CN105061590A - Bispecific antibody for hepatitis B surface protein, and use thereof - Google Patents
Bispecific antibody for hepatitis B surface protein, and use thereof Download PDFInfo
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention provides a bispecific antibody A3B5-BsAb for a hepatitis B surface protein. The bispecific antibody is formed by combining an antibody A3D5 with an antibody B5H6. The bispecific antibody has better HBV virus neutralization ability and HBsAg release inhibition ability than single use of the antibody A3D5 and the antibody B5H6, and has strong synergistic effects, so the bispecific antibody is likely to prevent HBV infection related hepatitis, hepatic cirrhosis and liver cancer. The bispecific antibody is a completely humanized antibody obtained through cloning HBsAg specific memory B cells in the peripheral blood of hepatitis B vaccine inoculated volunteer, has lower immunogenicity than murine, chimeric and humanized antibodies, and can be used to prepare hepatitis B virus related hepatopathy prevention or treatment drugs or diagnose reagents.
Description
Technical field
The invention belongs to biological technical field, more specifically, the invention discloses anti-hepatitis b surface protein bi-specific antibody technology of preparing and preventing the purposes in HBV infection and the relevant hepatocellular carcinoma for the treatment of HBV.
Background technology
Hepatitis B virus (HepatitisBvirus, HBV) is double-stranded DNA virus, and human body is infected easily causes chronic hepatitis B afterwards, and then causes liver cirrhosis and (or) liver cancer.About there are 3.5 hundred million HBV infection persons in the whole world in recent years according to statistics, and the liver failure about having 1,000,000 ~ 1,500,000 people to die from acute or chronic HBV infection every year to cause, liver cirrhosis and (or) liver cancer, thus prevent HBV infection to become world public health problem
[1].Hepatocellular carcinoma (Hepatocellularcarcinoma, HCC, be called for short liver cancer) in close relations with viral hepatitis, it is one of most important cause of death caused by viral hepatitis, also be one of modal malignant tumour clinically, be worldwide that the tumour occupying the 3rd is correlated with the cause of death, in annual new cases, China accounts for the whole world 42.5%, has become the deputy tumor etiology of China.
Research shows, in HBV infection associated HCC patient, the HCC incidence of HBVDNA carrying capacity height person (>105copies/ml) reaches 10.1%, and the HCC incidence of the low person of HBVDNA carrying capacity (<104copies/ml) is only 3.8%, baseline height virus load raises relevant with HCC incidence.Time high virus load (HBVDNA >=0.7mEq/ml), HCC risk of relapse ratio is 5.13, and the HCC risk of relapse ratio of the cutting edge positive is 2.14.Therefore, antiviral therapy is carried out to hepatitis B patient, contribute to delaying the progress of disease to HCC.Antiviral therapy being carried out to HBV associated HCC patient, improving survival rate by improving liver function.In recent years research display, high HBVDNA carrying capacity is relevant with high HCC recurrence rate and prognosis mala.Carrying out antiviral therapy to HBV infection associated HCC postoperative patient may be another kind of therapeutic choice except liver transplantation, and this treatment improves survival rate by improving liver function.
Mainly adopt the Antiviral Effect such as alpha-interferon and nucleotide analog to treat for HBV infection at present, but this two classes medicine life-time service curative effect is not good enough, is easy to cause virus mutation, brings extreme difficulties to treatment
[2].
Relative to the medicine of anti-virus infection, the antiviral effect of the specific immunoglobulin (Ig) of hepatitis B (HBIG) is well a lot.Give the specific immunoglobulin (Ig) of patient's hepatitis B (HBIG) treatment give by scholar's extensive concern of various countries.Such as, the research after the HBIG liver cancer of originating in hepatitis B after liver transplantation has reached good effect.HBIG is a kind of polyclone exogenous antibody of high-titer, and it screens from healthy blood donor, makes through bioconcentration technique.Accept this passive immunization preparation passively as human infection HBV, body can be made to neutralize rapidly in a short time and remove hepatitis B virus free in serum, avoiding hepatitis B virus localized infection.
But there is many non-safety factors as the source of therapeutic antibodies in current HBIG.Meanwhile, anti-hepatis B immunoglobulin complicated component, Hazard Factor are relatively uncertain, and the mode of production is limited, and these factors govern the Clinical practice of hepatitis B therapeutic antibody
[3].
Therefore Human Hepatitis B Immune Globulin is substituted in the urgent need to researching and developing a kind of safe and effective and that Hazard Factor are relatively controlled biological products, so research and development hepatitis B specificity human genetically engineered antibody more and more receives publicity.Because monoclonal antibody has high specificity, the highly sensitive and easy advantage such as scale operation, therefore research and development are for hepatitis B surface antigen (HepatitisBsurfaceAg, HBsAg) monoclonal antibody, to stoping the process of HBV infection and relevant hepatitis, liver cirrhosis and liver cancer safely and effectively.
Due to the multifactor property of disease, single target treatment often can not meet clinical demand
[4].The report of existing conjunctive use two kinds of mono-clonal preclinical laboratory in clinical trial
[5], but having of this combined utilization monoclonal antibody is much restricted
[6].Such as, combined utilization two kinds of monoclonal antibodies security and effect need to go respectively to evaluate, consequent long period, high development cost limit its development process, and bi-specific antibody solves this bottleneck just
[7].
Summary of the invention
The present invention, for solving the problem and carrying out, provides a kind of bi-specific antibody A for surface antigen protein
3b
5-BsAb, present invention employs following technical scheme:
Bi-specific antibody A for hepatitis B surface albumen provided by the invention
3b
5-BsAb, is characterized in that, has: heavy-chain variable domains, comprises hypervariable region CDR
1, CDR
2, CDR
3, CDR
4, CDR
5, CDR
6; And light variable domains, comprise hypervariable region CDR
1＇, CDR
2＇, CDR
3＇, CDR
4＇, CDR
5＇, CDR
6＇, the aminoacid sequence of heavy-chain variable domains is as shown in SEQIDNO.6, and the aminoacid sequence of light variable domains is as shown in SEQIDNO.8.
Wherein, CDR
1aminoacid sequence be: Met-Tyr-Ala-Phe-Ser; CDR
2aminoacid sequence be: Gly-Ile-Ile-Pro-Ser-Phe-Asp-Thr-Thr-Asn-Tyr-Ala-Gln-Lys-Phe-Gln-Cys; CDR
3aminoacid sequence be: Ser-Val-Ile-Thr-Asp-Leu-Asp-Thr-Val-Gly-Asn-Asp-Glu-Ser-Gly-Asp-Ala-Ser-Tyr-Tyr-Tyr-Met-Asp-Val; CDR
4aminoacid sequence be: Ser-Ser-Ala-Ile-Leu; CDR
5aminoacid sequence be: Trp-Ile-Val-Val-Gly-Ser-Gly-Asn-Ala-Lys-Tyr-Ala-Gln-Arg-Phe-Gln-Glu; CDR
6aminoacid sequence be: Arg-Gly-His-Ser-Phe-Thr-Ser-Pro-Phe-Asp-Ser;
CDR
1the aminoacid sequence of ＇ is: Arg-Ser-Ser-Glu-Ser-Leu-Gln-His-Ser-Asp-Gly-Thr-Tyr-Tyr-Leu-Asp; CDR
2the aminoacid sequence of ＇ is: Ser-Ala-Ser-Asn-Thr-Ala-Pro; CDR
3the aminoacid sequence of ＇ is: Met-Gln-Ala-Leu-Glu-Thr-Pro-Phe-Thr; CDR
4the aminoacid sequence of ＇ is: Arg-Ala-Ser-Gln-Ser-Val-Gly-Ser-Asn-Tyr-Leu-Ala; CDR
5the aminoacid sequence of ＇ is: Gly-Ala-Ser-Thr-Arg-Ala-Thr; CDR
6the aminoacid sequence of ＇ is: Gln-Lys-Tyr-Gly-Ser-Ser-Leu-Thr.
Meanwhile, bi-specific antibody A
3b
5-BsAb be selected from the bi-specific antibody of anti-hepatitis B virus surface antigen and described bispecific antibody fragment any one, this bispecific antibody fragment is the Fab fragment of described bi-specific antibody or F (ab ')
2fragment.
Further, present invention also offers a kind of antibody fragment for hepatitis B surface albumen, this antibody fragment is bi-specific antibody A
3b
5the Fab fragment of-BsAb or F (ab ')
2fragment.
Further, the invention provides a kind of encoding bispecific antibody A
3b
5the gene of-BsAb, the nucleotide sequence of encoding heavy chain variable domains is as shown in SEQIDNO.5, and the nucleotide sequence of coding light variable domains is as shown in SEQIDNO.7.
Further, the invention provides a kind of expression vector of the said gene containing at least one copy.
Further, the invention provides a kind of host cell containing at least one above-mentioned expression vector.
Further, the invention provides bi-specific antibody A
3b
5-BsAb or its Fab fragment or F (ab ')
2the purposes of fragment in the medicine preparing prevention or treatment hepatitis B virus related liver disease or diagnostic reagent.Described medicine or diagnostic reagent except comprising antibody or antibody fragment, also comprise in pharmaceutically acceptable vehicle, thinner and carrier any one.
Invention effect and effect
The invention provides a kind of bi-specific antibody A for hepatitis b surface antigen protein
3b
5-BsAb, this bi-specific antibody is by antibody A
3d
5and antibody B
5h
6combine, but more simple by antibody A with the ability of the ability of HBV virus and suppression HBsAg release in it
3d
5and antibody B
5h
6combined utilization is more excellent, shows stronger synergy, and then the process of hepatitis, liver cirrhosis and the liver cancer that the infection of HBV may be stoped to be correlated with.Meanwhile, because this antibody clones the human antibody obtained in the memory B cells that HBsAg is special from volunteer's peripheral blood of HB vaccination, it has compared with mouse source, the immunogenicity that chimeric and humanized antibody is lower.
Accompanying drawing explanation
Fig. 1 is bi-specific antibody A of the present invention
3b
5-BsAb specific binding AM;
Fig. 2 is the position view of synthetic peptide of the present invention at hepatitis B surface antigen (HBsAg);
Fig. 3 (A) is bi-specific antibody A of the present invention
3b
5-BsAb conjugated antigen epi-position P
3result figure;
Fig. 3 (B) is bi-specific antibody A of the present invention
3b
5-BsAb conjugated antigen epi-position P
2result figure;
Fig. 4 is bi-specific antibody A of the present invention
3b
5-BsAb affects the detection by quantitative result figure that in cell, HBsAg expresses;
Fig. 5 is bi-specific antibody A of the present invention
3b
5-BsAb affects HBVDNA in cell and copies several detection by quantitative result figure;
Fig. 6 is bi-specific antibody A of the present invention
3b
5-BsAb suppresses the result figure of HBsAg release in liver cancer cell.
Embodiment
Below in conjunction with embodiment, the present invention is further detailed, but should not be construed as limitation of the present invention.
Embodiment does not comprise the detailed description to traditional method, as: OverlappingPCR reacts, and restriction endonuclease reaction, is inserted into plasmid vector by gene, plasmid is introduced the method etc. of host cell.Such method is well-known for person having ordinary skill in the art, and described by having in many publications, such as Sambrook, J., Fritsch, E.F.andManiais, T. (1989) MolecularCloning:ALaboratoryManual, 2ndedition, ColdspringHarborLaboratoryPress.And material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Reagent and material
Lymphocyte separation medium, purchased from Beijing Ding Guo biotechnology limited liability company; Trizol reagent, purchased from Invitrogen company; Serum free medium EX-CELL302, purchased from SIGMA-ALDRICH company; Goatanti-humankappa-HRP is purchased from Invivogen company;
Embodiment one: bi-specific antibody A
3b
5the preparation of-BsAb
One, the clone of antibody light chain constant region, weight chain constant area gene and the structure of carrier thereof
With lymphocyte separation medium separating health human lymphocyte, extract total serum IgE with Trizol reagent, design according to the sequence that document [8] and document [9] are reported heavy chain and the light chain constant region gene that primer adopts QIAGENOneStepRT-PCRKit amplification people antibody respectively.The heavy chain of people's antibody and light chain constant region gene are connected respectively in expression vector AbVec plasmid, construct heavy chain constant region nucleotide carrier IgG-AbVec plasmid and antibody light chain constant region nucleotide carrier Ig κ-AbVec plasmid respectively, confirm after sequence verification to obtain correct clone.Wherein, human antibody heavy chain's constant region nucleotide sequence and aminoacid sequence are respectively SEQIDNO:1 and SEQIDNO:2, and human antibody light chain constant region nucleotide sequence and aminoacid sequence are respectively SEQIDNO:3 and SEQIDNO:4.
Two, antibody heavy chain variable region, light-chain variable sequence obtain and the structure of recombinant expression vector
Particularly, adopt ultra-high speed streaming separation system to be separated the memory B cells (HBsAg+IgG+CD19+) that in volunteer's peripheral blood of HB vaccination, HBsAg is special, and be prepared into unicellular sample.And then, adopt SingleCellRT-PCR technology to clone light chain of antibody and weight chain variabl area sequence in each sample, because the cDNA of individual cells synthesis is rare especially, the method for Chao Shi PCR must be adopted to increase its antibody gene contained.
Visible significantly PCR band amplification (size is about 400bp) after two-wheeled PCR.After analyzing its sequence, specific PCR amplification antibody variable region band, heavy chain introduces restriction enzyme A geI and restriction enzyme SalI site, and light chain introduces restriction enzyme A geI and restriction enzyme BsiwI site.Heavy chain of antibody and chain variable region gene are cloned into respectively IgG-AbVec plasmid and Ig κ-AbVec plasmid, plant method thus, build multiple total man sources hepatitis B surface protein monoclonal antibody, wherein choose the antibody that two strains have higher HBV Neutralization effect, respectively called after A
3d
5and B
5h
6.A
3d
5the weight of antibody, the carrier of light chain called after: IgG-AbVec-A respectively
3d
5, Ig κ-AbVec-A
3d
5; B
5h
6the weight of antibody, the carrier of light chain called after: IgG-AbVec-B respectively
5h
6, Ig κ-AbVec-B
5h
6.
Adopt the method for OverlappingPCR, by A
3d
5the C end of variable region of heavy chain is by Linker small peptide " ASTKGPSVFPLAP (nucleotides sequence is classified as: GCTAGCACCAAGGGACCTTCTGTGTTCCCTCTGGCCCCT) " and B
5h
6the N end of antibody heavy chain variable region is connected, and introduces restriction enzyme A geI and restriction enzyme SalI site, forms A
3b
5the variable region of heavy chain of-BsAb bi-specific antibody, its nucleotide sequence and aminoacid sequence are respectively SEQIDNO:5 and SEQIDNO:6, by A
3b
5-BsAb variable region of heavy chain is cloned into IgG-AbVec carrier and forms its heavy chain expression vector IgG-AbVec-A
3b
5-BsAb.Same method, by A
3d
5the C end of variable region of light chain is by Linker small peptide " TVAAPSVFIFPP (nucleotides sequence is classified as: ACCGTGGCTGCCCCTAGCGTGTTCATCTTCCCTCCT) " and B
5h
6the N end of antibody chain variable region is connected, and introduces restriction enzyme A geI and restriction enzyme BsiwI site, forms A
3b
5the variable region of light chain of-BsAb bi-specific antibody, its nucleotide sequence and aminoacid sequence are respectively SEQIDNO:7 and SEQIDNO:8, by A
3b
5-BsAb variable region of light chain is cloned into Ig κ-AbVec carrier and forms its light chain expression vector Ig κ-AbVec-A
3b
5-BsAb.
Three, bi-specific antibody A
3b
5-BsAb Expression and purification
5 × 10 are inoculated in 3.5cm Tissue Culture Dish
5cHO-K
1cell, cell cultures is carried out transfection when 90%-95% merges, and concrete steps are as follows: get 5 μ g plasmid IgG-AbVec-A
3d
5, 5 μ g plasmid Ig κ-AbVec-A
3d
5and 20 μ LLipofectamine2000Reagent, carry out transfection by Lipofectamine2000Reagent test kit specification sheets.After 24h is carried out in transfection, cell culture medium is changed to containing 600 μ g/mLG
418dMEM Screening of Media resistance clone.
The CHO-K of the stably express antibody obtained will be screened
1cell strain serum free medium EX-CELL302 enlarged culturing, use ProteinASepharose4FastFlow purification system, by specification affinity purification obtains bi-specific antibody A
3b
5-BsAb.The anti-second bi-specific antibody PBS obtained by purifying dialyses, and finally measures A with ultraviolet absorption method
3b
5the content of-BsAb antibody.
Adopt polyacrylamide gel electrophoresis method, the structure of qualification antibody.Antibody after purifying detects its molecular size range by polyacrylamide gel electrophoresis respectively under non-reduced and reductive condition.Electrophoresis result shows: under the reducing conditions, A
3b
5-BsAb antibody is rendered as heavy chain and the light chain bands that molecular size range is about 65KDa and 35KDa.Under non reducing conditions, A
3b
5-BsAb antibody presents the single band that molecular weight is about 200KDa.
The bi-specific antibody A for hepatitis B surface antigen can be inferred thus
3b
5the structure of-BsAb: this antibody is made up of the heavy chain that two identical light chains are identical with two, light chain is made up of variable region of light chain and constant region of light chain, heavy chain is made up of variable region of heavy chain and CH, wherein a light chain is connected by disulfide linkage with a heavy chain respectively, formation two near points, is connected to form described antibody by disulfide linkage between two heavy chains of two near points.So far, we build and give expression to complete bi-specific antibody A
3b
5-BsAb.
Embodiment two, bi-specific antibody A
3b
5the specificity of-BsAb
Bi-specific antibody A is detected by Elisa method
3b
5the ability of-BsAb specific binding HBsAg albumen.
Recombinant HBsAg albumen and reference protein cIg are wrapped by enzyme linked immunological plate, after confining liquid is closed, adds blank group, the A of the reference protein cIg of different concns and the different concns of expression and purification respectively
3d
5antibody incubation, adds Goatanti-humankappa-HRP after washing plate, and TMB nitrite ion develops the color, and microplate reader reads value under 450nm wavelength.
OD value according to antibody concentration and correspondence draws Fig. 1, can be obtained by Fig. 1 analysis, and along with increasing of antibody concentration, it wraps to be read by HBsAg protein groups correspondence that value is also corresponding to be increased.And bag is read value and blank group by reference protein group to read value close and do not change with antibody concentration and change, and reference protein cIg group is read value and is not relied on self change in concentration and change, therefore bi-specific antibody A
3b
5-BsAb can specific binding HBsAg albumen.
Embodiment three, bi-specific antibody A
3b
5-BsAb conjugated antigen Epitope Identification
The hepatitis B surface antigen (HBsAg) of comprehensive previous literature report has the antigenic epitopes region of Neutralization effect, synthesize biotin labeled hepatitis B surface antigen small peptide, measure the calmodulin binding domain CaM of total man source hepatitis B surface protein monoclonal antibody on HBsAg.As shown in the HBsAg mode chart in Fig. 2, four biotin labeling hepatitis B surface antigen small peptide P of synthesis
1(aa:104-120), P
2(aa:121-137), P
3(aa:139-148), P
4(aa:149-163), P
1-P
4aminoacid sequence as shown in SEQIDNO.9 ~ SEQIDNO.12.Wherein, antigen small peptide P
1and P
4for linear structure, P
2and P
3for ring texture
[10].
Previous experiments shows, is combined in conformational epitope section (P
2and P
3region) monoclonal antibody in and HBV activity be better than and be combined in linear epitope section (P
1and P
4region) monoclonal antibody.Adopt the methods analyst of ELISA A
3d
5, B
5h
6, A
3b
5the conjugated antigen epi-position of-BsAb bi-specific antibody, from Fig. 3 (A) and Fig. 3 (B), A
3d
5and B
5h
6be combined in conformational epitope section P respectively
3and P
2, A
3b
5-BsAb bi-specific antibody is incorporated into conformational epitope section P
2, P
3position, shows that this antibody can in conjunction with two epi-position.
Embodiment four, bi-specific antibody A
3b
5with the qualification of HBV activity during-BsAb is external
Nearly decades, slow to HBV Infection in Vitro Models, with chimpanzee be mainly the HBV In vivo infection model of host because of expensive and more difficult acquisition, hinder HBV further investigation always
[11].In recent years, along with the foundation of HepaRG clone, there is greater advance to the research of HBV
[12].HepaRG clone is current unique putative HBV Infection in Vitro model.Therefore, we adopt HepaRG raji cell assay Raji A
3d
5in monoclonal antibody and the ability of HBV infection HepaRG cell, comprehensive HBsAg and HBVDNA copy number quantitative detecting analysis A
3d
5with HBV ability in monoclonal antibody.
One, antibody A
3b
5-BsAb affects HBsAg detection by quantitative
Cultivate HepaRG cell with William ' sEmedium, add 2%DMSO and cultivate surrounding, within every three days, change liquid.After surrounding, differentiation-inducing good HepaRG cell presents typical liver cell sample tuftlet, and separates from the HepaRG cell of undifferentiated, and DMSO induction continued cultivation after two weeks, and cell about has half to present the change of liver cell sample.Differentiation-inducing good HepaRG cell continues with containing 10% foetal calf serum William ' sEmedium100units/mL penicillin, 100 μ g/mL Streptomycin sulphates, 5g/mL recombinant human insulin, 5 × 10
5mol/L hydrocortisone sodium salt continues to cultivate, for subsequent use.
Purifying degerming anti-hbs monoclonal antibodies (1 μ g/mL) and 2 × 10
6the virus quantity incubated at room of HBVDNA copy number 1 hour.Add in the differentiation-inducing good HepaRG cell of DMSO, and add PEG8000, final concentration is 4%, 37 DEG C of overnight incubation.Within second day, change liquid, wash three times with phosphate buffered saline buffer, add the fresh William ' sEmedium prepared, continue to cultivate.Collect the cell conditioned medium of after infecting the 6th day, the Electrogenerated chemiluminescent immunoassay instrument adopting Roche Holding Ag to produce and matched reagent weight per unit length thereof detect HBsAg.
As shown in Figure 4, infect the 6th day, same to control group (cIgG group) is compared, monoclonal antibody A
3d
5group and monoclonal antibody B
5h
6group effectively can reduce the expression amount of HBsAg in cell conditioned medium, and A
3d
5with B
5h
6there is stronger synergy in combined utilization group, the expression amount of the HBsAg in cell conditioned medium significantly reduces.But bi-specific antibody A
3b
5-BsAb group and A
3d
5, B
5h
6monoclonal antibody group and combined utilization group thereof are compared, and in cells and supernatant, very obviously declining (* P < 0.05) appears in HBsAg, and this bi-specific antibody A is described
3b
5-BsAb can suppress the expression of HBsAg better compared with its parental antibody and combination group thereof, is the effect of 1+1 > 2, so show its have extremely strong in and the ability of HBV virus.
Two, bi-specific antibody A
3b
5-BsAb affects HBVDNA copy number detection by quantitative
Detect HBVDNA copy number in the cell infecting latter 6th day according to HBVDNA PCR kit for fluorescence quantitative specification sheets, step is as follows:
(1) preparation work: sample treatment liquid A is placed in 70 DEG C of heating 5-10min, after melting, mixing is for subsequent use; In sample treatment liquid B, add dehydrated alcohol 6mL, mix for subsequent use; In sample treatment liquid C, add dehydrated alcohol 16mL, mix for subsequent use; Add 700 μ L sterilizing DEPC process water disinthibiting in agent, after dissolving, mixing is for subsequent use;
(2) nucleic acid extraction: draw 20 μ L and disinthibite agent in 0.5mL centrifuge tube; Add 100 μ L samples, repeatedly blow and beat 3 times with band filter core suction nozzle; Add 100 μ L sample treatment liquid A, vibration mixing, the brief centrifugation several seconds be placed on 70 DEG C of conditions under react 10min; The nucleic acid extraction column performing mark is put into 2ml centrifuge tube, all liquid in previous step is moved into nucleic acid extraction column.Centrifugal 10000rpm × 1min; Extraction column is put into a new 2mL centrifuge tube, add 500 μ L sample treatment liquid B, centrifugal 10000rpm × 1min; Extraction column is put into a new 2mL centrifuge tube, add 500 μ L sample treatment liquid C, centrifugal 10000rpm × 1min; Extraction column is put into a new 2mL centrifuge tube, centrifugal 14000rpmx1min; Extraction column is put into a new 2mL centrifuge tube, carefully 50uL sterilizing pure water is added in cylinder central authorities, leave standstill 1min; Centrifugal 10000rpm × 1min.The collection liquid 12 μ L got in 2mL centrifuge tube does PCR reaction template.Fluorescent value is detected according to HBVDNA PCR kit for fluorescence quantitative specification sheets.
As shown in Figure 5, HBV infection HePaRG the 6th day, antibody A
3d
5group and B
5h
6group effectively can reduce the copy number of HBVDNA in cell compared with control antibodies group (cIgG group); A
3d
5with B
5h
6there is stronger synergy in combined utilization group, after wherethrough reason, the copy number of HBVDNA decreases equally; And A
3d
5with B
5h
6combined utilization group is compared, bi-specific antibody A
3b
5after-BsAb processes HePaRG groups of cells, in cell, very significantly declining (* P < 0.05) appears in the copy number of HBVDNA, bi-specific antibody A
3b
5-BsAb has the ability that extremely excellent suppression HBVDNA copies.
Embodiment five, bi-specific antibody A
3b
5-BsAb suppresses the release of liver cancer cell HBsAg
Relation between HBV virus infection and primary hepatocellular carcinoma occur receives publicity day by day.Alexander in 1976 etc. are separated to a strain Bel7402 from Mozambique primary hepatocarcinoma male patient, called after PLC/PRF/5 (hereinafter referred to as PLC), this clone can produce HBsAg continually and steadily, since report, PLC has become the important experimental model studying HBV and Relations with Liver Cancer in the world.
HBsAg release in liver cancer cell whether can be suppressed with this hepatoma model research antibody.CIg, A
3d
5, B
2e
5, D
3f
6and F
2e
5antibody component other places reason PLC/RPF/5 cell, within 24 hours, recession is except antibody, and generation is with fresh substratum.3,6,12,24 hours points, detect the content of the HBsAg in cell conditioned medium.As shown in Figure 6, compared with control group (cIg group), after antibody is removed, pass in time, A
3d
5, B
5h
6, A
3d
5+ B
5h
6and A
3b
5in cell conditioned medium in-BsAb different treatment group, the concentration ascensional range of HBsAg slows down.Bi-specific antibody A
3b
5the content climbing speed of the HBsAg in-BsAb treatment group cell conditioned medium is starkly lower than A
3d
5, B
5h
6, A
3d
5and B
5h
6combined utilization treatment group (* p < 0.05), bi-specific antibody A
3b
5-BsAb is Be very effective in suppression HBsAg release.
The effect of embodiment and effect
Embodiment provides a kind of bi-specific antibody A for hepatitis b surface antigen protein
3b
5-BsAb, this bi-specific antibody is by antibody A
3d
5and antibody B
5h
6combine, but more simple by antibody A with the ability of the ability of HBV virus and suppression HBsAg release in it
3d
5and antibody B
5h
6combined utilization is more excellent, shows stronger synergy, and then the process of hepatitis, liver cirrhosis and the liver cancer that the infection of HBV may be stoped to be correlated with.Meanwhile, because this antibody clones the human antibody obtained in the memory B cells that HBsAg is special from volunteer's peripheral blood of HB vaccination, it has compared with mouse source, the immunogenicity that chimeric and humanized antibody is lower.
Reference
[1]GanemD,PrinceAM,Hepatitis.Bvirusinfection-naturalhistoryandclinicalconsequences.NEnglJMed2004；350:1118–29.
[2]YangPL,AlthageA,ChungJ,ChisariFV.HydrodynamicinjectionofviralDNA:amousemodelofacutehepatitisBvirusinfection.ProcNatlAcadSciUSA2002；99:13825–30.
[3]SandgrenEP,PalmiterRD,HeckelJL,etal.Completehepaticregenerationaftersomaticdeletionofanalbumin-plasminogenactivatortransgene.Cell1991；66:245–56.
[4]BrussV,LuX,ThomssenR,etal.Post-translationalalterationsintransm-embranetopologyofthehepatitisBviruslargeenvelopeprotein.EMBOJ1994；13:2273–9.
[5]PetitMA,DubanchetS,CapelF,etal.HepG2cellbindingactivitiesofdifferenthepatitisBvirusisolates:inhibitoryeffectofanti-HBsandanti-preS1(21-47).Virology1991；180:483–91.
[6]HeermannKH,GoldmannU,SchwartzW,etal.LargesurfaceproteinsofhepatitisBviruscontainingthepre-sequence.JVirol1984；52:396–402.
[7]LeSeyecJ,ChouteauP,CannieI,Guguen-GuillouzoC,GriponP.InfectionprocessofthehepatitisBvirusdependsonthepresenceofadefinedsequenceinthepre-S1domain.JVirol1999；73:2052–7.
[8]HieterPA，MaxEE，SeidmanJG，MaizelJVJr，LederP.ClonedhumanandmousekappaimmunoglobulinconstantandJregiongenesconservehomologyinfunctionalsegments.Cell.1980；22(1Pt1):197-207.
[9]EllisonJW，BersonBJ，HoodLE.ThenucleotidesequenceofahumanimmunoglobulinCgamma1gene.NucleicAcidsRes.1982Jul10；10(13)：4071-9.
[10]GriponP,CannieI,UrbanS.EfficientinhibitionofhepatitisBvirusinfectionbyacylatedpeptidesderi-vedfromthelargeviralsurfaceprotein.JVirol2005；79:1613–22.
[11]EngelkeM,MillsK,SeitzS,etal.CharacterizationofahepatitisBandhepatitisdeltavirusreceptorbindingsite.Hepatology2006；43:750–60.
[12]LeistnerCM,Gruen-BernhardS,GlebeD.RoleofglycosaminoglycansforbindingandinfectionofhepatitisBvirus.CellMicrobiol2008；10:122–33.
Claims (11)
1. the bi-specific antibody A for hepatitis B surface albumen
3b
5-BsAb, is characterized in that, has:
Heavy-chain variable domains, comprises hypervariable region CDR
1, CDR
2, CDR
3, CDR
4, CDR
5, CDR
6; And
Light variable domains, comprises hypervariable region CDR
1＇, CDR
2＇, CDR
3＇, CDR
4＇, CDR
5＇, CDR
6＇,
Wherein, described CDR
1aminoacid sequence be: Met-Tyr-Ala-Phe-Ser;
Described CDR
2aminoacid sequence be: Gly-Ile-Ile-Pro-Ser-Phe-Asp-Thr-Thr-Asn-Tyr-Ala-Gln-Lys-Phe-Gln-Cys;
Described CDR
3aminoacid sequence be: Ser-Val-Ile-Thr-Asp-Leu-Asp-Thr-Val-Gly-Asn-Asp-Glu-Ser-Gly-Asp-Ala-Ser-Tyr-Tyr-Tyr-Met-Asp-Val;
Described CDR
4aminoacid sequence be: Ser-Ser-Ala-Ile-Leu;
Described CDR
5aminoacid sequence be: Trp-Ile-Val-Val-Gly-Ser-Gly-Asn-Ala-Lys-Tyr-Ala-Gln-Arg-Phe-Gln-Glu;
Described CDR
6aminoacid sequence be: Arg-Gly-His-Ser-Phe-Thr-Ser-Pro-Phe-Asp-Ser;
Described CDR
1the aminoacid sequence of ＇ is: Arg-Ser-Ser-Glu-Ser-Leu-Gln-His-Ser-Asp-Gly-Thr-Tyr-Tyr-Leu-Asp;
Described CDR
2the aminoacid sequence of ＇ is: Ser-Ala-Ser-Asn-Thr-Ala-Pro;
Described CDR
3the aminoacid sequence of ＇ is: Met-Gln-Ala-Leu-Glu-Thr-Pro-Phe-Thr;
Described CDR
4the aminoacid sequence of ＇ is: Arg-Ala-Ser-Gln-Ser-Val-Gly-Ser-Asn-Tyr-Leu-Ala;
Described CDR
5the aminoacid sequence of ＇ is: Gly-Ala-Ser-Thr-Arg-Ala-Thr;
Described CDR
6the aminoacid sequence of ＇ is: Gln-Lys-Tyr-Gly-Ser-Ser-Leu-Thr.
2. the bi-specific antibody A for hepatitis B surface albumen according to claim 1
3b
5-BsAb, is characterized in that:
Wherein, the aminoacid sequence of described heavy-chain variable domains is as shown in SEQIDNO.6, and the aminoacid sequence of described light variable domains is as shown in SEQIDNO.8.
3. the bi-specific antibody A for hepatitis B surface albumen according to claim 2
3b
5-BsAb, is characterized in that:
Wherein, described bi-specific antibody A
3b
5-BsAb be selected from the bi-specific antibody of anti-hepatitis B virus surface antigen and described bispecific antibody fragment any one,
Described bispecific antibody fragment is the Fab fragment of described bi-specific antibody or F (ab ')
2fragment.
4., for an antibody fragment for hepatitis B surface albumen, it is characterized in that:
Described antibody fragment is the bi-specific antibody A described in claim 1 or 2
3b
5the Fab fragment of-BsAb or F (ab ')
2fragment.
5. the bi-specific antibody A for hepatitis B surface albumen described in claim 1 or 2 that encodes
3b
5the gene of-BsAb, is characterized in that:
Encode the nucleotide sequence of described heavy-chain variable domains as shown in SEQIDNO.5, and the nucleotide sequence of described light variable domains of encoding is as shown in SEQIDNO.7.
6. an expression vector, is characterized in that:
Described expression vector contains the gene according to claim 5 of at least one copy.
7. a host cell, is characterized in that:
Described host cell contains at least one expression vector according to claim 6.
8. the bi-specific antibody A for hepatitis B surface albumen described in any one of claims 1 to 3
3b
5the purposes of-BsAb in the medicine preparing prevention or treatment hepatitis B virus related liver disease or diagnostic reagent.
9. the purposes of antibody fragment in the medicine preparing prevention or treatment hepatitis B virus related liver disease or diagnostic reagent for hepatitis B surface albumen according to claim 4.
10. one kind contains the bi-specific antibody A for hepatitis B surface albumen described in any one of claims 1 to 3
3b
5the medicine of-BsAb or diagnostic reagent, is characterized in that:
Wherein, described medicine or diagnostic reagent also comprise any one in pharmaceutically acceptable vehicle, thinner and carrier.
11. 1 kinds, containing the medicine of the antibody fragment for hepatitis B surface albumen according to claim 4 or diagnostic reagent, is characterized in that:
Wherein, described medicine or diagnostic reagent also comprise any one in pharmaceutically acceptable vehicle, thinner and carrier.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107556381A (en) * | 2017-10-12 | 2018-01-09 | 南京京达生物技术有限公司 | A kind of hepatitis B virus surface antigen detection antibody IgM and application |
| WO2018184593A1 (en) * | 2017-04-07 | 2018-10-11 | 厦门大学 | Antibody for treating hepatitis b infection and related disease |
| CN111234012A (en) * | 2018-11-29 | 2020-06-05 | 清华大学 | Hepatitis B virus neutralizing antibody B432 and application thereof |
| CN113196057A (en) * | 2018-11-09 | 2021-07-30 | 积水医疗株式会社 | Method for detecting viral liver cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018184593A1 (en) * | 2017-04-07 | 2018-10-11 | 厦门大学 | Antibody for treating hepatitis b infection and related disease |
| KR20220126788A (en) * | 2017-04-07 | 2022-09-16 | 시아먼 유니버시티 | Antibodies for treatment of hepatitis B infection and related diseases |
| KR102561555B1 (en) | 2017-04-07 | 2023-07-31 | 시아먼 유니버시티 | Antibodies for treatment of hepatitis B infection and related diseases |
| CN107556381A (en) * | 2017-10-12 | 2018-01-09 | 南京京达生物技术有限公司 | A kind of hepatitis B virus surface antigen detection antibody IgM and application |
| CN113196057A (en) * | 2018-11-09 | 2021-07-30 | 积水医疗株式会社 | Method for detecting viral liver cancer |
| CN111234012A (en) * | 2018-11-29 | 2020-06-05 | 清华大学 | Hepatitis B virus neutralizing antibody B432 and application thereof |
| CN111234012B (en) * | 2018-11-29 | 2022-03-15 | 清华大学 | Hepatitis B virus neutralizing antibody B432 and application thereof |
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