CN105061590B - For the bispecific antibody and application thereof of hepatitis B surface albumen - Google Patents
For the bispecific antibody and application thereof of hepatitis B surface albumen Download PDFInfo
- Publication number
- CN105061590B CN105061590B CN201510468936.1A CN201510468936A CN105061590B CN 105061590 B CN105061590 B CN 105061590B CN 201510468936 A CN201510468936 A CN 201510468936A CN 105061590 B CN105061590 B CN 105061590B
- Authority
- CN
- China
- Prior art keywords
- antibody
- hepatitis
- bsab
- bispecific antibody
- drug
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The present invention provides a kind of bispecific antibody A for hepatitis b surface antigen protein3B5- BsAb, the bispecific antibody is by antibody A3D5And antibody B5H6It is composed, but its ability for neutralizing HBV virus and the ability for inhibiting HBsAg to discharge are more merely by antibody A3D5And antibody B5H6Use in conjunction is more excellent, shows stronger synergy, shows stronger synergy, and then may prevent the process for infecting relevant hepatitis, cirrhosis and liver cancer of HBV.Simultaneously, since the antibody is to clone the human antibody obtained from the special memory B cells of HBsAg in volunteer's peripheral blood of HB vaccination, it has compared with source of mouse, the chimeric and lower immunogenicity of humanized antibody, can be used for preparing prevention or treats the drug or diagnostic reagent of hepatitis B related liver disease.
Description
Technical field
The invention belongs to field of biotechnology, more specifically, the invention discloses anti-hepatitis B surface protein bispecifics to resist
Antibody Production Techniques and its purposes in prevention HBV infection hepatocellular carcinoma related to HBV is treated.
Background technique
Hepatitis B (Hepatitis B virus, HBV) is double-stranded DNA virus, easily causes chronic after human body is infected
Type hepatitis, and then lead to cirrhosis and (or) liver cancer.According to statistics in recent years the whole world there are about 3.5 hundred million HBV infection persons, every year there are about
1000000~1,500,000 people die of liver failure caused by acute or chronic HBV infection, cirrhosis and (or) liver cancer, thus prevent
HBV infection becomes world public health problem[1].Hepatocellular carcinoma (Hepatocellular carcinoma, HCC, abbreviation liver cancer)
It is in close relations with virus hepatitis, it is one of most important cause of death caused by virus hepatitis, is also clinically most often
One of malignant tumour seen is worldwide the tumour correlation cause of death for occupying third, China in annual new cases
Account for the whole world 42.5%, it has also become the deputy tumor etiology in China.
Studies have shown that the HCC of the high person of HBV DNA carrying capacity (> 105copies/ml) occurs in HBV infection associated HCC patient
Rate is up to 10.1%, and the HCC incidence of the low person of HBV DNA carrying capacity (< 104copies/ml) is only 3.8%, and the high virus of baseline carries
It measures related with HCC incidence raising.When high virus load (HBV DNA >=0.7mEq/ml), HCC risk of relapse ratio is 5.13, is cut
The HCC risk of relapse ratio of the peristoma positive is 2.14.Therefore, antiviral therapy is carried out to hepatitis B patient, help to delay disease to
The progress of HCC.Antiviral therapy is carried out to HBV associated HCC patient, survival rate can be improved by improving liver function.In recent years
The high HBV DNA carrying capacity of studies have shown that is related with high HCC recurrence rate and prognosis mala.To HBV infection associated HCC postoperative patient into
Row antiviral therapy may be another therapeutic choice in addition to liver transfer operation, which can improve life by improving liver function
Deposit rate.
At present for HBV infection mainly use the Antiviral Effects such as alpha-interferon and nucleotide analog treat, but this two
Class drug long-time service curative effect is not good enough, is easy to lead to virus mutation, carrys out extreme difficulties to treatment zone[2]。
Relative to the drug of viral infection resisting, the antiviral effect of the immunoglobulin (HBIG) of hepatitis B specificity is well very
It is more.The immunoglobulin (HBIG) for giving patient's hepatitis B specificity is treated to by scholar's extensive concern of various countries.Such as, HBIG
Research after the liver cancer in hepatitis B source after liver transfer operation has reached good effect.HBIG is a kind of polyclonal external source of high-titer
Property antibody, is to screen from healthy blood donor, is made by bioconcentration technique.Passively as human infection HBV
Receive this passive immunity preparation, body can be enable to neutralize rapidly in a short time and remove the hepatitis B to dissociate in serum, avoids
Hepatitis B localized infection.
However, current HBIG is as the sources of therapeutic antibodies, there are many non-safety factors.Meanwhile hepatitis B immune ball egg
White complicated component, risk factor is relatively uncertain, and production method is limited, these factors restrict the clinic of hepatitis B therapeutic antibody
It uses[3]。
Therefore there is an urgent need to research and develop biological products substitution people's hepatitis B that one kind is safe and effective and risk factor is relatively controllable to exempt from
Epidemic disease globulin, thus research and development hepatitis B specificity human genetically engineered antibody more and more attention has been paid to.Because monoclonal antibody has spy
The advantages that anisotropic strong, high sensitivity and easily large-scale production, therefore research and develop and be directed to hepatitis B surface antigen (Hepatitis B
Surface Ag, HBsAg) monoclonal antibody, to can safely and effectively prevent HBV infection and its relevant hepatitis, liver
The process of hardening and liver cancer.
Due to the multifactor property of disease, single target treatment tends not to meet the needs of clinical[4].In clinical test
The report of existing two kinds of monoclonal preclinical laboratories of conjunctive use[5], but this use in conjunction monoclonal antibody has many limits
Property processed[6].For example, the safety of two kinds of monoclonal antibodies of use in conjunction and effect need to go to evaluate respectively, resulting length
The bottleneck that period, high development cost limit its development process, and bispecific antibody solves just[7]。
Summary of the invention
The present invention is to carry out in order to solve the above problem, and it is anti-to provide a kind of bispecific for surface antigen protein
Body A3B5- BsAb, present invention employs following technical solutions:
Bispecific antibody A provided by the invention for hepatitis B surface albumen3B5- BsAb is comprising: weight
Chain variable domains include hypervariable region CDR1、CDR2、CDR3、CDR4、CDR5、CDR6；And light variable domains, include height
Become area CDR1', CDR2', CDR3', CDR4', CDR5', CDR6', the amino acid sequence of heavy-chain variable domains such as SEQ ID NO.6
Shown, the amino acid sequence of light variable domains is as shown in SEQ ID NO.8.
Wherein, CDR1Amino acid sequence are as follows: Met-Tyr-Ala-Phe-Ser；CDR2Amino acid sequence are as follows: Gly-
Ile-Ile-Pro-Ser-Phe-Asp-Thr-Thr-Asn-Tyr-Ala-Gln-Lys-Phe-Gln-Cys；CDR3Amino acid
Sequence are as follows: Ser-Val-Ile-Thr-Asp-Leu-Asp-Thr-Val-Gly-Asn-Asp-Glu-Ser- Gly-Asp-Ala-
Ser-Tyr-Tyr-Tyr-Met-Asp-Val；CDR4Amino acid sequence are as follows: Ser-Ser-Ala-Ile-Leu；CDR5Amino
Acid sequence are as follows: Trp-Ile-Val-Val-Gly-Ser-Gly-Asn-Ala-Lys-Tyr-Ala-Gln-Arg- Phe-Gln-Glu；
CDR6Amino acid sequence are as follows: Arg-Gly-His-Ser-Phe-Thr-Ser-Pro-Phe-Asp-Ser；
CDR1The amino acid sequence of ' are as follows: Arg-Ser-Ser-Glu-Ser-Leu-Gln-His-Ser-Asp-Gly-Thr-
Tyr-Tyr-Leu-Asp；CDR2The amino acid sequence of ' are as follows: Ser-Ala-Ser-Asn-Thr-Ala-Pro；CDR3The amino acid of '
Sequence are as follows: Met-Gln-Ala-Leu-Glu-Thr-Pro-Phe-Thr；CDR4The amino acid sequence of ' are as follows: Arg-Ala-Ser-
Gln-Ser-Val-Gly-Ser-Asn-Tyr-Leu-Ala；CDR5The amino acid sequence of ' are as follows: Gly-Ala-Ser-Thr-Arg-
Ala-Thr；CDR6The amino acid sequence of ' are as follows: Gln-Lys-Tyr-Gly-Ser-Ser-Leu-Thr.
Meanwhile bispecific antibody A3B5- BsAb is selected from the bispecific antibody of anti-hepatitis B virus surface antigen and described
Any one in bispecific antibody fragment, the bispecific antibody fragment are the Fab segment or F of the bispecific antibody
(ab’)2Segment.
Further, the present invention also provides a kind of antibody fragment for hepatitis B surface albumen, which is double
Specific antibody A3B5The Fab segment or F (ab ') of-BsAb2Segment.
Further, the present invention provides a kind of encoding bispecific antibody As3B5The gene of-BsAb, encoding heavy chain are variable
The nucleotide sequence of structural domain encodes the nucleotide sequence such as SEQ ID of light variable domains as shown in SEQ ID NO.5
Shown in NO.7.
Further, the present invention provides a kind of expression vectors of said gene containing at least one copy.
Further, the present invention provides a kind of host cells containing at least one above-mentioned expression vector.
Further, the present invention provides bispecific antibody A3B5- BsAb or its Fab segment or F (ab ')2Segment is being made
The standby drug for preventing or treating hepatitis B related liver disease or the purposes in diagnostic reagent.The drug or diagnostic reagent remove comprising
Outside antibody or antibody fragment, also comprising any one in pharmaceutically acceptable excipient, diluent and carrier.
Invention action and effect
The present invention provides a kind of bispecific antibody A for hepatitis b surface antigen protein3B5- BsAb, the bispecific
Antibody is by antibody A3D5And antibody B5H6It is composed, but its ability for neutralizing HBV virus and the energy for inhibiting HBsAg release
Power is more merely by antibody A3D5And antibody B5H6Use in conjunction is more excellent, shows stronger synergy, and then may prevent
The process for infecting relevant hepatitis, cirrhosis and liver cancer of HBV.Simultaneously as the antibody is the aspiration from HB vaccination
The human antibody of acquisition is cloned in person's peripheral blood in HBsAg special memory B cells, is had compared with source of mouse, chimeric and people
The lower immunogenicity of source antibody.
Detailed description of the invention
Fig. 1 is bispecific antibody A of the invention3B5- BsAb molecule of the antigen binding figure；
Fig. 2 is the position view of synthetic peptide of the invention in hepatitis B surface antigen (HBsAg)；
Fig. 3 (A) is bispecific antibody A of the invention3B5- BsAb combines epitope P3Result figure；
Fig. 3 (B) is bispecific antibody A of the invention3B5- BsAb combines epitope P2Result figure；
Fig. 4 is bispecific antibody A of the invention3B5- BsAb influences the quantitative detection result figure of HBsAg expression in cell；
Fig. 5 is bispecific antibody A of the invention3B5- BsAb influences the quantitative detection knot of HBV DNA replication dna number in cell
Fruit figure；
Fig. 6 is bispecific antibody A of the invention3B5- BsAb inhibits the result figure of HBsAg release in liver cancer cells.
Specific embodiment
The present invention is further detailed with reference to embodiments, but should not be construed as limiting the invention.
Embodiment does not include detailed descriptions of conventional methods, such as: Overlapping PCR reaction, restricted digestion are anti-
It answers, gene is inserted into plasmid vector, plasmid is introduced to the method etc. of host cell.Such method in this field for having
The personnel of ordinary skill are well-known, and are all described in many publications, such as Sambrook, J.,
Fritsch, E.F.and Maniais, T. (1989) Molecular Cloning:A Laboratory Manual,
2ndedition, Cold spring Harbor Laboratory Press.And material as used in the following examples, reagent
Deng being commercially available unless otherwise specified.
Reagent and material
Lymphocyte separation medium is purchased from Beijing Ding Guo biotechnology Co., Ltd；Trizol reagent, is purchased from
Invitrogen company；Serum free medium EX-CELL 302 is purchased from SIGMA-ALDRICH company；Goat anti-human
Kappa-HRP is purchased from Invivogen company；
Embodiment one: bispecific antibody A3B5The preparation of-BsAb
One, the building of antibody light chain constant region, the clone of weight chain constant area gene and its carrier
With lymphocyte separation medium separating health human lymphocyte, total serum IgE is extracted with Trizol reagent, according to document [8]
Primer is separately designed using the heavy chain of QIAGEN OneStep RT-PCR Kit amplification human antibody with the sequence of document [9] report
And light chain constant region gene.The heavy chain of human antibody and light chain constant region gene are connected respectively in expression vector AbVec plasmid,
Heavy chain constant region nucleotide carrier IgG-AbVec plasmid and antibody light chain constant region nucleotide carrier Ig are constructed respectively
κ-AbVec plasmid, confirmation obtains correct clone after sequence verification.Wherein, the constant region nucleotide sequence of human antibody heavy chain and
Amino acid sequence is respectively SEQ ID NO:1 and SEQ ID NO:2, human antibody light chain constant region nucleotide sequence and amino acid sequence
Column are respectively SEQ ID NO:3 and SEQ ID NO:4.
Two, antibody heavy chain variable region, light-chain variable sequence acquisition and the building of recombinant expression carrier
Specifically, special using HBsAg in volunteer's peripheral blood of ultrahigh speed streaming separation system separation HB vaccination
Different memory B cells (HBsAg+IgG+CD19+), and it is prepared into unicellular sample.And then, using Single Cell
RT-PCR technology clones antibody light chain and weight chain variabl area sequence in each sample, since the cDNA of individual cells synthesis is especially dilute
It is few, it is necessary to which that its antibody gene contained is expanded using the method for Chao Shi PCR.
Visible apparent PCR band amplification (size about 400bp) after two-wheeled PCR.After analyzing its sequence, specific PCR expands
Increase antibody variable region band, heavy chain introduces restriction enzyme A ge I and restriction enzyme Sal I site, and light chain introduces limit
Property restriction endonuclease Age I and restriction enzyme Bsiw I site processed.Heavy chain of antibody and light-chain variable region gene are cloned into respectively
Thus IgG-AbVec plasmid and Ig κ-AbVec plasmid plant method, construct multiple full source of people hepatitis B surface protein monoclonal antibodies,
The antibody for wherein choosing two plants of HBV neutralization activities with higher, is respectively designated as A3D5And B5H6。A3D5Weight, the light chain of antibody
Carrier be respectively designated as: IgG-AbVec-A3D5, Ig κ-AbVec-A3D5；B5H6The weight of antibody, the carrier of light chain are named respectively
Are as follows: IgG-AbVec-B5H6, Ig κ-AbVec-B5H6。
Using the method for Overlapping PCR, by A3D5The C-terminal of heavy chain variable region passes through Linker small peptide
" ASTKGPSVFPLAP (nucleotide sequence are as follows: GCT AGC ACC AAG GGA CCT TCT GTG TTC CCT CTG GCC
) " and B CCT5H6The N-terminal of antibody heavy chain variable region is connected, and introduces restriction enzyme A ge I and restriction enzyme Sal I
Point constitutes A3B5The heavy chain variable region of-BsAb bispecific antibody, nucleotide sequence and amino acid sequence are respectively SEQ ID
NO:5 and SEQ ID NO:6, by A3B5- BsAb heavy chain variable region is cloned into IgG-AbVec carrier and constitutes its heavy chain expression vector
IgG-AbVec-A3B5-BsAb.Same method, by A3D5The C-terminal of light chain variable region passes through Linker small peptide " TVAAPSVFIFPP
(nucleotide sequence are as follows: ACC GTG GCT GCC CCT AGC GTG TTC ATC TTC CCT CCT) " and B5H6Antibody light chain
The N-terminal of variable region is connected, and introduces restriction enzyme A ge I and restriction enzyme Bsiw I site, constitutes A3B5-BsAb
The light chain variable region of bispecific antibody, nucleotide sequence and amino acid sequence are respectively SEQ ID NO:7 and SEQ ID
NO:8, by A3B5- BsAb light chain variable region is cloned into Ig κ-AbVec carrier and constitutes its light chain expression vector Ig κ-AbVec-A3B5-
BsAb。
Three, bispecific antibody A3B5- BsAb expression and purifying
5 × 10 are inoculated in 3.5cm Tissue Culture Dish5CHO-K1Cell, by cell culture to 90%-95% merge when into
Row transfection, the specific steps are as follows: take 5 μ g plasmid IgG-AbVec-A3D5, 5 μ g plasmid Ig κ-AbVec-A3D5And 20 μ
LLipofectamine 2000Reagent is transfected by Lipofectamine 2000Reagent kit specification.Turn
After dye carries out for 24 hours, cell culture medium is changed to containing 600 μ g/mL G418DMEM Screening of Media resistance clone.
The CHO-K for stablizing expression antibody that screening is obtained1Cell strain is expanded with serum free medium EX-CELL 302 to be trained
It supports, with Protein A Sepharose 4Fast Flow purification system, by specification affinity purification obtains bispecific antibody
A3B5-BsAb.The anti-second bispecific antibody that purifying obtains is dialysed with PBS, finally measures A with ultraviolet absorption method3B5-
The content of BsAb antibody.
Using polyacrylamide gel electrophoresis method, the structure of antibody is identified.Antibody after purification respectively non-reduced and
Its molecular size range is detected with polyacrylamide gel electrophoresis under reducing condition.Electrophoresis result is shown: under the reducing conditions, A3B5-
BsAb antibody is rendered as the heavy chain and light chain bands of molecular size range about 65KDa and 35KDa.Under non reducing conditions, A3B5-
BsAb antibody shows the single band that molecular weight is about 200KDa.
It thereby it is assumed that out the bispecific antibody A for hepatitis B surface antigen3B5The structure of-BsAb: the antibody is by two
The identical light chain of item and two identical heavy chain compositions, light chain are made of light chain variable region and constant region of light chain, and heavy chain is by heavy chain
Variable region and heavy chain constant region composition, wherein a light chain is connect with a heavy chain by disulfide bond respectively, formation two near points
Son connects to form the antibody by disulfide bond between two heavy chains of two near points.So far, we construct and have given expression to
Whole bispecific antibody A3B5-BsAb。
Embodiment two, bispecific antibody A3B5The specificity of-BsAb
Bispecific antibody A is detected with Elisa method3B5The ability of-BsAb specific binding HBsAg albumen.
Recombinant HBsAg albumen and reference protein cIg coating enzyme linked immunological plate are separately added into sky after confining liquid is closed
White group, the A of the various concentration of the reference protein cIg of various concentration and expression and purification3D5Goat is added after board-washing in antibody incubation
The colour developing of anti-human kappa-HRP, TMB developing solution, microplate reader readings under 450nm wavelength.
Fig. 1 is drawn according to antibody concentration and corresponding OD value, can be obtained by Fig. 1 analysis, with increasing for antibody concentration, packet
Readings is corresponded to by HBsAg protein groups also accordingly to increase.And peridium pair is close with blank group readings according to protein groups readings and not with anti-
Bulk concentration changes and change, and reference protein cIg group readings and does not rely on itself concentration and change and change, therefore bispecific
Antibody A3B5- BsAb can specifically bind HBsAg albumen.
Embodiment three, bispecific antibody A3B5- BsAb is identified in conjunction with epitope
The hepatitis B surface antigen (HBsAg) of comprehensive previous literature report has the antigenic epitopes region of neutralization activity, closes
At the hepatitis B surface antigen small peptide of biotin labeling, to measure full source of people hepatitis B surface protein monoclonal antibody on HBsAg
Bond area.As shown in the HBsAg ideograph in Fig. 2, four biotin labeling hepatitis B surface antigen small peptide P of synthesis1
(aa:104-120)、P2(aa:121-137)、P3(aa:139-148)、P4(aa:149-163), P1-P4Amino acid sequence such as
Shown in SEQ ID NO.9~SEQ ID NO.12.Wherein, antigen small peptide P1And P4For linear structure, P2And P3For ring-type knot
Structure[10]。
Previous experiments show to be incorporated in comformational epitope section (P2And P3Region) monoclonal antibody in and HBV activity to get well
In being incorporated in linear epitope section (P1And P4Region) monoclonal antibody.A is analyzed using the method for ELISA3D5、B5H6、
A3B5The combination epitope of-BsAb bispecific antibody, by Fig. 3 (A) and Fig. 3 (B) it is found that A3D5And B5H6It is bonded respectively to structure
As epitope region P3And P2, A3B5- BsAb bispecific antibody is incorporated into comformational epitope section P2、P3Position shows the antibody energy
It is enough to combine double epitopes.
Example IV, bispecific antibody A3B5- BsAb neutralizes the active identification of HBV in vitro
Recent decades, it is slow to HBV Infection in Vitro Models, mainly with chimpanzee to feel in the HBV body of host
Model is contaminated because of expensive and more difficult acquisition, hinders HBV further investigation always[11].In recent years, building with HepaRG cell line
It is vertical, there is greater advance to the research of HBV[12].HepaRG cell line is currently the only putative HBV Infection in Vitro model.Cause
This, we use HepaRG raji cell assay Raji A3D5The ability of the neutralization HBV infection HepaRG cell of monoclonal antibody, comprehensive HBsAg
And HBV DNA copy number quantitative detecting analysis A3D5The neutralization HBV ability of monoclonal antibody.
One, antibody A3B5- BsAb influences HBsAg quantitative detection
HepaRG cell is cultivated with William ' s E medium, 2%DMSO is added and cultivates surrounding, changes liquid every three days.Four
Zhou Hou induces the HepaRG cell broken up that typical liver cell sample tuftlet is presented, and divides from the HepaRG cell of undifferentiated
It separates out and, DMSO induction continues culture after two weeks, and there are about half, and the variation of liver cell sample is presented for cell.Induce the HepaRG broken up
Cell, which continues to use, contains 10% fetal calf serum William ' s E medium 100units/mL penicillin, 100 μ g/mL strepto-s
Element, 5g/mL rh-insulin, 5 × 105Mol/L hydrocortisone sodium salt continues to cultivate, spare.
Purify the anti-hbs monoclonal antibodies (1 μ g/mL) and 2 × 10 of simultaneously degerming6The virus quantity room of HBV DNA copy number
Temperature is incubated for 1 hour.It is added in the HepaRG cell that DMSO induction has been broken up, and PEG8000 is added, final concentration of 4%, 37 DEG C incubate
It educates overnight.It changes liquid within second day, is washed three times with phosphate buffer, fresh prepared William ' s E is added
Medium continues to cultivate.The cell conditioned medium for collecting the 6th day after infecting, using immune point of the electrochemical luminescence of Roche Holding Ag's production
Analyzer and its matched reagent product quantitative detection HBsAg.
As shown in figure 4, infection the 6th day, compared with control group (cIgG group), monoclonal antibody A3D5Group and monoclonal are anti-
Body B5H6Group can be effectively reduced the expression quantity of HBsAg in cell conditioned medium, and A3D5With B5H6There is stronger association in use in conjunction group
The expression quantity of same-action, the HBsAg in cell conditioned medium significantly reduces.However bispecific antibody A3B5- BsAb group and A3D5、B5H6
Monoclonal antibody group and its use in conjunction group compare, and HBsAg appearance is extremely decreased obviously (* P < in cells and supernatant
0.05), illustrate bispecific antibody A3B5- BsAb can preferably inhibit the table of HBsAg compared with its parental antibody and its combination group
It reaches, is the effect of 1+1 > 2, and then show it with the extremely strong ability for neutralizing HBV virus.
Two, bispecific antibody A3B5- BsAb influences HBV DNA copy number quantitative detection
According to HBV DNA copy in the 6th day after the detection infection of HBV DNA PCR kit for fluorescence quantitative specification cell
Number, steps are as follows:
(1) preparation: sample treatment liquid A is placed in 70 DEG C of heating 5-10min, is mixed after thawing spare；At sample
It manages in liquid B plus dehydrated alcohol 6mL, mixing is spare；In sample treatment liquid C plus dehydrated alcohol 16mL, mixing are spare；It is disinthibiting
700 μ L sterilizing DEPC processing water is added in agent, is mixed after dissolution spare；
(2) nucleic acid extraction: 20 μ L are drawn and disinthibite agent in 0.5mL centrifuge tube；100 μ L samples are added, are inhaled with band filter core
Mouth is blown and beaten 3 times repeatedly；Be added 100 μ L sample treatment fluid A, oscillation mixes, the brief centrifugation several seconds be placed on 70 DEG C under the conditions of react
10min；The nucleic acid extraction column for performing label is put into 2ml centrifuge tube, all liq in previous step is moved into nucleic acid extraction column.
It is centrifuged 10000rpm × 1min；Extraction column is put into a new 2mL centrifuge tube, 500 μ L sample treatment fluid B, centrifugation is added
10000rpm×1min；Extraction column is put into a new 2mL centrifuge tube, 500 μ L sample treatment fluid C, centrifugation is added
10000rpm×1min；Extraction column is put into a new 2mL centrifuge tube, 14000rpm x 1min is centrifuged；Extraction column is put into
In one new 2mL centrifuge tube, 50uL sterilizing pure water is carefully added in cylinder center, stands 1min；It is centrifuged 10000rpm × 1min.
The 12 μ L of collection liquid in 2mL centrifuge tube is taken to do PCR reaction template.It is detected according to HBV DNA PCR kit for fluorescence quantitative specification
Fluorescent value.
As shown in figure 5, HBV infection HePaRG the 6th day, antibody A3D5Group and B5H6Group and control antibodies group (cIgG group) phase
Than the copy number that can be effectively reduced HBV DNA in cell；A3D5With B5H6Equally there is stronger collaboration and makees in use in conjunction group
With the copy number for being processed rear HBV DNA decreases；And A3D5With B5H6Use in conjunction group is compared, bispecific antibody
A3B5After-BsAb handles HePaRG groups of cells, there is extremely significantly decline (* P < 0.05) in the copy number of HBV DNA in cell,
Bispecific antibody A3B5- BsAb has the ability of extremely excellent inhibition HBV DNA replication dna.
Embodiment five, bispecific antibody A3B5The release of-BsAb inhibition liver cancer cells HBsAg
Relationship between HBV virus infection and primary hepatoma generation is of increasing concern.Alexander in 1976
It is separated to one plant of Bel7402 Deng from one primary carcinoma of liver male patient of Mozambique, it is (following to be named as PLC/PRF/5
Abbreviation PLC), which can continually and steadily generate HBsAg, and since report, PLC has become studies HBV and liver in the world
One important experimental model of cancer relationship.
It is discharged so that whether this hepatoma model research antibody is able to suppress HBsAg in liver cancer cells.cIg,A3D5、B2E5、
D3F6And F2E5PLC/RPF/5 cell is managed in antibody component other places, antibody is withdrawn from after 24 hours, generation is with fresh culture medium.3,6,
12,24 hour time point detects the content of the HBsAg in cell conditioned medium.As shown in fig. 6, resisting compared with control group (cIg group)
After body is removed, over time, A3D5、B5H6、A3D5+B5H6And A3B5HBsAg in cell conditioned medium in-BsAb different disposal group
Concentration ascensional range slow down.Bispecific antibody A3B5The content climbing speed of HBsAg in-BsAb processing group cell conditioned medium
Significantly lower than A3D5、B5H6、A3D5And B5H6Use in conjunction processing group (* p < 0.05), bispecific antibody A3B5- BsAb is inhibiting
HBsAg release aspect significant effect.
The action and effect of embodiment
Embodiment provides a kind of bispecific antibody A for hepatitis b surface antigen protein3B5- BsAb, the bispecific
Antibody is by antibody A3D5And antibody B5H6It is composed, but its ability for neutralizing HBV virus and the energy for inhibiting HBsAg release
Power is more merely by antibody A3D5And antibody B5H6Use in conjunction is more excellent, shows stronger synergy, and then may prevent
The process for infecting relevant hepatitis, cirrhosis and liver cancer of HBV.Simultaneously as the antibody is the aspiration from HB vaccination
The human antibody of acquisition is cloned in person's peripheral blood in HBsAg special memory B cells, is had compared with source of mouse, chimeric and people
The lower immunogenicity of source antibody.
Bibliography
[1]Ganem D,Prince AM,Hepatitis.B virus infection-natural history and
clinical consequences.N Engl J Med 2004；350:1118–29.
[2]Yang PL,Althage A,Chung J,Chisari FV.Hydrodynamic injection of
viral DNA:a mouse model of acute hepatitis B virus infection.Proc Natl Acad
Sci U S A 2002；99:13825–30.
[3]Sandgren EP,Palmiter RD,Heckel JL,et al.Complete hepatic
regeneration after somatic deletion of an albumin-plasminogen activator
transgene.Cell 1991；66:245–56.
[4]Bruss V,Lu X,Thomssen R,et al.Post-translational alterations
intransm-embrane topology of the hepatitis B virus large envelope
protein.EMBO J 1994；13:2273–9.
[5]Petit MA,Dubanchet S,Capel F,et al.HepG2 cell binding activities
of different hepatitis B virus isolates:inhibitory effect of anti-HBs and
anti-preS1(21-47).Virology 1991；180:483–91.
[6]Heermann KH,Goldmann U,Schwartz W,et al.Large surface proteins of
hepatitis B virus containing the pre-sequence.J Virol 1984；52:396–402.
[7]Le Seyec J,Chouteau P,Cannie I,Guguen-Guillouzo C,Gripon
P.Infection process of the hepatitis B virus depends on the presence of a
defined sequence in the pre-S1 domain.J Virol 1999；73:2052–7.
[8] HieterPA, Max EE, Seidman JG, Maizel JV Jr, Leder P.Cloned human and
mouse kappa immunoglobulin constant and J region genes conserve homology in
functional segments.Cell.1980；22(1Pt 1):197-207.
[9] Ellison JW, Berson BJ, Hood LE.The nucleotide sequence of a human
immunoglobulin C gamma1 gene.Nucleic Acids Res.1982 Jul 10；10 (13): 4071-9.
[10]Gripon P,Cannie I,Urban S.Efficient inhibition of hepatitis B
virus infection by acylated peptides deri-ved from the large viral surface
protein.J Virol 2005；79:1613–22.
[11]Engelke M,Mills K,Seitz S,et al.Characterization of a hepatitis B
and hepatitis delta virus receptor binding site.Hepatology 2006；43:750–60.
[12]Leistner CM,Gruen-Bernhard S,Glebe D.Role of glycosaminoglycans
for binding and infection of hepatitis B virus.Cell Microbiol 2008；10:122–33.
Claims (9)
1. a kind of bispecific antibody A for hepatitis B surface albumen3B5- BsAb comprising:
Heavy-chain variable domains and light variable domains,
Wherein, the amino acid sequence of the heavy-chain variable domains is as shown in SEQ ID NO.6, the light variable domains
Amino acid sequence is as shown in SEQ ID NO.8.
2. a kind of antibody fragment for hepatitis B surface albumen, it is characterised in that:
The antibody fragment is bispecific antibody A described in claim 13B5The Fab segment or F (ab ') of-BsAb2Segment.
3. a kind of encode the bispecific antibody A described in claim 1 for hepatitis B surface albumen3B5The gene of-BsAb,
It is characterized in that:
The nucleotide sequence of the heavy-chain variable domains is encoded as shown in SEQ ID NO.5, encodes the light chain variable domain
The nucleotide sequence in domain is as shown in SEQ ID NO.7.
4. a kind of expression vector, it is characterised in that:
The expression vector contains the gene as claimed in claim 3 of at least one copy.
5. a kind of host cell, it is characterised in that:
The host cell contains at least one expression vector as claimed in claim 4.
6. the bispecific antibody A described in claim 1 for hepatitis B surface albumen3B5- BsAb is in preparation prevention or treatment second
The drug of hepatovirus related liver disease or the purposes in diagnostic reagent.
7. the antibody fragment as claimed in claim 2 for hepatitis B surface albumen is in preparation prevention or treatment hepatitis B correlation liver
Purposes in the drug or diagnostic reagent of disease.
8. a kind of contain the bispecific antibody A described in claim 1 for hepatitis B surface albumen3B5The drug of-BsAb is examined
Disconnected reagent, it is characterised in that:
Wherein, the drug or diagnostic reagent also include any one in pharmaceutically acceptable excipient, diluent and carrier.
9. a kind of drug or diagnostic reagent containing the antibody fragment as claimed in claim 2 for hepatitis B surface albumen, special
Sign is:
Wherein, the drug or diagnostic reagent also include any one in pharmaceutically acceptable excipient, diluent and carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510468936.1A CN105061590B (en) | 2015-08-03 | 2015-08-03 | For the bispecific antibody and application thereof of hepatitis B surface albumen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510468936.1A CN105061590B (en) | 2015-08-03 | 2015-08-03 | For the bispecific antibody and application thereof of hepatitis B surface albumen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105061590A CN105061590A (en) | 2015-11-18 |
| CN105061590B true CN105061590B (en) | 2019-06-04 |
Family
ID=54491141
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510468936.1A Active CN105061590B (en) | 2015-08-03 | 2015-08-03 | For the bispecific antibody and application thereof of hepatitis B surface albumen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105061590B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2765878C2 (en) * | 2017-04-07 | 2022-02-04 | Сямэнь Юниверсити | 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 |
| WO2020096043A1 (en) * | 2018-11-09 | 2020-05-14 | 積水メディカル株式会社 | Method for detecting viral liver cancer |
| CN111234012B (en) * | 2018-11-29 | 2022-03-15 | 清华大学 | Hepatitis B virus neutralizing antibody B432 and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104592390A (en) * | 2013-10-30 | 2015-05-06 | 上海张江生物技术有限公司 | Bispecific recombinant anti-HBsAg antibody, and preparation method and application thereof |
-
2015
- 2015-08-03 CN CN201510468936.1A patent/CN105061590B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104592390A (en) * | 2013-10-30 | 2015-05-06 | 上海张江生物技术有限公司 | Bispecific recombinant anti-HBsAg antibody, and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Human combinatorial antibody libraries to hepatitis B surface antigen;SUZANNE L. ZEBEDEE;《Proc. Nati. Acad. Sci. USA》;19920430;第89卷;第3175-3179页 |
| 人源抗乙肝病毒表面抗原单克隆抗体的抗原结合活性;韩焕兴;《中华医学杂志》;19960630;第76卷(第6期);第467-468页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105061590A (en) | 2015-11-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20230073067A1 (en) | Humanized monoclonal antibody for 2019 novel coronavirus and use thereof | |
| CN105061590B (en) | For the bispecific antibody and application thereof of hepatitis B surface albumen | |
| CN110029168B (en) | Application of gene FGL1 in preparation of colorectal cancer and lung cancer diagnostic kit and kit | |
| CN113563475B (en) | Bispecific antibody for resisting novel coronavirus and application thereof | |
| CN109776678A (en) | A kind of humanization PD-L1 monoclonal antibody, preparation method and application | |
| RU2613421C2 (en) | HIGH-AFFINITY HUMAN ANTIBODIES TO CYTOMEGALOVIRUS (CMV) gB PROTEIN | |
| CN113185608B (en) | High-affinity rabies virus-resistant fully-humanized monoclonal antibody and application thereof | |
| CN107840889A (en) | The anti-CD123 antibody of high-affinity and its application | |
| CN109265559A (en) | Chimeric antigen receptor, utilizes the NK cell of its modification and the application for the treatment of HBV infection at preparation method | |
| CN105001325A (en) | Total-humanized anti-hepatitis B virus neutralizing antibody and preparing method and application thereof | |
| CN102875674A (en) | Anti-tetanotoxin antibody, and preparation method and application thereof | |
| CN103642792B (en) | A kind of preparation of neutralizing monoclonal antibody of anti-hepatitis C virus and application thereof | |
| CN111620944A (en) | Fully humanized anti-hepatitis B virus monoclonal antibody, preparation method and application thereof | |
| MX2011006722A (en) | Anti-hcv monoclonal antibody as a medicament for the therapeutic treatment and prevention of hcv infections. | |
| CN112661841B (en) | Fully human monoclonal antibody 17-2 for neutralizing neoepitope of new coronavirus and application thereof | |
| CN105061591A (en) | Anti-hepatitis B virus surface antigen completely humanized human antibody and use thereof | |
| CN105017415A (en) | Preparation method and application of completely humanized monoclonal antibody aiming at hepatitis B virus (HBV) surface protein | |
| CN104830902A (en) | Preparation method and application of monoclonal antibody | |
| CN109553680B (en) | Monoclonal antibody ZKns4B8 and application thereof | |
| CN109535249B (en) | Monoclonal antibody ZKns3G2 and application thereof | |
| CN109503712B (en) | Monoclonal antibody ZKns2E11 and application thereof | |
| US10888615B2 (en) | Neutralizing human monoclonal antibody 8D6 against HCV infection | |
| NL2029160B1 (en) | Anti-ev-d68 monoclonal antibody and preparation method and use thereof | |
| CN103044552B (en) | The monoclonal antibody of the anti-surface of dendritic cells DEC-205 molecule of humanization | |
| CN108101990A (en) | The monoclonal antibody and its encoding gene of blocking people's Tim-3 functions and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |