CN1560081A - Preparing human source monoclone antibody by mouse capable of producing human IgGl weight chain-k light chain and application thereof - Google Patents
Preparing human source monoclone antibody by mouse capable of producing human IgGl weight chain-k light chain and application thereof Download PDFInfo
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Abstract
The invention relates to using a mouse able to generate human IgGl heavy chain- k light chain to prepare humanized monoclonal antibody and its application. The characteristic: respectively knocking in huma k-chain and gl-chain constant gene at the sites of gene in mouse k- light chain constant region and heavy chain gl constant region; the humanized monoclonal antibody: the constant region is fully the same as that of human antibody and the variable region is mouse sourece, and the preparing steps: obtaining mouse k-chain and gl-chain gene groups; constructing a knoncking-in carrier; making homologous recombination in ES cell; producing the mouse with light-heave conversion; further mating to obtain a mouse with complete chimeric gene; the application: taking spleen and marrow cells of an immunized chemeric mouse to make fusion and culture; screening hybridomas of humanized monoclonal antibody generating antigenic specificity; preparing the humanized monoclonal antibody with antigenic specificity. The advantages is easy to obtain high-affinity human-mouse chimeric antibody.
Description
Technical field
The present invention relates to a kind of preparation and application of humanization mouse monoclonal antibody, belong to bioengineering field.
Background technology
Monoclonal antibody has broad application prospects gathering around aspect medical diagnosis on disease, treatment and the preventions such as tumour, viral infection, rheumatoid arthritis, cardiovascular disorder and organ transplantation, therefore becomes the most noticeable research focus in present biotech drug field.
Antibody is the protein that animal immune system produces under external antigenic stimulation, and its specificity depends on the determinant of antigen molecule.Because various antigen molecules have a lot of determinants, so the antibody that immune animal produces is actually the mixture of multiple antibody.It is infeasible will preparing antibody by this method, and reason is that the content of antibody is few, separation is also very difficult, and animal's antibody injection people knows from experience the generation severe anaphylactic reaction.By the hybridoma that mouse bone-marrow-derived lymphocyte and myeloma cell's hybridization obtain, can only discern a kind of specific antigen at the antibody of external mass production single component, be called monoclonal antibody.
Monoclonal antibody technique is reined in (Kohler) development for the first time in 1975 by Britain's biochemist Millstein (Milstein) and German immunologist section.Monoclonal antibody medicine comprises monoclonal antibody goods and monoclonal antibody conjugate, because monoclonal antibody has the specificity of height to respective target molecule (antigen), so monoclonal antibody medicine has been used for the immunologic rejection reaction of tumour, viral infection, rheumatoid arthritis, cardiovascular disorder and organ transplantation.Monoclonal antibody medicine in the research and development is mostly at tumour, and reason is:
The subject matter that monoclonal antibody medicine exists comprises immunology and two aspects of pharmacology.The immunology aspect, the monoclonal antibody medicine that is used for clinical study for many years uses the mouse monoclonal antibody mostly, often causes the immune response of human body; Pharmacology aspect, subject matter are the dose deficiencies that arrives tumour, viral infection position or other diseased region.The monoclonal antibody medicine process of transporting in vivo is subjected to multiple factor affecting, because it is a foreign protein, can be absorbed by reticuloendothelial system, there is respective numbers will accumulate in liver, spleen and marrow, simultaneously monoclonal antibody medicine is a macromolecular substance, by the capillary endothelium layer and penetrate the tumour cell external series gap and all be restricted.The main path that addresses the above problem is to reduce the monoclonal antibody medicine immunogenicity, makes mouse originality monoclonal antibody humanization or develops human body completely.
Some monoclonal antibody products embedded or that personalize are got permission to produce abroad at present.The monoclonal antibody humanization mainly prepares chimeric antibody or reshaping antibody by genetic engineering technique.The development of transgenic animal technology changes into all monoclonal antibody human sources and is possible, and can reduce immunological rejection greatly.
U.S. Abgenix and Medarex company have obtained the transgenosis strain of mouse at present.The former has developed two products, and one is to be used for the medicine of curing psoriasis just in clinical study, and another medicine that is used for the treatment of cancer still is in the clinical preceding development phase.First Humanized monoclonal antibodies that is used for the treatment of rheumatoid arthritis of Medarex has entered the first phase clinical study.
Since the mid-80 in last century, the humanization of mouse source antibody is the striving direction that addresses these problems just always, and up to the present, the humanization scheme of having reported mainly contains:
(1) people-mouse chimeric antibody: chimeric antibody is the scheme that mouse source antibody humanization takes the earliest, also is quite successful scheme.Its method is: the light chain of clonal antibody and heavy chain variable region gene, insert people's constant region of light chain (generally all using the k chain) and CH by the frame requirement respectively and (can select various CH, but from effector function certain g chain commonly used) the upstream, express getting final product again.The advantage of chimeric antibody is that technology is fairly simple, has had the supporting chimeric antibody of multiple and variable region cloning process (mainly being frame) to make carrier now, obtain the variable region gene of antibody with RT-PCR after, can give expression to chimeric antibody soon.In addition, make two purposes that chimeric antibody can reach the antibody humanization substantially: obtain effective effector function; Reduce significantly or basically eliminate HAMA reaction (appearance of rejection is lower than 5%).Yet from absolutely being eliminated, this is not ascribed to because the variable region of chimeric antibody remains mouse source property rejection.
(2) humanization of variable region: as mentioned above, chimeric antibody does not solve the rejection of patient's antagonist fully, and the variable region that a reason is ascribed to antibody is that mouse source property is.It is humanization modified that this viewpoint impels people that the variable region is proceeded.This transformation mainly contains two kinds of approach, and the one, make up reconstruct type (re-shape) antibody, promptly the framework region of the variable region of mouse endogenous antibody is changed the adult source, and be responsible for and antigen bonded hypervariable region still keeps mouse with engineered method.This is a kind of antibody modification scheme based on sequence, so usually can cause the reduction of affinity of antibody.Another kind of approach is based on antigen avidity, i.e. strand displacement (chain-shift) scheme.By successively changing light chain and heavy chain, under the guiding of avidity, from the phage antibody library of people source, obtain at antigenic be the antibody variable gene in people source fully.Add people's constant region gene again by genetically engineered, can give expression to the antigen-specific human antibody.In addition, also can directly in the phage antibody library of people source, screen the variable region gene that obtains humanized antibody with antigen.These variable region humanization schemes all can be the antibody gene in people source fully, and its shortcoming is just can give expression to antibody protein with practical value through more genetically engineered operation.
(3) most typical technology is Tomizuka K.et al.Double trans-chromosomic mice:Maintenanceof two individual human chromosome fragments containing Ig heavy and k loci andexpression of fully human antibodies.PNAS.97:722-727, and the genetic background of passing through the transformation mouse of 2000. reports makes mouse carry out immunne response with human antibody.Its brief introduction is as follows: this scheme in fact has not been that mouse source antibody is carried out humanization, but has directly obtained the method for human antibody.Its technological core is based upon in recent years mouse is carried out on the genetics method of operating, i.e. mouse gene knockout and transgenic mice are technical.At first remove JH gene and the Ck gene of mouse respectively, thereby light chain immunoglobulin k of deactivation mouse and heavy chain H gene locus make it to express self IgH and Igk with the method for gene knockout.By cultivating mouse, make two kinds of sudden changes enter same mouse again.This mouse can not produce bone-marrow-derived lymphocyte owing to there is antigen receptor to express.Simultaneously, by the transgenic mice technology with including human normal immunoglobulin k chain gene (Vk-Jk-Ck, about 1.5Mb) and human immunoglobulin heavy chain (VH-D-JH-CH, about 1.5Mb) yeast artificial chromosome dna is made transgenic mice (also can pass through the ES cell transfecting, the approach that gomphosis mouse is made in microinjection carries out).Once more by the mouse mating, can obtain mouse immuning ball protein k gene and heavy chain gene and be inactivated and have people's the immunoglobulin (Ig) k and the mouse of heavy chain gene.This mouse has normal B cell development.After being subjected to antigenic stimulation, can carry out normal antibody response, but the heavy chain of the immunoglobulin (Ig) that is produced is all for the people source, 95% of light chain is that (remaining 5% is 1 chain of mouse for people's k chain, if with the deactivation of gene knockout method, then all light chain of antibody all are people's k chain with 1 chain of mouse).Behind the antigen immune mouse, but obtain at antigenic specificity Humanized monoclonal antibodies with hybridoma technology during marrow.
This method can thoroughly solve having problems of humanized antibody theoretically.But it mainly has problems is that 1. technical difficulty are big; 2. not having problems technically though reject the immunoglobulin gene of mouse, intactly change people's immunoglobulin gene not a duck soup over to---the processing ease that reaches the dna molecular of 1.5Mb brings structural variation; 3. have or not reorganization, the influence of integration site after entering mouse cell to expressing; 4. whether the whole gene rearrangement pattern in reorganization back is normal, and this will influence amount and the antibody and the antigenic activity that combines of transgenic mouse expressing human antibody.These problems are all most important to successfully setting up this class mouse species.
Yet even humanized antibody completely, its variable region still has immunogenicity for the patient who accepts Antybody therapy.As mentioned above, antibody variable gene produces through gene rearrangement and sudden change in the somatocyte level, all be undoubtedly antigenicity at the new proteic aminoacid sequence that produces to body this every day stimulates, so in clinical use, also can produce anti-antibody at human antibody.There is bigger diversity in the skeleton district of antibody variable region in planting, and between planting higher homology is arranged, and between people and mouse, the conservative property of some antibody variable region sequence between planting will be higher than the sequence homology between the family of different variable region in planting.So if human body can tolerate for the immunogenicity of the antibody variable region of self, the immunogenicity to the antibody variable region of mouse also can have certain tolerance at least so.And the use of remaining immunogenicity and human antibody is the same, can be got rid of by the change of route of administration, scheme etc.The gone through clinical use or enter clinical trial of numerous people-mouse chimeric antibodies illustrates that at least at some antigenic antibody, the immunogenicity that bring the variable region is can be uncared-for.
By to the brief introduction of existing technology as can be known, at present, people also have the following disadvantages in the preparation of the humanized antibody that treatment obtained that is used for various diseases: will just can give expression to antibody protein with practical value through more genetically engineered operation 1.; 2. technical difficulty is big; 3. when developing transgenic mouse, the processing ease that reaches the dna molecular of 1.5Mb brings structural variation; 4. have or not whether reorganization, integration site are normal to the whole gene rearrangement pattern of expressing of influence and reorganization back after entering mouse cell, its technology acuracy requires very high.
Summary of the invention
The objective of the invention is to overcome that prior art exists: 1. will just can give expression to antibody protein with practical value through more genetically engineered operation; 2. technical difficulty is big; 3. the full genetic manipulation that reaches the dna molecular of 1.5Mb brings structural variation easily; 4. have or not reorganization, integration site to the influence of expressing and whether normally these deficiencies of the whole gene rearrangement pattern in back of recombinating after being difficult to clearly enter mouse cell; And provide mouse that a kind of usefulness can produce human IgG1's heavy chain-k light chain as preparation monoclonal antibody and application, special technical solution of the present invention proposed.
Basic ideas of the present invention are: the clone's mode that does not adopt full gene of Ig and the full gene of k light chain; But on the Ig of mouse light chain k constant region gene and Ig heavy chain g1 constant region gene locus, knock in people's Igk constant region and IgG1 constant region gene respectively; Resulting mouse directly produces people-mouse chimeric antibody when antigen is carried out antibody response; On this basis, set up from the chimeric antibody mouse that obtains and pass through cytogamy, obtain secreting the technological line of the hybridoma of chimeric antibody.
Usefulness proposed by the invention can produce human IgG1's heavy chain-k light chain mouse as the preparation Humanized monoclonal antibodies, comprise: gene clone, carrier construction, transformant, transformed mouse, screening expression, immune mouse, cytogamy, Antibody Preparation, it is characterized in that: the mouse of being adopted is on the Ig of mouse light chain k constant region gene and Ig heavy chain g1 constant region gene locus, knock in people's Igk constant region and IgG1 constant region gene respectively, and cultivate the mouse that produces human IgG1's heavy chain-k light chain; Prepared Humanized monoclonal antibodies is that the constant region of its antibody and people's antibody are identical, then are the people-mouse chimeric antibodies identical with mouse with antigen bonded variable region; The chimeric antibody mouse with human IgG1's heavy chain-k light chain is expressed in screening; By antigen immune, cytogamy, acquisition can be secreted the hybridoma cell line of humanization resistant chimeric monoclonal antibody; The chimeric antibody preparation process is:
The first step, Ig κ chain and the IgG1 chain gene group gene of clone mouse from the genome dna library of 129sv mouse;
In second step, gene is knocked in the structure of carrier
The light chain carrier---complete people Ig kappa gene fragment is inserted the Ig kappa gene site of mouse, make that a bit of coding region of mouse kappa gene 5 ' end is lacked, when inserting people's kappa gene, to guarantee deactivation mouse kappa gene, positive screening-gene neo (neomycinphosphotransferase) ceneme is inserted in people's kappa gene downstream, insert negative screening-gene diphtheria toxin A unit (DTA) ceneme in 3 ' homologous region downstream, the size of 5 ' and 3 ' homologous region is respectively 6kb and 8kb;
The heavy chain carrier---insert the film exon of mouse IgM in the downstream of people's secretor type IgG1 gene, insert positive screening-gene ceneme again in the downstream, this fragment that makes up is inserted the IgM gene coding region of mouse near 5 ' end place, the IgM gene of complete inactivation mouse when making this gene fragment of insertion, negative screening sign is inserted in the homologous region downstream, and the size of 5 ' and 3 ' homologous region is respectively 5kb and 7kb;
In the 3rd step, in the ES cell, carry out homologous recombination
Cultivating mouse ES cells is E14, respectively the gene that makes up is knocked in carrier with electroporation and is transfected in the ES cell, cultivates and screens in the presence of G418;
The 4th step, the generation of gomphosis mouse
Mating C57BL/6 mouse, collect blastaea from the Mouse Uterus of gestation at post-coitum in the time of 4.5 days, with the ES cell of homologous recombination by microinjection, be injected in the segmentation cavity of C57BL/6 mouse, blastaea is transplanted in the uterus of ICR mouse of false pregnancy, the mouse of birth is judged chimeric degree according to the color and the hair color of eye again;
In the 5th step, with light chain mouse and the further mating of heavy chain mouse, obtaining on light chain and the heavy chain gene site all is the mouse of the gene knocked in;
The 6th step, the preparation of Humanized monoclonal antibodies.
Use usefulness of the present invention can produce of the application of human IgG1's heavy chain-κ light chain mouse as the preparation Humanized monoclonal antibodies, it is characterized in that: prepared monoclonal antibody is at tumour antigen or virus antigen, or bacterial antigens, or people CD and cytokine antigen have specific antibody; The step of its application method is:
The first step adopts the immunity of tumour antigen or virus antigen or bacteriotoxin antigen or people's T cell differentiation antigen to produce the mouse of human IgG1's heavy chain-κ light chain strain;
Second step, to get immunized mice spleen and myeloma cell and merge, cloning is cultivated;
In the 3rd step, screening produces the hybridoma cell line of tumor-resistant antigen or antiviral antigen or antibacterium toxin antigen or the specific Humanized monoclonal antibodies of anti-people's T cell differentiation antigen;
Screen by enzyme-linked immunosorbent assay, immunofluorescence experiment, immunohistochemical experiment etc.;
In the 4th step, preparation produces tumor-resistant antigen or antiviral antigen or anti-thin toxin bacterium antigen or the specific Humanized monoclonal antibodies of anti-people's T cell differentiation antigen, is that legal system is equipped with ascites or cell culture method prepares Humanized monoclonal antibodies routinely;
The present invention gets major advantage: 1. operated dna molecular is little, its big or small less than hundred K, and simple to operate, technical difficulty is little; 2. because operated dna molecular is little, reorganization accuracy height seldom causes structural changes and influences antibody expression; 3. knock in the constant region of people Ig gene, and do not comprise the variable region of people Ig gene, so gene rearrangement pattern of the variable region of Ig gene when not influencing antibody expression, easier acquisition high-affinity antibody people-mouse chimeric antibody, can in the genetic stability that keeps mouse, make antibody obtain humanization like this.4. prepared humanized antibody has specificity at tumour antigen or virus antigen or bacteriotoxin antigen or people's T cell differentiation antigen.
Embodiment
Embodiment 1:
Supreme Being's grace biotechnology engineering corporation utilizes genetic engineering technique and transgenic technology, and foundation can produce the mouse of human IgG1's heavy chain-κ light chain as development monoclonal antibody platform, and its step is as follows:
The first step, Ig κ chain and the IgG1 chain gene group gene of clone mouse from the genome dna library of 129sv mouse.
In second step, gene is knocked in the structure of carrier
1. the light chain carrier---with mouse k chain gene group gene fragment is homologous region, complete people Igk gene fragment is inserted the Igk gene locus of mouse, make that a bit of coding region of mouse k gene 5 ' end is lacked, when inserting people k gene, to guarantee deactivation mouse k gene, positive screening-gene neo (neomycinphosphotransferase) ceneme is inserted in people k gene downstream, insert negative screening-gene diphtheria toxin A unit (DTA) ceneme in 3 ' homologous region downstream, the size of 5 ' and 3 ' homologous region is respectively 6kb and 8kb, makes homologous recombination to take place with satisfied efficient; 2. heavy chain carrier---insert the film exon of mouse Igm in the downstream of people's secretor type Igg1 gene, insert positive screening-gene ceneme again in the downstream, this fragment that makes up is inserted the Igm gene coding region of mouse near 5 ' end place, the Igm gene of complete inactivation mouse when making this gene fragment of insertion, negative screening sign is inserted in 3 ' homologous region downstream, and 5 ' and 3 ' homologous region size is respectively 5kb and 7kb;
In the 3rd step, in the ES cell, carry out homologous recombination
Cultivating mouse ES cells is E14, respectively the gene that makes up being knocked in carrier with electroporation is transfected in the ES cell, cultivate and screen in the presence of G418, after the about week, the cell clone picking that will survive under the G418 screening comes out, a part is frozen after the amplification cultivation, a part is used for extracting genomic dna, with carrying out Southern hybridization after the restriction enzyme digestion, after the clonal expansion of homologous recombination takes place, determine its genotype with Southern hybridization once more, frozen then;
The 4th step, the generation of gomphosis mouse
Mating C57BL/6 mouse, collect blastaea from the Mouse Uterus of gestation at post-coitum in the time of 4.5 days, after Southern hybridization proof being had the ES cell clone amplification of homologous recombination generation, by microinjection, be injected in the segmentation cavity of C57BL/6 mouse, blastaea is transplanted in the uterus of ICR mouse of false pregnancy, the mouse of birth is judged chimeric degree according to the color and the hair color of eye again;
The 5th step, the mouse mating
Get high mouse of chimeric degree and the mating of C57BL/6 mouse, in the mouse of birth, get filemot mouse, get tail extraction genomic dna and carry out Southern hybridization, the kind system that determines the gene knocked in transmits (germlinetransmission), with light chain mouse and the further mating of heavy chain mouse, obtaining on light chain and the heavy chain gene site all is the mouse of the gene knocked in;
In the 6th step, prepare Humanized monoclonal antibodies with ordinary method;
The 7th step, the evaluation that produces the mouse species of human IgG1's heavy chain-κ light chain
Purpose: identify whether the mouse species that produces human IgG1's heavy chain-κ light chain sets up success.
Gene is knocked in the observation of the B cytodifferentiation growth of mouse:
(1) gets bone marrow cells in mice, analyze the differentiation state of pro-B, pre-B and immature B cells with flow cytometer (FACS).
(2) histological structure of analysis mouse spleen compares with wild-type mice.Separating spleen B cell with facs analysis B cell surface marker, compares with wild-type.Stimulate mouse spleen B cell with anti-human normal immunoglobulin, observe its propagation and apoptotic response.
(3) enzyme-linked immunosorbent assay (ELISA) level and the classification of analyzing mice serum Ig.
Detected result shows that gene is knocked in the B cytodifferentiation of mouse and grown and the wild-type mice no significant difference.
Hepatitis B surface antigen(HBsAg) (HBSAg) protein immunization chimeric antibody mouse, the titre of observing anti-HBSAg antibody in the serum with ELISA changes and the classification variation.The gene titre of knocking in HBSAg antibody in mouse and the wild-type mice serum all can reach 10 as a result
-7, the equal IgG1 of antibody classification is main.Wherein gene knock in the IgG1 antibody-like that mouse produces constant region through identifying, the constant region of human IgG1 antibody-like is identical.It is successful fully to show that gene is knocked in mouse.
Embodiment 2:
Supreme Being's grace biotechnology engineering corporation uses the mouse that can produce human IgG1's heavy chain-κ light chain as the application for preparing anti-tumor monoclonal antibody.
When cell takes place to transform, often be accompanied by the change of surface of cell membrane glycolipid or glycoprotein candy chain.(NeuAc α 2 → 6GalNAc α 1 → O-Ser/Thr) is a kind of disaccharides structure to sTn, can be expressed in various adenocarcinoma tissue, then seldom or not expresses in healthy tissues.The monoclonal antibody mAb of the anti-carbohydrate antigen sTn of immunity preparation, purpose is to check the feasibility of mouse species aspect the preparation anti-tumor monoclonal antibody that produces human IgG1's heavy chain-κ light chain, and carry out targeted therapy with it as targeting antibodies for the method that set up to detect sTn reagent is provided, its concrete steps are as follows:
The first step, the antigen immune mouse and the associated materials that are adopted
Antigen-immunized animal is for producing the mouse species of human IgG1's heavy chain-κ light chain, mouse myeloma cell line X63Ag8.653.14 routine colorectum cancerous tissues, 10 routine breast cancer tissues are provided by the clinical disease natural sciences.All samples after the section, carry out immunohistochemical staining all through 100mL/L formalin fixed, dehydration, routine paraffin wax embedding.Anti-sTn standard antibody (anti-TAG72) is available from U.S. Neomarker company;
In second step, get that immunized mice spleen and myeloma cell are merged, cloning, cultivation
After being the immunogen immune transgenic mouse with the tumour antigen, fusion, the cloning of getting its splenocyte and X63Ag8.653 murine myeloma cell, enlarged culturing and preparation ascites are all according to a conventional method;
In the 3rd step, screening produces the hybridoma cell line of the humanized monoclonal antibody of tumor-resistant antigen specific antigens
After repeated screening, 3 time cloningizations, obtain anti-carbohydrate antigen sTn hybridoma 3 strains, difference called after 2B10,3F5 and 3H2;
The 4th step, method routinely, preparation produces the antigenic Humanized monoclonal antibodies of antineoplastic specificity;
The 5th step is to the evaluation and the analysis of mAb characteristic
1. titration: indirect elisa method is adopted in the titration of mAb in Hybridoma Cell Culture supernatant and the ascites that induces thereof; 2. the IgG classification is identified: adopt the experiment of agar bidirectional diffusion; 3. epitope analysis: adopt the ascites mAb mark HRP of ammonium sulfate precipitation method and DEAE52 purification by chromatography, do indirect ELISA, detect anti-sTn standard antibody (anti-TAG72) and prepared mAb encapsulation situations to enzymic-labelled antibody; Titration: the ascites of 3 strain mAb is tired and is respectively 10
-6, 10
-5, 10
-6Tiring of cells and supernatant is respectively 1: 128,1: 64 and 1: 128.The IgG classification is identified: 3 strain mAb are IgG1, the κ light chain.
The immunohistochemical staining of tumor tissues is anti-as one with mAb3H2, and 10 routine breast cancer tissues and 14 routine colorectum cancerous tissues have been carried out immunohistochemical staining, and positive person is respectively 5 example and 12 examples.Dyeing part is positioned at the endochylema and the after birth of tumour cell.As seen the normal mucous membrane of colon of part has the slight dyeing that is confined in the glandular tube.The The positive expression rate of sTn is 50% (5/10) in the breast cancer tissue, is 86% (12/14) in the colorectum cancerous tissue.
The detection that sTn expresses in the tumor tissues: adopt immunohistochemistry staining method.The paraffin section routine dewaxes to water, and PBS shakes and washes, and adds 30mL/LH2O2 effect 20min, the blocking-up endogenous peroxydase.After with 100mL/L sheep blood serum sealing 30min, add mAb (culture supernatant), with anti-TAG72 antibody as positive control, PBS as negative control, 4 ℃ of overnight incubation, PBS shakes and washes, and adds the sheep anti-mouse igg (room temperature 40min) of HRP mark, the same shake wash after, dye dehydration, transparent, mounting, om observation with DAB colour developing, phenodin lining.Tumour cell dyeing＞5% is judged to the positive.
The anti-sTnmAb4F10 of the present invention's preparation has stronger tumour-specific and higher to the recall rate of sTn in the tumor tissues, is expected to try out in clinical in-vivo diagnostic and treatment; Also available its purifying corresponding antigen, preparation tumor vaccine, and the clone of screening and foundation expression sTn, sTn is at tumour generation, developing biological significance etc. in research.
Embodiment 3:
Supreme Being's grace biotechnology engineering corporation utilizes the application of mouse species in preparation anti-hepatitis B virus monoclonal antibody that produces human IgG1's heavy chain-κ light chain.
Hepatitis B virus is to endanger very serious a kind of transmissible disease at present, and the antibody of resistance of hepatitis B surface antigen has provide protection to the infection of human body opposing hepatitis B virus.The monoclonal antibody that the present invention develops people source resistance of hepatitis B surface antigen will be applied to human body therapy the basis is provided for preparing antiviral Humanized monoclonal antibodies with the mouse species that produces human IgG1's heavy chain-κ light chain.Its method steps is as follows:
The first step, employing can produce human IgG1's heavy chain-κ light chain antigen immune mouse
Get hepatitis B surface antigen(HBsAg) (HBSAg) protein immunization chimeric antibody mouse, the titre of observing anti-HBSAg antibody in the serum with EIISA changes and the classification variation, and the gene titre of knocking in HBSAg antibody in mouse and the wild-type mice serum all can reach 10 as a result
-5, the equal IgG1 of antibody classification is main;
Second goes on foot, and gets the splenocyte of immune mouse, with mouse myeloma cell line X63Ag8.653 fusion, cloning, cultivation;
In the 3rd step, screening produces the Humanized monoclonal antibodies hybridoma cell line of anti-hepatitis B virus specific antigens;
The 4th step, according to a conventional method, the Humanized monoclonal antibodies of preparation anti-hepatitis B virus specific antigens;
In the 5th step, identify and interpretation of result
Select the high cell of antibody titers to carry out cloning, further the monoclonal antibody that is produced is identified.The anti-HBSAg monoclonal antibody of the 26 strains qualification result that is obtained is shown have the heavy chain of 18 strain monoclonal antibodies to belong to the human IgG1, and 17 strain light chains in this 18 strain monoclonal antibody are people k chains.This result provides the foundation for further carrying out clinical trial.The fused cell that screening can be survived in the presence of G418 detects the ability that it secretes people-mouse inosculating antibody HBSAg antibody with ELISA.Fusion rate reaches 70% as a result, and the positive rate that produces anti-HBSAg antibody is 28%.The result shows that it is successful utilizing the application of mouse species in the antiviral monoclonal antibody of preparation that produces human IgG1's heavy chain-κ light chain.
Embodiment 4:
Supreme Being's grace biotechnology engineering corporation utilizes the application of mouse species in the anti-people's T cell differentiation antigen monoclonal antibody of preparation that produces human IgG1's heavy chain-κ light chain.
Anti-people's T cell differentiation antigen, monoclonal antibody have resisting transplant rejection clinically, suppress application widely such as hemopathy develops, the development of control autoimmune disorder.
The human leukocyte factor 5 (hIL5) Humanized monoclonal antibodies preparation process is as follows:
The first step, the antigen immune of employing produce into the mouse of IgG1 heavy chain-κ light chain strain and associated materials employing can produce human IgG1's heavy chain-κ light chain antigen immune mouse,
Antigen adopts: hIL5 (the human lymphocyte factor 5 antigens)
Second step, get that immune mouse spleen cell and medullary cell merge, add peritoneal immunity in the preparation employing spleen of cloning, cultivation mAb, merge according to a conventional method, screening and cloning;
The 3rd step, screening produces the selection of the specific Humanized monoclonal antibodies hybridoma cell line of anti-people's T cell differentiation antigen TF1 cell density and collects the TF1 cell that is in logarithmic phase, after washing 3 times, adjusting cell count with the RPMI-1640 that contains 10%FCS is 1.6 * 1010/L, behind doubling dilution, be added on 96 well culture plates, every hole 100 μ L, each extent of dilution establish 3 multiple holes; Every hole adds 10 μ g/LhIL, 5 standard substance again.After cultivating 60h in the 37 CO2 incubators, every hole adds 20 μ lMTT liquid and puts in the CO2 incubator and cultivate 4h～6h; Every hole adds 100 μ l lysates spends the night, and goes up the absorbance value of measuring A570nm in microplate reader (BIO-RAD), selects the optimal cell stand density;
In the 4th step, prepare the specific Humanized monoclonal antibodies of anti-people's T cell differentiation antigen according to a conventional method;
In the 5th step, identify and interpretation of result
The selection of hIL5 concentration is added on 96 well culture plates with the dilution hIL5 standard substance of difference, and each extent of dilution is established 3 multiple holes, and every again hole adds 100 μ l2 * 10
9The TF1 cell suspension of/L cell density the samely carries out the MTT colorimetric estimation, selects the suitableeest working concentration of hIL5.
MAb reaches the suitableeest hIL5 working fluid to the restraining effect of TF1 cell proliferation with the suitableeest TF1 cell suspension, is added on 96 well culture plates, and every hole adds the different anti-hIL5mAb ascites of dilution 7 strains (every hole 20 μ l) again; Establish the contrast of hIL5 and substratum simultaneously, carry out the MTT colorimetric estimation, and calculate the inhibiting rate of mAb TF1 cell proliferation with method.
The preparation of mAb is an immunogen with 19 peptides of synthetic, carries out in the spleen and the abdominal cavity fundamental immunity, through fusion, screening and cloning, obtains the hybridoma (3D6,2H10,3E12, and 3D4) of the anti-hIL5mAb of 5 strains.7 strain of hybridoma cultured continuously are surplus February, and the characteristic of secretion mAb is constant, and can induce out mAb ascites in the mouse body.
Anti-hIL5mAb has the blocking-up inhibiting rate of 3 strains to surpass 50% to the restraining effect of TF1 cell proliferation among the 5 strain mAb, be respectively 81.3%, 67.8% and 57.1%
IL5 is mainly produced by the T lymphocyte.We prepare the mAb of anti-hIL5.Obtain 7 strains through cytogamy hIL 5 is had in conjunction with active mAb, wherein have 5 strain mAb to have the activity that suppresses TF1 cell proliferation, show that these mAb have specificity preferably, lay a good foundation for further using.
Embodiment 5:
Supreme Being's grace biotechnology engineering corporation utilizes the application of mouse species in preparation bacteriotoxin monoclonal antibody that produces human IgG1's heavy chain-κ light chain.
Alpha toxin is the main lethal factor of severe trauma gas gangrene due to the A type clostridium perfringens, does not still have the effectively preventing method so far, uses microbiotic and multivalence toxinicide DeGrain.For this reason, the present invention adopts cell-fusion techniques, has prepared the monoclonal antibody (mAb) of anti-alpha toxin, and poisoning for treatment gas gangrene patient's alpha toxin provides possibility.
Concrete steps are:
The first step, employing can produce human IgG1's heavy chain-κ light chain antigen immune mouse
Alpha toxin antigen is the expression product of recombinant bacterial strain BL21, extracts;
In second step, spleen and the medullary cell of getting this mouse merge cloning, cultivation
The preparation animal immune of anti-alpha toxin mAb, cytogamy screening, cloning and ascites preparation are ordinary method;
The 3rd step, the hybridoma cell line of the Humanized monoclonal antibodies of screening antibacterium toxin antigen-specific;
The 4th step, according to a conventional method, the Humanized monoclonal antibodies of preparation antibacterium toxin antigen specific antigen;
In the 5th step, identify and interpretation of result
1, the preparation immune mouse spleen cell of anti-alpha toxin mAb and myeloma cell's fusion rate is 63.19%, and antibody positive rate is 30.63%.After cloning, obtain 3 strain positive colony cell strains altogether, difference called after 1A8,2C3 and 3D2;
2, the culture supernatant and the ascites of titration 3 strain of hybridoma of mAb detect with indirect ELISA, and tiring is respectively 1: 512～1024 and 10
6～10
8
3, the Ig subgroup identification of mAb identifies that with two expansion methods the Ig subclass of mAb is IgG1, and the light chain of 3 strains is the κ type;
4, mAb in and determination of activity ascites is mixed with the alpha toxin of equivalent, behind 37 effect 1h, be inoculated in respectively on the dull and stereotyped and blood agar flat board of egg yolk agar, observe the phenomenon that has or not decomposition Yelkin TTS and zone of hemolysis; The protection test of mAb get 8 age in week the Balb/c mouse, be divided into 4 groups at random, 5 every group, respectively the abdominal cavity injects the alpha toxin of 1 LD100.At 0h, 4h and 8h inject the mAb1A8 of anti-alpha toxin through the abdominal cavity then, and 1C3 or 1F1 establish the physiological saline control group simultaneously, and continuous 1 week is observed the death condition of respectively organizing mouse.As a result, 1A8mAb in vivo, external all to have a protection active, and the endogenous protective rate reaches more than 80%.
Alpha toxin is the topmost virulence factor of A type clostridium perfringens, can cause human wound's gas gangrene.For exploring effective methods of treatment, we develop the mAb of anti-alpha toxin on the basis of the successful alpha toxin protective antigen gene of clone.Wherein mAb1A8 has the provide protection that mouse is attacked in opposing lethality alpha toxin abdominal cavity.Can be to try out clinically new approach is provided in the poisoning of treatment alpha toxin.
Claims (2)
1, with producing human IgG1's heavy chain-κ light chain mouse as the preparation Humanized monoclonal antibodies, comprising: gene clone, vector construction, transformant, transformed mouse, expression screening, mouse immune, cytogamy, Antibody Preparation is characterized in that:
A) mouse of being adopted is on the Ig of mouse light chain κ constant region gene and Ig heavy chain g1 constant region gene locus, knocks in people's Ig κ constant region and IgG1 constant region gene respectively, and cultivates the mouse that produces human IgG1's heavy chain-κ light chain;
B) prepared Humanized monoclonal antibodies is that the constant region of its antibody and people's antibody are identical, then are the people-mouse chimeric antibodies identical with mouse antibodies with antigen bonded variable region;
C) the chimeric antibody mouse with human IgG1's heavy chain-κ light chain is expressed in screening;
D) obtain to secrete the hybridoma cell line of humanization resistant chimeric monoclonal antibody by antigen immune, cytogamy;
E) preparation process of chimeric antibody is:
The first step, Ig κ chain and the IgG1 chain gene group gene of clone mouse from the genome dna library of 129sv mouse;
In second step, gene is knocked in the structure of carrier
The light chain carrier---complete people Ig kappa gene fragment is inserted the Ig kappa gene site of mouse, make that a bit of coding region of mouse kappa gene 5 ' end is lacked, when inserting people's kappa gene, to guarantee deactivation mouse kappa gene, positive screening-gene neo (neomycinphosphotransferase) ceneme is inserted in people's kappa gene downstream, insert negative screening-gene diphtheria toxin A unit (DTA) ceneme in 3 ' homologous region downstream, the size of 5 ' and 3 ' homologous region is respectively 6kb and 8kb;
The heavy chain carrier---insert the film exon of mouse IgM in the downstream of people's secretor type IgG1 gene, insert positive screening-gene ceneme again in the downstream, this fragment that makes up is inserted the IgM gene coding region of mouse near 5 ' end place, the IgM gene of complete inactivation mouse when making this gene fragment of insertion, negative screening sign is inserted in the homologous region downstream, and the size of 5 ' and 3 ' homologous region is respectively 5kb and 7bk;
In the 3rd step, in the ES cell, carry out homologous recombination
Cultivating mouse ES cells is E14, respectively the gene that makes up is knocked in carrier with electroporation and is transfected in the ES cell, cultivates and screens in the presence of G418;
The 4th step, the generation of gomphosis mouse
Mating C57BL/6 mouse, collect blastaea from the Mouse Uterus of gestation at post-coitum in the time of 4.5 days, with the ES cell of homologous recombination by microinjection, be injected in the segmentation cavity of C57BL/6 mouse, blastaea is transplanted in the uterus of ICR mouse of false pregnancy, the mouse of birth is judged chimeric degree according to the color and the hair color of eye again;
In the 5th step, with light chain mouse and the further mating of heavy chain mouse, obtaining on light chain and the heavy chain gene site all is the mouse of the gene knocked in;
The 6th step, the preparation of Humanized monoclonal antibodies.
2, use the described usefulness of claim 1 can produce of the application of human IgG1's heavy chain-κ light chain mouse, it is characterized in that as the preparation Humanized monoclonal antibodies:
A) prepared Humanized monoclonal antibodies is to have specific antibody at tumour antigen or virus antigen or bacteriotoxin antigen or people's T cell differentiation antigen;
B) its application method step is:
The first step adopts tumour antigen or virus antigen or bacteriotoxin antigen or the immunity of people's T cell differentiation antigen can produce the mouse of human IgG1's heavy chain-κ light chain;
In second step, get immunized mice spleen and myeloma cell and merge, cloning, cultivation;
In the 3rd step, screening produces the hybridoma cell line of tumor-resistant antigen or antiviral antigen or antibacterium toxin antigen or the specific Humanized monoclonal antibodies of anti-people's T cell differentiation antigen;
In the 4th step, adopt conventional method to prepare ascites or cell culture method preparation generation tumor-resistant antigen or antiviral antigen or antibacterium toxin antigen or the specific Humanized monoclonal antibodies of anti-people's T cell differentiation antigen.
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