CN101070346A - Method for preparing double-function antibody - Google Patents
Method for preparing double-function antibody Download PDFInfo
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- CN101070346A CN101070346A CN 200610050611 CN200610050611A CN101070346A CN 101070346 A CN101070346 A CN 101070346A CN 200610050611 CN200610050611 CN 200610050611 CN 200610050611 A CN200610050611 A CN 200610050611A CN 101070346 A CN101070346 A CN 101070346A
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Abstract
This invention relates to a method of preparing bifunctional antibody. Firstly small molecule medicine and etiological agent or tumor specific antigen proceed chemical couple, execute small molecule medicine idiosyncratic proteinaceous conjugate ; take splenocyte from immune animal and proceed fusion with corresponding myeloma cell, to preapare monoclonal antibody, then through ELISA to screen out monoclonal antibody that resist small molecule medicine, and select out monoclonal antibody that has good respound to specificity carrier protein from positive hybridoma. The bifunctional antibody, small molecule medicine and special pathogenic antigen all incorporate by non-covalent bond, and the incorporation is reversible, can oriented bring small molecule medicine to special positional, so the medicine concentration of nidus is higher than rest position.
Description
Technical field
Present technique relates to the preparation method of bifunctional antibody, and particularly portability is used for the treatment of micromolecular medicine in the immunology, also has the bifunctional monoclonal antibody preparation method of orientating function simultaneously.
Technical background
Antibody in the immunology has specificity, specifically recognition objective antigen.Development along with hybridoma technology, people just can obtain a large amount of specific monoclonal antibodies by the immuning hybridization knurl technology of routine at an easy rate, yet the acquisition of especially specific anti-tumor monoclonal antibody makes monoclonal antibody play important effect to the diagnosis and the treatment of tumour. because the monoclonal antibody majority is a mouse source antibody, thereby certainly exist the heterology problem, and also must cause the rejection of immunity. and the mouse endogenous antibody also is limited to the effect that excites people's cellular immunization vigor aspect.
People had invented immunotoxin or radioimmunotherapy technology again afterwards, toxalbumin or radioactivity coordination and specific monoclonal antibody are passed through covalent coupling, prepare the chemical coupling thing of toxin and monoclonal antibody, because the guide effect of monoclonal antibody, thereby take toxalbumin or radioactivity coordination to the target cell place, utilize toxalbumin toxicity or radio isotope ray that target cell is killed again. this immunotoxin or radioimmunoassay technique, though target cell is had certain lethal effect, but poisonous protein molecule itself is exactly an xenobiotic simultaneously, it is good immunogen, after immunotoxin performance for some time effect, body produces antitoxin proteic antibody, its result of treatment will be had a greatly reduced quality. and it is stronger because of isotopic contaminative to put off until some time later radioimmunity, to body and environment all is that very big harm is arranged, thereby has also limited the application of radioimmunoassay technique.
Though the related chemicals of many chemotherapy has great lethal effect to cancer cell in the actual life, but because the negative interaction of chemotherapy or radiotherapy process Chinese traditional medicine is very big, make normal cell during treating, also be injured greatly simultaneously, this mainly be since the drug level that can bear of normal tissue cell and tumour cell to treat used concentration very approaching, though killed tumour cell, kill and wound greatly but also normal cell is produced simultaneously, to such an extent as to phenomenons such as alopecia, sick body weakness all appear in a lot of radiotherapy and patients undergoing chemotherapy.
So people have expected bifunctional antibody, wish that antibody had both had the specific recognition function, also have simultaneously the treatment function. present bifunctional antibody, after majority concentrates on the antibody acquisition of antineoplastic specificity, again by inductor inducement crossbreeding knurl, obtain auxotrophic tumour cell, carry out cytogamy with anti-second kind of antigenic splenocyte again, and associated protein during second kind of antigen is normally treated, this bifunctional antibody research and development cycle is longer, and also there is the problem of stability in bifunctional antibody self, and result of treatment exists not enough in practice.
People be badly in need of to want a kind of bifunctional antibody, be used for the treatment of, and the R﹠D cycle appearance are to shorter, and good stability, result of treatment is strong, and toxicity and the little bifunctional antibody of negative interaction.
I just formed before the several years though prepare the design of this bifunctional antibody, to suffer from condition such as fund and limit and fail and implement, and also to have dropped into many energy in person in other respects in order raising money.Now related manufacturing processes is disclosed so that people can fundamentally solve a difficult problem medically, and treatment brings inspiration to other diseases.
Summary of the invention
A kind of method for preparing bifunctional antibody, its content is: with small-molecule drug as haptens with carry out chemical coupling with specific antigens as carrier proteins, wherein medicine is a micromolecular compound, having certain Chemical bond is haptens, and specific antigens is a macromolecular substance, directly as carrier proteins; Form the conjugate of small-molecule drug and differential protein by the chemical coupling preparation of routine, with its conjugate as immunogen, immune animal, yet get its splenocyte and carry out cytogamy with corresponding myeloma cell, select at HAT under the screening of substratum, the growth of can only fused cell could surviving, filter out through ELISA again, the micromolecular monoclonal antibody of antiradiation drug, the chemical coupling thing that used screening antigen is small-molecule drug and another kind of protein carrier, can obtain the monoclonal antibody of anti-small-molecule drug like this, this monoclonal antibody also is positive to carrier proteins simultaneously, filters out at specific carrier antigen more also to have good specific monoclonal antibody from these positive hybridomas.So just obtain the micromolecular specific monoclonal antibody of antiradiation drug, and this specific monoclonal antibody is that specific pathogen antigen also has good antigen antibody reaction ability to the carrier proteins composition in the immunogen simultaneously, promptly obtains to have the monoclonal antibody of difunctional characteristics!
The present invention is based upon on the basis of some conventional immunology theories or technology, and I am according to relevant immunological technique and then proposition " directed immunotherapy argument " simultaneously, and the substance of this argument is:
1, small molecules chemicals and the hapten-carrier albumen composition of doing the method formation that carrier can be by chemical coupling with the specific antigens of cause of disease, this mixture still has good immunogen
2, the mixture of resulting haptens-cause of disease specific antigens can bring out body, produces anti-small-molecule drug, the also bifunctional antibody of disease-resistant former specific antigens simultaneously
3, the bifunctional antibody that is produced can combine with the mode by non covalent bond of haptens small-molecule drug freely, also can carry out that Ag-Ab is affine to be combined simultaneously by the mode of non covalent bond with tumour specific antigen
4, such bifunctional antibody can combine in advance with the haptens medicine, and after the haptens combination, still can combine with the specific antigens in the specific pathogen, thereby take affected area to from drug molecule, because haptens drug molecule and antibodies are non covalent bonds, this combination is reversible reaction, thereby small-molecule drug can come off from antibody
5, since small-molecule drug can from bifunctional antibody, come off, it is a kind of dynamic reversible reaction, this bifunctional antibody has " Teat pipette " effect to a certain extent, with effective medicine, directionally take to around the specific antigens of cause of disease, make drug concentrations around the focus be far longer than the concentration of serum Chinese traditional medicine, so just can alleviate the toxic action of medicine body.
6, small-molecule drug is with after bifunctional antibody combines, do not come off during again in conjunction with the specific antigen of affected area small-molecule drug also can along with the specific antigen on the focus cell on cytolemma move and in the target approach cell.
When 7, adopting the cause of disease specific antigen as carrier and the coupling of inhibition virus replication small-molecule drug, resulting specific bifunctional antibody preparation the medicine orientation can be taken to virus infected cell around, improve the interference effect of chemotherapeutic agent to virus replication.
When 8, adopting the cause of disease specific antigens to make carrier, with have the cytotoxicity small-molecule drug and carry out coupling, resulting specific bifunctional antibody, can realize Cytotoxic medicine is taken to around the virus infected cell, make virus infected cell death, limited viral diffusion. but this class cell of body can not be the cell that the organa parenchymatosum organizes, and avoids bringing injury to the body organa parenchymatosum.
When 9, adopting the pathogenetic bacteria specific antigens as carrier, can be with the medicine of killing bacteria, thereby orientation bring the disease bacterium around improved the sterilization effect of medicine.
10, with other pathogenic micro-organisms or parasitic specific antigen as carrier, and also have roughly the same directed Teat pipette effect in conjunction with related drugs.
11, at the different pharmaceutical haptens respectively with same species-specific antigen coupling, prepare different bifunctional monoclonal antibodies, difunctionally do not combine in advance with different pharmaceutical respectively of the same race, in remix input patient's the body, help the raising of result of treatment.
12, this bifunctional antibody also can be in conjunction with external application, and the bifunctional antibody that will be combined with medicine with non covalent bond and on the condition near body temperature, is sent to the cause of disease place with medicine in damping fluid, pathogenic micro-organism is constituted kill and wound.
The basis of immunology theory involved in the present invention is: 1, haptens has only immunoreactivity and does not have immunogen, 2, with specific antigens as carrier proteins, resulting antibody is certain to have immunological response with specific antigens, just can therefrom filter out good bifunctional antibody hybridoma as long as further screen people.Related basic fundamental comprises: immunological techniques such as small-molecule drug and specific antigens chemical coupling technology, hybridoma technology, enzyme linked immunological adsorption technology.
Use the resulting bifunctional monoclonal antibody of the present invention, be that haptens carries out immune affine the combination preferably in advance before the use with small-molecule drug, yet the mode of this mixture by transfusion circulated through blood, utilize bifunctional antibody can carry out the characteristics of specific compatible reaction to specific antigens, bifunctional antibody is sent to certain location, simultaneously also will treat used medicine and take affected area to, thereby make that the drug level of affected area is very high, hang down the concentration of blood Chinese traditional medicine, thereby can not constitute toxic action the healthy of patient.Though small-molecule drug and this bifunctional monoclonal antibody in vivo also can in conjunction with, the effect of Teat pipette can be not as combining small-molecule drug with bifunctional monoclonal antibody in advance.With in small molecules medicine and the bifunctional monoclonal antibody input body, medicine also can orientation be delivered to affected area simultaneously, and the effect that transmits medicine may be much lower.
Though there is the problem of heterology in the present invention, but the method for production of the bifunctional antibody among the present invention, but can offer help for the research and development that the mankind start the bifunctional monoclonal antibody of a series of therapeutic, also provide the basis simultaneously for the humanized genetic engineering antibody of people's class formation, promote people's source hybridoma technology and external immunological technique maturation, thereby thoroughly solve the immunity rejection.
Specifically execute example
Embodiment 1
Chemicals with treatment people cancer of the stomach, 5-fluoro-uracil ribose and the specific antigen of people's cancer of the stomach carry out chemical coupling, the method of chemical coupling herein can adopt the sodium periodate oxidation style, an amount of 5-fluoro-uracil ribose and an amount of sodium periodate are carried out the oxidizing reaction of glycosyl, and then the adding proper amount of glycol reacts away excessive sodium periodate, add the specific antigen of an amount of cancer of the stomach again and carry out the chemical coupling reaction in the meta-alkalescence condition, the an amount of sodium borohydride of back adding spends the night, eliminate the enol-type structure that produces in the chemical coupling reaction, change liquid through dialysis and repeated multiple times again, can obtain the mixture of haptens-cancer of the stomach specific antigens, with this thing as immunogen, immunity BAL b/c mouse, immunity immunization method is routinely carried out, extracting spleen cell and mouse source myeloma cell carry out cytogamy then, obtain fused cell again through the ELISA screening, promptly can obtain difunctional authentic monoclonal antibody.This bifunctional monoclonal antibody, existing in conjunction with small molecules medicine 5 FU 5 fluorouracil ribose or small molecules 5 FU 5 fluorouracil ability, simultaneously also can be special
Opposite sex ground is in conjunction with the tumour specific antigen in the stomach cancer cell.
Embodiment 2
Utilize daunorubicin and leukemia specific antigens to carry out the mixture that chemical coupling obtains daunorubicin-leukemia specific antigens and carry out immunity, cytogamy, filter out specific bifunctional monoclonal antibody, the preparation monoclonal antibody method is with conventional hybridization knurl integration technology.Daunorubicin is as follows with the coupling method of leukemia specific proteins: an amount of daunorubicin is mixed with an amount of hydration carbodiimide, mix with the specific antigen of an amount of leukemia again, utilize the amino in the daunorubicin to carry out priming reaction, carry out covalent attachment with carrier proteins, obtain daunorubicin-leukemic specific antigens mixture.And then immunological method immunity routinely, carry out cytogamy, obtain hybridoma, and therefrom filter out specific bifunctional monoclonal antibody, because this antibody has anti-daunorubicin, the specific function of leukemia simultaneously, when chemotherapy, just the chemicals of being correlated with can be enriched to around the focus, why these medicines can be enriched to around the focus, be because another antigen that bifunctional antibody is discerned is exactly the antigen of tumour-specific, and this species specific antigen have only affected area more.There are many tumour cells in affected area, all there is specific tumour antigen in these tumour cells, and the healthy tissues of body is not have this species specific antigen, thereby medicine will be directed and is enriched to around the tumour cell, and just can reducing many, the drug level in the body blood can not influence the curative effect of medicine, so just can draw back the drug level that lesion tissue and body cell are touched, fall the negative interaction that subtracts medicine.Perhaps under original treatment concentration, the medicine orientation of greater concn is delivered to affected area, improve the result of treatment of medicine by the effect of directed drug delivery.
Embodiment 3
Ah's Mycinomycin II and lung cancer specific antigens carry out chemical coupling by carbodiimide, be prepared into the immunocomplex of Ah's Mycinomycin II and the chemical coupling of lung cancer specific proteins, after this immunocomplex immune mouse, can be able to hybridoma through cytogamy, in the Hybridoma Cell Culture supernatant that obtains, screen by the ELISA detection, the hybridoma of anti-Ah's Mycinomycin II is screened, further resulting hybridoma is carried out specificity screening and mono-clonal processing again, thereby filter out stable, anti-Ah's Mycinomycin II is the monoclonal antibody hybridoma of also anti-lung cancer specific antigens simultaneously, it will be two Ah's Mycinomycin IIs that this hybridoma is handled through the clone, also can resist the bifunctional monoclonal antibody of lung cancer simultaneously.
Embodiment 4
With microbiotic and the chemical coupling of bacterium specific antigens.Utilize the method for chemical coupling that specific curative microbiotic Streptomycin sulphate and the specific antigen of bacterium are carried out chemical coupling, obtain hapten-carrier albumen composition immune animal, prepare specific difunctional hybridoma and come secrete monoclonal antibody, resulting bifunctional antibody can react with microbiotic, also can combine simultaneously, possess bifunctional antibody with specific pathogenetic bacteria.
When using this bifunctional antibody, antibody along with blood circulation is taken medicine to the pathogenic bacteria place, makes that the antibiotic concentration around the specific germ is higher than environment far away, thereby has disturbed the growth of bacterium again with after microbiotic combines, promotes the curative effect of medicine.
Related drugs and carrier proteins are carried out chemical coupling, and to prepare the immunogen method be sophisticated, but people mainly consider immunologic detection low molecular compound content, and then carry out determination and analysis etc.And specific antigens early has supply on market, but people adopt its specificity to be used for the Position Research in cellularstructure of immunology detection analysis, immunology diagnosis and specific protein, consider a problem and low molecular medicine and specific antigens combined, prepare not seeing of direct bifunctional monoclonal antibody.
The curative medicine of using in the reality is a lot, the research and development of involved specific antigens are also a lot, people can adopt a kind of small molecules medicine and multiple specific antigens material to carry out chemical coupling, so just accomplish that a kind of disease adopts small-molecule drug of the same race to carry out the chemotherapy reaction, but specific protein carrier, people can a species specific carrier protein and multiple small-molecule drug carry out coupling, carry out the diversification of small-molecule drug, accomplish to resist a kind of disease, enumerate no longer one by one herein with multiple drug combination.
Claims (5)
1, a kind of method for preparing bifunctional antibody is characterized in that: small-molecule drug and specific antigens are carried out chemical coupling, and wherein medicine is a micromolecular compound, be haptens, and specific antigens is a macromolecular substance albumen, directly as carrier proteins; The method of the chemical coupling by routine prepares the conjugate of small-molecule drug and differential protein, with this conjugate as immunogen, immune animal, yet get the immune animal splenocyte and carry out cytogamy with corresponding myeloma cell, select at HAT under the screening of substratum, the growth of can only fused cell could surviving, filter out through ELISA again, the micromolecular monoclonal antibody of antiradiation drug, the chemical coupling thing that used screening antigen is small-molecule drug and another kind of protein carrier, can obtain the monoclonal antibody of anti-small-molecule drug like this, this monoclonal antibody also is positive to carrier proteins simultaneously, filters out at specific carrier antigen more also to have good specific monoclonal antibody from these positive hybridomas.So just obtain the micromolecular specific monoclonal antibody of antiradiation drug, and this specific monoclonal antibody is that specific pathogen antigen also has good antigen antibody reaction ability to the carrier proteins composition in the immunogen simultaneously, promptly obtains to have the monoclonal antibody of difunctional characteristics!
2, described according to claim 1, be used for haptens chemistry link coupled carrier proteins be specific antigens, they can be specific proteins antigen, virus-specific proteantigen, the parasitic specific proteins antigen of tumour specific antigen, bacterium fungi.
3, described according to claim 1, bifunctional monoclonal antibody at the medicine small molecules, be small-molecule drug or small molecules toxic substance, and small-molecule substance is the hapten compound with certain chemical structure, can realize covalent coupling by chemical means under the prerequisite of the primary structure that does not destroy micromolecular compound with protein carrier.
4, described according to claim 1, it all is the form combination of non covalent bond that bifunctional monoclonal antibody combines with small-molecule drug and specific antigens, at affected area reversibility combination and obscission can take place, small-molecule drug is transported to affected area by this mode.
5, described according to claim 1, bifunctional antibody can be discerned affected area specific antigen protein matter specifically, and other parts do not have this species specific antigenic component, thereby this bifunctional antibody maintenance directive action, and the medicine orientation is delivered to affected area.
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| CN 200610050611 CN101070346A (en) | 2006-05-08 | 2006-05-08 | Method for preparing double-function antibody |
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| CN 200610050611 CN101070346A (en) | 2006-05-08 | 2006-05-08 | Method for preparing double-function antibody |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103328512A (en) * | 2010-11-08 | 2013-09-25 | 诺瓦提斯公司 | CXCR2 binding polypeptides |
| CN104487456A (en) * | 2012-05-09 | 2015-04-01 | 诺华股份有限公司 | Biparatopic binding polypeptides for CXCR2 and uses thereof |
-
2006
- 2006-05-08 CN CN 200610050611 patent/CN101070346A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103328512A (en) * | 2010-11-08 | 2013-09-25 | 诺瓦提斯公司 | CXCR2 binding polypeptides |
| CN104487456A (en) * | 2012-05-09 | 2015-04-01 | 诺华股份有限公司 | Biparatopic binding polypeptides for CXCR2 and uses thereof |
| CN104487456B (en) * | 2012-05-09 | 2018-04-13 | 诺华股份有限公司 | Double paratope Binding peptides of CXCR2 and application thereof |
| US11466089B2 (en) | 2012-05-09 | 2022-10-11 | Ablynx N.V. | Chemokine receptor binding polypeptides |
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