capable of stably secreting antibodies, ascites was prepared using standard techniques (《Experimental Techniques in Cellular and Molecular Immunology (First Edition)》, pp. 9-17). Babl / c mice were used as the host. Antibodies were purified from the ascites using Protein A / G affinity chromatography, achieving a mAb purity of 95%. Identification of the antibody IgG subtype followed the instructions of the Sigma assay kit. Ig subclass determination of the secreted antibodies revealed them to be IgG1 subclass with a κ light chain.

[0041] 1.2 Titer determination of monoclonal antibodies against the RBD region of SARS-CoV-2 glycoprotein

[0042] The relative affinity of ascites fluid before purification and purified mAb was determined using an indirect ELISA method. The coating antigen was a recombinant immunogen expressing SARS-CoV-2 glycoprotein. The test samples were serially diluted ascites fluid and purified mAb. The detection antibody was goat anti-mouse HRP-labeled antibody, and ABTS was used as the substrate. The ascites fluid titers of the 53 selected monoclonal antibodies were all 1×10⁻⁶. -6 .

[0043] 1.3 Identification of the neutralizing activity of monoclonal antibodies against SARS-CoV-2 glycoprotein RBD

[0044] The SARS-CoV-2 pseudovirus uses the HIV virus as a backbone, coated with the SARS-CoV-2 spike protein, and can infect cells overexpressing human ACE2. It can be used to test and study neutralizing antibodies or other drugs that block viral invasion. The pseudovirus is a single-infection virus that cannot replicate, has high safety, and can be used in P2-level laboratories. The pseudovirus carries both Firefly Luc and EGFP markers, and infection efficiency can be detected by measuring Firefly Luc and EGFP.

[0045] Experimental steps:

[0046] 1) On the first day, 5000 Cos7-Hace2 cells were seeded in each well of a 96-well plate.

[0047] 2) On the second day, remove the culture medium from the culture plate, incubate the pseudovirus and antibody sample at 37 degrees for 1 hour, add to the culture well, and centrifuge the culture plate (1200g x 2 hours, 4 degrees).

[0048] 3) Centrifuge the infected cells, remove the culture medium, and wash once with PBS; add 30 μL of 0.25% trypsin to each well, incubate at 37°C for 5 minutes, add 100 μL of complete culture medium, and continue culturing.

[0049] 4) Continue culturing for 3 days after infection (changing the medium daily) and perform luc test.

[0050] The results showed that four of the antibodies had good neutralizing activity and could bind to the RBD region of the SARS-CoV-2 glycoprotein. The antibody numbers were: XA326.7, XA326.8, XA326.18, and XA326.49.

[0051] 2. Sequencing analysis of the light and heavy chain variable regions of the monoclonal antibody against SARS-CoV-2 glycoprotein RBD.

[0052] 2.1 Culture of XA326 mAbs hybridoma cells

[0053] Hybridoma cell lines XA326.7, XA326.8, XA326.18, and XA326.49 were cultured using conventional methods and cultured in RPMI 1640 containing 15% fetal bovine serum at 37°C in a 5% CO2 incubator until the logarithmic growth phase.

[0054] 2.2 Extraction of total RNA and synthesis of cDNA first strand

[0055] Total RNA was extracted using TRIZOL Reagent (purchased from GIBCO, USA), following the instructions. The cDNA first-strand synthesis kit was also purchased from GIBCO, USA. After obtaining the total RNA, cDNA first strand was synthesized by reverse transcription according to the instructions.

[0056] 2.3 RT-PCR amplification of the VL and VH genes of XA326.7, XA326.8, XA326.18, and XA326.49 mAb. A one-step RT-PCR amplification kit was purchased from TakaRa. The VL and VH genes of the hybridoma cell antibody were amplified according to the kit instructions.

[0057] The primers are as follows:

[0058] Primers for amplifying the antibody Fd fragment and the full-length light chain gene (bases in parentheses are degenerate).

[0059] 5' primer for the V region of mouse heavy chain:

[0060] VH1:5'-GGG GAT ATC CAC CAT GG(AG)ATG(CG)AG CTG(TG)GT(CA)AT(CG)CT CTT-3'

[0061] VH2:5'-GGG GAT ATC CAC CAT G(AG)A CTT CGG G(TC)T GAG CT(TG)GGT TTT-3'

[0062] VH3:5'-GGG GAT ATC CAC CAT GGC TGT CTT GGG GCT GCT CTT CT-3'

[0063] VH4:5'-GGG GAT ATC CAC CAT GAT(AG)GT GTT(AG)AG TCT T(CT)TGT(AG)CCT G3'

[0064] Fd 3':5'-AGG CTT ACT AGT ACA ATC CCT GGG CAC AAT-3';

[0065] Mouse light chain V region 5' end primer

[0066] VL1:5'-GGG GAT ATC CAC CAT GGA GAC AGA CAC ACT CCT GCT AT-3'

[0067] VL2:5'-GGG GAT ATC CAC CAT GGA TTT TCA AGT GCA GAT TTT CAG-3'

[0068] VL3:5'-GGG GAT ATC CAC CAT GGA G(AT)C ACA (GT)(AT)C TCG GGTCTT T(GA)TA -3'

[0069] VL4:5'-GGG GAT ATC CAC CAT G(GT)C CCC(AT)(AG)C TCA G(CT)TC(CT)CT(TG)G T-3'

[0070] VL5:5'-GGG GAT ATC CAC CAT GAA GTT GCC TGT TAG GCT GTT G-3'

[0071] Light chain 3' end primer:

[0072] MLC-3':5'-GCG CCG TCT AGA ATT AAC ACT CAT TCC TGT TGA A-3';

[0073] PCR program TOUCHDOWN: reaction volume 50 μL, reaction conditions: 98℃ 5 min; 98℃ 10 s, 57℃ 30 s (+0.7℃ / cycle, ×15 cycles), 98℃ 10 s, 57℃ 30 s - 72℃ 30 s (×28 cycles), 72℃ 10 min.

[0074] 2.4 Cloning and screening of PCR amplification products

[0075] The PCR products were subjected to 1.5% agarose gel electrophoresis, and the PCR amplified fragments were recovered. Using a DNA ligation kit (purchased from TakaRa), the fragments were inserted into the pMD-18T vector (purchased from TakaRa) with an A tail added, according to the manufacturer's instructions. The ligation product was transformed into E. coli and inoculated into Amp-resistant LB agar plates and incubated overnight at 37°C.

[0076] Clones were picked from LB agar plates and incubated overnight at 37°C in Amp-resistant LB medium. Using 1 μl of bacterial culture as a template, recombinant positive E. coli clones were screened by PCR using primers designed for the variable regions of the light and heavy chains. The obtained recombinant positive E. coli clones were then cultured and their gene sequences were determined. The protein sequence of the variable region of the antibody light chain is shown in SEQ ID NO.1, and the protein sequence of the heavy chain is shown in SEQ ID NO.2.

[0077] 3. Homology analysis of the light and heavy chain amino acid sequences of XA326.7, XA326.8, XA326.18, and XA326.49 mAb

[0078] After confirming that the sequencing was error-free, the variable region gene was translated into an amino acid sequence for amino acid sequence analysis. The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2.

[0079]

[0080] Amino acid sequence homology analysis (Blastp) was performed in the non-redundant Genbank CDS translations+PDB+SwissProt+PIR+PRF protein database.

[0081] 4. Using molecular docking technology, the variable region structures of three monoclonal antibodies, XA326.7, XA326.8, and XA326.49, were analyzed.

[0082] Determine the spatial structure that binds to the novel coronavirus, such as Figure 3 As shown in the figure, the spatial relationship between the antibody and the structural protein of the novel coronavirus can be seen.

[0083] Neutralizing antibodies targeting specific viral sites are derived from immunized mice. In these mice, B cells undergoing VDJ gene recombination exhibit high affinity for the B cell receptor (BCR) upon antigen stimulation, leading to the selection and dominant amplification of high-affinity B cells. Following somatic cell high-frequency mutation and affinity maturation, highly specific and high-affinity antibodies against various antigenic epitopes can be produced. Using in vitro methods, employing enzyme-linked immunosorbent assay (ELISA) and pseudovirus infection neutralization screening techniques, highly specific and high-affinity murine neutralizing antibodies against SARS-CoV-2 can be screened. The light and heavy chain variable region genes of these antibodies are cloned, representing unique and novel protein sequences. The uniqueness of these gene sequences and corresponding protein sequences, along with their CDR sequences, have been confirmed. This provides crucial technical support for the development of diagnostic and therapeutic reagents, and can be applied to research on the mechanisms of SARS-CoV-2 infection, as well as serving as an important diagnostic and immunotherapeutic technique, demonstrating significant practical value.

[0084] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A monoclonal antibody targeting the RBD region of the SARS-CoV-2 glycoprotein, characterized in that, Named XA326.8, it comprises a light chain and a heavy chain, wherein the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.1, and the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.

2.

2. The application of the monoclonal antibody against the RBD region of the SARS-CoV-2 glycoprotein as described in claim 1 in the preparation of SARS-CoV-2 detection products.

3. The use of the monoclonal antibody against the RBD region of the SARS-CoV-2 glycoprotein as described in claim 1 in the preparation of drugs that inhibit SARS-CoV-2.

4. The use of the monoclonal antibody against the RBD region of the SARS-CoV-2 glycoprotein as described in claim 1 in the preparation of an antibody drug formulation for the prevention or treatment of pneumonia caused by SARS-CoV-2.

5. A detection reagent or kit comprising the monoclonal antibody against the RBD region of the SARS-CoV-2 glycoprotein as described in claim 1.

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