CN110343173B

CN110343173B - High-functional-activity yellow fever virus humanized monoclonal antibody and application thereof

Info

Publication number: CN110343173B
Application number: CN201810302112.0A
Authority: CN
Inventors: 严景华; 高福; 李燕; 马素芳; 仵丽丽
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2018-04-04
Filing date: 2018-04-04
Publication date: 2021-05-04
Anticipated expiration: 2038-04-04
Also published as: CN110343173A

Abstract

The invention discloses a high-functional-activity yellow fever virus humanized monoclonal antibody and application thereof, belonging to the technical field of medicines. The invention uses yellow fever virus E protein expressed by colon bacillus as antigen, selects memory B cell which can specifically combine with yellow fever virus E protein from PBMCs of a convalescent patient by flow sorting, then carries out RT-PCR and PCR amplification on the selected single B cell to obtain variable region segment of antibody, and further connects the variable region segment and constant region into an expression vector. After mammalian cell expression and purification, a series of function tests are carried out to obtain the human monoclonal antibody with the function of protecting yellow fever virus infection. The affinity of the antibody and the antigen is 0.588nM, the antibody has strong yellow fever virus neutralization activity, the IC50 is 3.7ng/ml, the antibody can completely protect mice from being attacked by yellow fever virus with lethal dose, and the antibody has the application value of clinical treatment and prevention of the yellow fever virus.

Description

High-functional-activity yellow fever virus humanized monoclonal antibody and application thereof

Technical Field

The invention relates to a high-functional-activity yellow fever virus humanized monoclonal antibody and application thereof, belonging to the technical field of medicines.

Background

Yellow Fever Virus (YFV), a single-stranded positive-strand RNA virus, belonging to the flaviviridae family of flaviviridae, is a mosquito-borne pathogen that causes human morbidity, and includes zika virus (ZIKV), dengue virus (dengue virus, DENV), West Nile Virus (WNV), and the like. YFV is an important pathogen causing yellow fever, and in severe cases, it causes hemorrhagic fever with multiple organ failure, especially in the liver, spleen, lymph nodes, heart, and kidney.

In 1996, scientists estimated that yellow fever virus causes 20 million infections and 30 million deaths each year in africa and south america. In recent two years, yellow fever outbreaks occur in brazil, angora and Congo democratic republic, hundreds of people die with a mortality rate of 14%, and 11 cases are imported in 2016 in China. Currently, attenuated vaccines (YFV 17D) are available clinically, but vaccine shortages and inadequate vaccination rates lead to frequent outbreaks of disease, and non-immunized individuals remain at risk. There are no clinically effective specific drugs available to treat this disease after YFV infection.

To date, neutralizing antibodies have proven to be an effective method of treating viral diseases, including Human Immunodeficiency Virus (HIV), influenza, and other flaviviruses, among others. The flavivirus surface E protein (Envelope) recognizes receptors on the cell surface and facilitates membrane fusion of the viral membrane to the cell membrane, a process that is completed by three distinct domains (DI, DII and DIII). Therefore, the E protein is an important epitope for neutralizing antibody action generated by the immune system of the body.

Some human antibodies have been found to have neutralizing activity, and can neutralize some yellow fever virus strains before 2001, such as: 5A, 7A, R3(27) and the like can neutralize Central African Republic (CAR)1986, Ethiopia 1961, Senegal1990, Nigeria1987, Ghana 1927(Asibi) and the vaccine strain YFV 17D. However, RNA viruses are characterized by high mutations under antibody pressure, and although some neutralizing antibodies have been identified, more new antibodies directed against different epitopes are essential for therapy. The aim of the invention is to identify specific novel YFV neutralizing antibodies with protective effect.

Disclosure of Invention

In order to solve the problems, the invention firstly uses YFV-E protein expressed by Escherichia coli as antigen, selects memory B cells which can specifically bind to the YFV-E protein from PBMCs of a convalescent YFV patient through flow sorting, then carries out RT-PCR and PCR amplification on the selected single B cells to obtain variable region fragments of 1 strain of antibody, and further connects the variable region fragments and the constant region into an expression vector. After the sequencing is correct, a series of function detection including the detection of the binding force with YFV-E protein, the in vitro neutralization effect, the in vivo protective capability and the like is carried out through the expression and purification of mammalian cells.

It is a first object of the present invention to provide an antibody designated YD 73; the heavy chain variable region has an amino acid sequence shown in SEQ ID NO.1, and the light chain variable region has an amino acid sequence shown in SEQ ID NO. 3.

In one embodiment of the invention, the heavy chain of antibody YD73 comprises a heavy chain variable region and a heavy chain constant region, wherein the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 5.

In one embodiment of the invention, the light chain of antibody YD73 is a kappa chain; the light chain comprises a light chain variable region and a light chain constant region; the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 7.

The antibody can target the envelope protein E protein which is specific to yellow fever virus, and inhibit the infection of the virus to cells by inhibiting the receptor combination and/or the membrane fusion process which are mediated by the E protein.

The second purpose of the invention is to provide the application of the antibody in preparing medicines for treating and/or preventing yellow fever virus.

A third object of the present invention is to provide a pharmaceutical composition comprising antibody YD 73.

In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

The fourth purpose of the invention is to provide a kit for diagnosis, detection and the like, wherein the kit contains the antigen of the antibody, or the DNA molecule for coding the antigen, or the recombinant vector/expression cassette/transgenic cell line/recombinant bacteria for expressing the antigen.

It is a fifth object of the present invention to provide a DNA encoding said human monoclonal antibody YD 73.

In one embodiment of the invention, the nucleotide sequence of the heavy chain variable region of YD73 is SEQ ID No.2 and the nucleotide sequence of the light chain variable region is SEQ ID No. 4.

In one embodiment of the invention, the heavy chain of the antibody comprises a heavy chain variable region and a heavy chain constant region, wherein the nucleotide sequence of the heavy chain constant region is set forth in SEQ ID NO. 6.

In one embodiment of the invention, the light chain of antibody YD73 is a kappa chain; the light chain comprises a light chain variable region and a light chain constant region; the nucleotide sequence of the light chain constant region is shown as SEQ ID NO. 8.

In one embodiment of the present invention, the sequence encoding the heavy chain of the antibody sequentially comprises a CMV promoter sequence, a first enzyme cleavage site sequence, a leader sequence, a sequence encoding the heavy chain variable region, a sequence encoding the heavy chain constant region, and a second enzyme cleavage site sequence.

In one embodiment of the present invention, the sequence encoding the light chain of the antibody sequentially comprises a CMV promoter sequence, a first enzyme cleavage site sequence, a leader sequence, a sequence encoding the variable region of the light chain, a sequence encoding the constant region of the light chain, and a second enzyme cleavage site sequence.

In one embodiment of the present invention, the first or second cleavage site sequence includes but is not limited to EcoR I, Xho I, Sac I or Xho I cleavage site sequence.

In one embodiment of the invention, the leader sequence has an amino acid sequence shown as SEQ ID NO. 9 and a nucleotide sequence shown as SEQ ID NO. 10.

The invention also claims an expression vector and a cell containing the gene sequence or expressing the antibody.

The invention has the beneficial effects that:

the invention obtains 1 strain of human-derived YFV antibody-YD 73 with high neutralizing activity. The antibody is completely different from the reported YFV antibody sequence and is a newly discovered antibody. The humanized antibody has strong affinity for YFV-E, strong YFV neutralizing activity, 0.588nM affinity, and IC₅₀It was 3.7 ng/mL. Mice can be fully protected from challenge with a lethal dose of YFV China. The humanized antibody has the application value of clinically preventing and treating YFV infection.

Drawings

FIG. 1: the result of the YFV China-E protein purification molecular sieve and SDS-PAGE;

FIG. 2: after Protein A is purified, an antibody SDS-PAGE result;

FIG. 3: the kinetic curve result of the antibody and YFV China-E;

FIG. 4: results of neutralization curves of antibodies against YFV China;

FIG. 5: protective effect of antibody on YFV China infected mice; a is the body weight change of the surviving mice, and B is the survival rate of the mice.

Detailed Description

Example 1: expression and purification of yellow fever virus E protein

The DNA fragment of the extracellular domain of YFV CNYF01/2016(YFV-China) strain membrane protein E (the amino acid sequence is shown in SEQ ID NO:11, and the nucleotide sequence is shown in SEQ ID NO: 12) was digested with NdeI and XhoI, and then ligated to pET21a vector. Wherein the 3' end of the YFV E protein coding region is linked to a coding sequence for a 6 histidine-tag (hexa-His-tag) and a translation stop codon. The ligation product was then transformed into BL21 E.coli competent cells. The single clone was inoculated into 40mL of LB medium and cultured for 6-8 hours. Inoculating to 4L LB medium, and culturing at 37 deg.C to OD₆₀₀When the concentration was 0.4 to 0.6, IPTG was added to a final concentration of 1mM, and the culture was continued at 37 ℃ for 4 to 6 hours. The inclusion bodies were harvested and renatured by dilution. The renaturation solution is changed into 20mM Tris, 150mM NaCl, pH8.0 buffer solution and 10% glycerol after being concentrated. The concentrated protein solution was further purified by size exclusion chromatography using AKTA-purifier (GE) and superdex200Hiload 16/60 column (GE) using buffer A (20mM Tris, 150mM NaCl, pH8.0, 10% glycerol) while monitoring the UV absorbance at 280nm, and the protein of interest was recovered and the protein purity was identified by SDS-PAGE. Through identification, the high-purity E monomer protein with the size of 43kDa can be obtained. The results are shown in FIG. 1.

Example 2: isolation of specific memory B cells binding to YFV China-E protein

With patient informed consent, 20mL of blood was collected and PBMCs were isolated. Isolating the PBMCs at 10⁷density/mL with final concentration of 400nM YFV-E protein on iceBind for half an hour, then wash 2 times with PBS, and incubate with the following antibodies: anti-human CD3/PE-Cy5, anti-human CD16/PE-Cy5, anti-human CD235a/PE-Cy5, anti-human CD19/APC-Cy7, anti-human CD27/Pacific Blue, anti-human CD38/APC, anti-human IgG/FITC, and anti-His/PE. After half an hour incubation on ice, the antibodies were washed 2 times with PBS. PE-Cy5 collected by FACSAria III sorting^-APC^-APC-Cy7⁺Pacific Blue⁺FITC⁺PE⁺The cells of (4) were collected directly into a 96-well plate at 1 cell/well.

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

The B cells obtained in example 2 were reverse-transcribed by Superscript III reverse transcriptase (Invitrogen) primers shown in Table 1 (sequences shown by SED ID No.13 to SED ID No. 20), and reacted at 55 ℃ for 60 min.

TABLE 1 reverse transcription primers

Using this reverse transcription product as a template, PCR was performed using HotStar Tap Plus enzyme (QIAgen) to amplify an antibody variable region sequence (PCRa). Designing corresponding primers, wherein the reaction conditions are as follows: 95 ℃ for 5 min; 95 ℃ 30s, 55 ℃ (heavy chain/kappa chain)/50 ℃ (lambda chain) 30s, 72 ℃ 90s, 35 cycles; 72 ℃ for 7 min. This was used as template for a second round of PCR (PCRb) under the following conditions: 95 ℃ for 5 min; 95 ℃ 30s, 58 ℃ (heavy chain)/60 ℃ (kappa chain)/64 ℃ (lambda chain) 30s, 72 ℃ 90s, 35 cycles; 72 ℃ for 7 min.

1.2% agarose gel electrophoresis, and separating the PCR product. The size of the band is 400-500bp after the gel cutting recovery, and the band is sent to a sequencing company for sequencing. The sequencing results were analyzed using NCBI online software.

Analysis of the correct variable region sequences and the corresponding heavy and/kappa chain constant regions were ligated by bridge PCR and cloned into the expression vector pCAGGS. Wherein the heavy chain is EcoRI and XhoI linked and the kappa chain is SacI linked to XhoI. The strategy for B cell sequencing and expression plasmid construction is as follows:

the human antibody design strategy is as follows:

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

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

Wherein, the amino acid sequence of the Leader sequences is shown as SEQ ID NO. 9 (the nucleotide sequence is shown as SEQ ID NO. 10).

Example 4: expression and purification of antibodies

293T cells were cultured in DMEM with 10% FBS. 293T was co-transfected with plasmids containing genes encoding the light and heavy chains of the specific antibodies. After 4-6 hours of transfection, the medium was changed to serum-free DMEM for another 7 days, and the supernatant was collected.

The collected supernatant was centrifuged at 5000rpm for 30min, mixed with an equal volume of buffer containing 20mM sodium phosphate (pH 8.0), filtered through a 0.22 μm filter and bound to a protein A pre-column (5mL, GE Healthcare). Bound protein was eluted with 0.1M glycine (pH 3.0). The protein is collected, concentrated and then subjected to molecular sieve chromatography. The peak of interest was determined by SDS-PAGE, and the results are shown in FIG. 2, indicating that the target protein was normally expressed.

Finally, an antibody YD73 which can be combined with YFV China-E protein and has stronger neutralizing activity is obtained, and the specific gene rearrangement mode is shown in the following table 2.

TABLE 2 antibody Gene rearrangements

Example 5: performance testing of human antibodies

(1) Surface plasmon resonance technology detection and YFV-E binding capacity

Surface plasmon resonance analysis was performed using Biacore T100(Biacore Inc.). The method comprises the following specific steps:

first, an antibody against anti-human IgG was immobilized in an amino-coupled manner on a channel (flow cell, Fc) of a CM5 chip. The fixed amount is controlled around a response value (RU) of 10,000. The purified antibody is combined in an antibody capture mode, wherein the liquid flow speed is controlled at 10 mu L/min, the sample injection is carried out for 1min, and the antibody capture amount is about 60 RU. And diluting YFV China-E protein by 10mM HEPES, 150mM NaCl and pH 7.4 solution in a multiple ratio, regulating the flow rate to 30 mu L/min, sequentially passing through each channel, and loading the YFV-E protein one by one from low concentration. The curves constitute the kinetic curves shown in figure 3. The results are shown in Table 3. The calculation of binding kinetic constants was performed using BIAevaluation software T100(Biacore, Inc.). The results of SPR indicated that the YD73 antibody could bind to the YFV-E protein with an affinity of 0.588 nM.

TABLE 3 kinetic constants for binding of antibodies to YFV China-E protein

(2) Neutralization test

The purified antibody was diluted 3-fold, mixed with YFV (C6/36 amplified) diluted 1:60, and incubated at 37 ℃ for 60 minutes. The mixture was then added to 24-well plates, 300. mu.L/well, which had been plated with Vero cells. After incubation at 37 ℃ for 1 hour, each well was supplemented with 0.7mL of medium (DMEM, 10% FBS), and incubation was continued for 40 hours before staining. The cells were collected, treated with 4% paraformaldehyde, 0.05% soponin in PBS, and left on ice in the dark for 30 min. The cells were then washed 2 times with solution (PBS, 1% BSA, 0.01% soponin), incubated with 2. mu.g/mL Z6-FITC antibody on ice for 30min, washed 2 times with solution, and the cell positive ratio was determined using FACSCANTO. According to the positive proportion of different concentrations, the neutralizing capacity of the antibody to YFV is calculated, and the result is shown in figure 4, YD73 can neutralize YFV China of virus, and IC50 is 0.0037 ug/ml.

(3) Animal protection test

Female Balb/c mice (Witongli Hua) at 3 weeks of age were divided into 4-5 groups. Each mouse was injected intracranially with 80PFU virus YFV China, and mice were recorded for survival and weight change over 14 days, with weight change over 25% or sacrificed with signs of paralysis. 24 hours after infection, different doses of antibody were injected, and the protection rate of the 1mg/kg dose to mice was 100% (FIG. 5A, B and Table 4). After the mice were infected for 48h, 72h, 96h and 120h, the mice injected with 25mg/kg dose had 100% protection against the virus infection for 72h (FIG. 5C, D and Table 4). Control antibody injections began death on day 7 and all died on day 9.

Table 4 protective effect of different doses of YD73 on mice after injection at different time points

At present, the human antibodies against yellow fever virus have been found to be 5A, 7A, R3(27), 1A, 2A, R3(9), and the gene rearrangement mode is: VH4-59, VH3-11, VK1-12, VL1-19 (SEQ ID NO: AY661699, AY661700, AY661701, AY661702, AY661703 and AY661704 in GenBank): wherein 5A, 7A, R3(27) can neutralize partial strain before 2001 and vaccine strain YFV 17D. Neutralizing active IC₅₀About 1-10 ug/mL. No in vivo animal experiment verifies its function. IC of YD73 antibodies of the invention₅₀0.0037. mu.g/mL, which is much higher than the reported antibody. In addition, in animal protection experiments, the dosage of the antibody used by the invention is 1mg/kg, and mice which are attacked by lethal dose for 24 hours can be completely protected. Mice challenged with a lethal dose of 72h were fully protected with an antibody dose of 25 mg/kg.

In conclusion, the human-derived highly-neutralizing YFV antibody YD73 obtained by screening YFV China-E specific-binding memory B cells of rehabilitation patients is completely different from the reported yellow fever antibody sequence, is a newly discovered antibody, has strong binding capacity with antigen, has the affinity of 0.588nM, and has strong yellow fever virus neutralizing activity and IC (integrated Circuit) of yellow fever virus₅₀It was 3.7 ng/mL. Furthermore, mice can be completely protected from a lethal dose of yellow fever virus. The result indicates that the humanized antibody has the application value of clinical treatment and prevention of yellow fever.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Ala Thr Ile Arg Val Leu Ala Leu Gly Asn Gln Glu Gly Ser Leu Lys

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Thr Ala Leu Thr Gly Ala Met Arg Val Thr Lys Asp Glu Asn Asp Asn

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Asn Leu Tyr Lys Leu His Gly Gly His Val Ser Cys Arg Val Lys Leu

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Met Gln Val Lys Val Pro Lys Gly Ala Pro Cys Lys Ile Pro Val Ile

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Val Ala Asp Asp Leu Thr Ala Ala Val Asn Lys Gly Ile Leu Val Thr

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Val Asn Pro Ile Ala Ser Thr Asn Asp Asp Glu Val Leu Ile Glu Val

355 360 365

Asn Pro Pro Phe Gly Asp Ser Tyr Ile Ile Val Gly Thr Gly Asp Ser

370 375 380

Arg Leu Thr Tyr Gln Trp His Lys Glu Gly Ser Ser Ile Gly Lys His

385 390 395 400

His His His His His

405

<210> 12

<211> 1215

<212> DNA

<213> Artificial sequence

<400> 12

atggcacatt gcatcggcat taccgaccgc gatttcatcg agggtgtgca tggtggtaca 60

tgggtgagtg caaccctgga acaggataaa tgcgtgaccg tgatggcccc ggataagcct 120

agtctggata ttagcctgca gaccgtggcc attgatggtc cggcagaagc ccgtaaagtg 180

tgctacagcg ccgttctgac ccacgtgaag atcaacgaca agtgccctag cacaggcgaa 240

gcccatctgg cagaggagaa cgacggtgat aacgcctgta aacgcaccta cagcgaccgt 300

ggctggggta atggctgcgg cctgtttggc aagggtagca ttgtggcctg cgcaaaattc 360

acctgcgcca agagcatgag tctgttcgag gtggaccaga ccaagattca gtatgtgatc 420

cgcgcccagc tgcacgtggg cgcaaagcag gagaactgga acaccgacat caagaccctg 480

aagttcgatg ccctgagcgg cagccaagaa gccgagttta caggttacgg caaggcaacc 540

ctggagtgtc aagtgcagac cgcagtggat ttcggtaata gctatattgc cgagatggag 600

aaagacagct ggatcgtgga tcgccagtgg gcccaagatc tgaccctgcc gtggcagagc 660

ggtagtggtg gcatttggcg cgaaatgcat catctggtgg agtttgagcc gccgcatgcc 720

gcaaccattc gtgtgctggc cctgggcaat caggaaggca gcctgaaaac cgccctgaca 780

ggcgccatgc gcgtgaccaa agacgaaaac gataataatc tgtacaagct gcatggtggc 840

cacgtgagct gccgcgtgaa gctgagcgcc ctgaccctga aaggcaccag ctacaagatg 900

tgtacagaca aaatgagctt cgttaagaat ccgaccgata ccggccacgg caccgtggtg 960

atgcaggtta aggttccgaa aggcgcaccg tgcaaaatcc cggtgattgt tgccgatgac 1020

ctgaccgccg ccgtgaataa gggcattctg gtgaccgtga acccgatcgc aagcaccaac 1080

gatgatgagg tgctgatcga agtgaacccg ccttttggcg acagttacat catcgtgggt 1140

accggcgata gccgcctgac ctatcaatgg cacaaggaag gcagtagcat cggcaaacat 1200

catcaccacc accat 1215

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<400> 13

atggagtcgg gaaggaagtc 20

<210> 14

<211> 19

<212> DNA

<213> Artificial sequence

<400> 14

tcacggacgt tgggtggta 19

<210> 15

<211> 19

<212> DNA

<213> Artificial sequence

<400> 15

tcacggaggt ggcattgga 19

<210> 16

<211> 19

<212> DNA

<213> Artificial sequence

<400> 16

caggcgatga ccacgttcc 19

<210> 17

<211> 19

<212> DNA

<213> Artificial sequence

<400> 17

catgcgacga ccacgttcc 19

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<400> 18

aggtgtgcac gccgctggtc 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<400> 19

gcaggcacac aacagaggca 20

<210> 20

<211> 17

<212> DNA

<213> Artificial sequence

<400> 20

aggccactgt cacagct 17

Claims

1. An antibody aiming at yellow fever virus is characterized in that the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 3.

2. The antibody of claim 1, wherein the heavy chain of said antibody comprises a heavy chain variable region and a heavy chain constant region, said heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO. 5.

3. The antibody of claim 1 or 2, wherein the light chain of the antibody is a lambda chain; the light chain comprises a light chain variable region and a light chain constant region; the light chain constant region has an amino acid sequence shown in SEQ ID NO. 7.

4. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 3.

5. Use of an antibody according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment and/or prophylaxis of yellow fever virus.

6. A DNA encoding the antibody according to any one of claims 1 to 3.

7. The DNA of claim 6, wherein the DNA encodes the heavy chain of an antibody comprising a CMV promoter sequence, a leader sequence, a sequence encoding a heavy chain variable region, a sequence encoding a heavy chain constant region; sequences encoding the light chain of the antibody include a CMV promoter sequence, a leader sequence, a sequence encoding the variable region of the light chain, and a sequence encoding the constant region of the light chain.

8. The DNA of claim 7, wherein the nucleotide sequence of the leader sequence is set forth in SEQ ID NO 10.

9. An expression vector or cell comprising the DNA of claim 7 or 8 or expressing the antibody of any one of claims 1 to 3.