Human-derived monoclonal antibodies against coronavirus and uses thereof

Abstract

The present application relates to a human monoclonal antibody of coronavirus and application thereof. The antibody can specifically bind to SARS-CoV-2 RBD, block the combination of SARS-CoV-2 RBD and ACE2, and inhibit the infection of coronavirus.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a high- and high-activity human monoclonal antibody against coronavirus and its applications. Background Technology

[0002] As a newly emerging infectious disease, SARS-CoV-2 currently has no specific drugs for treatment.

[0003] Therapeutic antibody drugs play an important role not only in the treatment of cancer and autoimmune diseases, but are also effective in the treatment of infectious diseases. Currently marketed drugs for the treatment and prevention of viral infections include palilizumab (Synagis) for the prevention of respiratory syncytial virus (RSV) infection in children, elbalizumab (Trogarzo) for the treatment of HIV infection, and Rabishield for post-exposure prophylaxis against rabies. Meanwhile, monoclonal antibodies against numerous viruses are in various stages of clinical trials (https: / / clinicaltrials.gov / ).

[0004] SARS-CoV-2 belongs to the coronavirus family. Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), also belonging to the coronavirus family, caused outbreaks in 2002-2003 and 2012, respectively. On January 12, 2020, the World Health Organization (WHO) officially named SARS-CoV-2 "2019 Novel Coronavirus (2019-nCoV)". Subsequently, on February 11-12, 2020, the International Committee on Taxonomy of Viruses (ICTV) announced that the official classification name of the novel coronavirus (2019-nCoV) was severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). On the same day, at the Global Research and Innovation Forum held in Geneva, the WHO announced that the official name of the disease caused by this virus was "COVID-19".

[0005] For a virus to infect a cell, it must first bind to the host's receptors via envelope proteins. Antibodies, especially neutralizing antibodies, block viral binding to these receptors by binding to envelope proteins, thus preventing viral infection. Simultaneously, antibodies bind to envelope proteins, marking free viruses or infected cells. The Fc region of the antibody recruits macrophages or complement and other immune cells and molecules, thereby clearing free viruses and infected cells. Therefore, antibodies targeting the receptor-binding domain (RBD) not only neutralize viral infection but also function through their Fc region to promote the clearance of viruses and infected cells.

[0006] Based on research on other coronaviruses, especially SARS-CoV and MERS-CoV, the key envelope protein that binds to the receptor is the spike protein (S). S can be further divided into S1 and S2. S2 mediates membrane fusion. The N-terminus (NTD) and C-terminus (CTD) of S1 are both likely the receptor bound to the receptor ACE2. Through research on SARS-CoV-2, the team discovered that the CTD is the RBD of this coronavirus, binding to the receptor ACE2. Therefore, antibodies targeting the RBD, and specifically those that block the binding of S to ACE2, may become neutralizing antibodies that inhibit viral infection. The purpose of this invention is to identify specific, protective human neutralizing antibodies against SARS-CoV-2. Summary of the Invention

[0007] To obtain a protective human neutralizing antibody, this invention first used SARS-CoV-2 RBD expressed in insect cells as an antigen. Flow cytometry was used to screen memory B cells that specifically bind to the SARS-CoV-2 RBD protein from PBMCs of recovered patients who had been infected with SARS-CoV-2 RBD. Then, RT-PCR was performed on the selected single B cells to obtain the variable region sequence and fragment of the antibody, which was further ligated to an expression vector along with the constant region. After expression and purification in mammalian cells, a series of functional tests were performed, including the binding ability to the SARS-CoV-2 RBD protein, the blocking effect of SARS-CoV-2 RBD binding to ACE2, and the neutralizing effect against SARS-CoV-2 infection. A human monoclonal antibody neutralizing SARS-CoV-2 infection was obtained and named GH12.

[0008] Specifically, the present invention is achieved through the following aspects.

[0009] In one aspect, the present invention provides a humanized monoclonal antibody or an antigen-binding fragment thereof that specifically binds to SARS-CoV-2 RBD.

[0010] Its V H The CDR of the complementarity-determining region of the chain has an amino acid sequence selected from the following group:

[0011] As shown in SEQ ID NO: 1, CDR1,

[0012] As shown in SEQ ID NO: 2, CDR2 and

[0013] CDR3 as shown in SEQ ID NO: 3;

[0014] Its V L The CDR of the complementarity-determining region of the chain has an amino acid sequence selected from the following group:

[0015] As shown in SEQ ID NO:4, CDR1,

[0016] As shown in SEQ ID NO: 5, CDR2, and

[0017] CDR3 as shown in SEQ ID NO: 6.

[0018] In one embodiment, the human monoclonal antibody or its antigen-binding fragment contains:

[0019] As shown in SEQ ID NO: 7, the heavy chain variable region, and

[0020] As shown in SEQ ID NO: 8, the light chain variable region.

[0021] In one embodiment, the human monoclonal antibody or its antigen-binding fragment contains:

[0022] As shown in SEQ ID NO: 22, the heavy chain, and

[0023] Light chains as shown in SEQ ID NO: 23.

[0024] In one embodiment, the antigen-binding fragment is selected from Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2, and biantibody.

[0025] In another aspect, the present invention provides a polypeptide containing a sequence selected from SEQ ID NO: 7, 8, 22 or 23, wherein the polypeptide is a portion of a human monoclonal antibody that specifically binds to SARS-CoV-2RBD, and

[0026] When the polypeptide contains SEQ ID NO: 7, the human monoclonal antibody further contains the polypeptide shown in SEQ ID NO: 8;

[0027] When the polypeptide contains SEQ ID NO: 8, the human monoclonal antibody further contains the polypeptide shown in SEQ ID NO: 7;

[0028] When the polypeptide contains SEQ ID NO: 22, the human monoclonal antibody further comprises the polypeptide shown in SEQ ID NO: 23; or

[0029] When the polypeptide contains SEQ ID NO: 23, the human monoclonal antibody further contains the polypeptide shown in SEQ ID NO: 22.

[0030] In another aspect, the present invention provides a polynucleotide encoding any of the aforementioned human monoclonal antibodies or their antigen-binding fragments or polypeptides.

[0031] In another aspect, the present invention provides an expression vector comprising the aforementioned polynucleotides.

[0032] In another aspect, the present invention provides a host cell comprising the above-described expression vector.

[0033] In another aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned human monoclonal antibodies or antigen-binding fragments thereof and a pharmaceutical carrier.

[0034] In another aspect, the present invention provides the use of any of the above-described human monoclonal antibodies or antigen-binding fragments thereof in the preparation of medicaments for treating SARS-CoV-2 infection.

[0035] All references cited in this specification are incorporated herein by reference in their entirety.

[0036] definition

[0037] "Antigen-binding fragments" refer to antigen-binding fragments of antibodies and antibody analogs, which typically include at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Antigen-binding fragments include those selected from Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2, biantibodies, peptides containing CDRs, etc.

[0038] The “Fab fragment” consists of a light chain, a heavy chain, CH1, and a variable region.

[0039] The “Fc” region contains two heavy chain segments, including the CH1 and CH2 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.

[0040] The “Fab′ fragment” contains a light chain and a heavy chain portion containing the region between the VH domain and the CH1 domain or the region between the CH1 and CH2 domains. Interchain disulfide bonds are formed between the two heavy chains of the two Fab′ fragments to form the F(ab′)2 molecule.

[0041] The “F(ab′)2 segment” contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab′)2 segment consists of two Fab′ segments held together by disulfide bonds between the two heavy chains.

[0042] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0043] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment containing the VH and VL domains of the antibody, which are contained within a single polypeptide chain. Generally, scFv polypeptides contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding.

[0044] A "dual antibody" is a small antigen-binding fragment with two antigen-binding sites. The fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL or VL-VH). By using a linker so short as to prevent pairing between the two domains on the same chain, the domain pairs with a complementary domain of the other chain to form two antigen-binding sites.

[0045] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of the protein, for example, the binding reaction of the monoclonal antibody of this invention to the SARS-CoV-2 RBD protein, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0046] This invention also provides pharmaceutical compositions containing human-derived, highly neutralizing monoclonal antibodies or antigen-binding fragments thereof. To prepare the pharmaceutical compositions, various desired dosage forms can be prepared by mixing the antibody or antigen-binding fragment with a pharmaceutical carrier or excipient. Examples of dosage forms for the pharmaceutical compositions of this invention include, for example, oral dosage forms such as tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated tablets, pellets, sublingual tablets, and ointments; and non-oral dosage forms such as injections, suppositories, transdermal preparations, ointments, plasters, and topical liquids. Those skilled in the art can select appropriate dosage forms based on the route of administration and the target population.

[0047] The dosage of the active ingredient in the pharmaceutical composition of the present invention varies depending on the target patient, the target organ, symptoms, method of administration, etc. It can be determined based on the doctor's judgment, taking into account the type of dosage form, method of administration, patient's age and weight, patient's symptoms, etc.

[0048] The beneficial effects of this invention are:

[0049] This invention yields a human-derived, highly neutralizing antibody: GH12. This antibody is a human-derived antibody capable of neutralizing coronavirus infection such as SARS-CoV-2. The affinity of GH12 antibody for SARS-CoV-2 RBD is 5.87 nM. The human-derived, highly neutralizing antibody GH12 effectively blocks the binding of coronavirus such as SARS-CoV-2 RBD to hACE2 and exhibits good neutralizing activity against pseudovirus infection of coronavirus such as SARS-CoV-2. The GH12 of this invention has clinical application value in the treatment and prevention of coronavirus infection such as SARS-CoV-2. Attached Figure Description

[0050] Figure 1 : Identification of SARS-CoV-2 RBD by molecular sieve chromatography and SDS-PAGE;

[0051] Figure 2 Molecular sieve chromatography and SDS-PAGE identification of GH12 antibody;

[0052] Figure 3 : Kinetic curve of GH12 antibody binding to SARS-CoV-2 RBD;

[0053] Figure 4 GHl2 antibody blocks the binding of SARS-CoV-2 RBD to HEK293T-hACE2;

[0054] Figure 5 The effect of GH12 antibody in neutralizing VSV-SARS-CoV-2 infection. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0056] Example 1: Expression and purification of SARS-CoV-2 RBD

[0057] A six-histidine tag (hexa-His-tag) and a translation stop codon were added to the 3' end of the coding region of the SARS-CoV-2 RBD protein (amino acid sequence shown in SEQ ID NO: 9). This was then incorporated into the pFastBac1 vector (purchased from Invitrogen) by linking EcoRI and XhoI. The ligation product was then transformed into DH10Bac competent cells (purchased from Tiangen) for baculovirus recombination. The recombinant baculovirus was extracted, transfected into sf9 cells (purchased from Invitrogen) for baculovirus packaging, and after viral amplification, added to Hi5 cells (purchased from Invitrogen) for SARS-CoV-2 RBD protein expression.

[0058] Cell culture medium containing the target protein was purified by nickel affinity chromatography (HisTrap™ HP(GE)) and gel filtration chromatography (Superose™ 6Increase 10 / 300GL(GE)) to obtain a relatively pure target protein. SDS-PAGE analysis showed a size of 30 kDa, and the results are as follows: Figure 1 .

[0059] Example 2: Isolation of SARS-CoV-2 RBD protein-specific memory B cells

[0060] With informed consent, 15 mL of blood was collected from recovered patients discharged after SARS-CoV-2 RBD infection, and PBMCs were isolated. The isolated PBMCs were then processed at 10... 7 SARS-CoV-2 RBD protein at a density of / mL was incubated on ice for half an hour to a final concentration of 400 nM, followed by washing twice with PBS. Then, the mixture was incubated with the following antibodies (all purchased from BD): anti-human CD3 / PE-Cy5, anti-human CD16 / PE-Cy5, anti-human CD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-human CD38 / APC, anti-human IgG / FITC, and anti-His / PE. After incubation on ice for half an hour, PBMCs were washed twice with PBS.

[0061] After washing with PBS, PBMCs were sorted by FACSAria III. Cells with PE-Cy5-APC-APC-Cy7+Pacific Blue+FITC+PE+ (i.e., B cells) were collected and directly placed into 96-well plates at 1 cell / well.

[0062] Example 3: Single B-cell PCR, sequence analysis, and human antibody design

[0063] Following the method described by Qihui Wang et al. in Science Translational Medicine, Volume 8, Issue 369, December 2016, in "Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus", the B cells obtained in Example 2 were reverse transcribed using Superscript III reverse transcriptase (Invitrogen). The reverse transcription primers are shown in Table 1, and the reaction was carried out at 55°C for 60 min.

[0064] Table 1. Primers for reverse transcription reaction

[0065]

[0066] Using this reverse transcription product as a template, PCR was performed using HotStar Tap Plus enzyme (QIAgen) to amplify the antibody variable region sequence (PCRa). The corresponding primers were designed, and the reaction conditions were as follows: 95℃, 5 min; 95℃ 30 s, 55℃ (heavy chain) / 50℃ (λ chain) 30 s, 72℃ 90 s, 35 cycles; 72℃, 7 min. This product was then used as a template for another round of PCR (PCRb), under the following conditions: 95℃, 5 min; 95℃ 30 s, 58℃ (heavy chain) / 64℃ (λ chain) 30 s, 72℃ 90 s, 35 cycles; 72℃, 7 min, yielding the PCR product.

[0067] PCR products were separated by 1.2% agarose gel electrophoresis. Bands of 400-500 bp were excised from the gel, recovered, and sent to a sequencing company for sequencing. Sequencing results were analyzed using IMGT online software.

[0068] The correct variable region sequence obtained from the analysis was ligated to the corresponding heavy chain / λ chain constant region via bridging PCR and cloned into the expression vector pCAGGS (purchased from Addgene). The heavy chain and λ chain were ligated using EcoRI and XhoI. B cell sequencing and expression plasmid construction are as follows:

[0069] The human antibody design strategy is as follows:

[0070] Heavy chain: CMV promoter-EcoRI-Leader sequences-heavy chain variable region-CH-XhoI;

[0071] Light chain (λ): CMV promoter-EcoRI-Leader sequences-light chain variable region-CL (λ) -Xho I;

[0072] The amino acid sequence of the Leader sequence is shown in SED ID NO: 18, the amino acid sequence of CH is shown in SED ID NO: 19, and the amino acid sequence of CL is shown in SED ID NO: 20. Through sequencing, the sequence of an antibody was obtained, and this antibody was named GH12.

[0073] The heavy chain variable region sequence of GH12 is shown in SEQ ID NO: 7, the light chain variable region sequence is shown in SEQ ID NO: 8, the heavy chain sequence is shown in SEQ ID NO: 22, and the light chain sequence is shown in SEQ ID NO: 23.

[0074] The sequence identity of the GH12 antibody with the germline gene is compared as follows:

[0075] Table 2. Comparison of GH12 antibody heavy chain and germline genes

[0076]

[0077] Table 3. Comparison of GH12 antibody light chain and germline genes

[0078]

[0079] Example 4: Expression of GH12 antibody

[0080] 293T cells were cultured in DMEM containing 10% FBS. 293T cells were co-transfected with plasmids containing the light and heavy chain encoding genes of the specific antibody obtained in Example 3. 4-6 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 DMEM was added again, followed by another 4 days of culture. The supernatant was then collected again.

[0081] The collected supernatant was centrifuged at 5000 rpm for 30 min, then mixed with an equal volume of buffer containing 20 mM sodium phosphate (pH 8.0). After filtration through a 0.22 μm filter membrane, the mixture was bound to a pre-packed protein A column (5 mL, GE Healthcare). The bound protein was eluted with 10 mM glycine (pH 3.0). The collected protein was concentrated and subjected to molecular sieve chromatography. The target peak was determined by SDS-PAGE (reducing and non-reducing), and the results are shown below. Figure 2 Purified GH12 antibody was obtained.

[0082] Example 5: Detection of antibody binding ability to SARS-CoV-2 RBD using surface plasmon resonance technology

[0083] Surface plasmon resonance analysis was performed using a Biacore 8K (Biacore Inc.). The specific steps are as follows:

[0084] A protein A chip (purchased from GE Healthcare) was used. The purified antibody obtained in Example 4 was immobilized on the chip based on the affinity between protein A and antibody Fc. The antibody immobilization amount was approximately 5000 RU. The SARS-CoV-2 RBD protein was serially diluted with 10 mM HEPES, 150 mM NaCl, and pH 7.4 solution, and loaded sequentially from low to high concentrations. The kinetic curve of antibody binding to SARS-CoV-2 RBD is shown below. Figure 3 As shown in Table 4, the kinetic constants of antibody binding to SARS-CoV-2 RBD are kinetic constants. The calculation of these binding kinetic constants was performed using BIAevaluation software 8K (Biacore, Inc.). It is evident that the GH12 antibody can bind to SARS-CoV-2 RBD with a high affinity.

[0085] Table 4. Kinetic constants of GH12 antibody binding to SARS-CoV-2 RBD protein

[0086]

[0087] Example 6: Detection of GH12 blocking the binding of SARS-CoV-2 RBD to ACE2

[0088] The encoding gene for hACE2 (amino acid sequence shown in SEQ ID NO: 21) was constructed into the pEGFP-N1 vector (purchased from Addgene) using XhoI and BamHI, and then fused with GFP to form the pEGFP-hACE2 plasmid. HEK293T cells were transfected with the pEGFP-hACE2 plasmid, and GFP expression was observed under a fluorescence microscope after 24 hours. HEK293T-hACE2 cells were collected, and a reaction of 2x10⁵ cells was performed. The cells were incubated with SARS-CoV-2 RBD (200 ng / mL) at room temperature for 30 min. After centrifugation at 500xg for 5 min, the supernatant was removed, and the cells were washed twice with PBS. The cells were then incubated with anti-His / APC at room temperature for 30 min, washed twice with PBS, and the fluorescence on the cell surface was detected using BD FACSCanto.

[0089] To assess the blocking effect of GH12, the purified GH12 antibody obtained in Example 4 was incubated with 200 ng / mL SARS-CoV-2 RBD at a molar ratio of 10:1 at room temperature for 1 h, followed by incubation with HEK293T-hACE2 cells. The remaining steps were the same as above, and the binding of the protein to the cells was detected using anti-His / APC. The GH12 antibody's ability to block the binding of SARS-CoV-2 RBD to HEK293T-hACE2 cells is shown below. Figure 4 As shown, GH12 antibodies can block the binding of SARS-CoV-2 RBD to HEK293T-hACE2 cells.

[0090] Example 7: Detection of SARS-CoV-2 pseudovirus infection in GH12

[0091] The purified GH12 antibody obtained in Example 4 was serially diluted 3-fold from 50 μg / mL to the 10th gradient (2.5 ng / mL) with 1.6 x 10⁻⁶ oz / mL. 4 TCID 50 VSV-SARS-CoV-2 pseudovirus was mixed and incubated at 37°C for 1 hour, then added to 96-well plates pre-seeded with Huh7 cells (purchased from the Cell Center of Basic Medical Sciences, Peking Union Medical College). After 4 hours of incubation, the culture medium and virus solution were discarded, and the cells were added to DMEM culture medium containing 10% FBS and cultured for another 48 hours. The culture medium was discarded, the cells were washed once with PBS, and 1x lysis buffer (Promega, Luciferase Assay System) was added to lyse the cells. 10 μL of the lysis buffer was added to 50 μL of the reaction substrate, and the reaction was detected using Promega Luminometers. The neutralizing capacity of the antibody against VSV-SARS-CoV-2 pseudovirus was calculated based on the luciferase activity at different concentrations. The results are shown below. Figure 5 As shown in Table 5, the statistical results are presented.

[0092] Table 5 Neutralizing effect of GH12 antibody against SARS-CoV-2 pseudovirus

[0093]

[0094] a Half-inhibitory concentration

[0095] It is evident that the GH12 antibody can neutralize the SARS-CoV-2 pseudovirus with high neutralizing activity.

[0096] In summary, GH12 antibody can serve as a high- and high-activity human monoclonal antibody against the novel coronavirus (SARS-CoV-2).

[0097] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Human monoclonal antibodies or their antigen-binding fragments, which specifically bind to SARS-CoV-2 RBD. Its V H The CDR of the complementarity-determining region of the chain includes: CDR1 as shown in SEQ ID NO: 1, As shown in SEQ ID NO: 2, CDR2, and CDR3 as shown in SEQ ID NO: 3; Its V L The CDR of the complementarity-determining region of the chain includes: As shown in SEQ ID NO: 4, CDR1, As shown in SEQ ID NO: 5, CDR2, and CDR3 as shown in SEQ ID NO:

6.

2. The human monoclonal antibody or its antigen-binding fragment as described in claim 1, comprising: As shown in SEQ ID NO: 7, the heavy chain variable region, and The light chain variable region is shown in SEQ ID NO:

8.

3. The human monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2, comprising: As shown in SEQ ID NO: 22, heavy chains, and Light chains as shown in SEQ ID NO:

23.

4. The human monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3, wherein the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, and biantibody.

5. A polypeptide, wherein the polypeptide is a component of a human monoclonal antibody that specifically binds to the SARS-CoV-2 RBD, and The polypeptide contains the polypeptides shown in SEQ ID NO: 7 and SEQ ID NO:

8.

6. The polypeptide of claim 5, wherein the polypeptide contains the polypeptides shown in SEQ ID NO: 22 and SEQ ID NO:

23.

7. A polynucleotide encoding a human monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-4, or a polypeptide as described in claim 5 or 6.

8. An expression vector comprising the polynucleotide of claim 7.

9. A host cell comprising the expression vector of claim 8.

10. A pharmaceutical composition comprising the human monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-4 and a pharmaceutical carrier.

11. Use of the human monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-4 in the preparation of a medicament for treating SARS-CoV-2 infection.

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