CN1255378A - 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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- CN1255378A CN1255378A CN 99110806 CN99110806A CN1255378A CN 1255378 A CN1255378 A CN 1255378A CN 99110806 CN99110806 CN 99110806 CN 99110806 A CN99110806 A CN 99110806A CN 1255378 A CN1255378 A CN 1255378A
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Abstract
A proper process is used to obtain the active Fab' fragment of the monoclonal antibody 3A5 of human liver cancer cell BEL-7402. The glucosan T-40 is used as medium to couple Fab' with Pingyangmycin. The conjugate has killing action to human liver cancer cell BEL-7402 and oral cancer cell KB for the external experiment. For the internal experiment, colon cancer, tissue is homogenized by physiologic saline and then grafted to Balb/c mouse and the medicine is applied after 24 hrs. Its tumor suppression rate is 77-91%. The survival time of animal is elongated obviously. Its curative effect is prior to the Pingyang mycin.
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, and pH3.7. adds an amount of pepsin, and 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 inoculate sub-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
Heavy (g) tumour inhibiting rate of dosage number of animals body weight (g) tumor
(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 ± 0.4l 56* conjugate, 5 * 6 10/10 18.9/20.2,0.37 ± 0.07 77* △ subcutaneous vaccination tumor, intraperitoneal administration, two days are once; * P＜0.01 with compare △ △ 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, and is heavy by heavy (the mg)=l/2 major diameter * minor axis 2 calculating tumors of tumor, draws tumor growth curve and also 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
Heavy (g) tumour inhibiting rate of dosage number of animals body weight (g) tumor
(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* △
10×7 10/10 21.4/20.4 0.07±0.02 91*△△
The subcutaneous vaccination tumor, intravenously administrable, two days are once; * relatively △ △ P＜0.01 and Bleomycin A5 10mg/kg group be relatively with comparing △ 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 * 7 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 3A5Fab ' 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 and antitumor action thereof is characterized in that the enforcement of said antitumor action may further comprise the steps:
A. the preparation of monoclonal antibody 3A5 Fab ' active fragment;
B.3A5Fab '-preparation of Bleomycin A5 conjugate
C.3A5Fab '-detection of the antitumor action of Bleomycin A5 conjugate
2. according to described conjugate of claim 1 and antitumor action thereof, it is characterized in that said 3A5Fab ' produces F (ab) ' 2 with pepsin digestion monoclonal antibody 3A5, with DTT (DTT) reduction, produce Fab ' again, its structure comprises Fab and part hinge region; Monoclonal antibody 3D6 produces F (ab) ' 2 with the ficin enzymic digestion, obtains Fab ' through the beta-mercaptoethanol reduction.
3. according to described conjugate of claim 1 and antitumor action thereof, the preparation that it is characterized in that Fab '-Bleomycin A5 conjugate is that glucosan T-40 is oxidized to many aldehyde radicals glucosan (PAD), adds Bleomycin A5 earlier, adds Fab ' continuation reaction again, through sodium borohydride reduction, purification obtains product.
4. according to described conjugate of claim 1 and antitumor action thereof, it is characterized in that In vitro culture human hepatoma cell BEL-7402 and oral cavity squama glass KB cell, detect the cell killing effect of free Bleomycin A5 and conjugate thereof with clone forming method.
5. according to described conjugate of claim 1 and antitumor action thereof, it is characterized in that animal vivo test,, judge the tumour inhibiting rate of free Bleomycin A5 and conjugate thereof by intraperitoneal and intravenous administration in BALB/c mouse subcutaneous vaccination colon cancer 26.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105228645A (en) * | 2013-03-11 | 2016-01-06 | 建新公司 | By the site-specific antibodie-drug coupling of sugar engineering |
| US11186638B2 (en) | 2011-09-12 | 2021-11-30 | Genzyme Corporation | Anti-αβTCR antibody |
| US11697690B2 (en) | 2014-03-19 | 2023-07-11 | Genzyme Corporation | Site-specific glycoengineering of targeting moieties |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NO881077L (en) * | 1987-03-11 | 1988-09-12 | Univ Michigan | Chemo-RADIO IMMUNO conjugates. |
| US4952394A (en) * | 1987-11-23 | 1990-08-28 | Bristol-Myers Company | Drug-monoclonal antibody conjugates |
| US5010176A (en) * | 1988-11-10 | 1991-04-23 | Eli Lilly And Company | Antibody-drug conjugates |
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1999
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11186638B2 (en) | 2011-09-12 | 2021-11-30 | Genzyme Corporation | Anti-αβTCR antibody |
| CN105228645A (en) * | 2013-03-11 | 2016-01-06 | 建新公司 | By the site-specific antibodie-drug coupling of sugar engineering |
| CN105228645B (en) * | 2013-03-11 | 2019-06-07 | 建新公司 | Pass through site-specific antibodie-drug coupling of sugar engineering |
| CN110256560A (en) * | 2013-03-11 | 2019-09-20 | 建新公司 | Pass through site-specific antibodie-drug coupling of sugar engineering |
| US11807690B2 (en) | 2013-03-11 | 2023-11-07 | Genzyme Corporation | 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 |
| US11697690B2 (en) | 2014-03-19 | 2023-07-11 | Genzyme Corporation | Site-specific glycoengineering of targeting moieties |
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