CN1128157C - Conjugate of lidamycin with active fragment of monoclonal antibody - Google Patents
Conjugate of lidamycin with active fragment of monoclonal antibody Download PDFInfo
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- CN1128157C CN1128157C CN 01101937 CN01101937A CN1128157C CN 1128157 C CN1128157 C CN 1128157C CN 01101937 CN01101937 CN 01101937 CN 01101937 A CN01101937 A CN 01101937A CN 1128157 C CN1128157 C CN 1128157C
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- ldm
- lidamycin
- fab
- monoclonal antibody
- conjugate
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Abstract
The present invention adopts antitumor antibiotic lidamycin (LDM) as 'warhead' medicine coupled with a Fab'segment of a monoclonal antibody (single antibody). An anti-IV type collagenase single antibody 3D6 and an anti-human body liver cancer BEL-7402 cell single antibody 3A5Fab'segment are obtained by an appropriate method. N-hydroxy succinimide group-in-(N-maleimido) benzoic acid ester can be used as a coupling agent. Two kinds of single antibody Fab'segments are respectively coupled with the lidamycin, and two coupling substances Fab'-LDM can be obtained. The molecular weight of the coupling substances is measured as 65kDa by a polyacrylamide gel electrophoresis method, and the inhibiting action of KB and BEL-7402 cells cultured in vitro for the coupling substances is detected by a clone formation method. The result shows that the coupling substances have strong inhibiting action to target cells KB. Liver cancer 22 and colon cancer 26 can be used for in vivo experiment. The inhibiting action of Fab'-LDM coupling substances to liver cancer and colon cancer is obviously stronger than that of the lidamycin of the same dose. The present invention can determine that the lidamycin can be used as the 'warhead' medicine for preparing antitumor single antibody medicine coupling substances of miniaturization and high efficiency.
Description
The present invention relates to a kind of is " bullet " medicine with Lidamycin as antineoplastic antibiotic, the method for making conjugate and carrying out antineoplaston with monoclonal antibody (monoclonal antibody) Fab ' fragment.
Lidamycin (LDM) (lidamycin, LDM, claim C1027 again) be to separate an enediyne class antitumor antibiotics that screens the meta-bolites of a strain actinomycetes Streptomyces globisporus C1027 who obtains from Qianjiang county, China Hubei soil, (molecular weight is 10,500Da) forms with a nonatomic ring enediyne chromophoric group (molecular weight is 843Da) by a polypeptide chain.Lidamycin (LDM) has height lethal effect, its half-inhibition concentration (IC to tumour cell
50) 10
-17-10
-16Between the mol/L, stronger more than 10,000 times than conventional chemotherapy medicine Zorubicin etc., be the strongest peptide class antitumor antibiotics of the anti-tumor activity found so far (The Journal of Antibiotics 1989,42:1294).It is reported, lidamycin (LDM) and complete monoclonal antibody conjugate have obvious antitumor action (hi-tech communication 1993,3:4).The antitumor action of lidamycin (LDM) and rat anti human hepatocellular carcinoma BEL-7402 cell monoclonal antibody 3A5 Fab fragment conjugate is better than free lidamycin (LDM), show lidamycin (LDM) be make up the monoclonal antibody targeted drug comparatively ideal " bullet " medicine (Acta Pharmaceutica Sinica 1993,28:260).
For improving the penetration power of monoclonal antibody (monoclonal antibody) drug conjugates to tumor tissues, strengthen the lethal effect of conjugate to tumour cell, need the less conjugate of development molecular weight.Monoclonal antibody Fab ' fragment had both comprised the Fab fragment in structure, kept the part hinge region again, and was stronger to the penetration power of tumor tissues, can be used as the carrier of antitumor drug.It is reported that monoclonal antibody 3A5 Fab ' fragment and antitumor antibiotics Zhengguangmycin A5 conjugate have obvious antitumor action, its effect be better than free Zhengguangmycin A5 (Acta Pharmaceutica Sinica 2000,35:649).But do not see the report of lidamycin (LDM) and monoclonal antibody Fab ' fragment conjugate so far.Studies show that of this invention, lidamycin (LDM) and monoclonal antibody Fab ' fragment conjugate have the advantages that molecular weight is little, antitumor action is strong, may become the novel tumor medicine.
The objective of the invention is with lidamycin (LDM) is " bullet " medicine, and monoclonal antibody Fab ' fragment is that preparing carriers is efficient, the conjugate of monoclonal antibody medicine of miniaturization, is used for clinical treatment tumour.Content of the present invention and main points are:
1. monoclonal antibody 3A5 and the segmental preparation rat of 3D6 Fab ' anti-human liver cancer monoclonal antibody 3A5 are adjusted into 1.5mg/ml with citrate buffer solution, pH3.7.Add stomach en-by 1: 100 (w/v), 37 ℃ were reacted NaOH neutralization reaction liquid 6 hours.Reaction solution is collected F (ab ') 2 with Sephadex G-150 purifying.F (ab ') 2 fragments add final concentration 5mmol/L DTT, and room temperature reaction 1h removes excessive DTT with the PD-10 post, get the monoclonal antibody 3A5Fab ' fragment of sulfhydrylation.
Mouse type-IV collagenase-resisting monoclonal antibody 3D6 is adjusted into 4mg/ml, pH7.2 with the Tris-HCL damping fluid (containing 2mmol/L EDTA, the 1mmol/L aminothiopropionic acid) of 0.05mol/L.Add an amount of ficin, 37 ℃ of reactions 6 hours, product obtains F (ab ') 2 fragments through Sephadex G-150 chromatography purification.F (ab ') 2 fragments are through beta-mercaptoethanol reduction and PD-10 column purification, monoclonal antibody 3D6 Fab ' fragment that must sulfhydrylation.
2.Fab ' the preparation 2mg lidamycin (LDM) of fragment and lidamycin (LDM) conjugate is with 0.05M PB damping fluid, the pH6.8 dissolving.Add excessive 5 times N-hydroxy-succinamide base--(N-dimaleoyl imino) benzoic ether (MBS), room temperature lucifuge reaction 40 minutes, to mix with the monoclonal antibody 3A5 or the 3D6 Fab ' fragment of 1mg sulfhydrylation immediately after the desalination of PD-10 post, the oscillatory reaction of room temperature lucifuge is spent the night.Add 0.1mol/L N-ethylomaleimide (NEM), continue reaction 1 hour.Obtain monoclonal antibody Fab '-direct conjugate of LDM through Sephadex G-75 chromatography purification.
Fab '-LDM strengthens the preparation of conjugate and gets 1mg monoclonal antibody the Fab '-direct conjugate doubled amount of LDM LDM chromophoric group, mixes the back of spending the night and strengthens conjugate with Sephadex G-25 chromatography acquisition monoclonal antibody Fab '-LDM.
3. the conjugate molecular weight determination is measured the conjugate molecular weight with non-reduced type polyacrylamide gel electrophoresis.The result shows that monoclonal antibody Fab '-LDM conjugate molecular weight is 65kDa, and Fab ' fragment is 1: 1 (Fig. 1) with lidamycin (LDM) coupling molecule ratio.
4. conjugate immunocompetence conjugate immunocompetence detects with the ELISA method.Monoclonal antibody 3A5 Fab '-LDM conjugate and human hepatoma cell BEL-7402 and mouse junction cancer 26 (C26) cell all respond, but with human oral cavity squamous cell carcinoma KB reactionless (Fig. 2).
The Fab ' of monoclonal antibody 3D6-LDM conjugate and human oral cavity squamous cell carcinoma KB and rat liver cancer 22 (H22) cell all are the immunology positive reaction, but reactionless with liver cancer BEL-7402 cell, show that conjugate has selectivity (Fig. 3) to the reaction of tumour cell.
5. conjugate is measured drug effect 1 hour to the KB and the BEL-7402 cell of the effect vitro culture of the tumour cell of vitro culture with clone forming method.The Fab ' of monoclonal antibody 3A5-LDM conjugate is better than free lidamycin (LDM) to the effect of target cell BEL-7402, and the two suppresses the concentration (IC that 50% clone forms
50) be respectively 2.25 * 10
-17Mol/L and 2.64 * 10
-16Mol/L; Effect to non-target cell KB is suitable, IC
50Be respectively 2.49 * 10
-15Mol/L and 2.27 * 10
-15Mol/L (Fig. 4).
The Fab ' of monoclonal antibody 3D6-LDM conjugate is to the IC of target cell KB and non-target cell BEL-7402
50Be respectively 1.99 * 10
-15Mol/L and 1.04 * 10
-13Mol/L, and lidamycin (LDM) is to the IC of KB and BEL-7402 cell
50Be respectively 2.21 * 10
-15Mol/L and 2.0 * 10
-15Mol/L (Fig. 5).Conjugate has kept the strongly inhibited effect to target cell, but the effect of non-target cell is descended.Show that conjugate has selective killing effect to target cell.
6. the Fab ' of monoclonal antibody 3A5-LDM conjugate strips the colorectal carcinoma 26 knurl pieces that subcutaneous vaccination was gone down to posterity 10 days to the therapeutic action of animal subcutaneous vaccination colorectal carcinoma 26, add physiological saline by 1: 3 and make homogenate, it is subcutaneous to be inoculated in the BALB/c mouse armpit, begin treatment behind the 24h, the Fab ' of intravenous injection lidamycin (LDM) or monoclonal antibody 3A5-LDM conjugate, be administered once totally 3 times in per 3 days.Experimental session is weighed weekly and is measured knurl volume twice, with formula 1/2ab
2(a: tumour major diameter, b: the tumour minor axis) calculate the knurl volume.Record tumor growth situation and survival time.26 growths have obvious restraining effect to Fab '-LDM that the result shows monoclonal antibody 3A5 to BALB/c mouse subcutaneous vaccination colorectal carcinoma, intravenous injection 0.05mg/kg, 0.1mg/kg and 0.2mg/kg dosage, administration 3 times, conjugate can obviously suppress C26 growth of tumor (Fig. 6), and survival time of animals prolongs (Fig. 7).
7. the Fab ' of monoclonal antibody 3D6-LDM conjugate is to the therapeutic action kunming mice subcutaneous vaccination 1.5 * 10 of animal subcutaneous transplantation liver cancer 22
6Individual cell.Begin treatment behind the 24h, intravenous injection lidamycin (LDM) or Fab '-LDM conjugate was administered once totally 4 times in per 3 days.Experimental session was measured a tumour size in per two days, with formula 1/2ab
2(a: tumour major diameter; B: the tumour minor axis) calculate gross tumor volume and write down death time of animal.The result shows that treatment group tumor growth is slow, survival time of animals obviously prolong (Fig. 8, Fig. 9).
Advantage of the present invention and positively effect are: developed lidamycin (LDM) and monoclonal antibody Fab ' fragment conjugate.Lidamycin (LDM) is the strongest peptide class antitumor antibiotics of finding so far of antitumor action, be expected to become new anti-tumor medicine, and lidamycin (LDM) and monoclonal antibody Fab ' fragment conjugate have the advantages that molecular weight is little, antitumor action is strong, may be developed to new antitumoral monoclonal antibody targeted drug and be used for clinical cancer therapy, and obtain good efficacy.
：1 Fab’-LDM1 ( kDa ) 2Fab’-LDM3Fab’2 3A5Fab’-LDMBEL-7402、C26KB ( mg/ml ) 490nm1BEL-74022C263KB3 3D6Fab’-LDMKB、H22BEL-7402 ( mg/ml ) 490nm1H222KB3BEL-74024 3A5Fab’-LDMKBBEL-7402 ( mol/L ) ( ％ ) 1BEL-74022Fab’-LDMBEL-74023KB4Fab’-LDMKB5 3D6Fab’-LDMBEL-7402KB ( mol/L ) ( ％ ) 1KB2Fab’-LDMKB3BEL-74024Fab’-LDMBEL-74026 3A5Fab’-LDM26 ( ) ( cm3) 1 for contrast 2 for the lidamycins 3 of 0.1mg/kg dosage for the Fab'-LDM 4 of 0.05mg/kg dosage for the Fab'-LDM 5 of 0.1mg/kg dosage for the Fab'-LDM of Fab'-LDM Fig. 7 lidamycin of 0.2mg/kg dosage and monoclonal antibody 3A5 on the impact of subcutaneous vaccination colon cancer 26 mouse life spans wherein abscissa be days post inoculation (day) ordinate for survival percentage (%) 1 for contrast 2 for the lidamycins 3 of 0.1mg/kg dosage be the Fab'-LDM 4 of 0.05mg/kg dosage be 0.1mg/kg dosage Fab'-LDM 5 for the Fab'-LDM of Fab'-LDM Fig. 8 lidamycin of 0.2mg/kg dosage and monoclonal antibody 3D6 to the inhibitory action of mouse hypodermic inoculation liver cancer 22 wherein abscissa be days post inoculation (my god) ordinate is gross tumor volume (cm3) 1 for contrast 2 for the lidamycins 3 of 0.1mg/kg dosage for the Fab'-LDM 4 of 0.05mg/ dosage for the Fab'-LDM 5 of 0.1mg/kg dosage for the Fab'-LDM of Fab'-LDM Fig. 9 lidamycin of 0.2mg/kg dosage and monoclonal antibody 3D6 on the impact of subcutaneous vaccination liver cancer 22 mouse life spans wherein abscissa be days post inoculation (day) ordinate for survival percentage (%) 1 be to contrast 2 to be the Fab'-LDM of 0.2mg/kg dosage for the Fab'-LDM 4 of 0.05mg/kg dosage for the Fab'-LDM 5 of 0.1mg/kg dosage for the lidamycin 3 of 0.1mg/kg dosage
Claims (5)
1, lidamycin (LDM) (LDM) and monoclonal antibody (monoclonal antibody) active fragments conjugate is characterized in that said conjugate is made up of through chemical coupling by 1: 1 molecular ratio Fab ' fragment and the lidamycin (LDM) (LDM) of monoclonal antibody 3A5, and its molecular weight is 65kDa.
2, the preparation method of lidamycin (LDM) (LDM) and monoclonal antibody active fragments conjugate is characterized in that this method may further comprise the steps:
A. the preparation of the Fab ' active fragments of monoclonal antibody 3A5;
B.3A5Fab '-preparation of LDM conjugate.
3, preparation method as claimed in claim 2 is characterized in that 3A5Fab ' active fragments is to use pepsin hydrolysis rat anti-human liver cancer monoclonal antibody 3A5 earlier, and acquisition F (ab ') 2, add DTT again, obtain the 3A5Fab ' fragment of sulfhydrylation.
4, preparation method as claimed in claim 2, it is characterized in that 3A5Fab '-LDM conjugate be earlier with lidamycin (LDM) with the N-hydroxy-succinamide base--(N-dimaleoyl imino) benzoic ether modifies, with Fab ' the fragment coupling of monoclonal antibody 3A5, obtain again through Sephadex G-75 purifying.
5, lidamycin (LDM) and the monoclonal antibody active fragments conjugate application in the preparation antitumor drug.
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|---|---|---|---|
| CN 01101937 CN1128157C (en) | 2001-01-18 | 2001-01-18 | Conjugate of lidamycin with active fragment of monoclonal antibody |
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| CN1128157C true CN1128157C (en) | 2003-11-19 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101002947B (en) * | 2007-01-16 | 2010-08-25 | 湖南农业大学 | Target medicine used for treating bacteriosis, and its preparing method |
| CN101143902B (en) * | 2007-05-18 | 2010-06-02 | 中国医学科学院医药生物技术研究所 | Anti-HER2 single-chain antibody-cefuroxime sodium enhanced fusion protein HER2(Fv-LDM) |
| CN101475643B (en) * | 2009-01-16 | 2012-05-09 | 中国医学科学院医药生物技术研究所 | Double single chain antibody strengthened fusion protein dFv-LDP-AE, preparation and use thereof |
| CN108721641B (en) * | 2017-04-14 | 2021-05-11 | 中国医学科学院医药生物技术研究所 | Antibody drug conjugate of anti-CD30antibody and lidamycin, preparation method and application thereof |
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