CN110903386B - Fully human monoclonal antibody with high neutralizing activity and resisting chikungunya fever and application - Google Patents

Abstract

The invention discloses a fully human monoclonal antibody with high neutralizing activity against chikungunya virus, which is obtained by screening through a flow sorting-single cell PCR (polymerase chain reaction) technology and has a unique CDR (complementary deoxyribonucleic acid) partition. The monoclonal antibody disclosed by the invention has high-efficiency and specific chikungunya virus resistant activity, has the characteristics of high expression, full humanity and good stability, and is suitable for industrial production.

Description

Fully human monoclonal antibody with high neutralizing activity and resisting chikungunya fever and application

Technical Field

The invention discloses an antibody, and belongs to the fields of microbiology and immunology.

Background

Chikungunya fever (chikungunya feber), a mosquito-borne infectious disease caused by chikungunya virus (chikungunya virus), originally prevalent in tropical and subtropical regions of africa, and is continually expanding to southern asia, southeast asia, indian islands, and the americas. The major clinical signs of chikungunya fever are fever, headache, myalgia, rash and joint pain. Over the past decade, the number of outbreaks of chikungunya fever has increased, with an ever-expanding prevalence that has occurred in over 100 countries and regions worldwide, resulting in approximately 100 million cases per year worldwide. On 12/20/2015, the world health organization, based on the training of ebola epidemic response, identified eight dangerous pathogens (ebola, marburg virus, etc.) and three secondary dangerous pathogens (chikungunya fever, thrombocytopenia syndrome with severe fever and zika flavivirus) that may cause severe epidemic outbreaks, chikungunya virus being one of the three secondary dangerous pathogens. At the same time, chikungunya virus is classified as a class C biodefense pathogen by the american institute for allergy and infectious disease. The army of the united states considers chikungunya virus as a potential biological warfare/bioterrorism agent because it can potentially be infected by aerosol.

At present, chikungunya fever is mainly treated by symptomatic treatment such as fever reduction, pain relief and the like, and no specific treatment medicine exists clinically. Aedes aegypti and aedes albopictus are widely distributed in China, and China closely contacts with people in some chikungunya epidemic countries, so that the risk of chikungunya virus transmission exists.

The literature reports that antibodies have therapeutic effects: (1) multiple antibody: multivalent antibodies were isolated from chikungunya virus infection convalescent patients by the french pasteur institute, and showed strong neutralizing activity in vitro and potent protective effects in animal models. (2) Murine monoclonal antibodies: four murine antibodies, CHK-102, CHK-152, CHK-166, CHK-263, developed at the university of St.Louis Washington, USA, were effective in the mouse assay. Antibodies were raised against the glycoproteins E1 and E2, of which the most potent antibody CHK-152 was humanized and exhibited similar neutralizing properties to murine antibodies. (3) Human monoclonal antibodies: two human monoclonal antibodies 5F10 and 8B10 directed against viral envelope glycoproteins were isolated by the Singapore scientific research institute from patients infected with chikungunya virus, and have neutralizing effects against different chikungunya virus strains, and 100% protection was achieved in the test mice in the preventive experiment. A monoclonal antibody MAb C9 developed by the university of california, usa, was also isolated from chikungunya virus infected patients, and was administered to chikungunya virus E2 glycoprotein 1 day before infection in mice to prevent the development of viremia and arthralgia.

Polyclonal antibodies have antibody enhanced toxic effects, the mouse monoclonal antibodies are applied to human bodies, have immunogenicity, generate anti-mouse antibodies, and need immunogenicity modification, and the humanized monoclonal antibodies are the most preferable treatment strategies. The prior art for obtaining the human monoclonal antibody comprises a phage library technology and a single cell PCR technology, and compared with the phage library technology, the monoclonal antibody obtained by the single cell PCR technology is a natural pairing antibody and has better stability. Single cell PCR technology can be used to screen inactivated vaccine, attenuated live vaccine immunized volunteers and natural infectors, natural infectors are the preferred strategy, because the virus in vivo replication and stimulation, the antibody is natural. US9738704 discloses a monoclonal antibody against chikungunya virus, the IC of said antibody₅₀2.17ug/ml was reached.

The invention aims to obtain a monoclonal antibody with more excellent neutralizing and protecting activity from peripheral blood of a chikungunya virus infected person by adopting a single-cell PCR technology, and aims to provide a fully humanized anti-chikungunya virus monoclonal antibody with a good protecting effect.

Disclosure of Invention

Based on the aim, the monoclonal antibody against chikungunya virus is screened by a flow sorting-single cell PCR technology, and the amino acid sequences of CDR1, CDR2 and CDR3 regions in a heavy chain variable region of the monoclonal antibody are respectively shown as the sequences of 26 th to 33 th, 51 th to 58 th and 97 th to 109 th bits of SEQ ID NO: 1; the amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 of the monoclonal antibody are shown in the sequence of 27-32, 50-52 and 88-93 bits of SEQ ID NO. 5 respectively.

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

In a more preferred technical scheme, the amino acid sequence of the constant region of the heavy chain of the monoclonal antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the constant region of the light chain of the monoclonal antibody is shown as SEQ ID NO. 7 or SEQ ID NO. 9. One preferred monoclonal antibody of the invention is designated 8D 1.

Secondly, the invention also provides a polynucleotide for coding the heavy chain and/or the light chain of the monoclonal antibody, wherein the polynucleotide sequence for coding the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 2, and the polynucleotide sequence for coding the light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 6.

In a preferred technical scheme, the polynucleotide sequence for coding the constant region of the heavy chain of the monoclonal antibody is shown as SEQ ID NO. 4, and the polynucleotide sequence for coding the constant region of the light chain of the monoclonal antibody is shown as SEQ ID NO. 8 or SEQ ID NO. 10.

Thirdly, the present invention also provides a functional element for expressing the above polynucleotide encoding the heavy chain and/or the light chain of the monoclonal antibody, which can be a conventional expression vector.

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

In another preferred embodiment, the functional element is a mammalian expression vector.

Fourth, the present invention also provides a host cell containing the above functional element.

In a preferred embodiment, the cells are Expi 293F cells.

In another preferred embodiment, the cells are CHO-S cells.

Finally, the invention also provides the application of the monoclonal antibody in preparing a chikungunya heat treatment medicine.

The monoclonal antibody provided by the invention shows good neutralization protection effect on chikungunya virus infected cells. On a cell model, the neutralizing activity is 18 times that of monoclonal antibody Ab2 disclosed in US9738704 (refer to the variable region sequences of the heavy chain and the light chain of 8B10F8 disclosed in US9738704, the variable region genes are chemically synthesized, the full-length antibody genes are obtained by using a fusion PCR technology, cloned to an expression vector pMH vector, transfected to CHO-S cells, obtained by Protein A affinity chromatography purification and stored, and the invention is named as Ab 2). Research results show that the antibody provided by the invention has wide application prospect in preparing chikungunya heat treatment medicines. The monoclonal antibody disclosed by the invention also has the following technical advantages that (1) the monoclonal antibody is fully human, and in clinical application, the humanized modification is not needed to reduce human anti-mouse antibody reaction (HAMA reaction), namely low immunogenicity. (2) High expression, the expression amount in a transient expression system is close to 0.3g/L, and the industrialization is convenient to carry out later. (3) The epitope recognized by the monoclonal antibody is a conformation epitope, and the recognized target protein is E2 protein, so that the monoclonal antibody can be combined with the reported humanized monoclonal antibody for application, the generation of drug-resistant strains is avoided, and the curative effect is improved. (4) The stability is good, because the antibody gene is from the same cell of the human body and is naturally paired, the half-life of the IgG1 antibody in the human body is known to be 21-28 days, and theoretically, the disclosed monoclonal antibody has consistent half-life in the human body.

Drawings

FIG. 1 PCR amplification nucleic acid electrophoresis detection of heavy and light chain variable region genes;

FIG. 2 nucleic acid electrophoresis detection of the heavy and light chain linear expression cassettes;

FIG. 3 restriction enzyme digestion identification nucleic acid electrophoresis detection of expression vector;

FIG. 4 analysis of monoclonal antibody expression after transient transfection;

FIG. 5 SDS-PAGE detection of the purified monoclonal antibody by affinity chromatography;

FIG. 6 IC of monoclonal antibodies on cell models₅₀Measuring;

FIG. 7 detection of protective Activity of monoclonal antibodies on cell models;

FIG. 8 alignment of epitopes with structural and non-structural proteins of chikungunya virus.

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 exemplary only and are not intended to limit the scope of the invention as defined in the claims.

Example 1 screening and preparation of human anti-chikungunya Virus monoclonal antibodies

1.1 blood sample Collection

After informed consent, the eighth national hospital, guangzhou, provided peripheral blood of 1 chikungunya heat-infected patient 15 days after recovery for subsequent experiments.

1.2 flow cytometry sorting of Single cells

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

1.2.1 fresh anticoagulated whole blood (EDTA anticoagulated) was taken and the whole blood was diluted with an equal volume of PBS.

1.2.2 adding a certain volume of separating medium into the centrifuge tube, spreading the diluted blood sample above the liquid level of the separating medium, 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.

1.2.3 balance, room temperature, 800g (2000) of horizontal rotor, 2500rpm), 3-4 acc of acceleration, and 20-30min of centrifugation.

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

1.2.5 diluted to volume with PBS/1640 and mixed by inversion. At room temperature, 400g (1000-1500rpm) is horizontally rotated by a rotor, centrifuged for 5-10min, and the supernatant is discarded. The washing was repeated 1-2 times.

1.2.6 resuspend lymphocytes in PBS or appropriate medium for use.

PBMC were stained using flow antibody: anti-CD3-FITC, anti-CD20-FITC, anti-CD19-PE-Alexa 610, anti-CD27-PE, anti-CD38-PE-Cy5, per 5X 10⁶After incubating each PBMC cell with 5. mu.L of the above antibody for 30 minutes, washing was repeated 2-3 times using PBS containing 2% FBS. Selection of plasma cell-specific cell surface markers (CD 3) using MoFlo XDP flow sorting cytometer^neg/CD20 ^low/CD19 ^high/CD27 ^high/CD38 ^high) The plasma cells were sorted by directly sorting individual plasma cells into 96-well plates, each well being preliminarily filled with water containing 5U of RNase inhibitor and 19.8. mu.L of RNase-free water, and frozen at-80 ℃ for use.

1.3 amplification of the variable region Gene of the fully human monoclonal antibody by Single cell-PCR

1.3.1 reverse transcription PCR

With specific reference to the description (QIAGEN, 210212), the procedure is briefly described as follows:

1152 (total 12 cell plates, 96 cells each) single cells were sorted by flow cytometry, so there were 1152 reverse transcription reaction systems. All of the following specific primers for each subtype of heavy chain (heavy chain, H), Kappa light chain (Kappa chain, Kappa), and Lamda light chain (Lamda chain, lambda) were added simultaneously to each reaction system (see the primer sequences in Table 1).

Primer:

H：5′L-VH 1、L-VH 3、L-VH 4/6，5′L-VH 5、Hu IgG-const-anti、3′CμCH1

κ：5′L Vκ1/2、5′L Vκ3、5′L Vκ4、3′Cκ543–566

λ：5′L Vλ1、5′L Vλ2、5′L Vλ3、5′L Vλ4/5、5′L Vλ6、5′L Vλ7、5′LVλ8、3′Cλ

TABLE 1 reverse transcription PCR primers

The PCR reaction system comprises: 5 Xbuffer 6 u L, dNTP 1.2.2 u L, reverse transcriptase (all gold biotechnology limited, AT311)1.2 u L, primer, template for single cell, water to make up to 30L.

The PCR reaction conditions are as follows:

reverse transcription is carried out for 30min at the temperature of 50 ℃,

pre-denaturation at 95 ℃ for 15min,

then 40 cycles of 95 ℃ for 40s, 55 ℃ for 30s and 72 ℃ for 1min,

finally, extension is carried out for 10min at 72 ℃.

1.3.2 nested PCR

Taking the reverse transcription product as a template, and respectively carrying out nested PCR amplification on H, kappa and lambda, wherein the specific process is as follows:

primer:

H：VH3a-sense、VH3b-sense、Mu D、PW-Cgamma

5′Pan Vκ、3′Cκ494–516

λ：5′AgeI Vλ1、5′AgeI Vλ2、5′AgeI Vλ3、5′AgeI Vλ4/5、5′AgeI Vλ6、5′AgeI Vλ7/8、3′XhoI Cλ

TABLE 2 nested PCR primers

The PCR reaction system comprises: 10 Xbuffer 2.5 u L, 10mM dNTP 0.5 u L, DNA polymerase (all gold biotechnology limited, AP141)0.25 u L, primers as above, template for reverse transcription product 1 u L, water to 25L.

The PCR reaction conditions are as follows:

pre-denaturation at 94 ℃ for 4min,

then carrying out 40 cycles of 94 ℃ for 30s, 57 ℃ for 30s and 72 ℃ for 45min,

finally, extension is carried out for 10min at 72 ℃.

1.3.3 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.

1.3.4PCR amplification results are shown in FIGS. 1-A and 1-B, with FIG. 1-A showing the amplification of the heavy chain variable region gene, where lane 10 is the amplification of the 8D1 heavy chain variable region gene; FIG. 1-B is the result of amplification of the light chain variable region gene, wherein lane 4 is the result of amplification of the light chain variable region gene of 8D 1. The amplified fragments of the variable regions of both the light and heavy chains were about 400bp, as expected.

1.4. Construction of a Linear expression cassette

The antibody gene obtained by amplification is subjected to sequence analysis, and after the sequence analysis is expected, a large number of paired genes need to be expressed and subjected to function analysis. Thus, a linear expression cassette was first constructed. The linear expression cassette contains all elements expressed in eukaryotic cells, including promoters, enhancers, polyA tail termination structures, here we refer to a commercial expression vector in which a linear expression cassette comprising, in order from the 5' end, a CMV promoter sequence (Genbank accession number: X03922.1), an antibody variable region (amplified from a single cell), an antibody constant region (biosynthetic, heavy chain constant region sequence shown by SEQ ID NO:3, DNA coding sequence shown by SEQ ID NO:4, Kappa type light chain constant region sequence shown by SEQ ID NO:7, DNA coding sequence shown by SEQ ID NO:8, Lamda type light chain constant region sequence shown by SEQ ID NO:9, DNA coding sequence shown by SEQ ID NO: 10), a BGH poly A tail (Genbank accession number: X03896.1) is ligated and the linear form of DNA is transfected into cells for antibody expression.

The specific process is that each PCR fragment is connected and constructed by in vitro overlap extension PCR technology:

1.4.1 amplification of promoter-leader fragment and poly-A tail fragment using pcDNA3.4(ThermoFisher Scientific, A14697) as template. The PCR reaction system for amplifying the promoter-leader sequence fragment comprises: template plasmid pcDNA3.41ng, 10 × buffer solution 5 μ L, 10mM dNTP 1 μ L, DNA polymerase 0.5 μ L, primer 5'-CMV-forward (matching with CMV promoter upstream sequence) (5'-CGATGTACGGGCCAGATATACGCGTTG-3'), primer 3' -leader-sequence (5'-ACACTGGACACCTTTTAAAATTAG-3', nucleotide sequence 5'-ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTAATTTTAAAAGGTGT C-3' for fusion of heavy chain, signal peptide sequence), encoded amino acid sequence MNFGLSLIFLVLILKGV; the sequence of the fusion primer used for the light chain is 5'-GTCACCAGTGGAACCTGGAACCCA-3', the nucleotide sequence of the full-length signal peptide sequence is 5-ATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGGTACCTGTGGG, the amino acid sequence is MDSQAQVLMLLLLWVSGTCG, the signal peptide sequence is from the variable region of the murine monoclonal antibody), and the water is supplemented to 50 mu L.

The PCR reaction system for amplifying poly-A tail fragments comprises: the template plasmid pSecTag2(Invitrogen, V90020)1ng, 10 Xbuffer 5. mu.L, 10mM dNTP 1. mu. L, DNA polymerase 0.5. mu.L, primer 5'-BGH POLY- (A) (5'-GCCTCGACTGTGCCTTCTAGTT GC-3'), primer 3' -BGH-POLY (A) (5'-TCCCCAGCAT-GCCTGCTATTGTCT-3'), and water to 50. mu.L. The length of the amplified fragment is 215 bp.

And (3) PCR reaction conditions:

pre-denaturation at 94 ℃ for 4min,

then 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1min,

finally, extension is carried out for 10min at 72 ℃.

1.4.2 amplification of antibody constant regions.

The H chain constant region PCR system comprises: heavy chain constant region template 10ng, 10 × buffer 5 μ L, 10mM dNTP 1 μ L, DNA polymerase 0.5 μ L, primer 5'-CH1(5'-ACCAAGGGCCCATCGGTCTTCCCC-3'), primer 3' -CH3(5'-GCAACTAGAAGGCACAGTCGAGGCTTTACCCGGAGACAGGGA-3'), water to 50. mu.L.

The kappa chain constant region PCR system comprises: kappa chain constant region template 10ng, 10 Xbuffer 5. mu.L, 10mM dNTP 1. mu. L, DNA polymerase 0.5. mu.L, primer 5' -Ck (5'-ACTGTGGCTGCACCATCTGTCTTC-3'), primer 3' Ck (5' -GCAACTAGAAGGCACAGTCGAGGC)ACACTCTCCCCTGTTGAAGCT-3'), 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. (GCAACTAGAAGGCACAGTCGAGGC)TGAACATTCTGTAGGGGCCAC) And water to 50 μ L.

The above bold sequence portion GCAACTAGAAGGCACAGTCGAGGC is the complementary sequence to polyA for fusion amplification.

The PCR reaction conditions are as follows:

pre-denaturation at 94 ℃ for 4min,

then 30 cycles of 94 ℃ for 30s, 60 ℃ for 60s and 72 ℃ for 3min,

finally, extension is carried out for 10min at 72 ℃.

1.4.3 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.

And (3) PCR reaction conditions:

pre-denaturation at 94 ℃ for 4min,

then 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 3min,

finally, extension is carried out for 10min at 72 ℃.

TABLE 3 construction of PCR primers for Linear expression Frames

The scored portions are for fusion with the upstream and downstream segments, respectively.

And (3) recovering and purifying PCR products: the PCR products were subjected to 1% agarose gel electrophoresis, cut and recovered using an Omega recovery kit.

1.4.4 amplification of the Linear expression cassettes for the heavy and light chains, respectively.

The PCR reaction system comprises:

template: 10ng of purified promoter-leader 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. mu.L of 10 Xbuffer, 0.5. mu. L, DNA polymerase (all-open gold Biotechnology Co., Ltd., AP151-13) 0.25. mu.L of primer 5'-CMV-FORWARD (5'-CGATGTACGGGCCAGATATACGC GTTG-3') and 3' -POLY (A) (5'-TCCCCAGCATGCCTGCTATTGTCT-3', water to 25. 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.

1.4.5PCR product recovery purification and quantification:

the PCR reaction product was recovered directly with the recovery kit of OMEGA. DNA quantification: the PCR-recovered product was quantified using Nano (GE healthcare).

1.4.6 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.

1.4.7 cell Co-transfection: the next day, 1. mu.g each of the successfully constructed heavy and light chain linear expression cassette PCR products was added to 200. mu.L of serum-free MEM medium, mixed well, 4. mu.L of the transfection reagent Turbofect (Thermo Scientific, R0531) was added, incubated for 15-20min, and added dropwise to overnight-cultured 293T cell culture wells. In the presence of 5% CO₂Culturing at 37 deg.C for 48 hr in cell incubatorAnd collecting the cell culture supernatant for later use.

The results of the PCR fusion amplification nucleic acid electrophoresis detection of the linear expression cassette are shown in FIGS. 2-A and 2-B, and FIG. 2-A is the result of the nucleic acid electrophoresis of the heavy chain expression cassette, wherein lane 18 is the result of the electrophoresis of the heavy chain expression cassette of 8D1, and the amplified fragment 3700 is as expected; FIG. 2-B shows the results of electrophoresis of the light chain expression cassette, wherein lane 13 shows the results of electrophoresis of the light chain expression cassette of 8D1, and the amplified fragment 3000 or so, as expected.

1.5 construction of expression vector and enzyme digestion identification

The heavy chain linear expression frame is cut by Eco RI/Not I enzyme to recover heavy chain fragments with the size of about 1.4kb, an expression vector pCDNA3.4(ThermoFisher Scientific, A14697) is cut by Eco RI/Not I enzyme and then recovered, the heavy chain and the vector fragments are connected, DH5a is transformed, and clone is selected for PCR detection, double-enzyme digestion identification and sequence determination to construct an expression vector pCDNA3.4-8D1-H of a successful heavy chain. Similarly, the light chain linear expression frame is cut by using Eco RI/Not I enzyme to recover a light chain fragment with the size of about 0.7kb, the expression vector pCDNA3.4 is cut by using Eco RI/Not I enzyme and then recovered, the light chain and the vector fragment are connected, DH5a is transformed, and the clone is selected to carry out PCR detection, double-enzyme digestion identification and sequence determination, so that the expression vector pCDNA3.4-8D1-K of the light chain is successfully constructed. The results of the enzymatic cleavage are shown in FIG. 3, where lanes 1 and 4 represent the heavy chain results, lane 1 represents the heavy chain gene, lane 2 represents the empty vector pCDNA3.4, lane 3 represents the heavy chain-containing vector pCDNA3.4-8D1-H, and lane 4 represents the double cleavage results of pCDNA3.4-8D 1-H; lanes 5-8 show the light chain results, where lane 5 is empty vector, lane 6 is the light chain gene, lane 7 is the vector pCDNA3.4-8D1-K containing the light chain, and lane 8 is the double digestion results of pCDNA3.4-8D 1-K.

1.6 transient expression and affinity chromatography purification of monoclonal antibodies

An Expi293 expression system was used which combines a high expression Expi293 cell line, chemically defined serum free medium and a high efficiency transfection reagent with a proprietary enhancer. 15ug of heavy and 15ug of light chain were mixed and transfected into Expi 293F cells, the procedure was followed as described (ThermoFisher Scientific, A14635), and the culture was harvested after 5-6 days, centrifuged to give a supernatant of about 30ml and used in a volume of about5ml of prepackaged Protein A affinity chromatographic column, using 20mM PBS for balance before loading, injecting sample after conductance shows to baseline, using 20mM PBS for washing the chromatographic column after loading is finished till baseline is stable, using 0.1M glycine buffer solution with pH3.0 for eluting target Protein, and using OD to elute target Protein₂₈₀After near baseline, collection was stopped, the column was washed with at least 3 column volumes of 20mM PBS until baseline leveled off, and the column was washed with 20% ethanol. The content of the monoclonal antibody protein is determined by using an ultraviolet method, and the formula is as follows: protein content 1.54A₂₈₀-1.38A₂₆₀. The expression of the monoclonal antibody after transient transfection is shown in FIG. 4 (transient expression of the monoclonal antibody in 30mL serum-free suspension culture system on abscissa: mg, 28 different antibodies are listed on ordinate), and the SDS-PAGE result of the affinity chromatography purification is shown in FIG. 5 (lane 1 is the non-reduced SDS-PAGE electrophoresis result of monoclonal antibody 8D1, whole molecule (H2L2) is expected to have 150kDa, lane 2 is the molecular weight marker, lane 3 is the reduced SDS-PAGE electrophoresis result of monoclonal antibody 8D1, heavy chain (heavy chain, H) is expected to have 50kDa, and Light chain (Light chain, L) is expected to have 25 kDa). The total 28 monoclonal antibodies are expressed, purified and measured for protein concentration, and statistical analysis is carried out by Excel, the monoclonal antibody disclosed by the invention has good expression level, nearly 30ml of culture supernatant is collected, the amount of the purified antibody is 8.4mg, the purified antibody reaches the level of 280mg/L in proportion, and the expression level in the supernatant exceeds 300mg/L in proportion to 90% of purification yield. The expression quantity of transient transfection is the expression quantity of transient transfection, and the requirements of industrialization can be met by preparing a stably-transformed cell strain and optimizing a culture medium and a process.

Example 2 cell neutralization assay

2.1 day before experiment, Vero cells were diluted to 1.5X 10 with medium (MEM + 10% FBS)⁵cells/mL, inoculating to a 96-well cell culture plate, inoculating the cell culture plate with the volume of 200 mu L/well, and culturing in a 5% CO2 cell culture box at 37 ℃;

2.2 on the day of the experiment, purified monoclonal antibody was prepared by chemically synthesizing variable region gene from the initial concentration (initial concentration of 8D1 monoclonal antibody 100ug/ml, Ab2 control antibody 200ug/ml (refer to the variable region sequence of heavy and light chains of 8B10F8 disclosed in U.S. Pat. No. 4,9738704, chemically synthesizing variable region gene and using fusion PCR technique to obtain full length antibody gene, cloning to expression vector pMH vector, transfecting CHO-S cell, purifying by Protein A affinity chromatography, obtaining and storing, the laboratory name is Ab2), diluting 4 times, adding 96-well plate, volume is 120. mu.L/well, then adding 120. mu.L of chikungunya virus suspension per well (diluted with 1:1000 with MEM + 2% FBS), mixing well, incubating in cell culture box for 1h, reported patent monoclonal antibody 8B10 (refer to patent obtaining related sequence, prepared by cloning expression purification, laboratory preservation, label Ab2) as control;

2.3 discarding the cell culture supernatant in a 96-well plate, and adding 200 mu L of virus-antibody mixed suspension after co-incubation into each well; setting survival control (without adding virus and antibody) and death control (only adding virus), placing in 37 deg.C 5% CO2 cell culture box, and continuing culturing for 72 h;

discarding the cell culture supernatant after 2.472 h, adding 50 mu L of crystal violet, dyeing for 15min at room temperature, discarding, adding 200 mu L/hole pure water, and repeatedly washing for 5 times;

2.5 discard the washing solution, add 100. mu.L of destaining solution and dissolve it sufficiently to OD₆₂₀For reference, OD was measured with a microplate reader₅₇₀A value; cell viability was calculated using (OD sample well-OD death control)/(OD survival control-OD death control), and antibody IC was calculated using GraphPad Prism 5 fitted curves₅₀The value is obtained.

2.6 protective Effect and IC of monoclonal antibodies on cell models₅₀The results are shown in FIG. 6 (FIG. 6 shows the Log concentration of the monoclonal antibody measured in Log: ug/ml on the abscissa and the protection rate of the monoclonal antibody measured on the ordinate%) and FIG. 7 (published protection effect of monoclonal antibody 8D1 and reference control monoclonal antibody Ab2 on a VERO cell model, wherein negative indicates no virus added and the cells are all alive; positive indicates no monoclonal antibody added with virus and the cells are dead; Ab2 indicates the addition of virus and monoclonal antibody Ab 2; 8D1 indicates the addition of virus and monoclonal antibody Ab 2). The invention discloses the IC of monoclonal antibody 8D1₅₀Is 0.12ug/ml, IC of control mab Ab2₅₀Is 2.17ug/ml, and the activity of the monoclonal antibody disclosed by the invention is 18 times that of the monoclonal antibody reported in the literature.

Example 3 monoclonal antibody recognition epitope Studies

3.1ELISA screening of specific clones: 96-well enzyme-linked plate coated with monoclonal antibody against chikungunya virus(100. mu.g/mL, 150. mu.L/well), overnight at 4 ℃; discarding the coating liquid, adding 5mg/mL BSA blocking buffer (0.1mol/L NaHCO3, pH8.6) and blocking at 4 deg.C for 2 h; the liquid was decanted and washed 6 times with TBST (1mL/L Tween-20, TBS); add 100. mu.L of phage loop 7 peptide library (stock was diluted 1:10 with TBST, approximately 2X 10 phage-containing)¹¹pfu/100. mu.L), incubated with gentle shaking at room temperature for 30 min; discarding the liquid, washing 10 times with TBST, and washing away the unbound phage; adding 100 μ L eluent (1mg/mL BSA, 0.2mol/L Glycine-HCl, pH2.2) for elution, gently shaking at room temperature for 10min, quickly sucking out the liquid, and adding 15 μ L neutralizing solution (1mol/L Tris-HCl, pH9.1) for neutralization; taking 1 mu L of eluted phage for titer determination, infecting the remaining liquid with Escherichia coli ER2738, amplifying, purifying, and performing the next round of screening. Round 2 and round 3 selection with constant antibody coating concentration, and the phage amplified and purified in the previous round (2X 10)¹¹pfu/100. mu.L), wash second round 0.2% TBST and third round 0.5% TBST. After 3 times of screening, randomly selecting 50 well-separated positive clones, amplifying in Escherichia coli ER2738, purifying, and using for phage ELISA epitope fusion protein and ELISA detection

Monoclonal antibody coated 96-well enzyme-linked plates (2 ug/ml per well), incubated overnight at 4 ℃; discarding the liquid, adding blocking solution (5mg/mL BSA, 0.1mol/L NaHCO3, pH8.6), and incubating at 4 deg.C for 2 h; the liquid was discarded and separately screened and purified phage clones (1X 10)⁹pfu/well, 2 pairs of wells per gram), and incubating for 2h at room temperature; after washing 6 times with 0.5% TBST, 100. mu.L of HRP-labeled mouse anti-M13 phage antibody (0.82mg/ml, 1: 2000 dilution, as per the instructions) was added to each well and incubated at room temperature for 1 h; washing with TBST for 6 times, developing ABTS, and measuring A₄₅₀nM values. And 2 groups of negative controls are set, one group comprises a monoclonal antibody coated plate and 1% BSA, and the other group comprises an irrelevant antibody coated plate and phage clones. The result standard of positive cloning is A₄₅₀nM values are more than 2-fold higher than the negative control, and the negative control values are less than 0.1.

3.2 sequencing and alignment: through the identification of phage ELISA, 10 phage clones with high binding activity are selected, purified by PEG/NaCl, extracted with ssDNA, and subjected to sequence determination by Mimetai and company of Beijing. The sequencing primer is-96 gIII primer carried by the phage peptide library. Sequencing 10 clones, detecting 8 clones, and translating to contain 2 amino acid sequences, wherein the amino acid sequences of clones No. 1, 4, 7 and 10 are C-YFKWFWT-C; 2. the amino acid sequence of clones 3, 8 and 9 is C-TFNLYWT-C. Comparing epitope 1 (amino acid sequence TFNLYWT,8D1-C7-2) with structural protein S27 (S27-SP) of chikungunya virus standard strain, wherein the homologous sequence is located at amino acid positions 5-11 (homology 3/7) at the N end of E2 protein (S27-E2) as shown in FIG. 8-A; comparing epitope 1 with non-structural protein of chikungunya virus standard strain S27 (S27-NSP), only 1 amino acid of 7 amino acids is homologous, as shown in FIG. 8-B; comparing epitope 2 (amino acid sequence YFKWFWT,8D1-C7-5) with a structural protein ((S27-SP) of a standard strain S27 of chikungunya virus, wherein the homologous sequence is positioned at the position of 232-238 of the N end of the E2 protein (homology 2/7) is shown in figure 8-C, comparing epitope 2 with a non-structural protein (S27-NSP) of the standard strain S27 of the chikungunya virus, wherein 3 amino acids are homologous in 7 amino acids (shown in figure 8-D), wherein two amino acids KW in the three homologous sequences are also present in the structural protein E2, and preliminarily judging that the recognized conformational epitope is positioned on the E2 protein.

Sequence listing

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

<120> fully human monoclonal antibody with high neutralizing activity and anti-chikungunya fever and application

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 120

<212> PRT

<213> Homo sapiens

<400> 1

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly 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 Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Lys Glu Asp Gly Asp Tyr Val Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 2

<211> 360

<212> DNA

<213> Homo sapiens

<400> 2

caggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaataaggag 300

gacggtgact atgtaccctt tgactactgg ggccagggaa ccctggtcac cgtctcttca 360

<210> 3

<211> 330

<212> PRT

<213> Homo sapiens

<400> 3

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 Leu

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 Leu 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> 4

<211> 993

<212> DNA

<213> Homo sapiens

<400> 4

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag 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 gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcctccatc tcgggatgag 720

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagaagagcc tctccctgtc tccgggtaaa tga 993

<210> 5

<211> 103

<212> PRT

<213> Homo sapiens

<400> 5

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln 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 Phe Phe Gly Pro

85 90 95

Gly Thr Lys Val Asp Ile Lys

100

<210> 6

<211> 309

<212> DNA

<213> Homo sapiens

<400> 6

gacatcgtga tgacccagtc tccagccacc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttactttt tcggccctgg gaccaaagtg 300

gatatcaaa 309

<210> 7

<211> 107

<212> PRT

<213> Homo sapiens

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 8

<211> 324

<212> DNA

<213> Homo sapiens

<400> 8

cggaccgtgg cggcgccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggtaccgcta gcgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg ttag 324

<210> 9

<211> 106

<212> PRT

<213> Homo sapiens

<400> 9

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

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 10

<211> 321

<212> DNA

<213> Homo sapiens

<400> 10

ggtcagccca aggctgcccc ctcggtcact ctgttcccac cctcgagtga ggagcttcaa 60

gccaacaagg ccacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg 120

gcctggaagg cagatagcag ccccgtcaag gcgggagtgg agaccaccac accctccaaa 180

caaagcaaca acaagtacgc ggccagcagc tacctgagcc tgacgcctga gcagtggaag 240

tcccacaaaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 300

gcccctacag aatgttcata a 321

Claims (11)

1. A fully human monoclonal antibody with high neutralizing activity against chikungunya virus is characterized in that amino acid sequences of CDR1, CDR2 and CDR3 regions in a heavy chain variable region of the antibody are respectively shown as sequences 26-33, 51-58 and 97-109 of SEQ ID NO: 1; the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the antibody light chain variable region are shown in the sequence of 27-32, 50-52 and 88-93 bits of SEQ ID NO. 5 respectively.

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

3. The monoclonal antibody of claim 2, wherein the amino acid sequence of the antibody heavy chain constant region is set forth in SEQ ID NO. 3, and the amino acid sequence of the antibody light chain constant region is set forth in SEQ ID NO. 7 or SEQ ID NO. 9.

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

5. The polynucleotide of claim 4, wherein the polynucleotide encoding the antibody heavy chain constant region has the sequence shown in SEQ ID NO. 4, and the polynucleotide encoding the antibody light chain constant region has the sequence shown in SEQ ID NO. 8 or SEQ ID NO. 10.

6. A linear expression cassette for expressing the polynucleotides encoding the heavy and light chains of the monoclonal antibody of claim 5.

7. A mammalian expression vector expressing the polynucleotides encoding the heavy and light chains of the monoclonal antibody of claim 5.

8. A host cell comprising the linear expression cassette of claim 6 or the mammalian expression vector of claim 7.

9. The cell of claim 8, wherein the cell is an Expi 293F cell.

10. The cell of claim 8, wherein the cell is a CHO-S cell.

11. Use of a monoclonal antibody according to any one of claims 1 to 3 in the manufacture of a medicament for chikungunya heat treatment.

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