CN1110322C - Monoclonal antibody Fab'-pingyangmycin conjugate and its anticancer action - Google Patents
Monoclonal antibody Fab'-pingyangmycin conjugate and its anticancer action Download PDFInfo
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- CN1110322C CN1110322C CN99110806A CN99110806A CN1110322C CN 1110322 C CN1110322 C CN 1110322C CN 99110806 A CN99110806 A CN 99110806A CN 99110806 A CN99110806 A CN 99110806A CN 1110322 C CN1110322 C CN 1110322C
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Abstract
The present invention relates to a monoclonal antibody 3A5 for resisting human liver cancer BEL-7402 cells. An active Fab' segment of the monoclonal antibody is obtained by a proper method; the Fab' is coupled with pingyangmycin by using dextran T-40 as a mediator. For in-vitro experiments, cultivated human liver cancer BEL-7402 cells and oral squamous cell carcinoma KB cells are used and detected by a clone formation method to indicate that the conjugate has damage effect on both of the BEL-7402 cells and the KB cells, and concentrations for inhibiting 50% clone formation are respectively 0.02 mu mol/L and 0.5 mu mol/L. For in-vivo experiments, colon cancer 26 is used, the tumour tissue of colon cancer 26 is taken, physiological saline is added, and the mixture is homogenized and inoculated to the subcutaneous axilla of a Balb/C mouse, and the mouse is medicated after 24 hours. The antitumour rates are respectively 77%(ip 5mg/kg*6, one time every two days) and 89%(iv 5 mg/kg*7, one time every two days) or 91%(iv 10 mg/kg*7, one time every two days); meanwhile, the survival time of animals is obviously prolonged, and the effect of the monoclonal antibody 3A5 is superior to that of the isodose free bleomycin; thus, an evidence for using the conjugate of monoclonal antibody Fab' and pingyangmycin to cure tumours is provided.
Description
The present invention relates to a kind of monoclonal antibody as carrier, coupling is carried antitumor drug and is carried out the method that antitumor is controlled.
Monoclonal antibody (monoclonal antibody) 3A5 is to get its splenocyte and rat bone myeloma IR983F cell fusion behind human hepatocellular carcinoma BEL-7402 cell's immunity LOU/C rat, through screening, and the excretory antibody of clone's back gained hybridoma cell strain.
It is reported that mouse monoclonal antibody Fab ' fragment has the tissue penetration stronger than complete antibody, (Cancer Research 1991 51:6363), helps the tumor diagnosis and treatment to be easy to arrive the tumor tissues deep.But the rat monoclonal antibody is not seen similar report.With the dissimilar monoclonal antibodies of pepsin digestion rat, can obtain monoclonal antibody F (ab) '
2Fragment, but the segmental preparation method of its immunologic competence and Fab ' and with the research of drug conjugates (Journal of Immunologic methods 1983 64:141) there is no report.Have report to think that monoclonal antibody 3A5 and kinds of tumor cells have strong immunoreactivity, itself and the Bleomycin A5 conjugate is external that hepatocarcinoma BEL-7402 cell is had selective killing effect (Chinese microorganism and Journal of Immunology 1991; 11:230).Zoopery shows that this conjugate intracavitary administration can obviously prolong survival time of animals (Acta Pharmaceutica Sinica 1997; 32:669), but do not see the report of 3A5 active fragment and Bleomycin A5 conjugate.Anti-people's pulmonary carcinoma monoclonal antibody 3D3 and Bleomycin A5 are strong 5 times at external lethal effect specific ionization Bleomycin A5 to human lung adenocarcinoma cell after the coupling mutually with molecular proportion 1: 68, but do not see zooperal report (Chinese clinical tumor 1996; 23:437).3A5 can obtain Fab fragment (Acta Pharmaceutica Sinica 1993 with the papain degraded; 28:260) preparation of itself and Bleomycin A5 conjugate and Fab ' does not then appear in the newspapers.In sum, up to now, the segmental preparation of rat monoclonal antibody Fab ' and all do not have report both at home and abroad with the research of Bleomycin A5 conjugate.Studies show that of this invention can obtain activated monoclonal antibody Fab ' segment with suitable method, and itself and Bleomycin A5 conjugate have significant curative effect to the laboratory animal tumor, might become a kind of new anti-tumor medicine.
The objective of the invention is to prepare monoclonal antibody 3A5Fab ' fragment, and, be used for clinical treatment tumour this segment and Bleomycin A5 coupling mutually with suitable method.The content and the main points of this invention are: 1. the preparation monoclonal antibody 3A5 of monoclonal antibody 3A5Fab ' is adjusted into 1-2mg/ml with the citrate buffer solution of 0.2mol/L, pH3.7. add an amount of pepsin, 37 ℃ were reacted 6 hours, and product obtains F (ab) ' through the SephadexG-150 chromatography purification
26-10mg/ml can be through 5mmol/L DTT (DTT) reduction when pH7.5, and product gets Fab ' through the DE-52 anion-exchange chromatography, the about 55Kd of its molecular weight, and its structure comprises Fab and part hinge region.Through enzyme-linked immunosorbent assay (ELISA), kept immunoreactivity (Fig. 1) to the human hepatocellular carcinoma BEL-7402 cell.Mice type-IV collagenase-resisting monoclonal antibody 3D6 produces F (ab) ' with the ficin enzymic digestion
2, obtain Fab ' through the beta-mercaptoethanol reduction, also kept immunoreactivity (Fig. 2) to the IV collagenase.
2.Fab ' with Bleomycin A5 conjugate preparation with glucosan T-40 (Dextran T-40) with sodium periodate oxidation, dialysis, lyophilization, many aldehyde radicals glucosan (PAD).By weight getting PAD at 1: 2 with Bleomycin A5 is dissolved in an amount of phosphate buffer (PBS) altogether, 4 ℃ of lucifuges reactions are after 12 hours, add the Fab ' with molal quantity such as PAD, continue to react 12 hours.With right amount of boron sodium hydride reductase 12 hour.Product is through Sephadex G-100 chromatography purification, and collecting existing antibody activity has the bacteriostatic activity part again, is conjugate.
3. the human hepatoma cell BEL-7402 of the external training of lethal effect of conjugate pair cell and oral squamous cell carcinomas KB cell are measured drug effect 1 hour with clone forming method.Bleomycin A5 all has lethal effect to two kinds of cells as a result, suppresses the concentration (IC that 50% clone forms
50) be respectively 0.51 μ mol/L and 0.47 μ mol/L; Conjugate is to two kinds of cell IC
50Be respectively 0.02 μ mol/L and 0.50 μ mol/L.Show conjugate to the BEL-7402 cell have certain selective killing effect (Fig. 3, Fig. 4).
4. the conjugate lumbar injection is got colon cancer 26 tumor tissues to the curative effect of mouse junction cancer, adds normal saline by 1: 3 and grinds to form homogenate, and it is subcutaneous only to be inoculated in the BALB/c mouse axillary fossa by 0.2ml/; Inoculate 24 hours after intraperitoneal injection (ip) administration, matched group gives normal saline, puts to death animal after 14 days, gets tumor and weighs, and calculates tumour inhibiting rate.The result shows that Bleomycin A5 and conjugate are all effective in cure to colon cancer 26, and tumour inhibiting rate is respectively 56% and 77%, and there were significant differences (table 1) for the two.
The intraperitoneal injection of table 1 conjugate is to the inhibitory action of mouse junction cancer 26
Dosage
Number of animals Body weight (g) Tumor heavy (g)Tumour inhibiting rate
(mg/kg * number of times) beginning/end beginning/end x ± SD (%) contrast 10/10 18.8/21.7 1.59 ± 0.35 Bleomycin A5s 5 * 6 10/10 18.8/20.3,0.71 ± 0.41 56* conjugate 5 * 6 10/10 18.9/20.2,0.37 ± 0.07 77* Δ subcutaneous vaccination tumor, intraperitoneal administration, two days are once; * P＜0.01 with compare Δ Δ P＜0.01 and Bleomycin A5 group relatively
5. the conjugate intravenous administration is (iv) ditto described to the curative effect inoculation method of mouse junction cancer 26, and administering mode is intravenous injection.Animal is not put to death, and writes down body weight in per 3 days and surveys the tumor volume, by heavy (mg)=1/2 major diameter of tumor * minor axis
2The calculating tumor is heavy, draws tumor growth curve and observes survival time of animals.Result of study shows that the conjugate intravenous administration has significant curative effect to intestinal cancer 26, and its tumour inhibiting rate is respectively 89% (5mg/Kg) and 91% (10mg/Kg); And free Bleomycin A5 tumour inhibiting rate when same dose is respectively 70% and 79%.But conjugate is the significant prolongation animals survived time (table 2, table 3) also.
The intravenous injection of table 2 conjugate is to the inhibitory action of mouse junction cancer 26
Dosage
Number of animals Body weight (g) Tumor heavy (g) Tumour inhibiting rate
(mg/kg * number of times) beginning/end beginning/end x ± SD (%) contrast 10/10 21.6/22.8 0.77 ± 0.22 Bleomycin A5 5 * 7 10/10 20.9/20.4,0.23 ± 0.09 70*
10 * 7 10/10 21.1/19.3,0.16 ± 0.34 79* conjugate 5 * 7 10/10 20.9/20.3,0.09 ± 0.02 89* Δ
10 * 7 10/10 21.4/20.4,0.07 ± 0.02 91* Δ Δ subcutaneous vaccination tumor, intravenously administrable, two days are once; * relatively Δ Δ P＜0.01 and Bleomycin A5 10mg/kg group be relatively with comparing Δ P＜0.01 and Bleomycin A5 5mg/kg group in P＜0.01
Table 3 intravenous administration animal survival situation
Dosage number of animals median life cycle median life cycle
(mg/kg * number of times) (only) (my god) increase percent (%) contrast 10 17 Bleomycin A5 5 * 7 10 29 71
10 * 7 10 33 94 conjugate 5 * 71 10 30 76
10×7 10 38 124
6. conjugate toxicity conjugate and Bleomycin A5 intraperitoneal injection (5mg/kg * 6) are respectively 56% and 77% to the mouse junction cancer tumour inhibiting rate, and through histopathologic examination, the heart, liver, spleen, lung, kidney, stomach, small intestinal and femur there is no the toxicity pathological changes.
Advantage of the present invention and good effect are: determined rat monoclonal antibody Fab ' preparation method and provide necessary foundation for monoclonal antibody and Bleomycin A5 conjugate are used for the treatment of tumor.Bleomycin A5 has better curative effect in clinical practice for many years, and itself and monoclonal antibody Fab ' conjugate have the curative effect of the free Bleomycin A5s of dosage such as obviously being better than to the laboratory animal tumor, estimates to be used for clinical cancer therapy, and obtains good effect.
Description of drawings Fig. 1 monoclonal antibody 3A5Fab ' to the BEL-7402 cell immune response wherein abscissa be the AC ordinate be 490 light absorption values 1 for 3A52 be 3A5 Fab ' Fig. 2 monoclonal antibody 3D6Fab ' to the immunoreactivity of IV clostridiopetidase A wherein abscissa be the AC ordinate be 490 light absorption values 1 for 3D62 be 3D6Fab ' Fig. 3 bleomycin A5 and conjugate to the lethal effect of BEL-7402 cell wherein abscissa be drug concentration (umol/l) ordinate be cloning efficiency (%) 1 for bleomycin A5 2 for conjugate Fig. 4 bleomycin A5 and conjugate to the lethal effect of KB cell wherein abscissa be that drug concentration (umol/l) ordinate is that cloning efficiency (%) 1 is conjugate for bleomycin A5 2
Claims (5)
1, monoclonal antibody Fab '-Bleomycin A5 conjugate is characterized in that said conjugate is connected to form monoclonal antibody Fab ' through cross-linking agent glucosan T-40 with Bleomycin A5.
2, the preparation method of monoclonal antibody Fab '-Bleomycin A5 conjugate is characterized in that said preparation method comprises two steps:
A: the preparation of monoclonal antibody 3A5 Fab ' active fragment;
The preparation of B:3A5 Fab '-Bleomycin A5 conjugate.
3, preparation method as claimed in claim 2 is characterized in that said 3A5 Fab ' produces F (ab) ' with pepsin digestion monoclonal antibody 3A5
2, with DTT DTT reduction, producing Fab ' again, its structure comprises Fab and part hinge region; Monoclonal antibody 3D6 produces F (ab) ' with the ficin enzymic digestion
2, obtain Fab ' through the beta-mercaptoethanol reduction.
4, preparation method as claimed in claim 2, it is characterized in that said Fab '-Bleomycin A5 conjugate is that glucosan T-40 is oxidized to many aldehyde radicals glucosan PAD, add Bleomycin A5 earlier, add Fab again, continue reaction, obtain product through sodium borohydride reduction, purification.
5, the application of monoclonal antibody Fab '-Bleomycin A5 conjugate in the preparation antitumor drug.
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| Application Number | Priority Date | Filing Date | Title |
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| CN99110806A CN1110322C (en) | 1999-07-21 | 1999-07-21 | Monoclonal antibody Fab'-pingyangmycin conjugate and its anticancer action |
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| CN99110806A CN1110322C (en) | 1999-07-21 | 1999-07-21 | Monoclonal antibody Fab'-pingyangmycin conjugate and its anticancer action |
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| CN1110322C true CN1110322C (en) | 2003-06-04 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| UY34317A (en) | 2011-09-12 | 2013-02-28 | Genzyme Corp | T cell antireceptor antibody (alpha) / ß |
| BR112015020885A2 (en) | 2013-03-11 | 2017-10-10 | Genzyme Corp | hyperglycosylated binding polypeptides |
| CN103330937A (en) * | 2013-06-15 | 2013-10-02 | 济南环肽医药科技有限公司 | Monoclonal antibody-antigen binding segment-T-2 toxin conjugate |
| EP3129067B1 (en) | 2014-03-19 | 2023-01-04 | Genzyme Corporation | Site-specific glycoengineering of targeting moieties |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0282057A2 (en) * | 1987-03-11 | 1988-09-14 | The Board Of Regents Of The University Of Michigan | Chemo-radio-immuno-conjugates |
| EP0317957A2 (en) * | 1987-11-23 | 1989-05-31 | Bristol-Myers Squibb Company | Drug-monoclonal antibody conjugates |
| EP0368668B1 (en) * | 1988-11-10 | 1997-01-02 | Eli Lilly And Company | Antibody-drug conjugates |
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1999
- 1999-07-21 CN CN99110806A patent/CN1110322C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0282057A2 (en) * | 1987-03-11 | 1988-09-14 | The Board Of Regents Of The University Of Michigan | Chemo-radio-immuno-conjugates |
| EP0317957A2 (en) * | 1987-11-23 | 1989-05-31 | Bristol-Myers Squibb Company | Drug-monoclonal antibody conjugates |
| EP0368668B1 (en) * | 1988-11-10 | 1997-01-02 | Eli Lilly And Company | Antibody-drug conjugates |
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