A novel broad-spectrum coronavirus antibody embedded with a human ace2 engineered protein and applications thereof

Abstract

This invention provides a broad-spectrum COVID-19 antibody with a modified human ACE2 protein embedded within it. Using a COVID-19 antibody as a backbone, it exhibits good stability. By utilizing human ACE2 to combat variants of the novel coronavirus, it offers advantages such as broad-spectrum activity and high specificity. The antibody with the modified human ACE2 protein embedded within it can bind to COVID-19 mutants that use human ACE2 as a receptor, serving as a broad-spectrum antibody to neutralize persistently mutating mutants. This addresses the limitations of monoclonal antibodies in neutralizing immune-evading mutants, making it highly valuable for application.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and immunology, specifically relating to a broad-spectrum COVID-19 antibody with human ACE2 protein embedding and its applications. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) has caused the global pandemic of COVID-19 (2019-nCoV), prompting the unprecedented development of therapeutic antibodies, vaccines, and drugs to combat viral transmission. Coronaviruses, such as SARS and the novel coronavirus, infect hosts by binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2) molecule via their spike protein (S protein). The spike protein consists of two subunits, S1 and S2, responsible for host cell receptor recognition and membrane fusion during invasion, respectively. Furthermore, the C-terminus (CTD) of the S1 subunit, also known as the receptor-binding domain (RBD), is a crucial region for ACE2 receptor recognition. Therefore, the spike protein, especially the RBD region, is vital for viral infection. However, the spread of the virus and treatment have increased the probability of RBD region mutations. Mutations in the RBD region may enhance viral transmissibility, allow immune evasion, increase the risk of reinfection, and reduce the effectiveness of vaccines and existing treatments. Therefore, there is an urgent need to develop therapeutic antibodies and vaccines with broad-spectrum activity, or those that still offer protection / effectiveness against mutant strains, in response to the increasing number and variety of mutant strains.

[0003] The World Health Organization (WHO) has identified five variants of concern (VOCs) for the novel coronavirus based on their characteristics: Alpha, Beta, Gamma, Delta, and Omicron. Among them, the Alpha strain, especially the Delta strain, has spread globally at an alarming rate, becoming the dominant strain in 2021. The Beta strain, excluding the newly discovered Omicron strain, has the highest level of immune evasion among known mutations. These mutants have all caused varying degrees of reduced effectiveness of monoclonal antibodies and vaccines in clinical treatment. The newly discovered Omicron strain has 32 amino acid mutations in its spike protein, rendering many existing monoclonal antibodies and vaccines ineffective, which is worrying. It is worth noting that although VOCs exhibit varying degrees of immune evasion, they still utilize human ACE2 as a receptor, and the S protein of some VOCs shows an even stronger affinity for ACE2 [Han P, Li L, Liu S, et al. Receptor binding and complex structures of human ACE2 to spike RBD from omicron and delta SARS-CoV-2. Cell, 2022]. This suggests that modifying ACE2 can achieve a "remaining unchanged amidst change" approach, circumventing antibody escape caused by antigen mutations. Summary of the Invention

[0004] To address the aforementioned issues, this invention designs a broad-spectrum COVID-19 antibody with human ACE2 protein or a modified protein embedded within it. The antibody with the human ACE2 modified protein embedded within it acts as a competitor, competing with the host cell's ACE2 receptor to bind to the novel coronavirus mutant strain, thereby providing protection.

[0005] Based on the discovery made during malaria research, the inventors isolated common antibodies in malaria patients that can bind to various strains of Plasmodium falciparum. Sequence analysis revealed that these antibodies contain an extracellular segment of the human immunosuppressive receptor LAIR1, inserted in two ways: VDJ insertion and switch region insertion. These common antibodies can exert their anti-malarial function by binding to the Plasmodium protein RIFIN on the surface of infected red blood cells through the inserted LAIR1 fragment. This suggests that the human immune system, during its evolution, possesses a mechanism for inserting its own encoding gene fragments into the V region of antibodies, and this insertion mechanism is naturally occurring. Based on these two different forms of common antibodies found in malaria patients, the inventors attempted to modify a COVID-19 antibody. CB6 monoclonal antibody is an antibody targeting the S protein RBD of the COVID-19 virus, jointly developed by the teams of Academician Gao Fu and Yan Jinghua at the Institute of Microbiology (Rui Shi et al., A human neutralizing antibody targets the receptor binding site of SARS-CoV-2. Nature, May 26, 2020). However, studies have reported that many VOC mutant strains have escaped from CB6.

[0006] Using the CB6 monoclonal antibody as an example, the inventors inserted the hACE2 protein into the CB6 antibody through two insertion methods. First, hACE2 is inserted into CDR3 of the complementarity-determining region (CDR) in the variable region of the antibody heavy chain. Because the CDR is the antigen-binding site, the insertion of hACE2 disrupts the antigen-binding region, causing the antibody backbone to lose its ability to bind antigens. Second, hACE2 is inserted into the switch μ region between the variable and constant regions of the antibody heavy chain. Because the antigen-binding site of the antibody is not disrupted, the antibody backbone retains its ability to recognize antigens, thus becoming a bispecific antibody.

[0007] Therefore, the purpose of this invention is to provide a broad-spectrum COVID-19 antibody embedded with a modified human ACE2 protein. Using a COVID-19 antibody as a backbone, it exhibits good stability; using human ACE2 to combat variants of the novel coronavirus provides advantages such as broad-spectrum activity and high specificity. This also provides a novel method for constructing bispecific antibodies and can be used for the treatment of COVID-19 infected individuals.

[0008] The COVID-19 antibody backbone used can be any monoclonal antibody targeting different antigenic sites of the novel coronavirus S protein, while the embedded ACE2 itself can fight against different variants of the novel coronavirus. This novel bispecific antibody can maximize the selectivity and neutralizing activity of the maternal monoclonal antibody.

[0009] Furthermore, this invention uses different mutant versions of the hACE2 protein: hACE2-T27F and hACE2-T27F-R273Q. hACE2-T27F involves mutating amino acid 27T in the hACE2 protein to phenylalanine (hereinafter referred to as T27F) to enhance hydrophobic interactions and thus increase affinity. hACE2-T27F-R273Q involves mutating amino acid 273R in the hACE2 protein to glutamine (hereinafter referred to as R273Q), ensuring the modified protein retains the ability to bind to the SARS-CoV-2 virus but loses its enzymatic activity, such as its ability to lower blood pressure. Additionally, a full-length hACE2 protein and a truncated hACE2 peptidase domain (hereinafter referred to as hACE2-PD) were also designed. For this purpose, a total of 13 builds were designed, namely: Switch-ACE2, Switch-ACE2-T27F, Switch-ACE2-T27F-R273Q, Switch-ACE2-PD-T27F-R273Q, Switch-L-ACE2-PD-T27F-R273Q, VDJ-ACE2, VDJ-ACE2-T27F, VDJ-ACE2-T27F-R273Q, VDJ-GS-ACE2, VDJ-GS-ACE2-T27F, VDJ-GS-ACE2-T27F-R273Q, VDJ-GS-ACE2-PD-T27F-R273Q, and VDJ-ACE2-PD-T27F-R273Q. Eight constructs were expressed, and Switch-ACE2-T27F-R273Q and VDJ-GS-ACE2-T27F-R273Q were selected for further functional studies, which verified their effectiveness.

[0010] Therefore, the present invention provides a broad-spectrum COVID-19 antibody embedded from human ACE2 protein, characterized in that, using the COVID-19 antibody as a backbone, the human ACE2 protein is inserted into the CDR3 of the complementarity-determining region in the variable region of the COVID-19 antibody heavy chain, or inserted into the switch μ region between the variable region and the constant region of the antibody heavy chain.

[0011] In a preferred embodiment, the COVID-19 antibody is a monoclonal antibody, more preferably using a CB6 monoclonal antibody as its backbone. More specifically, the amino acid sequence of the CB6 antibody heavy chain is shown in SEQ ID NO: 2.

[0012] In a preferred embodiment, the human ACE2 protein is a full-length wild-type human ACE2 protein, the peptidase domain of the wild-type human ACE2 protein, and an amino acid-substituted modified human ACE2 protein. Preferably, the modified human ACE2 protein, relative to the wild-type human ACE2 protein, has a T27F mutation, or a T27F and R273Q mutation. More specifically, the amino acid sequence of the wild-type hACE2 protein is as shown in SEQ ID NO: 6; the amino acid sequence of the modified human ACE2 protein is as shown in SEQ ID NO: 8, or as shown in SEQ ID NO: 10; the amino acid sequence of the peptidase domain of the wild-type human ACE2 protein is as positions 1-597 in SEQ ID NO: 6.

[0013] Preferably, the human ACE2 protein is inserted into amino acid positions 139-140 of the monoclonal antibody CB6 heavy chain in a non-interval connection manner; more specifically, its amino acid sequence is as shown in SEQ ID NO: 12, 14, 16, 18; or the human ACE2 protein is inserted into amino acid positions 139-140 of the monoclonal antibody CB6 heavy chain using an L flexible hinge region as a linker (preferably the sequence: LDPGLQPGNFSAD); more specifically, its amino acid sequence is as shown in SEQ ID NO: 20.

[0014] In another preferred embodiment, the human ACE2 protein is inserted into amino acids 125-126 of the monoclonal antibody CB6 heavy chain in a non-interval connection manner, more specifically, the amino acid sequence of which is as shown in SEQ ID NO: 22, 24, 26, 36; or the human ACE2 protein is inserted into amino acids 125-126 of the monoclonal antibody CB6 heavy chain again using the GS flexible hinge region (preferably the sequence: GGGSGS) as a linker, more specifically, the amino acid sequence of which is as shown in SEQ ID NO: 28, 30, 32, 34.

[0015] Preferably, the light chain is the light chain of the CB6 antibody, specifically its amino acid sequence as shown in SEQ ID NO: 4.

[0016] The present invention also provides the encoding gene of the broad-spectrum COVID-19 antibody. Preferably, the encoding nucleotide sequence of the human ACE2 protein is shown in SEQ ID NO: 5, 7, 9; and the encoding nucleotide sequence of the monoclonal antibody CB6 antibody heavy chain is shown in SEQ ID NO: 1. Preferably, the heavy chain encoding nucleotide sequence of the broad-spectrum COVID-19 antibody is as follows: SEQ ID NO: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35; Preferably, the light chain encoding nucleotide sequence of the broad-spectrum COVID-19 antibody is shown in SEQ ID NO: 3.

[0017] The present invention further provides a vector containing the encoding gene of the broad-spectrum COVID-19 antibody. Preferably, the heavy chain and light chain encoding genes of the antibody are cloned into a eukaryotic cell expression vector to obtain different antibody construction vectors.

[0018] This invention also provides the application of the aforementioned broad-spectrum COVID-19 antibody in the preparation of drugs for the prevention or treatment of diseases caused by COVID-19 or SARS virus. Specifically, it is for the prevention or treatment of diseases caused by wild-type, Alpha, Beta, Gamma, Delta, Omicron, and SARS-CoV viruses.

[0019] The antibody embedded in the modified hACE2 protein provided by this invention can bind to SARS-CoV-2 mutants that use hACE2 as a receptor, serving as a broad-spectrum antibody to neutralize persistently mutating mutant strains. This compensates for the inability of monoclonal antibodies to neutralize immune-evading mutant strains, providing a "one-size-fits-all" approach. For example, studies have shown that Switch-ACE2-T27F-R273Q exhibits high neutralizing activity against VOCs strains. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structure constructed from different hACE2-intercalating antibodies of the present invention.

[0021] Figure 2 A schematic diagram illustrating the specific composition of different hACE2-embedded antibodies and their binding to RBD proteins.

[0022] Figure 3 Western blot results for constructing different hACE2 intercalation antibodies; the gel image is a reducing SDS-PAGE gel with DTT added. Figure 3 In A and B, 1 is VKD-ACE2; 2 is VDJ-ACE2-T27F; 3 is VDJ-ACE2-T27F-R273Q; 4 is Switch-ACE2; 5 is Switch-ACE2-T27F; 6 is Switch-ACE2-T27F-R273Q; 7 is a negative control; and 8 is a positive control. Figure 3In C, 1 is VKD-GS-ACE2; 2 is VDJ-GS-ACE2-T27F; 3 is VDJ-GS-ACE2-T27F-R273Q; 4 is Switch-ACE2-PD-T27F-R273Q; 5 ​​is Switch-L-ACE2-PD-T27F-R273Q; 6 is VDJ-GS-ACE2-PD-T27F-R273Q; 8 is the negative control; and 9 is the positive control.

[0023] Figure 4 DTT was added to the expression and reductive SDS-PAGE results of different hACE2 intercalation antibodies.

[0024] Figure 5 This is a schematic diagram illustrating the two-site binding characteristics of hACE2 intercalation antibody to RBD.

[0025] Figure 6 The results of kinetic curves for the binding of different hACE2 intercalation antibodies to different mutant RBD proteins are shown in the figure.

[0026] Figure 7 The figure shows the results of neutralizing activity of hACE2-intercalating antibodies against different mutant pseudoviruses. Detailed Implementation Plan

[0027] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0028] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods in the following examples that do not specify specific conditions are generally performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 4th edition), or as recommended by the manufacturer.

[0029] Example 1: Antibody Construction Design To ensure that the designed antibodies can be solublely expressed and have functional activity, this embodiment designed a large number of different antibody constructs in order to obtain soluble expression constructs with good functional activity.

[0030] The inventors designed a full-length hACE2 protein and a truncated version, hACE2-PD (the peptidase domain of hACE2 is truncated from amino acids 1-597, hereinafter referred to as hACE2-PD, amino acid sequence as shown in SEQ ID NO: 38). Wild-type hACE2 protein and amino acid-substituted modified hACE2 proteins, hACE2-T27F and hACE2-T27F-R273Q, were used. The monoclonal antibody CB6 was used as the backbone, and the hACE2 protein was embedded into the CB6 antibody using two insertion methods.

[0031] 1. Switch Insertion Mode Design In this type of insertion mode, we directly insert the gene sequences of wild-type hACE2 protein (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 5 and 6, respectively), hACE2-T27F protein (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 7 and 8, respectively), hACE2-T27F-R273Q protein (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 9 and 10, respectively), and the peptidase domain hACE2-PD-T27F-R273Q (sequence shown in SEQ ID NO: 37) of hACE2-T27F-R273Q protein into the monoclonal antibody CB6 heavy chain (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 1 and 2, respectively), with a switch between the variable and constant regions. The gene sequence of the μ region (amino acid sites 139-140 of the CB6 antibody heavy chain) was linked without gaps, and the resulting sequence was artificially synthesized (synthesis services provided by GenScript) to obtain the heavy chain gene sequence of the Switch series of constructing antibodies. The light chain adopted the CB6 antibody light chain gene sequence (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 3 and 4, respectively).

[0032] The heavy chain and light chain genes of the antibody were cloned into the pCAGGS eukaryotic cell expression vector to obtain different antibody construction vectors.

[0033] The heavy chain sequence is: Switch-ACE2 (encoding nucleotide and amino acid sequences are shown in SEQ ID NO: 11 and 12, respectively) Switch-ACE2-T27F (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 13 and 14, respectively) Switch-ACE2-T27F-R273Q (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 15 and 16, respectively) Switch-ACE2-PD-T27F-R273Q (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 17 and 18, respectively) In addition, hACE2-PD-T27F-R273Q was designed, using a short amino acid sequence (the amino acid sequence is LDPGLQPGNFSAD), named the L flexible hinge region, as the flexible hinge region connecting the hACE2 protein and the antibody, and then inserted into the gene sequence of the heavy chain switch μ region (amino acids 139-140 of the antibody heavy chain).

[0034] The heavy chain sequence is: Switch-L-ACE2-PD-T27F-R273Q (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 19 and 20, respectively) 2. VDJ Insertion Mode Design In this insertion mode, the gene sequences of wild-type hACE2 protein and hACE2-T27F, hACE2-T27F-R273Q, and hACE2-PD-T27F-R273Q protein were directly inserted into the complementarity-determining region (CDR3) of the variable region of the monoclonal antibody CB6 heavy chain (sequences shown in 1 and 2). These sequences were then linked without gaps, and the resulting sequences were artificially synthesized (synthesis services provided by GenScript) to obtain the heavy chain gene sequence of the VDJ series of constructing antibodies. The light chain uses the CB6 antibody light chain gene sequence (sequences shown in SEQ ID NO: 3 and 4).

[0035] The heavy chain and light chain genes of the antibody were cloned into the pCAGGS eukaryotic cell expression vector to obtain different antibody construction vectors.

[0036] The heavy chain sequence is: VDJ-ACE2 (encoding nucleotide and amino acid sequences are shown in SEQ ID NO: 21 and 22, respectively) VDJ-ACE2-T27F (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 23 and 24, respectively) VDJ-ACE2-T27F-R273Q (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 25 and 26, respectively) VDJ-ACE2-PD-T27F-R273Q (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 35 and 36, respectively) In addition, wild-type hACE2 protein and hACE2-T27F, hACE2-T27F-R273Q, and hACE2-PD-T27F-R273Q proteins were designed. The GS flexible hinge region (amino acid sequence GGGSGS) was used as the link between the hACE2 protein and the antibody, and then inserted into the gene sequence of the CDR3 region (amino acid position 126 of the CB6 antibody heavy chain).

[0037] The heavy chain sequence is: VDJ-GS-ACE2 (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 27 and 28, respectively) VDJ-GS-ACE2-T27F (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 29 and 30, respectively) VDJ-GS-ACE2-T27F-R273Q (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 31 and 32, respectively) VDJ-GS-ACE2-PD-T27F-R273Q (encoding nucleotide and amino acid sequences as shown in SEQ ID NO: 33 and 34, respectively) All construct gene diagrams are as follows: Figure 1 The specific antibody cartoon diagram, its specific composition, and the predicted combination pattern are shown below. Figure 2 As shown.

[0038] Example 2: Experimental expression of antibody recombinant expression vector HEK 293T cells (ATCC CRL-3216) were cultured in DMEM containing 10% FBS. Recombinant expression vectors encoding the heavy and light chains of the different antibodies described above were co-transfected into HEK 293T cells in six-well plates. Four to six hours after transfection, the cell culture medium was replaced with serum-free DMEM, and the cells were cultured for another 3 days. The supernatant was collected, and Western blotting was used to verify the soluble expression of the target protein (using goat anti-human antibody incubation). The expression results are shown below. Figure 3 As shown in the summary table, the results are as follows: Figure 1 As shown.

[0039] Of the 13 recombinant plasmid constructs, 8 were soluble and expressed, while VDJ-ACE2, VDJ-ACE2-T27F, VDJ-ACE2-T27F-R273Q, and VDJ-GS-ACE2-PD-T27F-R273Q were not expressed. Antibodies were selected for further functional studies based on the experimental objectives.

[0040] Example 3: Antibody Expression and Purification Based on the results of the expression trials, seven constructs were selected for expression and purification: Switch-ACE2, Switch-ACE2-T27F, Switch-ACE2-T27F-R273Q, Switch-ACE2-PD-T27F-R273Q, Switch-L-ACE2-PD-T27F-R273Q, VDJ-GS-ACE2, and VDJ-GS-ACE2-T27F-R273Q.

[0041] Recombinant expression vectors encoding the antibody heavy and light chains, respectively, were co-transfected into suspension 293F cells (Thermo) using a cationic polymer transfection reagent (SinoBiology), at a cell density of approximately 2 × 10⁻⁶ cells. 6 ml. Feed was added 24h and 72h post-transfection, and cell supernatant was collected on day 5. The collected supernatant was centrifuged at 6500 rpm for 40 min at 4°C, then filtered through a 0.22 μm filter to remove cell debris. The cell supernatant containing antibody proteins was loaded into a Protein A affinity chromatography column at 4°C using a peristaltic pump. Contaminating proteins were washed with 20 mM sodium phosphate buffer (pH 7.4) until the buffer was free of protein to remove non-specifically binding proteins. The target protein was eluted with 0.1 M glycine elution buffer (pH 2.0). 200 μL of 1 M Tris-HCl (pH 9.0) buffer was pre-added to the collection tube to prevent protein inactivation in an overly acidic environment. Finally, the buffer was changed to PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4).

[0042] The eluted antibody product was concentrated using a 100 kDa concentrator and then purified by Superdex 200 molecular sieve chromatography. Subsequently, the purified target protein was analyzed by SDS-PAGE (reducibility assay). Figure 4 As shown. Figure 4 The results showed that, under reducing conditions, the disulfide bonds between the heavy and light chains of the different antibody-constructed proteins obtained after purification were opened in the SDS-PAGE gel, thus appearing as two bands, and the antibody purity exceeded 95%.

[0043] Example 4: Evaluation of the dual-site binding ability of Switch series bispecific antibodies Using the Biofilm Interferometry (BLI) Octet RED system, Switch-ACE2 was selected to verify the antibody's ability to bind antigens at both CB6 and hACE2 sites. VDJ-ACE2 and hACE2-hFc were used as controls. The VDJ series antibodies, by inserting the hACE2 protein into CDR3, disrupted the antigen-binding region of the CB6 backbone, rendering the antibody backbone unable to bind antigens, leaving only hACE2 capable of binding the antigen RBD. The Switch series antibodies, by inserting the hACE2 protein into the Switch μ region, did not disrupt the antigen-binding site, thus preserving the antibody backbone's ability to recognize antigens, and thus becoming bispecific antibodies.

[0044] The specific experimental steps are as follows: a. First, three protein samples (10-15 μg / mL) were immobilized into the anti-human IgG capture (AHC) biosensor.

[0045] b. Block the hACE2 site of the antibody using 500mM 11B11 (the Fab fragment of the anti-hACE2 antibody). After blocking saturation, flow 500mM 11B11 and 500mM wild-type SARS-CoV-2 RBD (Prototype RBD). For bispecific antibodies, such as the Switch-hACE2 antibody, the RBD binding site of the CB6 antibody backbone can bind to the Prototype RBD, while VDJ-ACE2 and hACE2-hFc cannot bind to the Prototype RBD. All procedures were performed in a PBST buffer system (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 and 0.005% (v / v) Tween 20, pH 7.4).

[0046] The specific experimental design is shown in the table below: A PBST PBST PBST PBST PBST PBST B PBST sample PBST PBST PBST PBST C PBST sample PBST PBST RBD PBST D PBST sample PBST 11B11 PBST PBST E PBST sample PBST 11B11 11B11 PBST F PBST sample PBST 11B11 11B11+RBD PBST Experimental results are as follows Figure 5 As shown, Switch-ACE2, as a bispecific antibody, showed an upward trend in its binding curve after saturation of the hACE2 blocking site when the Prototype RBD was flown, proving that a second site binds to the RBD, demonstrating the feasibility of the bispecific antibody design. In contrast, VDJ-ACE2 and hACE2-hFc showed flat curves when the RBD was flown, without an upward trend, proving that no second site binds to the RBD, and thus served as negative controls for Switch-ACE2.

[0047] Example 5: Evaluation of the binding ability of antibodies to wild-type / variant strains of SARS-CoV-2 and SARS virus RBD Using surface plasmon resonance (SPR) technology, the affinity of three antibodies (CB6, Switch-ACE2-T27F-R273Q, and VDJ-GS-ACE2-T27F-R273Q), a fusion protein hACE2-T27F-R273Q-hFc, and seven strains of novel coronavirus (prototype, Alpha, Beta, Gamma, Delta, Omicron) and SARS-CoV RBD was detected.

[0048] The specific experimental steps are as follows: a. Three antibodies, CB6, Switch-ACE2-T27F-R273Q, and VDJ-GS-ACE2-T27F-R273Q, and a fusion protein hACE2-T27F-R273Q-hFc, were immobilized in the flow cells (Fc) of a Protein A chip. The amount of Switch-ACE2-T27F-R273Q and VDJ-GS-ACE2-T27F-R273Q antibodies immobilized was controlled at approximately 1,000 response units, and the amount of CB6 and hACE2-T27F-R273Q-hFc immobilized was controlled at approximately 400 response units (RU).

[0049] b. The experimental buffer system was PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), and the RBD protein was serially diluted using PBST. Then, the serially diluted RBD proteins (CB6 and hACE2-T27F-R273Q-hFc were diluted at concentrations of 3.125, 6.25, 12.5, 25, and 50 nM, while Switch-ACE2-T27F-R273Q and VDJ-GS-ACE2-T27F-R273Q were diluted at concentrations of 6.25, 12.5, 25, 50, and 100 nM) were sequentially passed through each channel.

[0050] c. Record the kinetic curves of each antibody binding to the RBD protein, and calculate the kinetic constants using BIAevaluation software 8K (Biacore, Inc.). The affinity kinetic curves of the RBD of the novel coronavirus wild-type, mutant strain, and SARS virus to the modified protein and different antibodies are shown below. Figure 6 Affinity constant (K) D As shown in the table below.

[0051] The results are as follows Figure 6 As shown in the table above, CB6 does not bind to the RBDs of SARS-CoV-2 Beta, Gamma, Omicron, and SARS-CoV strains. In contrast, Switch-ACE2-T27F-R273Q and VDJ-GS-ACE2-T27F-R273Q compensate for this deficiency and can bind to mutant strains that CB6 cannot bind to. Compared to Switch-ACE2-T27F-R273Q, VDJ-GS-ACE2-T27F-R273Q binds to the K of all RBDs. D The values ​​are higher. Switch-ACE2-T27F-R273Q, VDJ-GS-ACE2-T27F-R273Q and the K value of the mutant strain are higher. D The values ​​were all about twice as high as those of the wild type, while the K values ​​of the SARS-CoV strain were... D The value is similar to that of the Prototype strain.

[0052] Example 6: In vitro neutralizing activity assay of antibodies against wild-type / mutant strains of SARS-CoV-2 and pseudoviruses of SARS-CoV-2 The neutralizing effects of three antibodies, CB6, Switch-ACE2-T27F-R273Q, and VDJ-GS-ACE2-T27F-R273Q, on seven SARS-CoV-2 strains (Prototype, Alpha, Beta, Gamma, Delta, Omicron, and SARS-CoV) in Vero cells were determined.

[0053] Vero cells + culture medium (negative control group) and Vero cells + sham virus dilution + culture medium (positive control group) were set up.

[0054] Antibody stock solution preparation: CB6, Switch-ACE2-T27F-R273Q and VDJ-GS-ACE2-T27F-R273Q antibodies were dissolved in 10% FBS DMEM medium under sterile conditions to prepare solutions with a concentration of 120 μg / mL.

[0055] Preparation of gradient dilution buffers for each antibody stock solution: Using empty deep-well plates, each antibody stock solution was sequentially diluted 10-fold using DMEM medium with 10% FBS. In the initial gradient (i.e., the highest concentration) wells, the concentration of each antibody was 120 μg / ml (after adding an equal volume of virus diluent, the highest concentration was 60 μg / ml). Nine concentrations were set up for each protein, with three replicates for each concentration, for a total of three experiments.

[0056] Virus dilution: Dilute the pseudovirus solution with DMEM containing 10% FBS to the appropriate infection level according to the determined virus titer.

[0057] The specific experimental steps are as follows: a. The day before the sham toxicity neutralization experiment, Vero cells in the logarithmic growth phase were seeded into 96-well cell culture plates and cultured overnight in DMEM medium containing 10% FBS, 5% CO2, and at 37°C. After 24 hours, the cell confluence density reached over 90%.

[0058] b. Take the gradient dilutions of the above antibodies, add an equal volume of pseudovirus dilution to a deep well plate, mix well to obtain the mixed working solution, and incubate at 37°C for 1 hour before use.

[0059] c. Carefully discard the supernatant from the 96-well cell culture plate, and slowly and carefully add 100 μL / well of the above-mentioned mixed working solution to each Vero cell well using a pipette. Simultaneously, set up negative and positive controls for each plate. Incubate in an incubator for 15 hours.

[0060] d. Use a CQ1 instrument to read the fluorescence values ​​of each well, and calculate the half-maximum inhibition rate (IC50) using GraphPad 8.0. 50 Curve plotting as follows Figure 7 The numerical values ​​are summarized in the table below.

[0061] The results showed that CB6 antibody could not neutralize Beta, Gamma, Omicron, and SARS-CoV strains; however, Switch-ACE2-T27F-R273Q and VDJ-GS-ACE2-T27F-R273Q could effectively neutralize all SARS-CoV mutant strains and SARS virus strains. (IC50% inhibition rate...) 50 The neutralization values ​​reflect that the neutralization effectiveness of VDJ-GS-ACE2-T27F-R273Q is lower than that of Switch-ACE2-T27F-R273Q, IC 50 Values ​​below 150 ng / mL, and reduced to 300-400 ng / mL against SARS-CoV-2 Prototype and SARS-CoV strains. Switch-ACE2-T27F-R273Q can very effectively neutralize all SARS-CoV-2 mutant strains and SARS virus strains, with an IC50 value against mutant strains. 50The values ​​were below 10 ng / mL, higher than the Prototype. Furthermore, they exhibited excellent neutralizing efficacy against SARS-CoV strains. Switch-ACE2-T27F-R273Q and VDJ-GS-ACE2-T27F-R273Q compensated for the immune evasion of mutant strains by screening antibodies, demonstrating excellent broad-spectrum activity. Moreover, with viral mutation, the antibodies showed increasingly stronger or near-neutralizing activity against mutant strains compared to the Prototype strain.

[0062] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims. <110> Institute of Microbiology, Chinese Academy of Sciences <120> A novel broad-spectrum COVID-19 antibody with human ACE2 modified protein embedding and its application <130> <160> 38 <170> PatentIn version 3.5 <210> 1 <211> 1413 <212> DNA <213> Artificial sequence <400> 1 <210>2 <211> 470 <212>PRT <213>Artificial sequence <400>2 METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAEDTAVYYCARVLPMYGDYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 470 <210>3 <211> 717 <212>DNA <213>Artificial sequence <400>3 Atggagacggatacgctgctcctgtgggttttgctgctctgggttccaggttccactggtgacgacatcgtgatgacacagagccctagcagcctgagcgctagcgtgggcgacagagtgaccatcacctgcagagctagccaaagcatcagcagatacctgaactggtatcagcagaagcccggcaaggcccccaagctgctgatctacgccgctagcagcctgcagagcggcgtgcctagcagattcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgtcagcagagctacagcaccccccccgagtacaccttcggccaaggcaccaagctggagatcaagcgaactgtggctgcaccaagcgtgtttatcttccctcccagcgacgagcagctgaagagcggcaccgccagcgtggtctgtctcctgaacaacttctatcccagggaggccaaggtccagtggaaagtggacaacgccctgcaaagcggcaatagccaggagtccgtcacagagcaggacagcaaggacagcacctacagcctgtccagcaccctgaccctcagcaaggccgactacgagaagcacaaggtgtacgcttgcgaggtgacccatcagggcctgtccagccccgtgaccaagtccttcaacaggggcgaatgcagctaa 717 <210>4 <211> 238 <212>PRT <213>Artificial Sequence <400>4 METDTLLLWVLLLWVPGSTGDDIVMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPEYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECS 238 <210>5 <211> 2213 <212> DNA <213> Artificial Sequence <400>5 <210>6 <211> 737 <212>PRT <213>Artificial sequence <400>6 STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSYLDYWGQGTLVTVSS 737 <210>7 <211> 2213 <212>DNA <213>Artificial sequence <400>7 <210>8 <211> 737 <212>PRT <213>Artificial sequence <400>8 STIEEQAKFFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSYLDYWGQGTLVTVSS 737 <210>9 <211> 2213 <212>DNA <213>Artificial sequence <400>9< <210>10 <211> 737 <212>PRT <213>Artificial sequence <400>10 STIEEQAKFFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGQFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSYLDYWGQGTLVTVSS 737 <210>11 <211> 3579 <212>DNA <213>Artificial sequence <400>11 <210> 12 <211> 1192 <212> PRT <213> Artificial sequence <400> 12 <210> 13 <211> 3579 <212> DNA <213> Artificial sequence <400> 13 <210> 14 <211> 1192 <212> PRT <213> Artificial sequence <400> 14 <210> 15 <211> 3579 <212> DNA <213> Artificial sequence <400> 15 <210> 16 <211> 1192 <212> DNA <213> Artificial sequence <400> 16 <210> 17 <211> 3204 <212> DNA <213> Artificial sequence <400> 17 <210> 18 <211> 1067 <212> PRT <213> Artificial sequence <400> 18 <210> 19 <211> 3273 <212> DNA <213> Artificial sequence <400> 19 <210> 20 <211> 1090 <212> PRT <213> Artificial sequence <400> 20 <210> twenty one <211> 3579 <212> DNA <213> Artificial sequence <400> twenty one <210> twenty two <211> 1192 <212> PRT <213> Artificial sequence <400> twenty two <210> twenty three <211> 3579 <212> DNA <213> Artificial sequence <400> twenty three <210> twenty four <211> 1192 <212> PRT <213> Artificial sequence <400> twenty four <210> 25 <211> 3579 <212> DNA <213> Artificial sequence <400> 25 <210> 26 <211> 1192 <212> PRT <213> Artificial sequence <400> 26 <210> 27 <211> 3693 <212> DNA <213> Artificial sequence <400> 27 <210> 28 <211> 1212 <212> PRT <213> Artificial sequence <400> 28 <210> 29 <211> 3693 <212> DNA <213> Artificial sequence <400> 29 <210> 30 <211> 1212 <212> PRT <213> Artificial sequence <400> 30 <210> 31 <211> 3579 <212> DNA <213> Artificial sequence <400> 31 <210> 32 <211> 1212 <212> PRT <213> Artificial sequence <400> 32 <210> 33 <211> 3264 <212> DNA <213> Artificial sequence <400> 33 <210> 34 <211> 1087 <212> PRT <213> Artificial sequence <400> 34 <210> 35 <211> 3273 <212> DNA <213> Artificial sequence <400> 35 <210> 36 <211> 1090 <212> PRT <213> Artificial sequence <400> 36 <210> 37 <211> 1860 <212> DNA <213> Artificial sequence <400> 37 <210>38 <211> 620 <212>DNA <213>Artificial sequence <400>38 LDPGLQPGNFSADEEQAKFFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGQFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADGGGGSGGGGSGGS 620

Claims

1. A broad-spectrum SARS-CoV-2 antibody with human ACE2 protein embedded, characterized in that, Its heavy chain amino acid sequence is shown in SEQ ID NO: 16 or in SEQ ID NO: 32; The amino acid sequence of the light chain is shown in SEQ ID NO:

4.

2. The encoding gene of the broad-spectrum COVID-19 antibody as described in claim 1.

3. The encoding gene of the broad-spectrum COVID-19 antibody as described in claim 2, characterized in that, Its heavy chain nucleotide sequence is shown in SEQ ID NO: 15 or in SEQ ID NO: 31; The nucleotide sequence of the light chain is shown in SEQ ID NO:

3.

4. A vector containing the encoding gene of the broad-spectrum COVID-19 antibody as described in claim 3.

5. The carrier as described in claim 4, characterized in that, The heavy and light chain encoding genes of the antibody were cloned into eukaryotic cell expression vectors, respectively.

6. The use of the broad-spectrum COVID-19 antibody as described in claim 1 in the preparation of a medicament for treating diseases caused by COVID-19 or SARS virus.

7. The application as described in claim 6, characterized in that, Treatment of diseases caused by wild-type novel coronavirus, alpha mutant, beta mutant, gamma mutant, delta mutant, omega-jung mutant, and SARS virus.

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