CN1318565A - Humanized hapotitis A virus neutralizing genetically engineered total antibody - Google Patents
Humanized hapotitis A virus neutralizing genetically engineered total antibody Download PDFInfo
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- CN1318565A CN1318565A CN 01110720 CN01110720A CN1318565A CN 1318565 A CN1318565 A CN 1318565A CN 01110720 CN01110720 CN 01110720 CN 01110720 A CN01110720 A CN 01110720A CN 1318565 A CN1318565 A CN 1318565A
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Abstract
The total antibody originating from IgG1 lambda total antibody of bacteriophage antibody library and being recombined and expressed with humanized IgGFc gene, consists of two heavy chains and two lambda light chains and has a molecular weight of about 15 kd. The present invention features that this recombined antibody is neutral humanized complete antibody obtained through recombining baculovirus expression system in insect cell and with high affinity of high efficiency expression specific combination and recognizing hepatitis A virus VP1 protein. The potential application is that recombined baculovirus with the total antibody gene may be utilized to produce this antibody in insect cell and this antibody may be used to replace gamma globulin from blood clinically in preventing and treating hepatitis A.
Description
Preparation and the application, the especially specificity that the present invention relates to prevent and treat personnel selection source gene engineering monoclonal antibody are at the proteic neutrality gene engineering monoclonal antibody of hepatitis A virus (HAV) VP1.
Since B lymphocyte hybridoma cell-fusion techniques in 1975 came out, monoclonal antibody was used for fundamental research as a class, laboratory diagnosis, and the novel product of clinical treatment and prevention, its applying value and DEVELOPMENT PROSPECT have obtained general affirming.The researchdevelopment of molecular biology and molecular immunology has caused the generation and the development of genetic engineering antibody.Reorganization by the antibody molecule gene level can obtain diversified specific murine source and human antibody, and making has had breakthrough and more and more demonstrated its significance and practice prospect studies on Monoclonal Antibody.The phage antibody gene pool technology rise of rising the beginning of the nineties at the end of the eighties and the development in whole genetic engineering antibody technical study field make the development research of source, people from world today or genetic engineering antibody obtain remarkable progress and step into substantive applied research and development phase (1) by the fundamental research stage.At present in the biological products that drugs approved by FDA is gone on the market or awaited the reply, various forms of monoclonal antibodies and genetic engineering antibody account for certain proportion, in 67 kinds of biological products with the approval listing, treatment and prevention have 9 kinds with monoclonal antibody and genetic engineering antibody at present.The medium antibody that awaiting the reply has 35 kinds (2).Wherein in four kinds of humanized genetic engineering antibodies of approved listing and generation tremendous economic and social benefit, a kind of antiviral gene engineered antibody that is is wherein arranged, its commodity are called " SynagisTM ", be humanization preventing respiratory combination of syndromes poison (RSV) genetic engineering antibody (3), and that the anti-RSV virus gene engineering antibody in people source that derives from phage antibody library screening has gone through to enter the II phase is clinical, and another kind of antiviral antibody anti-HBs antibody is also among examining.
Up to now, most of virus diseases do not have the specific treatment medicine, the biological products that are used for clinical treatment and some virus disease of prevention are still based on blood product, as the haematogenous gamma-globulin that uses clinically for many years, be used for viral hepatitis that hepatitis A or hepatitis B virus cause and measles etc., not only non-specific foreign protein is many in these blood products, specific antibody content is very low, and maximum problem is the cause of disease pollution problem that blood source goods potential fails to detect, and considers and should abandon from long-term interest.Therefore become a general orientation of domestic and international research with human source gene engineering product Blood substitute goods, and progressively led to success.Still the precedent of not having any approval with pure mouse resource monoclonal antibody prevention and treatment virus disease in the world, domestic have a clinical treatment of filing an application to be used for anti-encephalitis and hemorrhagic fever, mouse source antibody human is a heterologous protein with maximum disadvantage, causes easily that in human body inherited immunity repels and antibody was lost efficacy and cause immunological disease.Therefore, specificity antivirus people source neutrality antibody is one of the most promising biological products of virus disease prevention and treatment.The research of people's source antivirus genetic engineering antibody is except that successful anti-rsv antibodies, present system by phage surface, gene engineering antibody library technology and molecular biology method unite utilization, made substantial progress people's source antivirus antibody of having succeeded in developing at present of the research in this field has the antibody of anti-following virus: respiratory syncytial virus (3), HIV (human immunodeficiency virus) (HIV) (4), hepatitis B virus (5), hepatitis C virus (6), hsv (HSV) (7), Hantaan virus (8), B19 virus (9), CMV virus (10), the anti-hepatitis A virus that VZV (10) etc. and this institute that does not deliver have as yet succeeded in developing, rabies virus antibodies etc.Anti-hepatitis A antibody preparation is succeeded in developing, and will be domestic and international initiative, might obtain a kind new medicine certificate.
The objective of the invention is anti-hepatitis A virus (HAV) Fab antibody gene of acquired neutrality and the gene recombination of humanized IgG constant region, by recombinant baculovirus-insect cell expression system, in insect cell, produce at hepatitis A virus (HAV) have obviously in and the complete humanized IgG antibody of secretor type of function, the specific antibody medicine that provides feasibility in the future possible clinical antiviral prevention and treatment, and be expected to replace haematogenous in the market and plant sphaeroprotein.The human source anti-hepatitis A virus neutrality genetically engineered whole antibody of the present invention's statement comprises:
For derive from phage antibody library screening and with humanized IgG Fc dna recombinant expression after IgG1 λ whole antibody, it is made up of two heavy chains and two lambda light chains.Molecular weight is about 150kd.
2. be in insect cell, to obtain the specificity combination that efficiently expresses and discern the proteic high-affinity neutrality of hepatitis A virus (HAV) VP1 people source complete antibody by the recombinant baculovirus expression system.
Its in conjunction with hepatitis A virus (HAV) VP1 albumen and in and the function of virus infection by complementary region (the Complementarity-Dertemining Regions of decision family that is present in antibody gene light chain and variable region of heavy chain, what CDRs) the specificity nucleotide sequence determined among CDR1, CDR2 and the CDR3, its amino acid sequence corresponding has constituted the specific antigens calmodulin binding domain CaM of antibody.
4. specific light chain and heavy chain gene derive from the rich long-pending screening expression of the specificity of human source anti-hepatitis A antiviral antibody gene pool, its corresponding three CDR region sequences are the distinctive brand-new sequence of this antibody, state in number of patent application is the patent application of CN00105698.0.
5. this antibody is the reorganization genetic engineering antibody, its genomic constitution feature: form by total length IgG1 heavy chain gene and total length lambda chain light chain, its weight chain leader and heavy chain Fc gene source be in carrier pAc-L-Fc, states in number of patent application is 00105698.0 patent application.Between light chain gene leader and VL gene, contain Sac I enzyme point of contact; Between heavy chain gene leader and VH gene, contain Xho I enzyme point of contact; Between heavy chain Fd gene and Fc gene, contain Spe I enzyme point of contact.
6. this antibody can pass through the scale production in insect cell of recombinant baculovirus expression system, is secretor type antibody.Antibody protein can obtain from cells and supernatant by affinity chromatography.
7. this antibody has very high avidity to hepatitis A virus (HAV) antigen, measures about 10-11M through ELISA
8. the proteic gene of described specific antibody of encoding is a human source anti-hepatitis A virus neutrality antibody gene, can be used to any expression system and express anti-hepatitis A virus (HAV) neutrality whole antibody.
9. utilize the recombinant baculovirus kind that has this antibody gene, can cultivate in the system at existing or in the future presumable insect cell and produce and this antibody protein of purifying.
10. the purposes of human source anti-hepatitis A virus neutrality genetically engineered whole antibody, it is characterized in that: during described antibody has on the identification hepatitis A virus (HAV) VP1 albumen and antigen, thereby the function that blocking-up hepatitis A virus (HAV) poison infects, utilize in its height and functional performance that hepatitis A virus (HAV) infects and with the similar whole antibody molecular characterization of natural antibody molecule, be expected to be used to prevent and treat by hepatitis A virus (HAV) and infect the hepatitis A that causes at Blood substitute goods gamma-globulin clinically.
Traditional hybridoma cell technology of utilizing obtains quite difficulty of human monoclonal antibody, and the clone of setting up is unstable usually, and gene is easily lost.The human source anti-hepatitis A virus neutrality genetically engineered whole antibody of the present invention's statement, it is the expression that on the basis that obtains antibody gene, obtains the full-antibody gene product, this antibody gene can be along with plasmid DNA duplicating and stable duplicating in bacterium, and be recombined in the baculovirus and obtain recombinant baculovirus.Utilize this recombinant baculovirus, can in insect cell, produce anti-hepatitis A virus (HAV) whole antibody, thereby obtain a kind of clinical treatment antibody preparation of feasibility.
Following preferential embodiment elaborates to the present invention, and load does not mean that restriction content of the present invention in these embodiments, is explanation the present invention, and the monoclonal antibody gene of employing and baculovirus IgG expression vector are all from this laboratory invention.Used main bacterial strain is commercial prod XLI-Blu (U.S. Strategene company).SF9 and H5 cell are available from American I nvitrogene company.Hepatitis A virus (HAV) is from the isolating imperial first strain of China patient.Embodiment 1-3 is the clone preparation method of human source anti-hepatitis A virus neutrality genetically engineered whole antibody HAIgG; Embodiment 4 is the protein specificity of human source anti-hepatitis A virus neutrality genetically engineered whole antibody HAIgG; Example 5-8 is a human source anti-hepatitis A virus neutrality genetically engineered whole antibody functional character.
Example 1, clone's reorganization of human source anti-hepatitis A virus neutrality genetically engineered full-antibody gene: will present the anti-hepatitis A virus (HAV) neutralizing antibody Fab gene clone that system obtains by phage surface and go in the baculovirus IgG expression vector of this laboratory invention.Wherein, light chain gene is cloned among the pAc-L-Fc with Sac I and EcoRV, places under the control of p10 promotor, obtains intermediate carrier pAc-HA-L, and heavy chain Fd gene is cloned among the pAc-HA-L with Xho I and Spe I, places under the control of polyhedron promotor; It is light that acquisition has anti-hepatitis A antibody, the expression vector pAc-HA-HL of heavy chain full-antibody gene.This vector plasmid DNA is used for transfection insect cell behind column chromatography purification (QiagenMidi Kit).
Example 2, the acquisition of recombinant baculovirus: with the plasmid DNA 2ug of above-mentioned purifying and Baculo-Gold (Pharmingen) linear DNA in the baculovirus transfection reagent box with the calcium phosphate precipitation method cotransfection SF9 insect cell of introducing in the test kit, cell was cultivated 5-6 days at 27C, collect supernatant, be first-generation recombinant baculovirus seed culture of viruses.This seed culture of viruses is picked out the stable recombinant baculovirus virus plaque that goes down to posterity and express through the plaque purifying, adapts on the SF9 cell and goes down to posterity, and makes virus titer reach 10-8, and preparation seed culture of viruses storehouse is for the expression of insect cell.
Example 3, the expression of human source anti-hepatitis A virus full-antibody gene in insect cell: the recombinant baculovirus of above-mentioned 10-8 is obtained the H5 cell with the virus inoculation amount Sf9 that each T75 cm culturing bottle infects 0.5ml, and results infect supernatant after 5-6 days.Detecting the expression of IgG1 antibody in the insect cell with immunofluorescence method, as shown in Figure 1, is the fluorescence of expressing humanized IgG antibody with the insect cell (H5 cell) of the anti-human IgG fluorescent antibody staining of FITC mark.Detect excretory IgG antibody in the infection supernatant with the ELISA sandwich assay, wrap by elisa plate with anti-human IgG Fab antibody (Sigma company), after adding different dilution insect cell infection supernatant to be detected, add the HRP mark and must resist human IgG Fc antibody, the 0.D value is detected in the colour developing back, and compare in the humanized IgG standard substance ELISA examination criteria curve with concentration known, calculate the IgG content of expressing in the supernatant.As shown in Figure 2, be that ELISA detects the secretor type IgG antibody in the insect cell expression supernatant, detect two batches and expressed sample, its expression level can reach 18mg/L.
Example 4, the affinitive layer purification of human source anti-hepatitis A virus neutrality genetically engineered whole antibody: the insect cell culture supernatant behind the collection recombinate shape virus infection, 2000G is centrifugal, with low protein adsorption membrane filtration supernatant, to filter the back supernatant and cross Protein G affinity chromatographic column, the wash-out neutralization is through the desalting column desalination.Fig. 3 is the human source anti-hepatitis A virus IgG antibody of SDS-PAGE electrophoretic analysis recombinant baculovirus at the affinitive layer purification of expressed in insect cells.Be respectively the protein molecular standard substance from left to right, anti-hepatitis A virus (HAV) IgG heavy chain of antibody of the purifying under the reductive condition (50KD) and light chain (25KD) electrophoresis band, the anti-hepatitis A virus (HAV) IgG antibody of the purifying under non-reduced condition H2L2 tetramer electrophoresis band.
Example 5, with the hepatitis A virus (HAV) bag of purifying by elisa plate, add 4 strain sites close neutrality monoclonal antibody people source, the anti-VP1 mouse source recombinant antibodies of identification simultaneously and carry out ELISA competition inhibition test, result such as Fig. 4 have confirmed that antibody HAIgG16 is that a strain is at the proteic neutrality genetically engineered of hepatitis A virus (HAV) primary structure albumen VP1 whole antibody.Simultaneously, as shown in Figure 5, with the purifying human antibody HAVIgG16 that obtains with from the gamma-globulin of national biological product calibrating institute and horseradish peroxidase (HRP) the inhibition ELISA that is at war with, purifying human antibody HAVIgG16 can well compete in the anti-VP1 mouse source of HRP mark monoclonal antibody, and its competition curve is similar to 78 international unit/ml gamma-globulin.
The hepatitis A virus (HAV) bag of example 6 usefulness purifying is by elisa plate, compare simultaneously with from the gamma-globulin of calibrating institute's SGG and Shanghai Vaccine and Serum Institute, real its affinity costant can reach 10-¨ M, shown in Fig. 6 elisa assay result, reorganization hepatitis A antibody, be anti-hepatitis A virus gene engineering antibody, high dilution (12800) protein concentration of concentration: 500ug/m1. is 3ng.Calibrating third ball is a SGG, contains anti-hepatitis A antibody: 78 international unit/ml.Shanghai third ball: 10% human of being produced is ground in the Shanghai life, about 200mg/and ml.Anti-hepatitis A antibodies purifying behind injection volume: the 3ml/ adult purifying.Compare with being used for clinical gamma-globulin now, the anti-hepatitis A virus (HAV) antibody of specificity is higher than the nearly 400-500 of gamma-globulin doubly, according to the injection of gamma globulin standard, with the anti-hepatitis A virus (HAV) antibody of this purifying only need about 1.5mg/everyone.
Analyze with the function of hepatitis A virus (HAV) among 7 pairs of purifying human antibodies of example HAVIgG16.Fig. 7 be purifying human antibody HAVIgG16 on cell in and hepatitis A virus (HAV) infect elisa assay.ELISA is a sandwich assay, and the sample of detection is that hepatitis A virus (HAV) infects the lysis supernatant after 21 days.Neutralization test result shows that this strain antibody not only has very high avidity, and same according to stronger neutralising capacity is arranged.When 100% external in and the 100TCID50 hepatitis A virus (HAV) only need about 0.6ug when infecting, and in 50% and the time, only need 0.08ug antibody.The anti-hantavirus genetic engineering antibody of similarity condition purifying then can not in and hepatitis A virus (HAV) infect.Confirmed the credibility of experiment.
Claims (4)
1, human source anti-hepatitis A virus neutrality genetically engineered whole antibody is characterized in that:
(1) for derive from phage antibody library screening and with humanized IgG Fc dna recombinant expression after IgG1 λ whole antibody, it is made up of two heavy chains and two lambda light chains.Molecular weight is about 150kd.
(2) be in insect cell, to obtain the specificity combination that efficiently expresses and discern the proteic high-affinity neutrality of hepatitis A virus (HAV) VP1 people source complete antibody by the recombinant baculovirus expression system.
(3) its in conjunction with hepatitis A virus (HAV) VP1 albumen and in and the function of virus infection by the complementary region (Complementarity-DerteminingRegions of decision family that is present in antibody gene light chain and variable region of heavy chain, what CDRs) the specificity nucleotide sequence determined among CDR1, CDR2 and the CDR3, its amino acid sequence corresponding has constituted the specific antigens calmodulin binding domain CaM of antibody.
(4) specific light chain and heavy chain gene derive from the rich long-pending screening expression of the specificity of human source anti-hepatitis A antiviral antibody gene pool, its corresponding three CDR region sequences are the distinctive brand-new sequence of this antibody, state in number of patent application is 00107424.5 patent application.
(5) this antibody is the reorganization genetic engineering antibody, its genomic constitution feature: form by total length IgG1 heavy chain gene and total length lambda chain light chain, its weight chain leader and heavy chain Fc gene source be in carrier pAc-L-Fc, states in number of patent application is 00105698.0 patent application.Between light chain gene leader and VL gene, contain Sac I enzyme point of contact; Between heavy chain gene leader and VH gene, contain Xho I enzyme point of contact; Between heavy chain Fd gene and Fc gene, contain Spe I enzyme point of contact.
(6) this antibody can pass through the scale production in insect cell of recombinant baculovirus expression system, is secretor type antibody.Antibody protein can obtain from cells and supernatant by affinity chromatography.
(7) this antibody has very high avidity to hepatitis A virus (HAV) antigen, measures about 10-11M through ELISA.
2, according to claim 1, human source anti-hepatitis A virus neutrality genetically engineered whole antibody, it is characterized in that: the proteic gene of described specific antibody of encoding is a human source anti-hepatitis A virus neutrality antibody gene, can be used to any expression system and express anti-hepatitis A virus (HAV) neutrality whole antibody.
3, according to claim 1, human source anti-hepatitis A virus neutrality genetically engineered whole antibody, it is characterized in that: utilize the recombinant baculovirus kind that has this antibody gene, can cultivate in the system at existing or in the future presumable insect cell and produce and this antibody protein of purifying.
4, according to claim 1,2,3, the purposes of human source anti-hepatitis A virus neutrality genetically engineered whole antibody, it is characterized in that: during described antibody has on the identification hepatitis A virus (HAV) VP1 albumen and antigen, thereby the function that blocking-up hepatitis A virus (HAV) poison infects, utilize in its height and functional performance that hepatitis A virus (HAV) infects and with the similar whole antibody molecular characterization of natural antibody molecule, be expected to be used to prevent and treat by hepatitis A virus (HAV) and infect the hepatitis A that causes at Blood substitute goods gamma-globulin clinically.
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| CN 01110720 CN1318565A (en) | 2001-04-17 | 2001-04-17 | Humanized hapotitis A virus neutralizing genetically engineered total antibody |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923882A (en) * | 2013-08-09 | 2014-07-16 | 北京科兴生物制品有限公司 | Hepatitis A virus monoclonal antibody and its application |
| CN103923881A (en) * | 2013-08-09 | 2014-07-16 | 北京科兴生物制品有限公司 | Hepatitis A virus monoclonal antibody and its application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923882A (en) * | 2013-08-09 | 2014-07-16 | 北京科兴生物制品有限公司 | Hepatitis A virus monoclonal antibody and its application |
| CN103923881A (en) * | 2013-08-09 | 2014-07-16 | 北京科兴生物制品有限公司 | Hepatitis A virus monoclonal antibody and its application |
| CN103923881B (en) * | 2013-08-09 | 2016-04-06 | 北京科兴生物制品有限公司 | Hepatitis A virus (HAV) monoclonal antibody and application thereof |
| CN103923882B (en) * | 2013-08-09 | 2016-09-28 | 北京科兴生物制品有限公司 | Hepatitis A virus (HAV) monoclonal antibody and application thereof |
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