CN1634582A - Crosslinker of anti-FcepsilonRIalpha monoclonal antibody Fab and tripterine - Google Patents
Crosslinker of anti-FcepsilonRIalpha monoclonal antibody Fab and tripterine Download PDFInfo
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- CN1634582A CN1634582A CN 200410084326 CN200410084326A CN1634582A CN 1634582 A CN1634582 A CN 1634582A CN 200410084326 CN200410084326 CN 200410084326 CN 200410084326 A CN200410084326 A CN 200410084326A CN 1634582 A CN1634582 A CN 1634582A
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Abstract
A cross linking body of monoclonal antibody of Fc epsilon R I alpha(Fab) with tripterine is disclosed. It can be applied for preparing biological formulation to treat mast cell and basophilic cell inducing disease. The preparation process is also provided.
Description
Technical field:
The invention belongs to biological technical field.Be specifically related to the disease mediated therapeutic biological preparation of mastocyte and basophilic granulocyte, anti-Fc ε RI alpha monoclonal antibodies Fab and crosslinked body of tripterine and preparation method.
Mastocyte and basophilic granulocyte are I metallergy disease and the diseases related main effects cell as allergic dermatitis, dermatomyositis, psoriasis etc. of some mastocytes.Anaphylactogen makes the IgE receptor that little gathering take place combine the complex that forms and cell surface IgE receptors bind such as mastocyte and basophilic granulocyte with its specific IgE after, and the triggering signal conduction comprises protein kinase activation, phosphatidylinositols hydrolysis and Ca
2+Flow etc., cause that cell medium and cytokine are synthetic, expression of adhesion molecule increases and cell stretches, film fold forms and finally causes cell degranulation, discharge vaso-active substance and some enzyme and cause that smooth muscle contraction, mucus secretion, blood pressure reduce and tissue injury.The IgE receptor has two kinds of high low-affinities, and low-affinity receptor Fc ε RII is distributed in mastocyte, has a liking for alkali, eosinophilic granulocyte, B cell and dendritic cell.High-affinity receptor Fc ε RI mainly is distributed in mastocyte and basophil, and also there is a small amount of expression on the eosinophilic granulocyte surface.Physiological condition Fc ε RII expression is higher than Fc ε RI, and the two expresses all increases pathologic condition, but Fc ε RI expression is significantly higher than Fc ε RII, and alpha subunit is the binding site of Fc ε RI and IgE.Therefore, IgE or its receptor blocking are the best means that stops allergy to take place.
Biotherapeutics according to the present allergic disease of above-mentioned principle concentrates on anti-IgE and anti-Fc ε RI alpha monoclonal antibodies two classes.The former purpose be by exogenous antibody combine with IgE and in and IgE or form the ability that big complex makes IgE forfeiture and its receptors bind with IgE, the latter then is by stoping combining of IgE and receptor with IgE competition receptor binding site, and the two all is to reach the purpose of alleviating or treating allergic disease by suppressing cell activation.The former is the most successful is to enter the medicine Omalizumab that the clinical I phase tests in calendar year 2001.This medicine is gene recombination humanized anti human IgE monoclonal antibody, antibody name rhuMAB-E25.It is reported that asthma and allergic rhinitis remission rate are apparently higher than the curative effect of placebo behind its subcutaneous injection, it can make free serum IgE concentration and IgE high-affinity receptor expression obviously reduce, report thinks that the Omalizumab concentration of therapeutic dose is very low, can not cause IgE bounce-back after the drug withdrawal.The mechanism of action of anti-IgE Mab Omalizumab is to enter in the body to combine the formation immune complex with IgE, and this big immune complex makes IgE lose binding ability with the IgE receptor, makes the mastocyte activation that is not triggered.
The segmental research report of the anti-Fc ε of humanization RI alpha monoclonal antibodies Fab comes from the laboratory of calendar year 2001 Japan, does not see further elaborate report so far.
But antibody is biological preparation, enters the body endogenous cause of ill and is degraded and exists biological half-life, therefore needs repetitively administered; In addition, the synthetic expression with Fc ε RI of IgE is a dynamic changing process, and these all directly influence administering effect and interval even effect; The bounce-back that IgE concentration and Fc ε RI express after the drug withdrawal also is the problem that needs consideration.
Technical problem to be solved by this invention is to overcome above-mentioned weak point, designs a kind of target biology guided missile, is used for the treatment of mastocyte and basophilic granulocyte is diseases related.
The inventor considers that under study for action mastocyte and basophilic granulocyte are the main effects cells of allergosis, and Fc ε RI is the IgE receptor that only is expressed in these two kinds of cells, play pivotal role in the allergic disease that the mediation anaphylactogen causes, alpha subunit is the IgE binding site.Therefore, utilize anti-Fc ε RI alpha monoclonal antibodies to combine and effectively to stop the activation of IgE mastocyte and basophilic granulocyte with mastocyte and basophilic granulocyte.Mastocyte and basophilic granulocyte are present in tissue or mucosa mostly, and the whole antibody molecular weight is excessive, enter to be difficult to infiltrate through tissue in the body, thereby are difficult to prove effective; Mastocyte can be triggered equally after the more important thing is whole antibody and Fc ε RI combining and basophilic granulocyte takes off granule, and utilize the Fab fragment of antibody, molecular weight reduces greatly on the one hand, solved the problem that whole antibody is difficult to permeate tissue, on the other hand, the Fab fragment has the mastocyte and the basophilic granulocyte that are equal to whole antibody and combines activity, but has lost triggering mastocyte and the degranulated ability of basophilic granulocyte.
In addition, apoptosis (apoptosis) is a kind of dead mode of cell, and the apoptotic body that apoptosis forms is finally engulfed by adjacent cells, does not cause the inflammatory reaction of surrounding tissue.Thereby inducing the mastocyte apoptosis is the desirable approach of a treatment I allergic reaction type.Be directed at the inducer of apoptosis of mastocyte, will directly and farthest bring into play the effect of medicine, and can limit toxicity effectively.
Radix Tripterygii Wilfordii (Tripterygium wilfor-dii Hook.f.) belongs to bejuco, and it has various active such as antiinflammatory, antitumor, antifertility, and its chemical constituent mainly is diterpene, triterpene, sesquiterpene, alkaloid etc.Diseases such as its treatment rheumatoid arthritis of motherland's medical use, systemic lupus erythematosus (sle) have irreplaceable pharmacological action.Tripterine (tripterine) is one of monomer of Radix Tripterygii Wilfordii, belongs to the pentacyclic triterpene pigment, molecular formula C29H38O4, molecular weight 450.Studies confirm that tripterine can induce the mastocyte apoptosis, apoptosis does not cause the inflammatory reaction of surrounding tissue, on the other hand, the mastocyte apoptosis can not cause yet as may bring behind other apoptosis with function damage or disorderly relevant side effect and complication.With regard to its pharmacological mechanism, tripterine should be the ideal medicament of treatment I allergic reaction type disease.Yet because the popularity of its effect target, using in the body has tangible toxicity, particularly immunity and reproduction inhibition etc., thus limited its clinical application range greatly.
Having competitive inhibition IgE from anti-Fc ε RI α Fab itself noted earlier and combine with mastocyte and basophilic granulocyte and suppress the two degranulated biological effect, also is the direction of Biotherapeutics.
Based on the above-mentioned mechanism of action, the inventor has designed a kind of target biology guided missile, and wherein guided missile is a tripterine, and target is mastocyte and basophilic granulocyte, and the target fairlead is anti-Fc ε RI alpha monoclonal antibodies Fab fragment.
The present invention is crosslinked with tripterine and Fc ε RI α Fab, make it to become medicine with targeting specific, the bonded tripterine of indivisible and anti-Fc ε RI α Fab enters in the body, directed combine, not only reached tripterine to induce the purpose of mastocyte apoptosis but also limited its toxicity with mastocyte.
The invention provides a kind of anti-Fc ε RI alpha monoclonal antibodies Fab and crosslinked body of tripterine and preparation method, this method is the rat basophilic granulocyte leukemia cell line RBL-2H3 immune mouse with Fc ε RI high expressed, getting high mouse spleen of serum antibody concentration and mouse myeloma cell line SP2/0 merges, prepare anti-Mus Fc ε RI alpha monoclonal antibodies, the Fab fragment of the antibody that obtains from ascites and cells and supernatant is also crosslinked with tripterine, obtains crosslinked body.
Fc ε RI alpha monoclonal antibodies Fab of the present invention and the crosslinked body of tripterine have carried out following experiment in vitro and zoopery:
1. the correctness and the feasibility of confirmatory experiment design: combining (immunofluorescence and ELISA) with mastocyte with crosslinked body by the relatively more simple Fc ε RI alpha monoclonal antibodies Fab fragment of experiment in vitro, the maxicell that causes fat takes off grain effect (the ELISA method is surveyed histamine release), release of cytokines (ELISA detects IL-4) and induce mastocyte apoptosis (DNA electrophoresis, dna content, TNUNEL marker detection dna break point and Annexin V detect and distinguish apoptosis and necrosis) difference of aspect;
2. effectiveness of application in the body: clearly hand over the curative effect (effective, invalid) of parabiotic pairs asthma by zoopery, further determine administering mode, time (before or after the outbreak, dosing interval and the course of treatment), route of administration (suck, subcutaneous) and administration concentration;
3. mechanism of action and approach: detect serum and bronchoalveolar lavage fluid histamine and IL-4 concentration and determine the clear and definite action pathway of serum total Ig E concentration before and after the influence of mast cell degranulation and cytokine, the treatment of enzyme immunofluorescence assay respectively, lung tissue and bronchus section detect mastocyte and basophilic granulocyte quantity, apoptosis detect (TUNEL, HE dyeing, Electronic Speculum) clear and definite effect;
4. the animal survival phase observes and the toxic and side effects observation.
Above-mentioned experimental result shows:
Rat basophil granules cell is that the BALB/c mouse splenocyte and the myeloma cell SP2/0 of RBL-2H3 immunity merges the antibody that ELISA screening Mus serum and fused cell produce, limited dilution cloning cultivation.SDS-PAGE gel electrophoresis, immunodiffusion are identified antibody character, and IgE competition combination, histamine release detect the antibody biological activity,
125The antibody recognition antigenic component is identified in the autoradiography of I-cell membrane antibody complex immunoprecipitation.Obtain the anti-Fc ε of 2 strains RI alpha monoclonal antibodies and produce cell strain ER-E5.3, ER-C7.4 is IgG1.The antibody titer that culture supernatant and mouse ascites obtain is all greater than 10
5Flow cytometer (flow cytometry, FCM) analyze antibody and RBL-2H3 combination rate all greater than 95%, with rat chest cell and peripheral blood lymphocyte (peripheral blood lymphocyte, PBL) no cross reaction, and combining with mastocyte by competitive inhibition IgE.Antibody triggers RBL-2H3 and takes off granule, and the histamine release effect is identical to the activation effect of mastocyte with antigenic specificity IgE.The Fab fragment that the Fructus Chaenomelis enzyme action obtains can combine with RBL-2H3, also can compete and suppress combining of IgE and mastocyte, but unlikely RBL-2H3 takes off granule.The cell membrane component of antibodies is an alpha subunit.
With the aldehyde radical is that cross-linking agent connects tripterine and anti-Fc ε RI alpha monoclonal antibodies Fab segment, and the mixture of acquisition is removed unconjugated tripterine through column chromatography, obtains crosslinked body and the blending constituent that does not combine tripterine.Through external and mastocyte effect, the TUNEL labelling, HE dyeing, DNA electrophoresis and dna content analysis have all confirmed the mastocyte apoptosis, confirm crosslinked success.So, also can't determine crosslinking rate because the tripterine molecular weight only 450 can't separate with the anti-Fc ε RI alpha monoclonal antibodies Fab fragment of uncrosslinked tripterine crosslinked.But, as long as have crosslinked and can induce the mastocyte apoptosis just can achieve the goal, because only anti-Fc ε RI alpha monoclonal antibodies Fab fragment also has its therapeutic effect.
By experiment, the invention solves following problem
1), preparation mouse anti rat Fc ε RI alpha monoclonal antibodies, and identify its biological activity and immunocompetence
2), obtain mouse anti rat Fc ε RI alpha monoclonal antibodies Fab fragment, identification of organism activity
3), Fc ε RI alpha monoclonal antibodies Fab fragment and tripterine be crosslinked, identifies the external evoked mastocyte apoptosis of crosslinked body characteristic
4), set up the rat asthmatic model, establish the mode used in the crosslinked body body, time, administration interval, curative effect
5), confirmation and tripterine be in conjunction with using different effect, dosage with simple antibody, and causes apoptosis effect behind the crosslinked tripterine
6), determine that application has no side effect in the crosslinked body body
Characteristics of the present invention are as follows:
1), the present invention utilized immunology principle with antibody and traditional Chinese Medicine and Clavicular, the main effect of antibody is a guiding; 2), one aspect of the present invention can stop combining of IgE and mastocyte and basophilic granulocyte, can also induce the mastocyte apoptosis in addition; 3), not only to the I metallergy disease of IgE mediation, and also effective in cure to the tissue fibering of the disease of mastocyte and basophilic granulocyte mediation such as allergic dermatitis, psoriasis, dermatomyositis even mast cell mediated; 4), since curative effect realize by apoptosis, so be hopeful to eradicate the disease of mast cell mediated, and be not only relief of symptoms; 5) probability of SERUM IgE concentration even Fc ε RI expression bounce-back is very little after biological half-life, the drug withdrawal.
Example one, the anti-Fc ε of the present invention RI alpha monoclonal antibodies Fab and tripterine cross-linking agent act on rat, bring out asthma with ovalbumin immunity SD rat, set up asthmatic model, and asthma is brought out rate 80%.Respectively at giving tripterine and the crosslinked spray body of anti-Fc ε RI alpha monoclonal antibodies Fab before and after bringing out once more.The result is administered once before bringing out once more, can obviously alleviate the asthma attack degree, rechallenge 2-4 time, and asthma is not reaccessed.Bringing out the average administration in back once more no longer replys anaphylactogen for 7 times.Serum and bronchoalveolar lavage fluid IL-4, total IgE, histamine concentration significantly reduce the two difference highly significant (p＜0.001) with not treatment group.Various dose group comparison 10-20 μ g/ml/ day promptly reaches therapeutic effect, the continuous use heart in January,, organs such as liver, kidney, lung do not have pathology and change.
Example two, the apoptosis effect of the anti-Fc ε of the present invention RI alpha monoclonal antibodies Fab and tripterine cross-linking agent.Fc ε RI alpha monoclonal antibodies after cutting, papain is obtained micromolecule Fab fragment, itself and tripterine are obtained anti-Fc ε RI alpha monoclonal antibodies Fab and the crosslinked body of tripterine with the coupling of aldehyde radical coupling agent, this crosslinked body enters in the body and combines on the one hand with mastocyte and basophil cellular surface Fc ε RI, stoped combining of IgE and mastocyte and basophilic granulocyte, thereby made IgE can not trigger mastocyte and basophilic granulocyte activation; On the other hand, tripterine is oriented to mastocyte, or enters cell by its surperficial receptor, because tripterine is that the minimum five rings terpenoid pigment of molecular weight can freely enter cell, promptly start the apoptosis signal in case enter cell, make apoptosis.
Example three, the present invention will resist Fc ε RI alpha monoclonal antibodies Fab and the crosslinked body of tripterine to compare with simple tripterine and simple Fc ε RI alpha monoclonal antibodies Fab, inducing fertilizer to reach aspect the apoptosis: crosslinked body and tripterine are at dosage (molar concentration) and do not have significant difference on the time, and simple Fc ε RI alpha monoclonal antibodies Fab is without any inducing the mastocyte apoptotic effect, illustrate Fc ε RI alpha monoclonal antibodies Fab and tripterine crosslinked be successful; Aspect the specificity of target: tripterine can inducing T cell be the 6T-CEM apoptosis, and crosslinked body and simple Fc ε RI alpha monoclonal antibodies Fab do not cause apoptotic effect to the 6T-CEM cell, illustrate that crosslinked body is oriented to mastocyte.
Claims (3)
1, a kind of anti-Fc ε RI alpha monoclonal antibodies Fab and the crosslinked body of tripterine, it is characterized in that this crosslinked body is the targeting specific biological preparation, wherein guided missile is that tripterine, target are that mastocyte and basophilic granulocyte, target fairlead are anti-Fc ε RI alpha monoclonal antibodies Fab fragment.
2, the preparation method of a kind of anti-Fc ε RI alpha monoclonal antibodies Fab and the crosslinked body of tripterine, it is characterized in that this method is the rat basophilic granulocyte leukemia cell line RBL-2H3 immune mouse with Fc ε RI high expressed, getting high mouse spleen of serum antibody concentration and mouse myeloma cell line SP2/0 merges, prepare anti-Mus Fc ε RI alpha monoclonal antibodies, the Fab fragment of the antibody that obtains from ascites and cells and supernatant is also crosslinked with tripterine, obtains crosslinked body.
3, a kind of anti-Fc ε RI alpha monoclonal antibodies Fab and the crosslinked body of the tripterine application in preparing the disease mediated biological preparation of treatment mastocyte and basophilic granulocyte.
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| CN1634582A true CN1634582A (en) | 2005-07-06 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102156113A (en) * | 2010-12-13 | 2011-08-17 | 中国中医科学院医学实验中心 | Direct monitoring method of mast cell degranulation |
| CN103257237A (en) * | 2013-05-09 | 2013-08-21 | 中国农业大学 | In-vitro detection method of allergen in food |
| CN104212770A (en) * | 2013-09-11 | 2014-12-17 | 李莉 | Anti-human FcepsilonRIalpha subunit monoclonal antibody and application thereof |
| CN104293738A (en) * | 2013-09-11 | 2015-01-21 | 李莉 | Anti-human Fc epsilon RI alpha subunit monoclonal antibody and application thereof |
| CN104800164A (en) * | 2015-04-13 | 2015-07-29 | 上海市第一人民医院 | Common threewingnut root immunonanoparticles and application |
| CN108324735A (en) * | 2017-01-20 | 2018-07-27 | 李莉 | For the extracellular body preparation of disease treatment and its application |
-
2004
- 2004-11-18 CN CN 200410084326 patent/CN1274364C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102156113A (en) * | 2010-12-13 | 2011-08-17 | 中国中医科学院医学实验中心 | Direct monitoring method of mast cell degranulation |
| CN102156113B (en) * | 2010-12-13 | 2012-10-03 | 中国中医科学院医学实验中心 | Direct monitoring method of mast cell degranulation |
| CN103257237A (en) * | 2013-05-09 | 2013-08-21 | 中国农业大学 | In-vitro detection method of allergen in food |
| CN103257237B (en) * | 2013-05-09 | 2015-05-20 | 中国农业大学 | In-vitro detection method of allergen in food |
| CN104212770A (en) * | 2013-09-11 | 2014-12-17 | 李莉 | Anti-human FcepsilonRIalpha subunit monoclonal antibody and application thereof |
| CN104293738A (en) * | 2013-09-11 | 2015-01-21 | 李莉 | Anti-human Fc epsilon RI alpha subunit monoclonal antibody and application thereof |
| CN104800164A (en) * | 2015-04-13 | 2015-07-29 | 上海市第一人民医院 | Common threewingnut root immunonanoparticles and application |
| CN104800164B (en) * | 2015-04-13 | 2017-11-07 | 上海市第一人民医院 | A kind of tripterygium wilfordii immunonanoparticles and purposes |
| CN108324735A (en) * | 2017-01-20 | 2018-07-27 | 李莉 | For the extracellular body preparation of disease treatment and its application |
| CN108324735B (en) * | 2017-01-20 | 2024-02-09 | 李莉 | Exosome preparation for treating diseases and application thereof |
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| CN1274364C (en) | 2006-09-13 |
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