CN113292649B

CN113292649B - Human monoclonal antibodies to novel coronaviruses and uses thereof

Info

Publication number: CN113292649B
Application number: CN202010114282.3A
Authority: CN
Inventors: 王奇慧; 严景华; 史瑞; 高福; 马素芳
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-02-24
Filing date: 2020-02-24
Publication date: 2022-08-12
Anticipated expiration: 2040-02-24
Also published as: CN113292649A

Abstract

The invention relates to a novel human monoclonal antibody of coronavirus and application thereof. The antibody can specifically bind to 2019-nCoV RBD, block the binding of 2019-nCoV RBD and ACE2, and inhibit 2019-nCoV infection.

Description

Human monoclonal antibodies to novel coronaviruses and uses thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a novel coronavirus (2019-nCoV) humanized monoclonal antibody with high neutralizing activity and application thereof.

Background

The novel coronavirus 2019-nCoV is used as a new emergent infectious disease pathogen, and no specific medicine aiming at the virus is approved to be on the market at present.

Therapeutic antibody drugs not only play an important role in the treatment of tumors and autoimmune diseases, but also are effective in the treatment of infectious diseases. Currently marketed drugs for treatment and prevention of viral infections are palivizumab (Synagis) for prevention of Respiratory Syncytial Virus (RSV) infection in children, abalizumab (Trogarzo) for treatment of HIV infection, and Rabishield for post-exposure prophylaxis of rabies virus. Monoclonal antibodies against a number of viruses are also in different stages of clinical research (https:// clinicaltralals. gov /).

2019-nCoV belongs to coronavirus. Severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV) of the same genus coronavirus have also caused epidemic in 2002-. According to the World Health Organization (WHO), it was counted that SARS-CoV co-caused 8000 human infections and 794 human deaths (https:// www.who.int /). The MERS-CoV infection virus cases are continuously increasing from 2012 to date, and 2499 infection cases and 861 death cases are diagnosed globally by the end of 2019. On 12.1.2020, the world health organization formally named "2019 novel coronavirus (2019-nCoV)", and thereafter on 11.12.2.2020, the International Committee on Taxonomy of virues, ICTV, announced the formal classification of novel coronavirus (2019-nCoV) named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the World Health Organization (WHO) announced on the global research and innovation forum on the same day as the japanese tile, and the disease caused by this virus was formally named "covi-19".

To infect a cell, the virus first needs to bind to the host's receptor via the envelope protein. Antibodies, particularly neutralizing antibodies, block viral infection by binding to envelope proteins, blocking binding of the virus to cellular receptors. At the same time, the antibody binds to the envelope protein, thereby labeling the free virus or infected cells, recruiting immune cells and immune molecules such as macrophages or complement through the Fc region of the antibody, and removing the free virus and infected cells. Thus, antibodies that target the Receptor Binding Domain (RBD) not only have the ability to neutralize viral infection, but also act through the Fc region to facilitate viral and infected cell clearance.

Based on studies of other coronaviruses, particularly SARS-CoV and MERS-CoV, the important envelope protein for receptor binding is spike protein (S). S can be further divided into two parts, S1 and S2. The role of S2 is to mediate membrane fusion. Both the N-terminal (NTD) and C-terminal (CTD) of S1 may be RBDs. Through the research on 2019-nCoV, the team finds that CTD is RBD of the coronavirus and binds to the receptor ACE 2. Antibodies that target RBD, and block S binding to ACE2, may therefore be neutralizing antibodies that inhibit viral infection. The invention aims to identify specific human neutralizing antibodies with protective effect aiming at 2019-nCoV.

Disclosure of Invention

In order to obtain humanized neutralizing antibody with protective effect, 2019-nCoV RBD expressed by insect cells is used as antigen, memory B cells capable of specifically binding 2019-nCoV RBD protein are screened from PBMCs of hospital-discharged persons who recover after 2019-nCoV RBD infection through flow sorting, then RT-PCR is carried out on the screened single B cells to obtain variable region sequences and fragments of the antibody, and the variable region sequences and the fragments are further connected with a constant region into an expression vector. After mammalian cell expression and purification, a series of function tests are carried out, including the binding capacity with 2019-nCoV RBD protein, the blocking effect of blocking the binding of 2019-nCoV RBD and ACE2, the neutralization effect of inhibiting 2019-nCoV infection and the like, and the human monoclonal antibody for neutralizing 2019-nCoV infection is obtained and is named as GH 4.

Specifically, the present invention is achieved by the following aspects.

In one aspect, the invention provides a human monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to a 2019-nCoV RBD,

wherein the CDRs of the complementarity determining regions of the VH chain have an amino acid sequence selected from the group consisting of:

as shown in SEQ ID NO:1 of the CDR1 shown in FIG. 1,

as shown in SEQ ID NO:2, and a CDR2, and

as shown in SEQ ID NO:3, CDR 3;

wherein the CDR of the complementarity determining region of the VL chain has an amino acid sequence selected from the group consisting of:

as shown in SEQ ID NO:4 of the CDR1 shown in figure 4,

as shown in SEQ ID NO: CDR2 shown in FIG. 5, and

as shown in SEQ ID NO:6, CDR3 shown.

In one embodiment, the human monoclonal antibody or antigen-binding fragment thereof comprises:

as shown in SEQ ID NO:7, and

as shown in SEQ ID NO:8, or a light chain variable region.

as shown in SEQ ID NO:22, and

as shown in SEQ ID NO:23, or a light chain as shown.

In one embodiment, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab '-SH, Fv, scFv, F (ab')2, diabody.

In another aspect, the invention provides a polypeptide comprising a sequence selected from SEQ ID NOs: 7. 8, 22 or 23.

In another aspect, the present invention provides a polynucleotide encoding any one of the human monoclonal antibodies or antigen-binding fragments or polypeptides thereof described above.

In another aspect, the present invention provides an expression vector comprising the polynucleotide described above.

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

In another aspect, the present invention provides a pharmaceutical composition comprising the human monoclonal antibody or antigen-binding fragment thereof of any of the preceding claims and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a use of any one of the above human monoclonal antibodies or antigen-binding fragments thereof in the preparation of a medicament for treating 2019-nCoV infection.

All documents mentioned in this specification are herein incorporated in their entirety by reference.

Definition of

"antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of an antibody, which typically include at least a portion of the antigen-binding or variable region, e.g., one or more CDRs, of the parent antibody. Fragments of an antibody retain at least some of the binding specificity of the parent antibody. Antigen binding fragments include those selected from the group consisting of Fab, Fab '-SH, Fv, scFv, F (ab') ₂ Diabodies, CDR-containing peptides, and the like.

A "Fab fragment" consists of one light and one heavy chain of CH1 and the variable domains.

The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.

A "Fab ' fragment" contains a portion of one light chain and one heavy chain comprising the VH domain and the CH1 domain or the region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chains of the two Fab ' fragments to form F (ab ') ₂ A molecule.

“F(ab′) ₂ Fragment "heavy chain comprising two light chains and two parts comprising a constant region between the domains CH1 and CH2, thusAn interchain disulfide bond is formed between the two heavy chains. Thus, F (ab') ₂ The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks the constant region.

"Single chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, which domains are comprised in a single polypeptide chain. Generally, scFv polypeptides comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

A "diabody" is a small antigen-binding fragment having two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain. By using linkers that are too short to pair between two domains of the same chain, the domains pair with complementary domains of another chain and form two antigen binding sites.

"specific" binding, when referring to ligand/receptor, antibody/antigen or other binding pairs, refers to determining whether a binding reaction of a protein, e.g., a monoclonal antibody of the invention, to a 2019-nCoV RBD protein is present in a heterogeneous population of proteins and/or other biological agents. Thus, under the conditions specified, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The invention also provides a pharmaceutical composition containing the human-derived highly neutralizing monoclonal antibody or antigen-binding fragment thereof specifically binding to 2019-nCoV RBD of the invention. To prepare a pharmaceutical composition, the antibody or antigen-binding fragment thereof can be prepared into various desired dosage forms by mixing with a pharmaceutically acceptable carrier or excipient. Examples of the dosage form of the pharmaceutical composition of the present invention include tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated preparations, pellets, sublingual tablets, and ointments, which are oral preparations, and examples of non-oral preparations include injections, suppositories, transdermal preparations, ointments, plasters, and external liquid preparations, and those skilled in the art can select an appropriate dosage form according to the administration route and the administration target.

The dose of the active ingredient of the pharmaceutical composition of the present invention varies depending on the administration target, the target organ, the symptom, the administration method, and the like, and can be determined according to the judgment of the doctor in consideration of the type of the formulation, the administration method, the age and weight of the patient, the symptom of the patient, and the like.

The invention has the beneficial effects that:

the invention obtains the antibody with human source high neutralizing activity: GH 4. The antibody is the first known human antibody with 2019-nCoV infection neutralization. The binding constants of the GH4 antibody and the 2019-nCoV antibody are respectively 1.44E-09M. The humanized antibody GH4 with high neutralizing activity can effectively block the combination of 2019-nCoV RBD and hACE2, and has good neutralizing activity for inhibiting 2019-nCoV pseudovirus infection. The GH4 has an application value in clinical treatment and prevention of 2019-nCoV infection.

Drawings

FIG. 1: 2019-nCoV RBD molecular sieve chromatography and SDS-PAGE identification;

FIG. 2: molecular sieve chromatography and SDS-PAGE identification of GH4 antibody;

FIG. 3: kinetics of binding of the GH4 antibody to the 2019-nCoV RBD;

FIG. 4: the GH4 antibody blocked binding of 2019-nCoV RBD to HEK293T-hACE 2;

FIG. 5: the GH4 antibody neutralizes the effects of VSV-2019-nCoV infection.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

Example 1: expression and purification of 2019-nCoV RBD

The coding sequence of 6 histidine-tag (hexa-His-tag) and the translation stop codon were ligated to the 3' end of the 2019-nCoV RBD protein (amino acid sequence shown in SEQ ID NO: 9) coding region and constructed into pFastBac1 vector (purchased from Invitrogen) by ligation of EcoRI and XhoI. The ligation products were then transformed into DH10Bac competent cells (purchased from Tiangen) for baculovirus recombination. Recombinant baculovirus was extracted, transfected into sf9 cells (ex Invitrogen) for packaging of baculovirus, amplified by virus, added into Hi5 cells (ex Invitrogen) for expression of 2019-nCoV RBD protein.

The cell culture fluid containing the target protein is purified by nickel ion affinity chromatography (HisTrap TMHP (GE)) and gel filtration chromatography (Superose TM 6 Incrase 10/300GL (GE)), so that the target protein can be obtained in a relatively pure form. SDS-PAGE identified a size of 30kD, and the results are shown in FIG. 1.

Example 2: isolation of 2019-nCoV RBD protein specific memory B cells

With the informed consent of the discharge personnel that healed after 2019-nCoV RBD infection, 15mL of blood was collected and PBMCs were isolated. Isolating the PBMCs at 10 ⁷ The density of/mL combined with ice incubation of the 2019-nCoV RBD protein at a final concentration of 400nM for half an hour, followed by washing 2 times with PBS, and incubation with the following antibodies (all 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 half an hour incubation on ice, PBMCs were washed 2 times with PBS.

PBMCs washed by PBS are sorted by FACSAria III, cells (namely B cells) of PE-Cy 5-APC-Cy 7+ Pacific Blue + FITC + PE + are collected, and the cells are directly collected into a 96-well plate at 1 cell/well.

Example 3: single B cell PCR, sequence analysis and design of humanized antibody

The B cells obtained in example 2 were reverse transcribed by Superscript III reverse transcriptase (Invitrogen) following the method described by Qiaui Wang et al at 2016, 12 months in Science Translational Medicine, Vol.8, published at Molecular inhibitors of human neutral antibodies isolated from a patient infected with the with Zika virus, and the reverse transcription primers were reacted for 60min at 55 ℃.

TABLE 1 reverse transcription primers

Using this reverse transcription product as a template, PCR was performed using HotStar Tap Plus enzyme (QIAgen) to amplify an antibody variable region sequence (PCRa). Designing corresponding primers, wherein the reaction conditions are as follows: 95 ℃ for 5 min; 95 ℃ 30s, 55 ℃ (heavy chain/kappa chain) 30s, 72 ℃ 90s, 35 cycles; 72 ℃ for 7 min. This product was used as template for 1 more round of PCR (PCRb) under the following conditions: 95 ℃ for 5 min; 95 ℃ 30s, 58 ℃ (heavy chain)/60 ℃ (kappa chain) 30s, 72 ℃ 90s, 35 cycles; PCR products were obtained at 72 ℃ for 7 min.

The PCR products were separated by electrophoresis on a 1.2% agarose gel. The size of the band is 400-500bp after the gel cutting recovery, and the band is sent to a sequencing company for sequencing. Sequencing results were analyzed using IMGT online software.

The correct variable region sequence analyzed was ligated to the corresponding heavy/kappa chain constant region by bridge PCR and cloned into the expression vector pCAGGS (purchased from addge). Wherein the heavy chain is EcoRI and XhoI linked and the kappa chain is SacI linked to XhoI. B cell sequencing and expression plasmid construction were as follows:

the human antibody design strategy is as follows:

heavy chain: CMV promoter-EcoR I-Leader sequences-heavy chain variable region-CH-Xho I;

light chain (κ): CMV promoter-Sac I-Leader sequences-light chain variable region-CL _(κ) -Xho I；

Wherein, the amino acid sequence of Leader sequence is as shown in SED ID NO: 18, the amino acid sequence of CH is shown as SED ID NO: 19, the amino acid sequence of CL is as defined in SED ID NO: 20, and sequences of three antibodies named CA1, CB6 and GH4 were obtained by sequencing.

Wherein the heavy chain variable region sequence of GH4 is shown in SEQ ID NO:7, the light chain variable region sequence is shown as SEQ ID NO:8, and the heavy chain sequence is shown as SEQ ID NO:22, and the light chain sequence is shown as SEQ ID NO: shown at 23.

Wherein the sequence identity of the GH4 antibody and the germline gene is compared as follows:

TABLE 2 comparison of GH4 antibody heavy chain and germline genes

TABLE 3 comparison of GH4 antibody light chain and germline genes

Example 4: expression of GH4 antibody

293T cells were cultured in DMEM with 10% FBS. 293T was co-transfected with a plasmid containing the genes encoding the light and heavy chains of the specific antibodies obtained in example 3. And (3) after 4-6 hours of transfection, replacing the cell culture solution with serum-free DMEM, continuing to culture for 3 days, collecting the supernatant, supplementing the DMEM, continuing to culture for 4 days, and collecting the supernatant.

The collected supernatant was centrifuged at 5000rpm for 30min, mixed with an equal volume of buffer containing 20mM sodium phosphate (pH 8.0), filtered through a 0.22 μm filter and bound to a protein A pre-column (5mL, GE Healthcare). Bound protein was eluted with 10mM glycine (pH 3.0). The protein is collected, concentrated and then subjected to molecular sieve chromatography. The peak of interest was determined by SDS-PAGE (reducing and non-reducing) and the results are shown in FIG. 2. Purified GH4 antibody was obtained.

Example 5: detection of binding capacity of antibody and 2019-nCoV RBD by surface plasmon resonance technology

Surface plasmon resonance analysis was performed using Biacore 8K (Biacore Inc.). The method comprises the following specific steps:

the purified antibody obtained in example 4 was immobilized on a protein A chip (purchased from GE Healthcare) at an antibody immobilization amount of about 5000RU by affinity of protein A to antibody Fc, and the 2019-nCoV RBD protein was diluted by 10mM HEPES, 150mM NaCl, pH 7.4 at a double ratio, and loaded one by one from low to high concentrations. The kinetic curves for antibody binding 2019-nCoV RBD are shown in FIG. 3. Kinetic constants for antibody binding 2019-nCoV RBD are shown in Table 4. The calculation of binding kinetic constants was performed using BIAevaluation software 8K (Biacore, Inc.). It can be seen that the GH4 antibody can bind with high affinity to 2019-nCoV RBD.

TABLE 4 kinetic constants for binding of antibodies to 2019-nCoV RBD protein

Example 6: detection of GH4 blocking binding of 2019-nCoV RBD and ACE2

The gene encoding hACE2 (amino acid sequence shown in SEQ ID NO: 21) was constructed into pEGFP-N1 vector (purchased from Addgene) by XhoI and BamHI, and expressed in fusion with GFP to form pEGFP-hACE2 plasmid. The plasmid pEGFP-hACE2 was transfected into HEK293T cells, and GFP expression was observed 24h under a fluorescent microscope. Collecting HEK293T-hACE2 cells, 2X10 ⁵ For one reaction, the cells were incubated with 2019-nCoV RBD (200ng/mL) at room temperature for 30 min. After centrifugation at 500Xg for 5min, the supernatant was removed and washed 2 times with PBS. And incubating with anti-His/APC for 30min at room temperature, washing with PBS for 2 times, and detecting the fluorescence on the cell surface by using BD FACSCAnto.

To examine the blocking effect of GH4, the purified GH4 antibody obtained in example 4 was mixed with 200ng/mL 2019-nCoV RBD at a molar ratio of 10: 1 at room temperature, and then incubated with HEK293T-hACE2 cells. The remaining steps were as above, and the binding of the protein to the cells was detected using anti-His/APC. The GH4 antibody blocked binding of 2019-nCoV RBD to HEK293T-hACE2 cells as shown in FIG. 4. As can be seen, the GH4 antibody can block the binding of 2019-nCoV RBD and HEK293T-hACE2 cells.

Example 7: GH4 neutralization 2019-nCoV pseudovirus infection detection

The purified GH4 antibody from example 4 was diluted 3-fold from 50. mu.g/mL to a 10 th gradient (2.5ng/mL) and 1.6X10 ⁴ TCID ₅₀ VSV-2019-nCoV pseudovirus mixes were incubated at 37 ℃ for 1h with mixing and then added to 96-well plates pre-seeded with Huh7 cells (purchased from the basic medicine cell center, university of coordination and medicine). After 4 hours of incubation, the culture and virus solutions were discarded and 10% F was addedThe culture was continued for 48 hours in DMEM medium containing BS. The culture medium was discarded, washed once with PBS, and after lysis of the cells by addition of 1 Xlysate (Promega, Luciferase Assay System), 50. mu.L of reaction substrate was added to 10. mu.L of lysate and detected by Promega luminescence meters. The neutralizing capacity of the antibody to VSV-2019-nCoV pseudovirus was calculated according to the luciferase activity at different concentrations, and the results are shown in FIG. 5, and the statistics are shown in Table 5.

TABLE 5 neutralizing Effect of GH4 antibody on different sources of viruses

^a Half inhibitory concentration

The GH4 antibody can be seen to neutralize 2019-nCoV pseudovirus with high neutralizing activity.

Taken together, the GH4 antibody can be a novel coronavirus (2019-nCoV) human monoclonal antibody with high neutralizing activity.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A human monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to a 2019-nCoV RBD,

the VH chain has:

CDR1 shown in SEQ ID NO. 1,

CDR2 as shown in SEQ ID NO. 2, and

CDR3 shown in SEQ ID NO. 3; and is provided with

The VL chain thereof has:

CDR1 shown in SEQ ID NO. 4,

CDR2 as shown in SEQ ID NO. 5, and

CDR3 shown in SEQ ID NO. 6.

2. The human monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising:

the variable region of the heavy chain as shown in SEQ ID NO 7, and

the variable region of the light chain as shown in SEQ ID NO 8.

3. The human monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequence of the heavy chain is set forth as SEQ ID NO. 22, and the amino acid sequence of the light chain is set forth as SEQ ID NO. 23.

4. The human monoclonal antibody or antigen-binding fragment thereof of any of claims 1-2, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab '-SH, Fv, scFv, F (ab')2, diabody.

5. A polypeptide comprising the sequences shown as SEQ ID NO 7 and 8 or the sequences shown as SEQ ID NO 22 and 23.

6. A polynucleotide encoding the human monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4 or the polypeptide of claim 5.

7. An expression vector comprising the polynucleotide of claim 6.

8. A host cell comprising the expression vector of claim 7.

9. A pharmaceutical composition comprising the human monoclonal antibody or antigen-binding fragment thereof of any of claims 1-4 and a pharmaceutically acceptable carrier.

10. Use of the human monoclonal antibody or antigen-binding fragment thereof of any of claims 1-4 in the manufacture of a medicament for treating a 2019-nCoV infection.