Novel coronavirus omicron mutant specific antibody and application thereof

Abstract

The application relates to the technical field of antibodies, in particular to a novel coronavirus Omicron mutant specific antibody and application thereof. The application provides an antibody specifically combined with the novel coronavirus Omicron mutant, the antibody can be used as a specific antibody for antigen detection of the Omicron mutant, is used for identification of a novel coronavirus vaccine designed for the Omicron mutant, and is used for quantitative detection of the content of Spike RBD protein expressed by the vaccine, or is used for quality control of a novel coronavirus vaccine and immunogenicity detection of clinical and preclinical research, or is used as a quality control antibody for detection of protective antibodies in serum after vaccination.

Description

Technical Field

[0001] This invention is a divisional application of patent application No. 2022106272663, filed on June 6, 2022, entitled "Specific Antibody Against the Omicron Mutant Strain of Novel Coronavirus and Its Application". This invention relates to the field of antibody technology, specifically to a specific antibody against the Omicron mutant strain of novel coronavirus and its application. Background Technology

[0002] The Omicron mutant strain of the novel coronavirus is a highly contagious and insidious variant that can circumvent existing vaccines and cause infection. It has been designated as the fifth "Variant of Concern" by the World Health Organization. Multivalent vaccines are considered the most effective means of combating constantly mutating viruses. Vaccine development has stringent requirements regarding immunogenicity and efficacy; therefore, antigen content detection is an indispensable and crucial step in the vaccine development process. Furthermore, the development of multivalent vaccines requires the establishment of quantitative detection methods for antigens against various mutant strains.

[0003] Currently, several vaccine companies, including Pfizer, Moderna, and AstraZeneca, are conducting extensive research on mutant strains of the novel coronavirus to advance the development of next-generation vaccines. A next-generation vaccine targeting the Omicron mutant strain is also under development.

[0004] Simplifying the development process for novel coronavirus vaccines is beneficial to accelerating vaccine research and development. However, due to the lack of long-term monitoring of the evolutionary history of the novel coronavirus and the level of population immunity, the World Health Organization has not yet established a rigorous process comparable to that for influenza vaccines to cope with the evolution of novel coronavirus strains. Convenient and efficient vaccine development tools will play a crucial role in enabling more effective vaccines to be available more quickly. However, currently, the antibodies detected for novel coronavirus vaccines are all universal antibodies and cannot specifically detect and identify vaccines designed against the Omicron mutant strain of the novel coronavirus. Summary of the Invention

[0005] The purpose of this invention is to provide a specific antibody against the Omicron mutant strain of the novel coronavirus and its application.

[0006] This invention involves immunizing mice with the Spike RBD protein of the Omicron mutant strain of the novel coronavirus as an immunogen. Following cell fusion and screening, and hybridoma cell subcloning, a hybridoma cell line expressing the antibody was obtained. Experimental verification showed that the antibody specifically recognizes the antigen recognition site of the Omicron mutant strain of the novel coronavirus. Furthermore, this invention obtained the amino acid sequence of the antibody and the nucleotide sequence of its encoding gene through hybridoma sequencing.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] The present invention provides an antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or the antigen-binding fragment thereof are shown in SEQ ID NO: 1, 2, and 3, respectively; and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO: 4, 5, and 6, respectively.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8.

[0010] Preferably, the antibodies or antigen-binding fragments thereof described above are selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies.

[0011] In some embodiments of the present invention, an antibody with clone number 3A7C12 is provided, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1, 2, and 3, respectively; the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO: 4, 5, and 6, respectively; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0012] Based on the above-mentioned antibody or its antigen-binding fragment, the present invention provides a nucleic acid molecule that encodes the antibody or its antigen-binding fragment.

[0013] Based on the amino acid sequence of the antibody or its antigen-binding fragment described above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the antibody or its antigen-binding fragment. Due to the degeneracy of codons, the nucleotide sequence encoding the nucleic acid molecule of the antibody or its antigen-binding fragment is not unique, and all nucleic acid molecules capable of encoding the antibody or its antigen-binding fragment are within the scope of protection of this invention.

[0014] Furthermore, the present invention provides a biomaterial containing the aforementioned nucleic acid molecules; the biomaterial is a carrier or a host cell.

[0015] The aforementioned vectors include, but are not limited to, plasmid vectors, bacteriophage vectors, viral vectors, and artificial chromosome vectors.

[0016] The host cells mentioned above include microbial cells, insect cells, or other animal cells.

[0017] The present invention also provides an antibody conjugate, which is obtained by conjugating the antibody or its antigen-binding fragment with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

[0018] Based on the functionality of the antibody or antigen-binding fragment thereof of the present invention, the present invention provides any of the following applications of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the biological material, or the antibody conjugate:

[0019] (1) Application in the identification or immunogenicity testing of novel coronavirus vaccines;

[0020] (2) Application in the quality control of novel coronavirus vaccines;

[0021] (3) Application in the preparation of reagents for the detection of the novel coronavirus;

[0022] (4) Application in the preparation of reagents for detecting the content of Spike RBD protein expressed by the novel coronavirus or its vaccines;

[0023] (5) Application as a quality control antibody for detecting protective antibodies in serum after immunization with novel coronavirus vaccine.

[0024] In the above applications, the identification of the novel coronavirus vaccine specifically involves using the antibody or its antigen-binding fragment provided by this invention to identify whether the novel coronavirus vaccine contains the antigen of the novel coronavirus (especially the Spike protein of the novel coronavirus Omicron mutant strain) and its content level, or to identify the authenticity of the novel coronavirus vaccine, that is, whether the vaccine is a vaccine against the novel coronavirus (especially the novel coronavirus Omicron mutant strain).

[0025] Identification methods include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, and immunochromatography.

[0026] In the above applications, immunogenicity testing specifically involves detecting the performance of the novel coronavirus vaccine in evoking an immune response in animals, including evaluating the humoral immune function of immunized animals (e.g., neutralizing antibodies and their levels, antibody affinity, etc.). The antibodies or antigen-binding fragments provided by this invention can be used as standard control antibodies for the immunogenicity testing of vaccines.

[0027] In the above applications, the quality control of the novel coronavirus vaccine specifically involves detecting whether the quality, content, and stability of the antigens in the novel coronavirus vaccine are up to standard. The antibody or antigen-binding fragment provided by this invention can be used as a binding antibody for antigen detection methods (such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, and immunochromatography) to detect the quality, content, and stability of the antigens in the vaccine.

[0028] In the above applications, the detection of the novel coronavirus specifically involves using the antibody or its antigen-binding fragment provided by this invention to detect the presence or level of the novel coronavirus (especially the Omicron mutant strain) or its Spike protein or the RBD of the Spike protein in a sample. The detection can be for diagnostic purposes (the sample is from a subject, including the subject's excrement, nasal or oral secretions, etc.) or non-diagnostic purposes (the sample is a cultured cell sample). Detection methods can include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, and other detection methods.

[0029] The reagents for detecting the novel coronavirus include the antibodies or antigen-binding fragments of the present invention, preferably the antibodies or antigen-binding fragments of the present invention further include a detectable label, and may also include a second antibody carrying the detectable label to detect the antibodies or antigen-binding fragments of the present invention.

[0030] In the above applications, detecting the Spike protein content expressed by the novel coronavirus or its vaccine specifically involves using the antibody or its antigen-binding fragment provided by this invention to detect the level of Spike protein or its RBD in a sample. The detection is for diagnostic purposes (the sample comes from a subject, including the subject's excrement, nasal or oral secretions, etc.) or non-diagnostic purposes (the sample is a cultured cell sample). Detection methods can include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assays, and similar methods.

[0031] In the above applications, the antibody or its antigen-binding fragment provided by the present invention can also be used as a quality control antibody for detecting protective antibodies in serum after immunization with a novel coronavirus vaccine. Specifically, it can be used as a standard control antibody in the process of detecting protective antibodies using detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, and immunochromatography.

[0032] Preferably, in the above applications, the novel coronavirus is the Omicron mutant strain of the novel coronavirus.

[0033] The present invention provides a test kit for the novel coronavirus or its vaccine, which contains the antibody or its antigen-binding fragment, or contains the antibody-drug conjugate.

[0034] The present invention provides a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof.

[0035] The above-described pharmaceutical compositions are used for the prevention or treatment of novel coronavirus infection or diseases related to novel coronavirus infection. The antibodies or antigen-binding fragments provided by this invention can be used as the sole active ingredient in the pharmaceutical composition, or in combination with other active pharmaceutical ingredients. The pharmaceutical compositions may also contain pharmaceutically permissible excipients.

[0036] The beneficial effects of this invention include at least the following: This invention provides an antibody that specifically binds to the Omicron mutant strain of the novel coronavirus. This antibody binds to a specific spatial epitope and specifically binds only to the Spike RBD of the Omicron mutant strain of the novel coronavirus, exhibiting high affinity, while not binding to wild-type or other mutant antigens (Alpha, Beta, Gamma, Delta). It is an ideal antibody for detecting Omicron mutant antigens and can be used for the detection and identification of Omicron mutant strains or novel coronavirus vaccines or multivalent vaccines designed against Omicron mutant strains. It can also be used for the quantitative detection of the Spike RBD protein expressed in the vaccine, or for the quality control and immunogenicity detection of novel coronavirus vaccines or multivalent vaccines in clinical and preclinical studies. Furthermore, it can be used as a quality control antibody for detecting protective antibodies in serum after vaccination. This antibody can serve as a convenient and efficient vaccine development tool, which is beneficial for accelerating the research and development process of next-generation vaccines. Attached Figure Description

[0037] Figure 1 The above are the SDS-PAGE identification results of the specific novel coronavirus Omicron mutant antibody in Example 3 of this invention. The protein molecular weight marker bands are 116.0, 66.2, 45.0, 35.0, 25.0, 18.4, and 14.4 kDa from top to bottom.

[0038] Figure 2 This is the SEC-MALS identification result of the specific novel coronavirus Omicron mutant antibody in Example 3 of the present invention.

[0039] Figure 3 This is the result of ELISA specific binding analysis of the antibody against the Omicron mutant strain of the novel coronavirus in Example 3 of the present invention.

[0040] Figure 4 The results of BLI analysis of the specific novel coronavirus Omicron mutant antibody in Example 3 of this invention are shown.

[0041] Figure 5 The results of quantitative analysis of SpikeRBD protein in the antibody against the Omicron mutant strain of the novel coronavirus in Example 3 of this invention. Detailed Implementation

[0042] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0043] Example 1: Preparation of Omicron Antibody Specific to Novel Coronavirus

[0044] In this embodiment, a novel coronavirus Omicron mutant specific antibody was prepared according to the following method:

[0045] 1. Mouse Immunization: Mice were immunized with the Spike RBD protein of the Omicron mutant strain of the novel coronavirus (purchased from Acrobiosystems). After immunization, the serum of the immunized animals was detected by ELISA. If the immunized animals achieved an immune response to the immunogen after routine immunization, cell fusion was performed.

[0046] 2. Screening: The supernatant of fused cells was screened using the ELISA method, and cells that showed positive specific binding to the Spike RBD protein of the Omicron mutant strain of the novel coronavirus and did not bind to wild-type, Alpha, Beta, Gamma, or Delta Spike RBD proteins were selected.

[0047] 3. Clonal expansion culture: Transfer positive maternal clones to 24-well plates for expansion culture. Collect the supernatant from each expanded culture clone and perform ELISA detection.

[0048] 4. Subcloning: Positive maternal clones were subcloned using the limiting dilution method, and subcloning was screened using the ELISA method.

[0049] 5. Hybridoma cell antibody gene sequencing: Total RNA was extracted from hybridoma cells and reverse transcribed into cDNA using RT-PCR. The antibody light and heavy chain sequences were cloned and constructed into a T vector, followed by DNA sequencing analysis to obtain the antibody gene sequence.

[0050] 6. Antibody production and purification: The antibody gene sequence obtained in step 5 was cloned into the expression vector and transfected into HEK293 cells for amplification culture. The antibody was purified by protein A / G affinity chromatography and the purified antibody was stored in phosphate-buffered saline (PBS) by dialysis.

[0051] Example 2: Antibody Specificity Analysis of the Omicron Mutant of Novel Coronavirus

[0052] In this embodiment, nine monoclonal antibodies with different sequences were obtained according to the method in Example 1 above, and the specificity of the antibodies against the Omicron mutant strain of the novel coronavirus was analyzed by enzyme-linked immunosorbent assay (ELISA). The analysis method is as follows:

[0053] 1. Dilute the wild-type, Alpha, Beta, Gamma, Delta, and Omicron SPIKE RBD proteins of the novel coronavirus to 2 μg / mL using CBS (0.015 mol / L Na2CO3, 0.035 mol / L NaHCO3, 0.0077 mol / L NaN3, pH 9.59), and add 100 μL to each well of an ELISA plate. Seal the plate with sealing film and incubate overnight at 4°C.

[0054] 2. Discard the liquid in the wells, blot dry the microplate, wash the plate with PBST washing buffer (300 μL / well), blot dry the plate, and perform the next washing. Repeat the washing process 3 times in total.

[0055] 3. Add 100 μL of blocking agent (PBST washing buffer containing 1.5% BSA) to each well, seal the plate with sealing film, incubate at 37°C, and then wash.

[0056] 4. Dilute the above-mentioned novel coronavirus Omicron mutant antibody to 1 μg / mL with sample dilution buffer (PBST washing buffer containing 0.5% BSA). Add 100 μL to each well of the ELISA plate. Seal the plate with sealing film and incubate at 37°C, then wash.

[0057] 5. Dilute HRP-Anti-Human IgG to 0.05 μg / mL with sample dilution buffer, add 100 μL to each well, seal with sealing film, incubate at 37°C, and then wash.

[0058] 6. Add 100 μL of colorimetric solution to each well. Seal the plate with sealing film and incubate at 37°C in the dark.

[0059] 7. Add 50 μL of stop solution to each well and gently shake the microplate until the color development is uniform.

[0060] 8. Read the absorbance values ​​at 450nm and 630nm using an ELISA reader, and use OD... 450 Deduct OD 630 The absorbance value (OD value) is obtained.

[0061] The absorbance values ​​(OD values) of each monoclonal antibody are shown in Table 1.

[0062] Table 1. OD values ​​of different antibodies detected by ELISA

[0063]

[0064] The above experimental results show that among the nine novel coronavirus antibodies, clones 1B12G6, 3A7C12, 3C6E1, and 9A5C5 exhibited high specificity for the Omicron mutant strain of the novel coronavirus and showed no cross-reactivity with other mutant strains.

[0065] Example 3: Analysis, identification, and functional analysis of specific antibodies against the Omicron mutant strain of the novel coronavirus.

[0066] In this embodiment, the novel coronavirus Omicron mutant specific antibody (clone number 3A7C12) screened in Example 2 was analyzed, identified, and functionally analyzed using methods known in the art, as follows:

[0067] 1. SDS-PAGE identification results ( Figure 1 The results showed that the two bands in the reduction electrophoresis of the 3A7C12 clone antibody were approximately 25kDa and 50kDa, respectively, with a purity greater than 99%.

[0068] 2. SEC-MALS identification results ( Figure 2 The results showed that the 3A7C12 clone antibody had a purity greater than 99% and a molecular weight of 150 kDa.

[0069] 3. ELISA combined with experimental results ( Figure 3 The results showed that the antibody of clone 3A7C12 could specifically recognize the novel coronavirus Omicron mutant antigen (the Spike RBD protein of the novel coronavirus Omicron mutant strain), but did not bind to the Spike RBD protein of the novel coronavirus wild type and the Alpha, Beta, Gamma, Delta mutant strains.

[0070] 4. BLI analysis data ( Figure 4The results show that the seven fitted lines from top to bottom represent the changes in affinity and dissociation over time between the Omicron mutant antigen and the antibody of clone 3A7C12 at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 0 nM. The results indicate that the antibody of clone 3A7C12 has an affinity of up to 9.07 nM for the Omicron mutant antigen of the novel coronavirus.

[0071] 5. Results of quantitative detection of novel coronavirus Omicron mutant antigen ( Figure 5 This indicates that the antibody from clone 3A7C12 can be used to quantitatively detect the Omicron mutant antigen of the novel coronavirus using a double-antibody sandwich method, thereby obtaining the content of the Spike RBD protein in the vaccine.

[0072] 6. The subtype identification results of the antibody clone number 3A7C12 showed that the subtype of the antibody was IgG1 kappa as detected by the Ig Isotyping Mouse Instant ELISA Kit (catalog number: 88-50660, Invitrogen).

[0073] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against the Spike RBD protein of the Omicron mutant strain of the novel coronavirus, characterized in that, The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 1, 2, and 3, respectively; the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO: 4, 5, and 6, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, single-chain antibody, animal-derived antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody.

4. A nucleic acid molecule, characterized in that, It encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3.

5. A biomaterial, characterized in that, It contains the nucleic acid molecule as described in claim 4, and the biological material is a carrier or host cell.

6. An antibody conjugate, characterized in that, It is obtained by conjugating the antibody or its antigen-binding fragment according to any one of claims 1 to 3 with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

7. Any of the following applications of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the biological material according to claim 5, or the antibody conjugate according to claim 6: (1) Application in the identification of novel coronavirus vaccines; (2) Application in the immunogenicity testing of novel coronavirus vaccines; (3) Application in the quality control of novel coronavirus vaccines; (4) Application in the preparation of reagents for the detection of the novel coronavirus; (5) Application in the preparation of reagents for detecting the content of Spike RBD protein expressed by the novel coronavirus or its vaccines; (6) Application in the preparation of quality control antibodies for the detection of protective antibodies in serum after immunization with novel coronavirus vaccine.

8. The application according to claim 7, characterized in that, The novel coronavirus is the Omicron mutant strain of the novel coronavirus.

9. A test kit for the novel coronavirus or its vaccine, characterized in that, It comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, or the antibody conjugate as described in claim 6.

10. A pharmaceutical composition, characterized in that, It comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3.

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