CN111138531B

CN111138531B - Monoclonal antibody 8F9 specifically bound to GP1 subunit of EBOV and application

Info

Publication number: CN111138531B
Application number: CN201811315415.2A
Authority: CN
Inventors: 陈薇; 范鹏飞; 于长明; 迟象阳; 张冠英; 侯利华; 房婷; 吴诗坡; 陈旖; 陈郑珊; 王美荣; 刘渝娇
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2018-11-06
Filing date: 2018-11-06
Publication date: 2021-07-30
Anticipated expiration: 2038-11-06
Also published as: CN111138531A

Abstract

The invention discloses a monoclonal antibody 8F9 of an anti-Ebola virus glycoprotein GP1 subunit, which has a unique CDR region, good binding activity with EBOV GP and EC₅₀The value was 0.015. mu.g/mL. 8F9 was effective in neutralizing EBOV pseudoviruses in vitro, as compared to control antibodies. The neutralizing activity of 8F9 is enhanced with the increase of the antibody concentration, and 90% of protection can be realized on EBOV pseudovirus infected cells at the concentration of 1 mu g/mL. 8F9 also has good in vitro neutralizing activity on EBOV, can be used as a candidate therapeutic drug for Ebola virus, and the binding epitope of 8F9 is positioned at the 227 th and 295 th amino acids of GP antigen, is different from the neutralizing antibody in the prior art, and has the potential of composing cocktail combination therapy with other neutralizing antibodies.

Description

Monoclonal antibody 8F9 specifically bound to GP1 subunit of EBOV and application

Technical Field

The invention discloses an antibody, and belongs to the technical field of polypeptides.

Background

Ebola virus (EBOV) can cause acute severe hemorrhagic fever in human and non-human primates, is one of the viruses with the highest fatality rate discovered so far, has the lethality rate as high as 90 percent, can be directly transmitted by contact, and has extremely strong infectivity and lethality rate. Glycoprotein (GP) spikes on the surface of the ebola virus envelope mediate almost all links of viral entry into cells, and thus ebola GP is an important target for virus-neutralizing antibodies. The ebola virus GP gene is processed into two proteins, one being secreted, non-structural GP (sGP); the other is structural GP, Ebola GP is firstly synthesized as a polypeptide and then is cut by Furin enzyme to be GP1 (amino acids 1-501) and GP2 (amino acids 502-676), the two subunits are connected by disulfide bonds, the formed trimer is fixed on the surface of an envelope by a transmembrane region in GP2, and GP1 comprises structural domains such as Mucin (Mucin), Glycan cap (Glycan cap), Head (Head), Base (Base) and the like. The GP structure formed after Furin enzyme digestion cannot directly stimulate the fusion process of the Ebola virus and the host cell membrane. After the ebola virus enters the body, the ebola virus is combined with receptors on the surface of cell membranes, but the adhesion factors on the surface of the host cell membranes are not the real receptors for the ebola virus to generate membrane fusion and enter the host cells. Upon binding of the virus to cell surface adhesion factors, endocytosis and pinocytosis are mediated by clathrin, and primary and secondary endosomal trafficking occurs. GP is cleaved by both histone B and histone L in endosomes, removing 60% of the amino acids including mucin and glycan cap from GP1, forming the active GP (GPcl), which activates the critical membrane fusion process. GPcl is combined with endosome membrane protein Niemann-Pick C1(NPC1) to generate membrane fusion, so that virus RNA enters cytoplasm to complete virus genome replication and transcription, and new virus protein is synthesized to assemble progeny virus particles and bud from the surface of a host cell. The endosomal membrane protein NPC1 is an essential host infectious agent for EBOV infection.

Ebola virus is classified as a class a biological/bioterrorism agent by NIH and CDC in the united states, and is classified as one of the pathogens that presents significant risks and risks to national safety and public health, and there is currently no approved drug for the prevention or treatment of ebola virus. After the ebola virus was discovered in 1967, a total of 12 larger scale transmissions were experienced. The western africa in 2014 has developed the largest-scale and most difficult-to-control ebola epidemic, which is identified as zaire type ebola virus, the epidemic causes more than 1 ten thousand deaths and more than 2.5 ten thousand infections (according to the ebola epidemic report issued at 2.17.2.2016. in WHO), the epidemic is spread out of the african continent for the first time, and great panic is caused in the world. The high level of public and authorities attention has rapidly pushed the search for ebola vaccines and antiviral drugs.

Several promising clinical trials of ebola vaccines have been rapidly conducted at home and abroad: gorilla adenovirus type 3 vector vaccines, adenovirus type 5 vector vaccines, vesicular stomatitis virus vector vaccines, and the like. Admittedly, the development of effective prophylactic-like drugs is an important goal, and prophylactic measures are considered to save a large number of lives, whereas vaccines are difficult to work in certain situations, such as: 1) the host individuals may have differences (e.g., weak immunity in the elderly, the young, and the immunocompromised); 2) the immune effect of the vaccine is gradually weakened after the vaccine is immunized for a long time; 3) vaccines elicit immunity in the host that is not resistant to high doses of ebola virus exposure; 4) most patients seek medical assistance only after the occurrence of ebola virus infection signals, and vaccines have difficulty eliciting an effective immune response in the host for a short treatment window. Therefore, the development of therapeutic drugs for ebola virus diseases in the process of developing vaccines is also not overlooked. Currently ebola virus neutralizing antibodies may exert protective effects through three pathways: 1) blocking GP from being digested and hydrolyzed by cathepsin; 2) block binding of the activated GP to the NPC1 receptor; 3) influencing the allosteric processes of GP before membrane fusion after binding to NPC1 receptor. Of these, the first two pathways have been documented, and the third pathway is a presumption in some documents, and there is no direct evidence.

Although there are no currently approved therapeutic agents for ebola virus, there are several experimental anti-ebola virus agents under investigation, including small interfering RNAs, antisense oligonucleotide drugs, nucleotide analogs, antibody drugs, and the like. In 2018, in 5 months, the Ebola epidemic situation outbreaks again in the Congo fruit democratic republic, and by 12 days in 8 months, 57 people are infected and 41 people die. After this outbreak, the WHO approved 5 drugs in the study phase that could be used for the emergency treatment of ebola virus disease, including 3 antibody drugs (Zmapp, REGN3470-3471-3479 and mAb114) and 2 small molecule drugs (Remdesivir and Favipiravir). The antibody drug Zmapp formed by mixing three monoclonal antibodies and the optimized strain MIL77 successfully saves lives of a plurality of people during outbreak of West Africa Ebola epidemic situation in 2014, the effect and the treatment window period length of the antibody drug are far longer than those of other antiviral drugs, the drug safety and the patient cure rate are greatly improved, and people are greatly encouraged. The application of antibody drugs to treat filovirus attracts worldwide attention and becomes a hotspot in the research field of ebola virus disease treatment drugs.

Zmapp is a cocktail treatment strategy consisting of three human murine chimeric mabs (C2G4, C4G7, C13C6) expressed in the tobacco system, where C2G4 (epitopes C511, N550, G553, C556) and C4G7 (epitopes C511, D552, C556) bind to GP2 subunit with overlapping epitopes; C13C6 (epitope T270, K272) bound to the glycan cap region at an approximately perpendicular angle. MIL77 is an antibody combination optimized for Zmapp, MIL77-1/-2/-3 contains the variable regions of C2G4, C4G7 and C13C6, respectively, and the framework regions are humanized and engineered to be expressed in CHO cells. Cocktail combination therapy of two antibodies, MIL77-1 and ML77-3, was able to achieve 100% protective activity against nonhuman primates 72 hours after infection.

The excellent effect shown by the cocktail combination therapy brings new revelation to the treatment of the ebola virus, and the development of anti-ebola virus monoclonal antibodies capable of resisting unique epitopes becomes a great technical demand in the field, because if more anti-ebola virus monoclonal antibodies different from the anti-unique epitopes already appeared in the prior art are provided, more choices are provided for the treatment scheme of the ebola virus, especially the scheme design of the cocktail combination preparation. Therefore, the invention aims to provide the monoclonal antibody of the anti-Ebola virus glycoprotein, which can aim at the unique epitope, and further provides the application of the monoclonal antibody in preparing the medicine for treating the Ebola virus.

Disclosure of Invention

In view of the above objects, the present invention provides, in a first aspect, a monoclonal antibody 8F9 that specifically binds to GP1 subunit of EBOV, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the antibody are represented by the amino acid sequences at positions 27-32, 50-52 and 89-97 of SEQ ID No.1, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody are represented by the amino acid sequences at positions 26-33, 51-58 and 97-118 of SEQ ID No.5, respectively.

In a preferred embodiment, the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO. 5.

In a more preferred embodiment, the amino acid sequence of the constant region of the antibody light chain is shown in SEQ ID NO.3 and the amino acid sequence of the constant region of the antibody heavy chain is shown in SEQ ID NO. 7.

Secondly, the invention also provides the gene coding sequences of the light chain and the heavy chain of the monoclonal antibody, wherein the gene coding sequence of the light chain variable region of the antibody is shown by SEQ ID NO.2, and the gene coding sequence of the heavy chain variable region of the antibody is shown by SEQ ID NO. 6.

In a preferred embodiment, the gene coding sequence for the light chain constant region of the antibody is represented by SEQ ID No.4 and the gene coding sequence for the heavy chain constant region of the antibody is represented by SEQ ID No. 8.

Furthermore, the present invention provides a functional element capable of expressing the coding sequence of the gene encoding the heavy and/or light chain of the monoclonal antibody described above.

In a preferred embodiment, the functional element is a linear expression cassette.

Still further, the present invention provides a host cell comprising the above linear expression cassette, wherein the cell is a 293T cell.

Finally, the invention provides the application of the monoclonal antibody in preparing the Ebola virus disease treatment medicine.

In a preferred embodiment, the monoclonal antibody is used as a component in an ebola virus cocktail formulation.

The monoclonal antibody 8F9 for resisting the envelope glycoprotein GP1 subunit of the Ebola virus has a unique CDR region, has good binding activity with EBOV GP and has EC₅₀The value was 0.015. mu.g/mL. 8F9 was effective in neutralizing EBOV pseudoviruses in vitro, as compared to control antibodies. The neutralizing activity of 8F9 is enhanced with the increase of the antibody concentration, and 90% of protection can be realized on EBOV pseudovirus infected cells at the concentration of 1 mu g/mL. 8F9 also has good in vitro neutralizing activity on EBOV, can be used as a candidate therapeutic drug for Ebola virus, and the binding epitope of 8F9 is positioned at the 227 th and 295 th amino acids of GP antigen, is different from the neutralizing antibody in the prior art, and has the potential of composing cocktail combination therapy with other neutralizing antibodies.

Drawings

FIG. 1 is a graph of flow sort memory B cell results;

FIG. 2 shows the electrophoretic identification map of nested PCR amplified antibody gene;

FIG. 3 is a schematic diagram of the splicing sequence of the linear expression boxes;

FIG. 4 is a schematic diagram of antibody binding screening;

FIG. 5.8F 9 antibody binding activity profile with four antigens;

FIG. 6.8 graph of neutralizing activity of F9 antibody against pseudovirus;

FIG. 7 is a schematic diagram of a truncated GP construction;

FIG. 8.8 binding activity profile of F9 with each truncated antigen;

FIG. 9.8 binding activity profile of F9 with each truncated antigen;

FIG. 10 is a graph showing the competitive binding values between antibodies.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

Example 1 antibody preparation

1. Collection of blood samples

After obtaining the informed consent, 5mL of blood sample of the recombinant ebola vaccine clinical trial subject after the second immunization for 28 days was collected for subsequent experiments.

FITC-labeled protein GPdM

Specific memory B cells need to be sorted by fluorescence labeled antigen, and the FITC labeled truncated antigen protein GPdM method is as follows:

1) fluoroescein Isothiocyanate _ FITC (SIGMA, F4274) was dissolved in DMSO at a concentration of 20 mg/mL.

2) mu.L of GPdM (3.3mg/mL) was taken and the buffer (pH9.6 carbonate buffer) was added to 400. mu.L.

3) Add 8. mu.L FITC to GPdM solution and incubate at 4 ℃ for 3h protected from light.

4) The solution was changed with PBS using a 50kD centrifuge tube until the filtrate was clear and colorless. And (4) wrapping the marked protein with tin foil paper, and storing at 4 ℃ for later use.

3. Flow sorting memory B cells

The collected blood samples were used for PBMC isolation by Ficoll density gradient centrifugation as follows:

1) taking fresh anticoagulated whole blood, and performing EDTA anticoagulation. Whole blood was diluted with an equal volume of PBS.

2) And (3) adding a certain volume of separation liquid into the centrifugal tube, flatly spreading the diluted blood sample above the liquid level of the separation liquid, and keeping the interface between the two liquid levels clear. The volume of the separating medium, anticoagulated undiluted whole blood and PBS (or physiological saline) is 1:1: 1.

3) Trim, room temperature, horizontal rotor 800g, acceleration 3acc, centrifuge for 30 min.

4) After centrifugation, the tube bottom is red blood cells, the middle layer is separation liquid, the uppermost layer is a plasma/tissue homogenate layer, and a thin and compact white membrane is arranged between the plasma layer and the separation liquid layer, namely: a layer of mononuclear cells (including lymphocytes and monocytes). Carefully aspirate the buffy coat into another centrifuge tube.

5) Dilute to volume with PBS and mix by inversion. At room temperature, the rotor was rotated horizontally at 300g, centrifuged for 10min, and the supernatant was discarded. The washing was repeated 2 times.

6) The lymphocytes were resuspended in PBS for use.

7) Counting the cells used for sorting, taking 1X 10⁶The cells, 100. mu.L in volume, were incubated with 6 fluorochromes as recommended in the following table at 4 ℃ in the dark for 1 h.

8) The washing was repeated 2-3 times with PBS containing 2% FBS, the 400. mu.L FPBS was resuspended, the cell pellet was removed with a 40 μm cell sieve, and stored at 4 ℃ in the dark for sorting.

9) GP-specific individual memory B cells were sorted using a cell sorter (SONY, SH 800S). The sorting strategy is as follows: IgG +/CD3-/CD38-/CD19+/CD27+/GPdM +, single plasma cells were directly sorted into 96-well plates, each well of which contained 5U RNase inhibitor and 19.8. mu.L of RNase-free water and stored at-80 ℃.

FIG. 1 is a graph of the results of cell sorting, in which the cells in the circled portion of the box are characterized as IgG +/CD3-/CD38-/CD19+/CD27+/GPdM +, i.e., the desired GP-specific memory B cells.

4. Single cell PCR amplification fully human source monoclonal antibody

1) Reverse transcription PCR

80 GPdM-specific memory B cells were obtained by sorting, and the following specific primers for each subtype of heavy chain (H), kappa light chain and lambda light chain were added simultaneously to each reaction system (see the primer sequences in Table 1).

TABLE 1 reverse transcription PCR primers

The PCR reaction system comprises: 5 Xbuffer 6 u L, dNTP 1.2.2 u L, reverse transcriptase 1.2 u L, primer, template for single cell, water to make up to 30L.

The PCR reaction conditions are as follows: reverse transcription at 50 deg.C for 30min, pre-denaturation at 95 deg.C for 15min, followed by 95 deg.C for 40s, 55 deg.C for 30s, 72 deg.C for 1min, 40 cycles, and final extension at 72 deg.C for 10 min.

2) Nested PCR

Taking the reverse transcription product as a template, and respectively carrying out nested PCR amplification on H, kappa and lambda for 3 times (the primer sequences are shown in Table 2), wherein the specific process is as follows:

TABLE 2 nested PCR primers

The PCR reaction system comprises: 2.5. mu.L of 10 Xbuffer, 0.5. mu.L of 10mM dNTP, 0.5. mu. L, DNA polymerase, 1. mu.L of the reverse transcription product as the template, and 25. mu.L of water.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min, followed by 94 ℃ for 30s, 57 ℃ for 30s, 72 ℃ for 45min, 40 cycles, and finally extension at 72 ℃ for 10 min.

3) Agarose gel electrophoresis

Clones with successful amplification of both heavy and light chain genes in a single cell were considered successful clones. And (3) carrying out 1% agarose gel electrophoresis on 5 mu L of nested PCR amplification products, sequencing the paired positive clones, analyzing the antibody variable region sequence obtained by sequencing by using Vector NTI software and an IMGT website, and carrying out antibody protein expression and function verification. FIG. 2 is the identification map of agarose gel electrophoresis after nested PCR amplification of three chain genes of H, kappa and lambda. Only when the heavy and light chain variable region genes were amplified from the same memory B cell, were naturally paired antibody genes considered and the corresponding nested PCR products sequenced for further study.

5. Linear expression cassette expression antibodies

Through the reverse transcription reaction, 35 paired antibody sequences are obtained from single cell cloning, and if the traditional cloning expression method is time-consuming and labor-consuming, the antibody can be quickly expressed by a method for constructing a linear expression frame. The basic principle of this method is to directly link a promoter sequence (GENBANK accession number: X03922.1), a coding sequence of an antibody leader peptide, an antibody variable region (amplified from a single cell), an antibody constant region (biosynthetic), and a poly-A tail (GENBANK accession number: X03896.1) by overlap extension PCR, and to transfect the linear form of DNA into cells for antibody expression.

The specific process is as follows:

1) a promoter-leader sequence fragment and a poly-A tail fragment were amplified using pSec Tag2(Invitrogen) as a template.

The PCR reaction system for amplifying the promoter-leader sequence fragment comprises: template plasmid pSec Tag2(Invitrogen)1ng, 10 Xbuffer 5. mu.L, 10mM dNTP 1. mu. L, DNA polymerase 0.5. mu.L, primer 5'CMV-FORWARD (CGATGTACGGGCCAGATATACGCGTTG), primer 3' leader-sequence (GTCACCAGTGGACCTGGAACCCCCA), water to 50. mu.L.

The PCR reaction system for amplifying poly-A tail fragments comprises: template plasmid pSec Tag2(Invitrogen)1ng, 10 Xbuffer 5. mu.L, 10mM dNTP 1. mu. L, DNA polymerase 0.5. mu.L, primer 5'POLY (A) (GCCTCGACTGTGCCTTCTAGTTGC), primer 3' POLY (A) (TCCCCAAGCATGCCTGCTATTGTCT), water to 50. mu.L.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min, followed by 94 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 1min, 30 cycles, and final extension at 72 ℃ for 10 min.

2) Amplifying the antibody constant region.

The H chain constant region PCR system comprises: heavy chain constant region template 10ng, 10 x buffer 5u L, 10mM dNTP 1 u L, DNA polymerase 0.5 u L, primer 5'CH (ACCAAGGCCCATCGGGTCTTCCCC), primer 3' CH (GCAACTAGAGGCACAGGTCGAGGCTTTACCCGGAGACAGGGGA), water to 50 u L.

The kappa chain constant region PCR system comprises: mu.L of kappa chain constant region template 10ng, 5. mu.L of 10 Xbuffer, 0.5. mu.L of 10mM dNTP 1. mu. L, DNA polymerase, 5 'C.kappa.primer (ACTGTGGCTGCACCATCTGTCTTC), 3' C.kappa.primer (GCAACTAGAAGGCACAGTCGAGGCACACTCTCCCCTGTTGAAGCT), and water to 50. mu.L.

The lambda chain constant region PCR system comprises: lambda chain constant region template 10ng, 10 Xbuffer 5. mu.L, 10mM dNTP 1. mu. L, DNA polymerase 0.5. mu.L, primer 5 'C.lambda. (GAGGAGCTTCAAGCCAACAAGGCCACA), primer 3' C.lambda. (GCAACTAGAAGGCACAGTCGAGGCTGAACATTCTGTAGGGGCCAC), water to 50. mu.L.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min, followed by 94 ℃ for 30s, 60 ℃ for 60s, 72 ℃ for 3min, 30 cycles, and final extension at 72 ℃ for 10 min.

3) Amplification of antibody variable regions

The PCR system comprises: the template was 10ng of the reverse transcription PCR product, 5. mu.L of 10 Xbuffer, 0.5. mu.L of 10mM dNTP 1. mu. L, DNA polymerase, the primers shown in Table 3 (heavy chain and light chain primers were mixed and added to the system), and water was added to make up to 50. mu.L.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min, followed by 94 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 3min, 30 cycles, and final extension at 72 ℃ for 10 min.

TABLE 3 construction of PCR primers for Linear expression Frames

Kappa chain primer sequence

Lamda chain primer sequence

4) And (3) recovering and purifying PCR products: the PCR products were subjected to electrophoresis on a 1% agarose gel, cut and recovered using a recovery kit from OMEGA.

5) Amplification of Linear expression cassettes for heavy and light chains, respectively

The schematic diagram of the splicing sequence of the linear expression boxes is shown in FIG. 3. In FIG. 3, A is an H chain linear expression cassette, B is a kappa chain linear expression cassette, and C is a lambda chain linear expression cassette.

The PCR reaction system comprises:

template: 10ng of purified promoter-leader sequence fragment, 10ng of heavy chain/light chain variable region fragment, 10ng of heavy chain/light chain constant region fragment, 10ng of poly-A tail fragment, 2.5 μ L of 10 × buffer solution, 0.5 μ L of 10mM dNTP 0.5 μ L, DNA polymerase, 0.25 μ L of primer 5'CMV-FORWARD (CGATGTACGGGCCAGATATACGCGTTG) and 3' POLY (A) (TCCCCAGCATGCTATTGTCT), water-supplemented to 25 μ L.

6) And (3) recovering and purifying PCR products: the PCR reaction product was recovered directly with the recovery kit of OMEGA.

7) DNA quantification: the PCR-recovered product was quantified using Nano (GE healthcare).

8) Cell inoculation: 293T cells at 2X 10⁵Perml in 24-well cell culture plates in 5% CO₂The cells were incubated at 37 ℃ overnight in an incubator.

9) Cell co-transfection: the next day, 200. mu.L of serum-free MEM was culturedTo the medium, 1. mu.g each of the successfully constructed heavy and light chain linear expression cassette PCR products was added, mixed well, 4. mu.L of transfection reagent Turbofect (Thermo Scientific, R0531) was added, incubated for 15-20min, and then added dropwise to 293T cell culture wells grown overnight. In the presence of 5% CO₂The cells were cultured at 37 ℃ for 48 hours in the cell incubator, and then the cell culture supernatant was collected for use.

ELISA screening for antibodies with binding Activity

1) One day prior to the experiment 96 well ELISA plates were coated with 1. mu.g/mL EBOV GP, 100. mu.L per well. The coated enzyme-linked plate was placed in a wet box at 4 ℃ overnight.

2) The experiment was washed 5 times on the day with a plate washer (BIO-TEK, 405_ LS).

3) Add 100. mu.L of blocking solution to each well and let stand at room temperature for 1 hour.

4) Washing with plate washing machine for 5 times.

5) 100. mu.L of the transfected cell culture supernatant was added and allowed to stand at room temperature for 1 hour.

6) Washing with plate washing machine for 5 times.

7) Diluting HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) with a diluent at a ratio of 1:10000, adding 100. mu.L of the diluted antibody to corresponding wells of an ELISA plate, and incubating at room temperature for 1 hour;

8) washing with plate washing machine for 5 times.

9) Adding 100 mu L of TMB single-component developing solution into each hole, developing for 6min, keeping the room temperature away from light, and then adding 50 mu L of stop solution into each hole to terminate the reaction.

10) Detecting OD value at the position of 450-630nm on a microplate reader, and storing and recording original data.

As a result: screening 35 monoclonal antibodies, and identifying the specificity of the combined antibody of the Ebola virus GP. There are 14 strains that specifically bind ebola GP, including 8E3, 8E5, 8B6, 8D7, 8H7, 8a8, 8G8, 8E9, 8F9, 8H10, 8C12, 8D12, 8F12, and 8G 12. (the detection results are shown in FIG. 4).

7.8F9 antibody sequence description

The 8F9 was identified to have good binding activity and neutralizing activity against EBOV. The sequence is described as follows:

the amino acid sequence of the light chain variable region is shown in SEQ ID NO.1, the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in amino acid sequences at positions 27-32, 50-52 and 89-97 of SEQ ID NO.1, and the gene coding sequence of the light chain variable region is represented by SEQ ID NO: 2, the amino acid sequence of the light chain constant region is shown as SEQ ID NO.3, and the gene coding sequence of the light chain constant region is shown as SEQ ID NO. 4;

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.5, the amino acid sequences of the CDR1, the CDR2 and the CDR3 of the heavy chain variable region are respectively shown as the amino acid sequences at positions 26-33, 51-58 and 97-118 of the SEQ ID NO.5, the gene coding sequence of the heavy chain variable region is shown as SEQ ID NO.6, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO.7, and the gene coding sequence of the heavy chain constant region is shown as SEQ ID NO. 8.

8. Expression plasmid construction and antibody preparation

8F9 is used for constructing an expression plasmid, and the monoclonal antibody is expressed and prepared. The method comprises the following steps:

1) the full-length genes of the linear expression cassettes of 8F9H and 8F9K were double digested with EcoRI (NEB, R3101) and NotI (NEB, R3189) and ligated into pcDNA3.4 expression plasmid.

2) 15. mu.g each of pcDNA3.4-8F9H and pcDNA3.4-8F9K was transfected into 30mL Expi293 systems (Life, A14524) at 125rpm with 5% CO₂And culturing for 72 h.

3)3000 Xg, centrifugating for 10min to collect the expression supernatant, filtering with 0.22 μm needle filter, and affinity purifying with rProtein A.

4) The collected antibodies were subjected to a solution change with PBS, and then the antibody concentration was determined with BCA protein quantification kit (Thermo Scientific, 23225).

Example 2 ELISA detection of antibody binding Activity

1) One day before the experiment, 96-well enzyme-linked plates were coated with 1. mu.g/mL of EBOV GP, BDBV GP, SUDV GP and RESTV GP, and each well was coated with 100. mu.L. The coated enzyme-linked plate was placed in a wet box at 4 ℃ overnight.

2) The experiment was washed 5 times with a plate washer on the day.

4) Washing with plate washing machine for 5 times.

5) 150 μ L of 8F9 mAb at 10 μ g/mL was added to the first well and 100 μ L of the dilution was added to the remaining wells. Aspirate 50 μ L from the first well and add to the second well and so on, dilute each well in a 1:3 gradient to a final volume of 100 μ L. The mixture was allowed to stand at room temperature for 1 hour.

6) Washing with plate washing machine for 5 times.

7) The HPR-labeled secondary goat anti-human IgG antibody was diluted at a ratio of 1:10000 in a diluent, 100. mu.L of each well was added to the corresponding well of the ELISA plate, and the plate was incubated at room temperature for 1 hour.

8) Washing with plate washing machine for 5 times.

As a result: the 8F9 monoclonal antibody and EBOV GP have good combination activity, EC₅₀The value was 0.015. mu.g/mL. In addition, 8F9 can also cross-bind to BDBV GP, EC₅₀The value was 0.108. mu.g/mL. 8F9 did not bind to RESTV GP and SUDV GP. (the detection results are shown in FIG. 5).

Example 3 pseudovirus neutralization experiment evaluation of 8F9 neutralizing Activity

The neutralizing activity of 8F9 was evaluated in vitro with EBOV pseudovirus packaging HIV backbone. The evaluation method is as follows:

1) 8F9 monoclonal antibody is diluted by DMEM medium, 75 mu L of antibody diluent with the concentration of 100 mu g/mL is added into the first hole of a 96-hole cell culture plate, and 50 mu L of DMEM medium is added into the other holes.

2) Sucking 25 mul of liquid from the first hole, adding the liquid into the second hole, mixing uniformly, and diluting according to the ratio of 1:3 by analogy, wherein the final volume of each hole is 50 mul.

3) The pseudovirus was diluted 1:5 with DMEM medium (control well fluorescein readings were controlled between 20000-100000) and added to each antibody well at 50. mu.L per well. Mix well and incubate at 37 ℃ for 1 h.

4)293T cell count, 2X 10⁵cells/mL, 100. mu.L per well.

5) And (3) putting the 96-hole cell culture plate into a 37 ℃ incubator for culturing for 36-48 hours.

6) The cell culture plate was removed and the culture broth carefully aspirated and discarded. Add 100. mu.L of cell lysate to each well and shake for 15min at 350rpm on a shaker.

7) Centrifuge at 3000rpm for 10min at room temperature.

8) A detection substrate lyophilizate of a luciferase detection system (Promega, E1501) and a detection buffer solution are mixed uniformly, and a detection pipeline of GLOMAX 96 MICROPLATE LUMINOMETER (Promega) is filled.

9) And (4) sucking 20 mu L of reading values of the lysis supernatant, reading fluorescence values, and calculating the protection rate of the antibody on the cells.

As a result: 8F9 was effective in neutralizing EBOV pseudoviruses in vitro, as compared to control antibodies. The neutralizing activity of 8F9 is enhanced with the increase of the antibody concentration, and 90% of protection can be realized on EBOV pseudovirus infected cells at the concentration of 1 mu g/mL. (see FIG. 6 for the detection results).

Example 4 truncated antigen analysis of the 8F9 binding region

8F9 exhibited good in vitro neutralization activity, and it was hoped to analyze the approximate binding region by constructing a truncated GP. Besides GPdTM, GPdM, GP1, sGP and GPcl which are common in the literature, GP is designed and constructed according to GP structural characteristics and analysis targets_{33-310；463-558}、GP_33-227、GP_33-158、GP_95-295、GP_158-295And GP_227-295。GP_{33-310；463-558}The latter heptad repeat region is removed on the basis of GPdM; GP_33-227The glycan cap is removed on the basis of sGP; GP_33-158Most of the Base area and the Head area are included; GP_95-295The Base region was deleted compared to sGP; GP_158-295Compared with sGP, the Base region and the Head region are basically deleted; GP_227-295The majority of the glycan cap region is contained. GPcl is obtained by cleaving the glycan cap with thermolysin (thermolysin) on the basis of GPdM, without constructing an expression plasmid. The schematic structure of each truncated GP is shown in FIG. 7.

The truncated GP assay 8F9 binding region method is as follows:

1) according to the schematic diagram, each truncated GP gene segment is amplified from the GP full-length gene, and His is added at the carboxyl terminal₆Tags, gene fragmentsLigated into pcDNA3.4 expression plasmid.

2) Each truncated GP was expressed in the Expi293 system and the protein of interest was obtained by NI-NTA purification.

3) The binding activity of 8F9 to each truncated GP was analyzed by ELISA as described in example 2.

As a result: 8F9 with GPdM, sGP, GP1, GP_95-295And GP_{33-310,463-558}Several truncated GP's are well combined, EC₅₀The values were 0.0062. mu.g/mL, 0.0088. mu.g/mL, 0.0095. mu.g/mL, 0.0138. mu.g/mL and 0.0052. mu.g/mL, respectively. The binding activity of 8F9 to these truncated GPs was close to or even better than that of GPdTM, possibly with partial region deletion to make the binding epitope more exposed. 8F9 and GP_158-295And GP_227-295Slightly weaker binding activity, EC₅₀The values were 0.273. mu.g/mL and 0.102. mu.g/mL, respectively. The binding epitope for 8F9 should be located in the region shared by these truncated GPs, i.e.,

amino acids

227 and 295. The binding activity of 8F9 to each truncated antigen was measured as shown in fig. 8-9.

Example 5 Competition experiments

The epitope of 8F9 was analyzed by competition binding ELISA. Using MIL77-1/2/3 and the remaining several non-neutralizing antibodies known to bind epitopes as control antibodies, the presence of overlap in the binding epitopes of these antibodies was reflected by examining whether the binding of the test antibody to GP was blocked by competing antibodies incubated therewith. The method comprises the following steps:

1) 4mg of biotin (Thermo Scientific, 21335) was weighed and dissolved in 2mL of ultrapure water to a concentration of 2 mg/mL.

2) Antibodies 8F9, 2E5, 5D7, 5G11, 5B12, 1B3, MIL77-1, MIL77-2 and MIL77-3 are respectively taken at 200 mu G, and the volume is controlled to be about 200 mu L.

3) The antibodies were labeled at a molar ratio of biotin to antibody of 20: 1. The antibody and biotin were mixed, incubated at room temperature for 1 hour, and the solution was changed with a 50kD 0.5mL spin filter, about 400. mu.L of PBS each time, 3 more times.

4) The final PBS filter liquid to all the filter to about 100 u L (in order to make the antibody concentration similar), the antibody concentration determination.

5) The 96-well ELISA plates were coated with GPdM at a concentration of 1. mu.g/mL overnight at 4 ℃.

6) The plate washer washes the ELISA plates, adds 100. mu.L of blocking solution per well, and incubate for 1 hour at 37 ℃.

7) In the experiment, the detection antibody is a biotin-labeled antibody, and the competitive antibody is a non-biotin-labeled antibody. Diluting the competitive antibody to 2.5 mu g/mL by using the diluent; dilutions dilute the detection antibody to 25 ng/mL.

8) The 96-well plate was washed. Each well was loaded with 50. mu.L of detection antibody and 50. mu.L of competitor antibody, 100. mu.L per final well volume.

9) Washing, streptavidin antibody (Thermo scientific, 21126) diluted 1:1000, 100. mu.L per well, and incubation at 37 ℃ for 1 hour.

10) Washing, adding 100 mu L of TMB single-component color development liquid into each hole, developing for 6 minutes in a dark place, and adding 50 mu L of stop solution.

11) Read 450 and 630nm OD values.

As a result: a competition binding value of less than 30 is considered to be strong; greater than 30 and less than 60 are considered weak; above 60 no competition is considered. The antibodies compete well for binding with each other and can be divided into four groups according to the results. Two groups of the protein are combined with GP1 subunit and GP2 subunit. Competition for 8F9 with control antibodies 2E5, 1B3, and MIL77-3 that bound to the GP1 subunit suggests that the 8F9 binding epitope is spatially close to or overlaps with the binding epitope of these antibodies. 8F9 did not compete for binding with 5D7 and 5G11 bound to the GP1 subunit, nor with the control antibody bound to the GP2 subunit, indicating that the 8F9 binding epitope is different from these antibodies. 8F9 has good in vitro neutralizing activity on EBOV, can be used as a candidate therapeutic drug for Ebola virus, has a binding epitope different from other neutralizing antibodies, and has the potential of composing cocktail combination therapy with other neutralizing antibodies (the detection result is shown in figure 10).

Sequence listing

<110> military medical research institute of military science institute of people's liberation force of China

<120> monoclonal antibody 8F9 specifically binding to GP1 subunit of EBOV and application

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 107

<212> PRT

<213> Homo sapiens

<400> 1

Ala Ile Arg Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Lys Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Met

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 2

<211> 321

<212> DNA

<213> Homo sapiens

<400> 2

gccatccgga tgacccagtc tccattctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtcg gaccattagc agctatttaa attggtatca gaagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ctcccatgac cttcggccaa 300

gggacacgac tggagattaa a 321

<210> 3

<211> 106

<212> PRT

<213> Homo sapiens

<400> 3

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 4

<211> 318

<212> DNA

<213> Homo sapiens

<400> 4

actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60

actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120

aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180

aaggacagca cctacagcct cagcagtacc ctgacgctga gcaaagcaga ctacgagaaa 240

cacaaagtct acgcctgcga agtcacccat cagggcctga gttcgcccgt cacaaagagc 300

ttcaacaggg gagagtgt 318

<210> 5

<211> 129

<212> PRT

<213> Homo sapiens

<400> 5

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Ser Asn Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Thr Arg Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Leu Ser Val Phe Gly Val Val Leu Ile Ser Ser Asp

100 105 110

Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120 125

Ser

<210> 6

<211> 387

<212> DNA

<213> Homo sapiens

<400> 6

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctgaatt cacctttagc aactatgcca tgaactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcaggt attagtacta gaggtagtag tacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agtcgaggac acggccgtat attactgtgc gaaagatcgt 300

ctttcggttt ttggagtagt tcttataagt tctgactact acggtatgga cgtctggggc 360

caagggacca cggtcaccgt ctcctca 387

<210> 7

<211> 330

<212> PRT

<213> Homo sapiens

<400> 7

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Pro

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Pro Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 8

<211> 990

<212> DNA

<213> Homo sapiens

<400> 8

gcgtcgacca agggcccatc ggtcttcccc ctggcacctt cttccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcccgcacc aggactggct gaatggcaaa 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc cgcccccatc ccgggatgag 720

ctgaccaaga accaggtcag cctgacctgc ctggtcaagg gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagaagagcc tctccctgtc tccgggtaaa 990

Claims

1. A monoclonal antibody for resisting the subunit GP1 of Ebola virus glycoprotein is characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of a light chain of the antibody are respectively shown as the amino acid sequences at positions 27-32, 50-52 and 89-97 of SEQ ID NO.1, and the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of a heavy chain of the antibody are respectively shown as the amino acid sequences at positions 26-33, 51-58 and 97-118 of SEQ ID NO. 5.

2. The monoclonal antibody according to claim 1, wherein the amino acid sequence of the antibody light chain variable region is represented by SEQ ID No.1, and the amino acid sequence of the antibody heavy chain variable region is represented by SEQ ID No. 5.

3. The monoclonal antibody of claim 2, wherein the amino acid sequence of the antibody light chain constant region is set forth in SEQ ID No.3 and the amino acid sequence of the antibody heavy chain constant region is set forth in SEQ ID No. 7.

4. A polynucleotide encoding the light and heavy chains of the monoclonal antibody of any one of claims 1-3, wherein the polynucleotide encoding the light chain variable region of the monoclonal antibody has the sequence shown in SEQ ID No.2 and the polynucleotide encoding the heavy chain variable region of the monoclonal antibody has the sequence shown in SEQ ID No. 6.

5. The polynucleotide of claim 4, wherein the polynucleotide encoding the light chain constant region of the monoclonal antibody has the sequence shown in SEQ ID No.4 and the polynucleotide encoding the heavy chain constant region of the monoclonal antibody has the sequence shown in SEQ ID No. 8.

6. A linear expression cassette comprising polynucleotides encoding the heavy and light chains of a monoclonal antibody according to claim 5.

7. A host cell comprising the linear expression cassette of claim 6, wherein said cell is a 293T cell.

8. Use of a monoclonal antibody according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of ebola virus disease.

9. The use according to claim 8, wherein the monoclonal antibody is used as a component in an ebola virus cocktail formulation.