CN1268377A - Antimatrix metalloprotease monovalent antibody Fab' medicine conjugate and its tumor-resisting action - Google Patents
Antimatrix metalloprotease monovalent antibody Fab' medicine conjugate and its tumor-resisting action Download PDFInfo
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- CN1268377A CN1268377A CN 00103497 CN00103497A CN1268377A CN 1268377 A CN1268377 A CN 1268377A CN 00103497 CN00103497 CN 00103497 CN 00103497 A CN00103497 A CN 00103497A CN 1268377 A CN1268377 A CN 1268377A
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Abstract
Anti-matrix metal protease monoanti 3D6 uses appropriate method to obtain Fab' segment and uses N-hydroysuecinimide-m-(N-maleimide) benzoate or glucan T-40 as intermediate to couple Fab' with LDM to obtain two kinds couplet Fab'-LDM and Fab'-PYM. Clone formation method and tetrazoblue method are used to detect the killing action of couplet against tumour BEL-7402, KB and PG cell in vitro, and results show that couplet possesses rather strong killing effect against in vitro cultured tumour cells which provides basis for using LDM to treat tumour.
Description
The present invention relates to a kind of monoclonal antibody as carrier, the method that antitumor drug carries out antineoplaston is carried in coupling.
Monoclonal antibody (monoclonal antibody) 3D6 is to get its splenocyte and mouse myeloma SP2/0 cell fusion after the IV Collagen Type VI enzyme immunity BALB/c mouse, through screening, and the excretory antibody of clone's back gained hybridoma cell strain.
It is reported that malignant cell secretes multiple matrix metalloproteinase, degraded extracellular matrix, the growth of regulate tumor cell and transfer.IV Collagen Type VI enzyme is the important member of matrix metalloproteinase family, degradable extracellular matrix main component IV Collagen Type VI, the growth of promotion tumor cell and transfer (Journal of theNational Cancer Institute 1997,89:1260).Suppress that matrix metalloproteinase produces and active small-molecule drug have in vivo obvious antineoplastic (Trends in Molecular Medicine1999,31:34).But at the monoclonal antibody of matrix metalloproteinase and less with the research of drug conjugates.Lidamycin LDM (Lidamycin, LDM claims C1027 again) be a kind of antitumor antibiotics that China develops voluntarily, obvious antineoplastic (Journal of Antibiotics 1989 is arranged in vivo and in vitro, 42:1294), its structure has detachable and the characteristics of rebuilding (Acta Pharmaceutica Sinica 1992,27:486).Lidamycin LDM and the segmental conjugate of rat anti human hepatocellular carcinoma BEL-7402 cell monoclonal antibody 3A5 Fab (Acta Pharmaceutica Sinica 1993,28:260) and Lidamycin LDM and type-IV collagenase-resisting monoclonal antibody 3D6 conjugate (foreign medical oncology credit volume 1998 5:68) all has obvious antitumor action in vivo and in vitro.But the active fragment of 3D6 and do not appear in the newspapers as yet with the Lidamycin LDM conjugate.Bleomycin A5 (Pingyangmycin PYM) is clinical a kind of antitumor antibiotics commonly used, Bleomycin A5 and monoclonal antibody 3A5 conjugate external to hepatocarcinoma BEL-7402 cell have selective killing effect (Chinese microorganism and Journal of Immunology 1991,11:230).Zoopery shows and can obviously prolong survival time of animals by this conjugate intracavitary administration (Acta Pharmaceutica Sinica 1997,32:669), but Bleomycin A5 and anti-matrix metalloproteinase monoclonal antibody and active fragment conjugate thereof do not appear in the newspapers.In sum, up to now, anti-matrix metalloproteinase monoclonal antibody active fragment and there is no report both at home and abroad with Lidamycin LDM and Bleomycin A5 conjugate.Studies show that of this invention, can obtain activated mice type-IV collagenase-resisting monoclonal antibody 3D6 Fab ' fragment with suitable method, itself and Lidamycin LDM and Bleomycin A5 conjugate have remarkable antitumor action in vivo and in vitro, might become new anti-tumor medicine.
The objective of the invention is to prepare monoclonal antibody 3D6 Fab ' fragment, and, obtain conjugate, be used for clinical treatment tumour this fragment and Lidamycin LDM and Bleomycin A5 coupling with suitable method.Content of the present invention and main points are:
1. the preparation of monoclonal antibody 3D6 Fab ' utilizes anti-matrix metalloproteinase monoclonal antibody 3D6 Fab ' fragment as the drug targeting carrier.At first prepare monoclonal antibody 3D6 Fab ' fragment with proper method.Monoclonal antibody 3D6 is with the 0.05mol/LTris-HCL buffer, and pH7.2 (containing 2mmol/L EDTA) fully is adjusted into 4mg/ml with same buffer in the dialysis back.Add the ficin that is dissolved in same buffer in advance by 0.06 unit enzyme/milligram antibody, again with 1mmol/L cysteine activating reaction, 37 ℃ were reacted 6 hours, and product obtains F (ab) ' 2 through Sephadex G-150 chromatography purification.6-10mg/ml F (ab) ' 2 reduces with beta-mercaptoethanol when pH7.5, product is through 0.05mol/L phosphate buffer (PB), after pH6.8 fully dialyses immediately with Lidamycin LDM crosslinked or with N-ethylomaleimide (NEMI) sealing sulfydryl after Sephadex G-75 chromatography purification obtains Fab ' is used for and the Bleomycin A5 coupling.Through enzyme-linked immunosorbent assay (ELISA), Fab ' maintenance
2 milligrams of Lidamycin LDMs of Fab ' and Lidamycin LDM conjugate preparation and N-hydroxy-succinamide base in right amount--(N-dimaleoyl imino) benzoate (MBS) mixes, room temperature lucifuge reaction 40 minutes, to mix with the 3D6 Fab ' of sulfhydrylation immediately after the desalination of PD-10 post, the reaction of room temperature lucifuge is spent the night.Add N-ethylomaleimide (NEMI), continue reaction 1 hour.Obtain the direct conjugate of Fab '-LDM through Sephadex G-75 chromatography purification.The direct conjugate doubled amount of Fab '-LDM chromophore mixes the back of spending the night and strengthens conjugate with Sephadex G-25 chromatography acquisition Fab '-LDM.
The preparation of Fab ' and Bleomycin A5 conjugate 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 12 hours add the 3D6 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.
4. the immunoreactivity of Fab '-LDM and Fab '-PYM conjugate and tumor cell is carried out with the ELISA method.Fab '-LDM and Fab '-PYM conjugate has kept and IV Collagen Type VI enzyme, human mouth scale cancer KB and pulmonary carcinoma PG cell immune response, with the human hepatocellular carcinoma BEL-7402 cell reactionless (Fig. 2, Fig. 3).
5. conjugate is measured conjugate to the human hepatoma cell BEL-7402 of In vitro culture and the lethal effect of oral squamous cell carcinomas KB cell, drug effect 1 hour to the lethal effect of tumor cell with clone forming method.Lidamycin LDM all has lethal effect to two kinds of cells as a result, and the concentration (IC50) that suppresses 50% clone's formation is respectively 2.0 * 10
-15Mol/L and 2.21 * 10
-15Mol/L; Fab '-LDM conjugate is respectively 1.04 * 10 to two kinds of cell IC50
-13Mol/L and 1.99 * 10
-15Mol/L.Show that Fab '-LDM has certain selective killing effect to target cell KB.Fab '-LDM and Lidamycin LDM are measured with the tetrazole blue laws the lethal effect of pulmonary carcinoma PG cell, and Lidamycin LDM and Fab '-LDM is respectively 3.7 * 10 to the IC50 of pulmonary carcinoma PG cell
-10Mol/L and 4.2 * 10
-10Mol/L (Fig. 4, Fig. 5, table 1).Similarly, PYM all has lethal effect to KB cell and the growth of BEL-7402 cell, and the concentration (IC50) that suppresses 50% clone's formation is respectively 0.42 μ mol/L and 0.17 μ mol/L; Fab '-PYM to two kinds of cell IC50 be respectively 0.012 μ mol/L and 0.48 μ mol/L (Fig. 6, Fig. 7).
Table 1MTT method mensuration Lidamycin LDM and Fab '-LDM are at external lethal effect to pulmonary carcinoma PG cell
Drug level (mol/L * 10
-10) A560 (% contrast)
Lidamycin LDM 88.16 21.97
8.816 34.89
0.882 67.95
0.088 77.00
0.009 101.82
Fab′-LDM 88.16 22.30
8.816 39.09
0.882 75.64
0.088 79.39
0.009 93.41
6. Fab '-LDM conjugate intravenous injection hepatocarcinoma 22 ascites that the curative effect of rat liver cancer 22 is got interior generation are diluted to 7.5 * 10 with normal saline
6Cell/ml, it is subcutaneous only to be inoculated in the kunming mice axillary fossa by 0.2ml/; Inoculate 24 hours posterior veins and inject (iv) administration, matched group gives normal saline, is administered once totally 3 times in per 3 days.Test and put to death animal on the 10th day, get tumor and weigh, calculate tumour inhibiting rate.The result shows that 3D6 Fab '-LDM has obvious curative effects to hepatocarcinoma 22,0.05mg/kg, 0.1mg/kg with 0.2mg/kg dosage, tumour inhibiting rate is respectively 86%, 89% and 91%, and 0.1mg/kg Lidamycin LDM tumour inhibiting rate is 77%, dosage conjugate (table 2) such as is starkly lower than.
Table 2 3D6 Fab '-LDM intravenous administration is to heavy (mg) tumour inhibiting rate of inhibitory action drug dose number of animals body weight (g) tumor of rat liver cancer 22
(mg/kg * number of times) beginning/end beginning/end x ± SD (%) contrast 10/10 19.4/27.4 1400 ± 373 Lidamycin LDMs 0.1 * 3 10/10 20.0/26.9 329 ± 97 77
*Fab '-LDM 0.05 * 3 10/10 19.7/27.3 203 ± 56 86
*, Δ
0.1×3 10/10 18.8/26.9 155±34 89
**，ΔΔ
0.2 * 3 10/10 19.4/27.1 129 ± 25 91
*, the Δ ΔThe subcutaneous vaccination tumor, intravenous administration, three days are once;
*P＜0.01 with compare
ΔCompare with the Lidamycin LDM group P＜0.05
The Δ ΔCompare with the Lidamycin LDM group P＜0.01
7. Fab '-LDM conjugate intravenous administration is (iv) got the subcutaneous Lewis cancerous tissue that goes down to posterity of C57/BL6 mice to the curative effect that Mice Bearing Lewis cancer lung shifts, and adds normal saline with 1: 3 and makes homogenate, and the C57/BL6 mice is pressed 0.2ml/ axillary fossa subcutaneous vaccination tumor liquid.Inoculate the 8th day and the 15th day each intravenous administration once.Test and handled animal on the 21st day, strip Subcutaneous tumor and weigh, get lungs, liquid-solid fixed with BouinShi, counting lung surface tumor knot calculates lung metastasis inhibition rate.The result shows, 3D6 Fab '-LDM does not have obvious influence to the growth of subcutaneous tumors, but transfer has obvious inhibitory action to Lewis cancer lung, 0.05mg/kg and 0.1mg/kg dosage, suppression ratio is respectively 91% and 94%, and 0.1mg/kg Lidamycin LDM suppression ratio is 88%, dosage conjugate (table 3) such as is starkly lower than.
Heavy (g) lung metastasis of inhibitory action group dosage number of animals body weight (g) subcutaneous tumors that the intravenous injection of table 3 conjugate is shifted subcutaneous vaccination Mice Bearing Lewis cancer lung
The sum suppression ratio
(mg/kg * number of times) beginning/end beginning/end x ± SD x ± SD (%) contrasts 10/10 18.9/22.1 8.53 ± 1.21 20.1 ± 2.2 Lidamycin LDMs 0.1 * 2 10/9 18.6/21.4 6.63 ± 1.17 2.4 ± 0.7 88
*Fab '-LDM 0.05 * 2 10/9 18.5/20.9 7.71 ± 1.41 1.9 ± 0.7 91
*
0.1 * 2 10/10 18.2/22.0 7.53 ± 0.63 1.2 ± 0.4 94
*, the Δ ΔThe subcutaneous vaccination tumor, intravenously administrable, seven days are once;
*P＜0.01 with compare
The Δ ΔCompare with the Lidamycin LDM group P＜0.01
Advantage of the present invention and good effect are: determined anti-matrix metalloproteinase monoclonal antibody 3D6Fab ' produced in fragments method and provide necessary foundation for anti-matrix metalloproteinase monoclonal antibody and Lidamycin LDM and Bleomycin A5 conjugate are used for the treatment of tumor.Bleomycin A5 is clinical antitumor antibiotics commonly used, and 3D6Fab '-PYM has the lethal effect stronger than Bleomycin A5 external to target cell; Lidamycin LDM is that a kind of cytotoxicity of report up to now is the strongest and the peptide class antitumor antibiotics of obvious anti-tumor in vivo effect arranged, as antitumor drug great development potentiality is arranged, and Lidamycin LDM and 3D6 Fab ' conjugate have the curative effect of the free Lidamycin LDMs of dosage such as obviously being better than to the laboratory animal tumor, estimating that monoclonal antibody 3D6 Fab ' fragment and Bleomycin A5 and Lidamycin LDM conjugate may be developed becomes the new antitumoral targeted drug and is used for clinical cancer therapy, and obtains good effect.
Description of drawings:Fig. 1 monoclonal antibody 3D6 Fab ' and IV Collagenase Type; KB; PG; BEL-7402 cell immune response wherein abscissa is that AC (μ g/ml) ordinate is that 490nm light absorption value 1 is BEL-7402 cytological map 2 monoclonal antibody 3D6 Fab '-LDM and IV clostridiopetidase As for KB cell 3 for PG cell 4 for IV Collagenase Type 2; KB; PG; BEL-7402 cell immune response wherein abscissa is that AC (μ g/ml) ordinate is that 490nm light absorption value 1 is BEL-7402 cytological map 3 monoclonal antibody 3D6 Fab '-PYM and IV clostridiopetidase As for KB cell 3 for PG cell 4 for IV Collagenase Type 2; KB; PG; BEL-7402 （μg／ml）490nm1IV2KB3PG4BEL-740243D6 Fab′-LDMKB （mol／L） （％）123D6 Fab′-LDM53D6 Fab′-LDMBEL-7402 （mol／L） （％）123D6 Fab′-LDM63D6 Fab′-PYMKB （μmol／L） （％）123D6 Fab′-PYM73D6 Fab′-PYMBEL-7402 （μmol／L） （％）123D6 Fab′-PYM。
Claims (6)
1. anti-matrix metalloproteinase monoclonal antibody Fab ' and drug conjugates 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 3D6 Fab ' active fragment;
B. the preparation of 3D6 Fab '-Lidamycin LDM conjugate;
C. the preparation of 3D6 Fab '-Bleomycin A5 conjugate;
D. the detection of the antitumor action of 3D6 Fab '-Lidamycin LDM conjugate;
E. the detection of the antitumor action of 3D6 Fab '-Bleomycin A5 conjugate;
2. according to described conjugate of claim 1 and antitumor action thereof, it is characterized in that utilizing anti-matrix metalloproteinase monoclonal antibody 3D6 to prepare Fab ' fragment.
3. according to described conjugate of claim 1 and antitumor action thereof, the preparation that it is characterized in that Fab '-Lidamycin LDM conjugate be with Lidamycin LDM earlier with special-shaped bifunctional reagent N-hydroxy-succinamide base--(N-dimaleoyl imino) benzoate (MBS) modifies, the Fab ' and the chromophore that add sulfhydrylation again, the purified product that obtains.
4. 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 mixed with Bleomycin A5 earlier, again with 3D6 Fab ' reaction, the purified product that obtains.
5. according to described conjugate of claim 1 and antitumor action thereof, it is characterized in that In vitro culture human hepatoma cell BEL-7402, oral squamous cell carcinomas KB cell and pulmonary carcinoma PG cell, with clone forming method or tetrazole blue laws detect free Lidamycin LDM or Bleomycin A5 and with the cell killing effect of 3D6 Fab ' conjugate.
6. according to described conjugate of claim 1 and antitumor action thereof, it is characterized in that animal vivo test, kunming mice subcutaneous vaccination hepatocarcinoma 22 or C57/BL6 mouse hypodermic inoculation Lewis cancer are judged free Lidamycin LDM and conjugate thereof the suppression ratio to tumor growth or neoplasm metastasis by intravenous administration.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103675262A (en) * | 2013-12-27 | 2014-03-26 | 步荣发 | Method of stable fluorescence labeling of living cells of oral squamous cell carcinoma |
| CN103948923A (en) * | 2014-05-12 | 2014-07-30 | 中国医学科学院医药生物技术研究所 | Pharmaceutical composition having antitumor collaborative synergistic effect |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9150659B2 (en) * | 2011-02-01 | 2015-10-06 | Gillian Murphy | Anti-tace antibody molecules and their uses |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103675262A (en) * | 2013-12-27 | 2014-03-26 | 步荣发 | Method of stable fluorescence labeling of living cells of oral squamous cell carcinoma |
| CN103948923A (en) * | 2014-05-12 | 2014-07-30 | 中国医学科学院医药生物技术研究所 | Pharmaceutical composition having antitumor collaborative synergistic effect |
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