Human SARS-CoV-2 antibody and application thereof

Abstract

The invention discloses a human SARS-CoV-2 antibody and application thereof. The amino acid sequence of a heavy chain variable region of the antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of a light chain variable region of the antibody is as shown in SEQ ID NO: 2. The antibody is subjected to humanized transformation based on a mouse monoclonal antibody, so that on one hand, the binding site and reactivity of an original antibody are maintained, and on the other hand, the immunogenicity when the antibody is used in a human body can be effectively reduced, and subsequent clinical transformation is facilitated; a powerful tool is provided for broad-spectrum prevention, treatment and diagnosis of diseases caused by infection of the new coronavirus SARS-CoV-2.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more particularly to a human SARS-CoV-2 antibody and its applications. Background Technology

[0002] ARS-CoV-2 is an enveloped beta-coronavirus whose spike protein (S protein) is crucial for viral invasion of host cells. The S protein consists of two functional subunits: S1 and S2. The S1 subunit recognizes and binds to the ACE2 receptor on the host cell surface, a process that determines the virus's cell tropism. The S2 subunit mediates the fusion of the viral envelope with the host cell membrane, allowing viral genetic material to enter the cell. Because the S1 subunit, particularly its receptor-binding domain (RBD), is the primary target of most neutralizing antibodies and vaccines, the novel coronavirus has evaded immunity by accumulating mutations in this region, leading to decreased efficacy of many antibody drugs and vaccines designed based on the original strain.

[0003] Therefore, the S2 subunit, with its more conserved sequence and higher homology among different coronaviruses, has become a popular target for broad-spectrum neutralizing antibodies. However, no monoclonal antibodies targeting the S2 subunit have yet been approved for marketing. Although Chinese patent CN114703147A discloses a broad-spectrum neutralizing monoclonal antibody against SARS-CoV-2, this antibody is a murine antibody and exhibits strong immunogenicity in humans, making it difficult to advance its subsequent clinical translation. Summary of the Invention

[0004] Objectives of the invention: The objective of this invention is to provide a human SARS-CoV-2 antibody with low immunogenicity, high affinity, and good specificity; the second objective is to provide nucleic acid molecules, related biological materials, and compositions encoding the human SARS-CoV-2 antibody; and the third objective is to provide applications containing the human SARS-CoV-2 antibody.

[0005] Technical solution: In order to achieve the above objectives, the human SARS-CoV-2 antibody or its antigen-binding fragment of the present invention has the following amino acid sequence in the heavy chain variable region as shown in SEQ ID NO: 1, or the amino acids that have more than 80% similarity to the sequence shown in SEQ ID NO: 1 and are different from it by conservative substitutions; the amino acid sequence in the light chain variable region is shown in SEQ ID NO: 2, or the amino acids that have more than 80% similarity to the sequence shown in SEQ ID NO: 2 and are different from it by conservative substitutions.

[0006] Preferably, the antibody or its antigen-binding fragment further includes a portion or all of the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, and a portion or all of the κ-type or λ-type light chain constant region.

[0007] Preferably, the species source of the constant region is human; more preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 3, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 4.

[0008] Preferably, the antigen-binding fragment includes any one or more of F(ab')2, Fab', Fab, Fv, ScFv, and dsFv.

[0009] The nucleic acid molecule described in this invention encodes the aforementioned human SARS-CoV-2 antibody or its antigen-binding fragment.

[0010] Preferably, the sequence of the nucleic acid molecule is shown in SEQ ID NO: 5.

[0011] The biomaterial described in this invention is any one of the following:

[0012] (a) An expression cassette containing the aforementioned nucleic acid molecules;

[0013] (b) A vector containing the aforementioned nucleic acid molecule or the expression cassette described in (a);

[0014] (c) A host cell containing the aforementioned nucleic acid molecule or the expression cassette described in (a) or the vector described in (b).

[0015] In one specific embodiment of the present invention, the host cell is a 293T cell. Other conventional eukaryotic host cells in the art, such as 293F cells and CHO cells, can also be used in the implementation of the present invention.

[0016] The composition of the present invention contains the aforementioned human SARS-CoV-2 antibody or its antigen-binding fragment, or the aforementioned nucleic acid molecule, or the aforementioned biological material as an active ingredient.

[0017] Preferably, the composition further includes pharmaceutically acceptable excipients.

[0018] The use of the human SARS-CoV-2 antibody or its antigen-binding fragment, nucleic acid molecule, biological material, or composition described in this invention in any of the following:

[0019] (1) Detect SARS-CoV-2 or the S2 subunit of the SARS-CoV-2 spike protein in the sample;

[0020] (2) Prepare products for detecting SARS-CoV-2 or the S2 subunit of the SARS-CoV-2 spike protein;

[0021] (3) To prepare drugs for the prevention of diseases caused by SARS-CoV-2 infection;

[0022] (4) To prepare drugs for relieving symptoms of disease caused by SARS-CoV-2 infection;

[0023] (5) Prepare drugs for treating diseases caused by SARS-CoV-2 infection.

[0024] This invention is based on the G5 murine monoclonal antibody disclosed in CN114703147A. After designing a humanized antibody, specific reverse mutations were performed, and finally, a humanized G5 antibody hG5 was obtained that can be highly expressed and effectively targets the highly conserved spike protein S2 subunit epitope in all SARS-CoV-2 variants. Subsequently, based on the sequence of this antibody, the heavy chain and light chain were linked with a linker to construct an expression vector, resulting in a human SARS-CoV-2 antibody hG5 single plasmid expression vector, which was successfully expressed.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0026] This human SARS-CoV-2 antibody is modified from a murine monoclonal antibody, maintaining the original antibody's binding site and reactivity while effectively reducing its immunogenicity when used in humans. This facilitates subsequent clinical translation and provides a powerful tool for the broad-spectrum prevention, treatment, and diagnosis of diseases caused by SARS-CoV-2 infection. Attached Figure Description

[0027] Figure 1 Western blot results for expression of different combinations of humanized antibody light and heavy chain mutants;

[0028] Figure 2 A statistical graph showing the reactivity of different combinations of humanized antibody light and heavy chain mutants to SARS-CoV-2 broad-spectrum polypeptide tandem fusion protein;

[0029] Figure 3 The image shows the gel electrophoresis results of the PCR amplification products during the construction of the human SARS-CoV-2 antibody hG5 single plasmid expression vector;

[0030] Figure 4 The image shows the results of Western blot identification of human SARS-CoV-2 antibody hG5 expression.

[0031] Figure 5 A statistical graph showing the reactivity of human SARS-CoV-2 antibody hG5 to SARS-CoV-2 broad-spectrum polypeptide tandem fusion protein. Detailed Implementation

[0032] The technical solution of the present invention will be further described below.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0034] The SARS-CoV-2 antigen was disclosed in the applicant's previous application (application number 202110631147.0).

[0035] 293T cells (ATCC: CRL-3216) were preserved and provided by Yangzhou University.

[0036] Example 1: Humanization of mouse monoclonal antibodies

[0037] 1. Construction of original humanized antibodies

[0038] The heavy and light chains of the murine monoclonal antibody G5 were compared and analyzed using the International Immunogenetic Information System (IMGT), and the complementarity-determining regions (CDRs) and backbone regions (FRs) of the variable regions of the light and heavy chains were annotated.

[0039] Human light and heavy chain variable region sequences with the highest homology to the G5 sequence were obtained by alignment in the IMGT database. The CDR region of G5 was transplanted into the human sequence to form the original humanized antibody light and heavy chain variable region sequences. Nanjing Qingke Biotechnology Co., Ltd. was commissioned to optimize the codons and synthesize the corresponding light chain (SEQ ID NO: 13) and heavy chain (SEQ ID NO: 14) variable region nucleic acid fragments. These fragments were cloned into Abvec-Igκ (addgene, Plasmid#183702) and Abvec-Igγ (addgene, Plasmid#80795) expression vectors through Age1 and Bswi1 restriction sites, respectively. After transfection into 293T cells, the original humanized antibody was expressed. The expression of the original humanized antibody was verified by Western blot, and the antibody reactivity was verified by indirect ELISA.

[0040] The results showed that the original humanized antibody could not recognize the triple SARS-CoV-2 broad-spectrum polypeptide tandem fusion protein (His-3×P4 protein, prepared according to the method described in CN113354717A) with the sequence shown in SEQ ID NO: 6.

[0041] 2. Mouse reversion mutation of the original humanized antibody

[0042] Using the plasmids expressing the original humanized antibody light and heavy chains obtained in step 1 as templates, one or more amino acid sites near the CDR region were selected for site-directed mutagenesis. The heavy chain amino acid mutation sites included positions 35, 50, 61, and 62, and the light chain amino acid mutation sites included positions 39, 58, and 59. The amino acid sequence of the hL1 variable region was the same as that of the original light chain (SEQ ID NO:15), and the amino acid sequence of the hH2 variable region was the same as that of the original heavy chain (SEQ ID NO:16). The specific information of the constructed reversion mutant is shown in Table 1.

[0043] Table 1. Amino acid mutation information of the recovered mutants

[0044]

[0045] After transfection with the heavy and light chain plasmids of each mutant in pairs, the expression of the mutants was verified by Western blot.

[0046] The results are as follows Figure 1 As shown, the combination of light chain variant 2 (hL2) and heavy chain variant 6 (hH6) showed better expression.

[0047] 3. Verification of the reactivity of mutants.

[0048] Reactivity was verified using an indirect ELISA method, as detailed below:

[0049] (1) Dilute His-3×P4 protein with 0.05M carbonate buffer at pH=9.6 to a protein concentration of 5 μg / mL. Add 100 μL to each well of a 96-well microplate and incubate overnight at 4℃ for coating. The coating amount is 0.5 μg / well. After coating, block with PBST containing 1% newborn calf serum and 5% skim milk powder at 37℃ for 2 h.

[0050] (2) One day before transfection, 293T cells were seeded in 12-well plates. After 16 hours, when the cell density was about 70%, the heavy and light chain plasmids expressing each mutant were combined in pairs and transfected by PEI. The transfection amount of each plasmid was 1 μg. After 6 hours of transfection, the medium was replaced with 5% FBS DMEM medium for maintenance culture. After 3 days, the cell supernatant of each well was collected as the sample to be tested.

[0051] (3) The sample to be tested was diluted with PBST containing 5% skim milk powder and 1% newborn calf serum at a ratio of 1:2 and added to the aforementioned 96-well microplate, and incubated at 37°C for 1 h;

[0052] (4) After incubation, wash each well with 360 μL of PBST three times, and then add goat anti-human IgG secondary antibody diluted with PBST containing 5% skim milk powder and 1% newborn calf serum at a ratio of 1:40000 for incubation again.

[0053] (5) After incubation, wash with PBST, repeat washing 3 times with 360 μL per well, add 100 μL TMB colorimetric solution and react at 37℃ for 10 min;

[0054] (6) After the reaction, terminate the reaction with 50 μL of 2% sulfuric acid and read the OD value at a wavelength of 450 nm to evaluate the reactivity of the sample to be tested.

[0055] The results are as follows Figure 2 As shown, compared to other variants, the combined expression of light chain variant 3 (hL3) and heavy chain variant 6 (hH6) or light chain variant 2 (hL2) and heavy chain variant 6 (hH6) can recognize the SARS-CoV-2 broad-spectrum polypeptide tandem fusion protein. Further dilution analysis showed that the combination of light chain variant 2 (hL2) and heavy chain variant 6 (hH6) exhibited better reactivity. A reactive human SARS-CoV-2 antibody (humanized G5, hG5) was finally obtained. The amino acid sequence of the heavy chain variable region of this antibody is shown in SEQ ID NO: 1, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 4. The plasmid expressing hL2 was named Abvec-Igκ-hG5, and the plasmid expressing hH6 was named Abvec-Igγ-hG5.

[0056] Example 2: Construction and expression of human SARS-CoV-2 antibody hG5 single plasmid expression vector

[0057] 1. Construction of human SARS-CoV-2 antibody hG5 single plasmid expression vector

[0058] (1) Amplification of linearized vector and antibody fragment:

[0059] PCR amplification was performed using pcDNA3.1 plasmid, Abvec-Igγ-hG5, and Abvec-Igκ-hG5 as templates, with the corresponding primers listed in Table 2. The PCR amplification reaction system consisted of 19 μL ultrapure water, 2 μL upstream primer, 2 μL downstream primer, 2 μL template plasmid (1 ng / μL), and 25 μL commercially available 2× Phata Max Master Mix high-fidelity enzyme premix. The PCR amplification cycle parameters were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 3 min, for 35 cycles, followed by a final extension at 72℃ for 10 min. After PCR, the products were analyzed by 1% agarose gel electrophoresis.

[0060] Table 2 Primer sequence information used in the construction of humanized monoclonal antibody expression vectors against SARS-CoV-2

[0061]

[0062] The results are as follows Figure 3 As shown, lane M is the DNA Marker, lane 1 is the linearized pcDNA3.1, lane 2 is the antibody heavy chain gene amplification fragment, and lane 3 is the antibody light chain gene amplification fragment.

[0063] (2) Ligation and transformation of PCR products:

[0064] Using Ezmax from Tolo Harbour Bio ® Recombinant cloning was performed using the Ultra Universal CloneMix kit. The reaction mixture consisted of 10 μL 2×Ezmax® Ultra Universal CloneMix, 100 ng of linearized vector, 20 ng of light chain amplification fragment, 40 ng of heavy chain amplification fragment, and ultrapure water to a final volume of 20 μL. The reaction conditions were 37°C for 20 min. The recombinant product was then transformed into DH5α competent cells and plated. The following day, single colonies were picked for colony PCR and sequencing verification, yielding the human SARS-CoV-2 antibody hG5 single plasmid expression vector pc-P2A-hG5. The nucleotide sequences of the light and heavy chain tandem coding regions of hG5 are shown in SEQ ID NO: 5.

[0065] 2. Expression and validation of human SARS-CoV-2 antibody hG5

[0066] The aforementioned pc-P2A-hG5 and empty vector pcDNA3.1 plasmids were transfected into 293T cells at 4 μg each. After 3 days, the cell culture supernatant was collected, and protein loading buffer was added to the collected supernatant. The cells were boiled at 98°C for 10 min. After separation by SDS-PAGE gel electrophoresis, the samples were transferred to nitrocellulose (NC) membranes. The membranes were then blocked with skim milk and incubated with HRP-labeled goat anti-human IgG secondary antibody at room temperature for 1 h. After washing three times with PBST, ECL chromogenic solution was added, and the cells were detected using a chemiluminescence analyzer.

[0067] The results are as follows Figure 4 As shown, lane M is the protein marker, lane 1 is the culture supernatant of 293T cells transfected with the empty vector plasmid, and lane 2 is the culture supernatant of 293T cells transfected with pc-P2A-hG5. It can be seen that the size of the heavy and light chain bands is as expected.

[0068] Example 3: Reactivity detection of human SARS-CoV-2 antibody hG5 against the SARS-CoV-2 S2 subunit

[0069] The cell culture supernatant obtained in Example 2 after transfection with pc-P2A-hG5 plasmid was used as the test sample, and the reactivity was verified by the indirect ELISA method described in Example 1.

[0070] The results are as follows Figure 5 As shown, the humanized monoclonal antibody hG5 can effectively recognize the His-3×P4 protein, which means it can effectively recognize the S2 subunit of the SARS-CoV-2 spike protein.

Claims

1. A human SARS-CoV-2 antibody or its antigen-binding fragment, characterized in that, 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: 1, or the amino acids that have more than 80% similarity to the sequence shown in SEQ ID NO: 1 and are different from it are conservative substitutions; the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 2, or the amino acids that have more than 80% similarity to the sequence shown in SEQ ID NO: 2 and are different from it are conservative substitutions.

2. The human SARS-CoV-2 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes a portion or all of the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as a portion or all of the κ-type or λ-type light chain constant region.

3. The human SARS-CoV-2 antibody or its antigen-binding fragment according to claim 2, characterized in that, The species of the constant region is human, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

4.

4. The human SARS-CoV-2 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragments include any one or more of F(ab')2, Fab', Fab, Fv, ScFv, and dsFv.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the human SARS-CoV-2 antibody or its antigen-binding fragment as described in any one of claims 1-4.

6. The nucleic acid molecule according to claim 5, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO:

5.

7. A biomaterial, characterized in that, The biomaterial is any one of the following: (a) An expression cassette containing the nucleic acid molecule of claim 5 or 6; (b) A vector containing the nucleic acid molecule of claim 5 or 6 or the expression cassette of (a); (c) A host cell containing the nucleic acid molecule of claim 5 or 6, or the expression cassette of (a) or the vector of (b).

8. A composition, characterized in that, The composition contains the human SARS-CoV-2 antibody or its antigen-binding fragment as described in any one of claims 1-4, or the nucleic acid molecule as described in claim 5 or 6, or the biomaterial as described in claim 7 as the active ingredient.

9. The composition according to claim 8, characterized in that, The composition also includes pharmaceutically acceptable excipients.

10. The use of the human SARS-CoV-2 antibody or its antigen-binding fragment according to any one of claims 1-4, or the nucleic acid molecule according to claim 5 or 6, or the biological material according to claim 7, or the composition according to claim 8 or 9, in any one of the following: (1) Detect SARS-CoV-2 or the S2 subunit of the SARS-CoV-2 spike protein in the sample; (2) Prepare products for detecting SARS-CoV-2 or the S2 subunit of the SARS-CoV-2 spike protein; (3) To prepare drugs for the prevention of diseases caused by SARS-CoV-2 infection; (4) To prepare drugs for relieving symptoms of disease caused by SARS-CoV-2 infection; (5) Prepare drugs for treating diseases caused by SARS-CoV-2 infection.

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