CN113461810B - Fully human monoclonal antibody for resisting novel coronavirus spike protein and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a fully human monoclonal antibody for resisting novel coronavirus spike protein and application thereof. The invention firstly provides a monoclonal antibody combination for treating the new coronary pneumonia by combining three antibodies at present, and the combination has the following three advantages: 1) the two antibodies aiming at the RBD in the formula can be non-competitively combined with different epitope of the RBD, can block the combination of the RBD and ACE2 receptor, and plays a role in blocking virus from entering cells; 2) the three antibodies of the invention have ADCC effects of different degrees, and can help to remove infected cells so as to achieve the purpose of completely removing focuses; 3) the three antibody combination positions do not interfere with each other, and the combined antibody has a superimposed antiviral effect and can enhance the capacity of coping with future virus mutation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a fully human monoclonal antibody for resisting novel coronavirus spike protein and application thereof.
Background
After a person is infected with the new coronavirus, the clinical manifestations are presented, the main common symptoms comprise fever, respiratory symptoms, cough, shortness of breath and the like, the continuous persistence of infection can develop into severe pneumonia, severe complications such as respiratory failure, ARDS, shock, organ failure and the like are caused, and the disease recurrence occurs after the recovery of some mild symptoms or patients who are discharged from hospital. To date, there is no specific therapeutic for new coronavirus infections. The eighth new coronavirus pneumonia diagnosis and treatment scheme issued by the national Weijian Commission (trial eighth edition) mainly treats antivirus and symptomatic treatment for mild infectors, wherein antiviral drugs comprise alpha-interferon, ritonavir, ribavirin, chloroquine phosphate and arbidol. The method has the advantages that the complications of severe patients are actively prevented and treated on the basis of symptomatic treatment, the plasma treatment therapy of convalescent patients is increased in the aspect of antivirus, and the clinical condition of new coronary patients can be improved after the new coronary patients are infused with plasma in the convalescent period in part of non-control preliminary case studies, so that the potential of neutralizing antibodies in the aspect of treating new coronavirus pneumonia is shown.
The neutralizing antibody is always an effective therapy for treating infectious diseases, and although plasma therapy of a rehabilitee has certain effect on treating critically ill patients, the plasma source is difficult to obtain, the product components are complex, and uncertain factors exist in plasma infusion, so that the development of the monoclonal antibody which has strong neutralizing activity, single component and easy mass production is a key direction for the research of new crown virus treatment medicines.
However, the prior art still has the following disadvantages: the course of disease after infection with new coronavirus is long, and the recurrence of disease in some patients may be due to the fact that the virus still develops at low levels in different organs. Therefore, the removal of free virus and various types of infected cells in the patient becomes the key to complete the cure of the patient. The new coronavirus spike (S) protein is a key protein for infecting host cells, is widely expressed on the surfaces of infected cells, and a Receptor Binding Domain (RBD) of the protein mediates the binding of the virus and an ACE2 receptor, and most anti-new coronavirus antibodies entering clinical trials are targeted to the region. The recombinant full-human anti-SARS-CoV-2S protein RBD monoclonal antibody developed by Junshi organism and the institute of microbiology of Chinese academy of sciences has been administered to all subjects in the Chinese phase I clinical trial at 7 months and 7 days of 2020. In addition to a single antibody formulation, the antibody cocktail therapy REGN-COV2 developed in conjunction with the national institute for allergy and infectious diseases, usa, entered phase III clinical studies at 6.7/2020 and was granted Emergency Use Authorization (EUA) by the us FDA in 11/2020, that two antibodies could bind two different epitopes of RBD noncompetitively, attenuating escape of the virus due to mutation. After the new coronavirus is epidemic for one year, different mutant strains are found in a plurality of regions or countries, and as long as 2021, 2 months and 2 days, in the S sequence mutation analysis result of a new coronavirus information base of the national biological information center 2019, 2028 mutations are found in the S protein, wherein 268 mutations are located in RBD, so that the design of an antibody targeting the RBD region is not enough to cope with the possible future mutation of the virus.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a fully human monoclonal antibody for resisting novel coronavirus spike protein and application thereof. The invention screens out two antibodies aiming at different epitopes of RBD and one antibody aiming at the NTD of S protein as a combined formula, and emphatically uses the killing function mediated by the antibody to eliminate infected cells on the basis of neutralizing free viruses, thereby preventing the infection of multiple organs caused by the further diffusion of the viruses in the body of a patient. Meanwhile, the combination of three antibodies aiming at different epitopes can also cope with the variation of the virus in the future, and the effectiveness of antibody protection is enhanced.
In order to achieve the purpose, the invention adopts the technical scheme that: an antibody specifically binding to an epitope of a novel coronavirus is provided,
the antibody is 24C7, and the amino acid sequence of CDR-H1 in the heavy chain variable region of the antibody is shown in SEQ ID NO: 1, and the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3 is shown in the specification; the amino acid sequence of CDR-L1 of the variable region of the antibody light chain is shown in SEQ ID NO: 5, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 6, the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 7 is shown in the specification; or:
the antibody is 24G1, and the amino acid sequence of CDR-H1 in the heavy chain variable region of the antibody is shown in SEQ ID NO: 9, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 10, the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 11 is shown in the figure; the amino acid sequence of CDR-L1 of the variable region of the antibody light chain is shown in SEQ ID NO: 13, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 14, the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 15 is shown in the figure; or:
the antibody is 24G2, and the amino acid sequence of CDR-H1 in the heavy chain variable region of the antibody is shown in SEQ ID NO: 17, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 18, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 19 is shown in the figure; the amino acid sequence of CDR-L1 of the variable region of the antibody light chain is shown in SEQ ID NO: 21, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 22, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: shown at 23.
As a preferred embodiment of the antibody of the present invention, the amino acid sequence of the heavy chain variable region of the antibody 24C7 is as shown in SEQ ID NO: 4, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 8.
As a preferred embodiment of the antibody of the present invention, the amino acid sequence of the heavy chain variable region of the antibody 24G1 is as shown in SEQ ID NO: 12, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 16.
As a preferred embodiment of the antibody of the present invention, the amino acid sequence of the heavy chain variable region of the antibody 24G2 is as shown in SEQ ID NO: 20, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: as shown at 24.
As a preferred embodiment of the antibody of the present invention, the heavy chain constant region sequence of said antibody is the heavy chain constant region sequence of human IgG 1.
As a preferred embodiment of the antibody of the present invention, the light chain constant region sequence of the antibody is that of human IgK.
The invention also provides nucleic acid molecules encoding the antibodies.
The invention also provides a vector comprising the nucleic acid molecule.
The invention also provides a host cell comprising said nucleic acid molecule or said vector.
The invention also provides a pharmaceutical composition containing the antibody and a pharmaceutically acceptable carrier.
The invention also provides application of the antibody or the combination of the antibodies in preparing a medicament for preventing and/or treating novel coronavirus pneumonia.
Interpretation of terms:
novel coronaviruses: the new coronavirus is a chimeric virus particle with new coronavirus recombinant membrane protein (Spike) expressed on the surface of replication-defective virus, and the virus particle loses the virus self-replication capacity and can only carry out infection of a single cell cycle.
New coronaviruses: means that a specimen taken from a patient with new corona infection is inoculated in a specific cell line, and new corona virus with continuous replication capacity is separated from the specimen.
The invention has the beneficial effects that:
compared with the traditional chemical medicine, the humanized monoclonal antibody has the advantages of definite action mechanism, small toxic and side effect, easy large-scale production and the like. The invention firstly provides a monoclonal antibody combination for treating the new coronary pneumonia by combining three antibodies at present, and the combination has the following three advantages:
(1) the two antibodies aiming at the RBD in the formula can be non-competitively combined with different epitopes of the RBD to prevent the combination of the RBD and an ACE2 receptor, thereby playing a role in neutralizing free viruses;
(2) the three antibodies screened by the invention have ADCC effects of different degrees, and can clear infected cells on the basis of neutralizing free viruses so as to achieve the purpose of thoroughly clearing focuses;
(3) the three antibody combination positions do not interfere with each other, and the combined antibody has a superposition effect and can effectively cope with possible mutation of the virus in the future.
Drawings
FIG. 1: single-cell sorting SARS-CoV-2S1 protein RBD region-specific memory B cell by flow cytometry. And (3) capturing RBD-specifically bound memory B cells by using FITC labeled S1-RBD-his after the enriched B cells are enriched, wherein the surface molecule of the RBD-specifically bound memory B cells is labeled as CD3-CD19+ CD27+ CD38int S1-RBD +. Cells selected in frame in the figure were sorted in 96-well plates for subsequent cloning of single B cells.
FIG. 2: single-cell sorting SARS-CoV-2S1 protein non-RBD region specific memory B cell by flow cytometry. And capturing the S1 specific binding memory B cells by using FITC labeled S1-his after the enriched B cells, wherein the surface molecule of the cells is labeled as CD3-CD19+ CD27+ CD38int S1 +. Cells selected in boxes in the figure were sorted in 96-well plates for subsequent cloning of single B cells.
FIG. 3: the results of the measurement of the affinity activity of the neutralizing antibodies 24C7, 24G1 and 24G2 for RBD and S1 proteins in the invention are shown in the figure.
FIG. 4: epitope competition results of the antibodies.
FIG. 5: structural analysis and epitope analysis of a cryoelectron microscope of the three antibodies.
FIG. 6: results of the neutralization activity assay of the three antibodies are shown.
FIG. 7: and (4) a graph of the results of the determination of the neutralizing activity after antibody combination.
FIG. 8: the result of the test of the blocking effect of the antibody on ACE2 protein is shown.
FIG. 9: the ADCC effect of the three antibodies is measured.
Detailed Description
To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.
The present invention relates to the sequence of an antibody:
EXAMPLE 1 SARS-CoV-2S-RBD protein and S1 protein specific memory B cell isolation
CoV-spike RBD (40591-V05H) and CoV-spike-S1 (40591-V08H) from Chiense were fluorescently labeled with ReadLinkFITC antibody labeling kit (AATBioquest,1299) to obtain fluorescent antigens S1-RBD-his-FITC and S1-his-FITC. Fresh blood samples of patients in the new coronary convalescence were collected from the first subsidiary hospital of Shantou university college of medicine, and B cells were obtained by enrichment using RosetteSep kit (Cat #: 15064, STEMCELL) and Lymphoprep reagent (Cat #: 07861, STEMCELL). On the basis, fluorescent antigen S1-RBD-his-FITC labeling, cell surface molecule staining and single cell sorting of new crown RBD specific memory B cells (CD 3)-CD19+CD27+CD38int S1-RBD+) The results are shown in FIG. 1.
In order to obtain the S1 specific memory B cells of the novel coronary virus except RBD, the enriched B cells are firstly incubated with the RBD antigen, then captured by fluorescent antigen S1-his-FITC and stained by cell surface molecules, and finally flow single cell sorting is carried out to obtain the novel crown S1 specific memory B cells (CD 3)- CD19+CD27+CD38int S1+) The results are shown in FIG. 2. All antigen-specific B cells were sorted in 96-well plates for subsequent antibody variable region sequence acquisition.
Example 2 antibody variable region sequence acquisition
The 96-well plate used for antigen-specific B cell sorting in example 1 was pre-loaded with 5 μ l TCL (Qiagen) per well; after the single B cell is sorted, adding 10 mu l of ultrapure water and 33 mu l of RNA Beads (Nanjing Nozan) into each hole, blowing, sucking, uniformly mixing, standing at room temperature for 10 minutes, placing on a magnetic frame for 5 minutes, then removing the supernatant, rinsing the magnetic Beads twice by 200ul of 80% ethanol, removing the supernatant, air-drying the magnetic Beads, adding 10 mu l of Mix A into each hole, blowing, uniformly mixing, and standing at room temperature for 5 minutes; after placing the magnetic frame for 2 minutes, transferring 5 mu l of the magnetic frame to a new 96-well plate per well, centrifuging for 10s at 500g, and operating the program 1; after completion of procedure 1, 5. mu.l Mix B (ThermoFisher) was added to each well, and after mixing and centrifugation, procedure 2 was run to complete cDNA synthesis.
Mix A：H2O, dNTP, Random 6, Oligo _ dT are mixed according to the ratio of 31:5:2: 2.
Mix B：H2O, Buffer, DTT, RNase I and RTase IV are mixed according to the ratio of 17:16:4:2: 1.
Procedure 1: 5min at 65 ℃ → 4 ℃infinity
Procedure 2: 10min at 23 ℃ → 30min at 50 ℃ → 10min at 80 ℃ → infinity at 4 ℃ ∞
The method of nested PCR is adopted to amplify antibody heavy chain (IgG) and light chain (IgK) variable region genes. In the first round of PCR, cDNA is taken as a template to simultaneously amplify antibody IgG and IgK variable region genes in a reaction system (20 mu l); and in the second round of PCR, the product of the first round of PCR is used as a template to respectively amplify the antibody IgG and IgK variable region genes in two reaction systems. The DNA polymerases were used in Dream Taq Green (ThermoFisher), the primer sequences were derived from Human Monoclonal Antibodies at pages 114 to 115, and the reaction conditions were as described at pages 130 and 131. The second round of PCR products were analyzed by agarose gel electrophoresis, and the light and heavy chain positive pairs were selected and purified using VAHTS DNA Clean Beads (nuozin) followed by antibody light and heavy chain variable region Sanger sequencing (ThermoFisher).
Example 3 transient expression of antibodies
RNA was extracted from PBMC of one volunteer, cDNA was synthesized and used as a template, Human-IgG1 and Human-IgK constant region genes were PCR-amplified with primers having the designed forward signal peptide (MGWSCIILFLVATATGVHS, SEQ ID NO: 25) coding sequence, and the PCR products were purified and inserted into the multiple cloning sites of the expression vector pcDNA3.4(ThermoFisher), respectively. After transformation of large intestine bacillus (DH5 alpha), picking up single colony, amplification culture, plasmid extraction, sequencing confirmation, the antibody expression vectors pcDNA3.4-hIgG1 and pcDNA3.4-hIgK are obtained.
The light and heavy chain variable region genes of the antibody are amplified by selecting proper primers (pages 114 to 115 in the Human Monoclonal Antibodies) and taking the products of the first round of PCR as templates through analyzing, comparing and classifying the sequences of the light and heavy chain variable regions of the antibody (IgBLAST), and the light and heavy chain variable region genes are respectively cloned into the upstream of the coding genes of the constant regions of eukaryotic transient expression vectors pcDNA3.4-hIgK and pcDNA3.4-hIgG1 and are transferred into enterobacter coli (DH5 alpha) for amplification, and the plasmids of the light and heavy chain of the antibody are obtained through extraction. According to Expi293TMThe Expression System (ThermoFisher) protocol states that the light and heavy chain plasmids of the antibodies were expressed in Expi293F cells. On the third day after transfection, the supernatant was harvested and the antibody concentration was determined using an Octet Red96e protein interactor (ForteBio).
EXAMPLE 4 Primary screening of neutralizing antibodies
Cloning the S gene of the new coronavirus synthesized after codon optimization into a multiple cloning site in a PiggyBac transposable System transposon vector (System Biosciences), and constructing an S protein expression vector pMie-Swtd 18S. The GFP gene is cloned into a multiple cloning site of a second generation lentivirus system transfer vector (Addgene,85133) to construct a pLV-GFP reporter gene vector. pMie-Swtd18S, pLV-GFP and a second generation lentivirus system packaging vector psPAX (Addgene, 12260) are co-transfected into 293T cells, cell culture supernatants which are cultured for 48 hours and 72 hours are respectively harvested, and the new coronavirus pseudovirus is obtained after concentration and purification.
Diluting the initial concentration of the harvested cell culture supernatant to 10ug/ml, 1ug/ml and 0.1ug/ml respectively, diluting the new corona pseudovirus with a GFP reporter gene to 1500TU, uniformly mixing the new corona pseudovirus with the diluted antibody in equal volume, after 1H of action at 37 degrees, transferring all the cells to H1299 cells over-expressing hACE2, after 48H of culture, measuring all the cells expressing GFP fluorescence under the full field of view of each well by using an OperaPhenix high content imaging system of Perkinelmer company, and calculating the inhibition rate of each antibody under different concentrations, wherein the inhibition rate is (average GFP positive cell number of blank control wells-positive cell number of antibody wells)/(average GFP positive cell number of blank control wells) 100.
The inhibition results showed that the inhibition of 4 total strains of RBD antibody was greater than 75% at a concentration of 0.1ug/ml, and the inhibition of 5 strains of antibody bound to non-RBD was greater than 50% at a concentration of 0.1ug/ml (table 1). Further determination of IC for antibodies binding to RBD50As a result, as shown in Table 2, two strains of antibodies having the best neutralizing activity against the IC of the novel coronavirus were selected503.51ng/ml (24G1) and 16.45ng/ml (24C7), respectively. Although the neutralizing activity of the antibody against S1 non-RBD was inferior to that of the antibody binding to RBD, the above-screened 5-strain antibody was combined with 24G1 and 24C7 at a ratio of 1:1, and it was found that the inhibition rate of the antibody at high concentration was improved when 5-strain binding S1 was combined with 24G1, and IC was also improved when 24G2 was combined with 24C750Reducing to 9.66ng/ml, combining 24G1, 24C7 and 24G2 at a ratio of 1:1:1, and obtaining IC506.16ng/ml (Table 3), suggesting that the combination of these three antibodies can effectively inhibit the infection of new coronavirus.
TABLE 1 results of inhibition rates of candidate antibodies at different concentrations
TABLE 2 binding of RBD candidate antibody IC50Comparison
| Candidate antibodies | IC50(ng/ml) |
| 24G1 | 3.51 |
| 24C7 | 16.45 |
| 24G5 | 94.29 |
| 24E10 | 56.51 |
TABLE 3 IC after combination of different antibodies50Comparison
| Antibody combinations | Antibody ratio | IC50(ng/ml) |
| 24G1+24C7 | 1:1 | 4.50 |
| 24G1+24G2 | 1:1 | 6.52 |
| 24C7+24G2 | 1:1 | 9.66 |
| 24G1+24C7+24G2 | 1:1:1 | 6.16 |
Example 5: neutralizing antibody affinity assay
The primary antibody clones were expressed as supernatants and affinity purified using a MabSelectPrismA column (Cat:17-5498-53, GE) from AKTA pure. The affinity of the antibodies obtained by screening for binding to the S1-RBD-His and S1-His protein antigens (purchased from Qianshen, Y.) was determined using the Octet Red96e protein interactor from Fortebio. The method mainly comprises the following steps:
binding of the antibody to S1-RBD-His and S1-His was determined by capturing the Fc fragment of the antibody with an AHC probe (Cat: 18-5060). Purified 24C7, 24G1, and 24G2 recombinant antibodies (4ug/mL) were passed over the AHC probe surface for 120 s. Different concentrations of diluted S1-RBD-His and S1-His were used as mobile phases. The binding time was 240s and the dissociation time was 240 s. After the experiment, blank control response values were deducted, and the software was run for 1: 1Langmuir binding pattern fitting, Global minus control line analysis results, calculation of antigen antibody binding kinetic constants, the results are shown in Table 4 and FIG. 3.
As can be seen from FIG. 3, all three antibodies had nM-grade affinity for the S1 recombinant protein, but the 24G2 antibody had no significant binding curve with the RBD protein, indicating that the binding site is in the non-RBD region of the S1 protein.
TABLE 4 affinity results of candidate antibodies to S1-RBD-His and S1-His recombinant proteins
Example 6: neutralizing antibody epitope competition binding activity assay
The epitope analysis was performed by Sandwich method (Sandwich) using an Octet protein interactor with 3 antibody clones 24C7, 24G1 and 24G2 that were screened out initially. According to the Biotin marker kit EZ-LinkTMDescription of NHS-PEG12-Biotin (Cat: A35389, ThermoFisher) antibodies 24C7, 24G1, 24G2 were labeled with Biotin. Using an Octet Red96e instrument from Fortebio, a Streptavidin (SA) bioprobe (Cat) was used18-5019) method for capturing biotins the recognition epitopes of the different antibodies were determined. For the assay, the SA probe was equilibrated in a Kinetics Buffer (PBS + 0.02% tween20+1mg/mL BSA, pH 7.4) for 60s, and then Biotin-labeled 24C7, 24G1, 24G2 recombinant antibodies (5ug/mL) were passed over the surface of the SA probe for 90s, each antibody loading 4 SA probes. The mobile phase was 100nM of S1-His protein (purchased from Cassia, Chinesen), bound for 180S; after the Kinetics Buffer was equilibrated for 60s again, the mobile phase was changed to recombinant antibodies 24C7, 24G1, 24G2 at a concentration of 100nM for 120s binding. And after the experiment is finished, observing whether the recognition epitopes of the antibodies have competition relations.
The experimental design is shown in table 5 and the results are shown in fig. 4. As can be seen from fig. 4, after binding the S1-His antigen, 24C7 could not bind 24C7 again, but could bind 24G1 and 24G2 well, which suggests that 24C7 recognizes different epitopes from 24G1 and 24G2 antibodies. The same applies to the 24G1 and 24G2 antibodies. This indicates that the 3 antibodies 24C7, 24G1 and 24G2 recognize 3 different epitopes on S1-His antigen and have no competition relationship with each other.
TABLE 5 Loading sequence for recombinant antibody binding epitope analysis
| Sensor location | Loading Sample ID | Sample ID |
| A4 | 24C7-Biotin | 24C7-Biotin |
| B4 | 24C7-Biotin | 24G1-Biotin |
| C4 | 24C7-Biotin | 24G2-Biotin |
| D4 | 24C7-Biotin | K Buffer |
| A5 | 24G1-Biotin | 24C7-Biotin |
| B5 | 24G1-Biotin | 24G1-Biotin |
| C5 | 24G1-Biotin | 24G2-Biotin |
| D5 | 24G1-Biotin | K Buffer |
| A6 | 24G2-Biotin | 24C7-Biotin |
| B6 | 24G2-Biotin | 24G1-Biotin |
| C6 | 24G2-Biotin | 24G2-Biotin |
| D6 | 24G2-Biotin | K Buffer |
Example 7 recognition epitopes of three candidate antibody molecules
To further explore the epitope information of three candidate neutralizing antibodies, we analyzed the epitope recognized by the antibody by analyzing the immune complex structure of the S protein and the antibody Fab fragment by cryo-electron three-dimensional reconstruction (cryo-EM). The method comprises the following specific steps:
(1) the purified three candidate antibodies (24C7, 24G1 and 24G2) were subjected to papain digestion to prepare Fab fragments. The papain and the antibody are mixed according to the concentration ratio of 1: 400-1: 1600 by mass, the mixture is reacted for 10-12 hours at 37 ℃, iodoacetamide (the final concentration is 30mmol/L) is finally added for reaction in a dark place for 45min, and the enzyme digestion reaction is stopped. The mixture was dialyzed overnight into a buffer of 20mM Tris-HCl pH 8.0, and purified by an anionic DEAE-HPLC column to obtain Fab fragments.
(2) The Fab fragments obtained by purification were reacted with the S protein at a mass ratio of 1.2:1 at 37 ℃ for 1 hour, and a quantifoil commercial Holey Carbon copper mesh (specification R2/2 or 2/1) was selected for frozen sample preparation. The samples were snap frozen using a Thermo Fisher Mark IV Vitrobot automatic prototype. Sucking about 3 mu L immune complex, dripping the immune complex on a copper net, sucking redundant liquid drops by using filter paper, setting the filter paper blot time to be 5s and the blot force to be 0, sucking residual liquid drops, quickly freezing the residual liquid drops into liquid ethane by Vitrobot, and transferring the liquid ethane into liquid nitrogen for storage for later use.
(3) The copper mesh was transferred to Gatan 626 type frozen sample rods. The frozen sample rod was cooled to near liquid nitrogen temperature (-196 deg.C), and rapidly placed in the sample chamber of a Thermo Fisher F30 cryoscope at 93,000 times magnification and pixel size in liquid nitrogen environment
A camera for data acquisition, a Thermo Fisher falcon3 electronic direct detection camera, uses Thermo Fisher EPU software to automatically acquire the data of a cryo-electron microscope.
(4) Carrying out picture drift correction on the acquired data by utilizing Motioncorr2 software; performing CTF evaluation by using Gctf software, and determining under-focus level and resolution evaluation; and (3) carrying out particle selection, 2D classification, initial model reconstruction and three-dimensional reconstruction by using CryoSPARC software to finally obtain the three-dimensional structure of the compound.
The results of the immune complex structure analysis are shown in fig. 5, and the resolution of the immune complexes corresponding to the three antibodies is: s, 24C7, and the preparation method comprises the steps of,
S:24G1，
S:24G2，
antibodies 24C7 and 24G1 bound RBD, with each S trimer binding to three antibody Fab fragments, respectively; antibody 24G2 then bound NTD, binding three 24G2 Fab fragments per S trimer.
24C7 is mainly directed to the epitopes from amino acids 444 to 448 of the S protein (aa444-448) and from amino acids 484 to 505 (aa 484-505); 24G1 is directed primarily to epitopes from amino acids 343 to 347 (aa343-447) and from amino acids 440 to 450 (aa440-450) of the S protein; the 24G2 is mainly directed against the epitope S protein from amino acids 144 to 153 (aa144-153) and from amino acids 246 to 255 (aa 246-255). As can be seen from the complex structure of the 3 candidate neutralizing antibodies and the S protein, the epitopes of the two RBD antibodies 24C7 and 24G1 have a certain degree of overlap, but do not overlap with the epitope of the NTD antibody 24G 2.
Example 8 neutralizing Activity of antibodies against New coronavirus (IC)50) Measurement of
Evaluation of neutralizing Activity of three neutralizing antibodies against New coronavirus Using plaque reduction assay. The purified antibody was diluted to 63.2ug/ml at the initial concentration and diluted 3.16 fold at 10 dilutions. And (3) respectively mixing the antibody dilution with 100PFUSARS-CoV-2 virus in equal volume, incubating at 37 ℃ for 1 hour, and adding the antibody dilution to a blank control to replace the antibody. 1 hour later, transferring all virus antibody mixture into Vero cells, adsorbing at 37 deg.C for 1 hour, discarding the adsorption solution, adding semisolid culture medium (DMEM/0.9% low melting point agarose gel) into Vero cells, standing at 4 deg.C for half an hour, transferring to 37 deg.C, and adding 5% CO2The cells were cultured in an inverted state for 3 days. After 3 days of culture, adding 4% of formaldehyde into the cells for fixation, removing the formaldehyde and the semi-solid culture medium after 2 hours, adding 0.1% of crystal violet staining solution for dyeing for half an hour, finally removing the crystal violet staining solution, washing and airing, counting the number of virus plaques in each cell well, calculating the virus inhibition rate under each antibody dilution degree, wherein the inhibition rate (%) [ (average number of plaques in blank control wells-number of plaques in antibody wells)/(average number of plaques in blank control wells)]*100. The IC of each antibody was calculated by nonlinear fitting analysis of the inhibition at each concentration using Prism 7 software50The value is obtained.
The results are shown in FIG. 6 and Table 6. The results showed that the neutralizing activity of the two strains against RBD antibody (24G1 and 24C7) was superior to that against S1 non-RBD antibody 24G2, IC thereof500.0138ug/ml (24G1) and 0.077ug/ml (24C7), respectively, and the inhibition rate is 100% at the concentration of 6.32 ug/ml. According to the inhibition results, the inhibition of 24G2 was about 50% at a concentration of about 2ug/ml and 100% at a concentration of 63.2 ug/ml.
The neutralizing activity of the combination of the three neutralizing antibodies was also evaluated using a plaque reduction assay. The purified three antibodies are combined pairwise and three antibodies, and the antibodies in the formula are mixed in equal proportion. The results are shown in fig. 7 and table 6, the neutralizing activity of the two antibodies is remarkably improved after the two antibodies are combined, and the main effect is that the concentration of each antibody combination reaching 100% inhibition rate is reduced by 10-30 times. Although the neutralization activity of 24G2 was weaker than that of 24G1 and 24C7, the neutralization activity was improved in the case of 24G2 in combination with 24G1 and 24C7, respectively. Epitope competition results show that three antibodies bind to three different epitopes, suggesting that the increased neutralizing activity after two-by-two combination may be due to additive effects.
TABLE 6 neutralizing Activity (IC) of each antibody combination50) Comparison
Example 9: antibody ACE2 blocking activity assay
Of the 3 high affinity antibodies, 24C7 and 24G1 bound to the S1-RBD antigen. Using an Octet Red96e protein interactor, a Pre-mix method (Pre-mix) was used to determine whether the two antibodies could block the binding of the S1-RBD antigen to the SARS-CoV-2 receptor protein ACE 2. First, antibodies 24G1, 24C7 and CR3022 (control antibody) at a concentration of 75. mu.g/mL and PBS were mixed with 100nM S1-RBD antigen 1:1 and incubated at room temperature for 30 min. During the determination, the His1K probe (Cat: 18-5120) was balanced in a Kinetics Buffer for 60s, and then recombinant ACE2-His protein (purchased from Qiao Shenzhou) with the concentration of 20 μ g/mL was flowed through the surface of the His1K probe for 180 s; the mobile phase was again equilibrated in a kinetic Buffer for 180S, and then replaced with a premixed antibody and S1-RBD antigen mixture to bind 180S. After the experiment, the premixed antigen-antibody mixture was observed for binding signals.
The result is shown in fig. 8, the recombinant antibody 24C7 is mixed with S1-RBD antigen and then combined with ACE2 protein, and no obvious combination signal exists, which indicates that the 24C7 antibody blocks the combination of S1-RBD and ACE 2; after the recombinant antibody 24G1 and the control antibody CR3022 were bound to S1-RBD, a significant binding signal was observed with ACE2 protein, indicating that the two antibodies could not block the binding of S1-RBD to ACE 2. The above results indicate that, although both 24C7 and 24G1 bind to the S1-RBD antigen, only 24C7 can block the binding of S1-RBD to ACE 2.
Example 10 antibody dependent cell-mediated cytotoxicity (ADCC) of three candidate antibody molecules
ADCC is an ideal mechanism for the use of antibody-mediated clearance of infected cells (target cells). Antibodies bind to the antigen of interest on the surface of target cells, and if the Fc portion of the antibody binds simultaneously to Fc g RIIIa (CD16) receptor on the surface of effector cells (mainly natural killer cells, NK cells), both cell types react, resulting in activation of the ADCC mechanism pathway.
To test the ADCC effect of three candidate molecules, we selected H1299 cells overexpressing the ACE2 receptor and co-incubated with S antigen as target cells. Antibodies 24G1, 24C7, 24G2 were diluted with a 3-fold gradient of nine dilutions at an initial concentration of 300ug/ml and 5X 10, respectively4H1299-S cells/well were incubated for 1 hour, followed by 1X 10 cells5One/well Jurkat cells were co-cultured for 10 hours as effector cells. Supernatants were removed to examine effector cell activation. Activation of gene transcription mediated by the NFAT (activated T-cell nuclear factor) pathway in effector cells is an earlier event in the activation of the ADCC mechanism pathway. In addition, Jurkat cells stably expressed the Fc g RIIIa (CD16) receptor (and firefly luciferase expressed driven by NFAT response elements. the biological activity of antibodies in the ADCC mechanism of action was quantified by luciferase produced by NFAT pathway activation, while the luciferase activity in effector cells was quantified by bioluminescence readings.
The results are shown in FIG. 9. We found that 24G2 has a greater ADCC effect than 24G1 and 24C7, and in particular that ADCC effect is maintained at lower antibody levels.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Shenzhen Shentian region lattice intelligence and recovery pathogeny research institute
<120> fully human monoclonal antibody for resisting novel coronavirus spike protein and application thereof
<130> 2021.04.28
<160> 25
<170> PatentIn version 3.3
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Claims (11)
1. An antibody specifically binding to an epitope of a novel coronavirus,
the antibody is 24C7, and the amino acid sequence of CDR-H1 in the heavy chain variable region of the antibody is shown in SEQ ID NO: 1, and the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3 is shown in the specification; the amino acid sequence of CDR-L1 of the variable region of the antibody light chain is shown in SEQ ID NO: 5, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 6, the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 7 is shown in the specification; or:
the antibody is 24G1, and the amino acid sequence of CDR-H1 in the heavy chain variable region of the antibody is shown in SEQ ID NO: 9, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 10, the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 11 is shown in the figure; the amino acid sequence of CDR-L1 of the variable region of the antibody light chain is shown in SEQ ID NO: 13, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 14, the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 15 is shown in the figure; or:
the antibody is 24G2, and the amino acid sequence of CDR-H1 in the heavy chain variable region of the antibody is shown in SEQ ID NO: 17, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 18, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 19 is shown in the figure; the amino acid sequence of CDR-L1 of the variable region of the antibody light chain is shown in SEQ ID NO: 21, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 22, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: shown at 23.
2. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of antibody 24C7 is as set forth in SEQ ID NO: 4, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8.
3. The antibody of claim 1, wherein the variable region of the heavy chain of antibody 24G1 has the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 16.
4. The antibody of claim 1, wherein the variable region of the heavy chain of antibody 24G2 has the amino acid sequence set forth in SEQ ID NO: 20, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: as shown at 24.
5. The antibody of claim 1, wherein the heavy chain constant region sequence of said antibody is the heavy chain constant region sequence of human IgG 1.
6. The antibody of claim 1, wherein the light chain constant region sequence of said antibody is a light chain constant region sequence of human IgK.
7. A nucleic acid molecule encoding the antibody of claim 1.
8. A vector comprising the nucleic acid molecule of claim 7.
9. A host cell comprising the nucleic acid molecule of claim 7 or the vector of claim 8.
10. A pharmaceutical composition comprising the antibody of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.
11. Use of an antibody or a combination of antibodies as claimed in any one of claims 1 to 6 in the manufacture of a medicament for the prophylaxis and/or treatment of novel coronavirus pneumonia.
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