CN1240721C - Conjugate of lidamycin, monoclonal antibody and its Fab'segment and its application in targeting treatment of colon cancer and other tumors - Google Patents
Conjugate of lidamycin, monoclonal antibody and its Fab'segment and its application in targeting treatment of colon cancer and other tumors Download PDFInfo
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Abstract
The present invention relates to a conjugate of lidamycin, monoclonal antibodies and Fab' segments thereof, and an application of the conjugate in a targeted therapy for the tumours such as colon cancer, etc. 2-imidogen tetrahydrothiophene (2-IT) and N-hydroxysuccinimide base-m-(N-maleimide base) benzoate (MBS) or other cross linking agent molecules are used as intermediates to couple monoclonal antibody 3G11 and the Fab' segments thereof for resisting type IV collagenase with lidamycin(LDM), and therefore, monoclonal antibody 3G11-LDM conjugate and Fab'-LDM conjugate are obtained. The immunohistochemical staining is carried out with colon cancer, lung cancer, oesophagus cancer, gastric cancer, mammary cancer and malignant tumors by using the monoclonal antibody 3G11, and expressions of the increase of the activity of type IV collagenase are displayed in the digestive tract tumours. The indirect ELISA method mensurates that both the monoclonal antibody 3G11 and Fab' segments thereof have a strong immunity combining capability when combined with human colon cancer HT-29, lung cancer PG, squamous cancer KB, colon cancer 26 (C26) and liver cancer 22 (H22), and the monoclonal antibody 3G11 can generate a clear immunization video picture for the human FG lung cancer transplanted into a nude mouse. Experiments in vivo are used for curing mouse colon cancer and liver cancer, compared with free LDM in the same dosage, both monoclonal antibody 3G11-LDM conjugate and the Fab'-LDM conjugate show a stronger tumor growth inhibiting function. Therefore, 3G11-LDM conjugate and Fab'-LDM conjugate are possibly used for a targeted therapy for malignant tumors such as colon cancer, etc.
Description
Technical field:
The present invention relates to a kind of immune conjugate and preparation method and application of forming by lidamycin (LDM) and monoclonal antibody and fragment thereof in neoplasm targeted therapies such as colorectal carcinoma.
Background technology:
Monoclonal antibody (monoclonal antibody) has high degree of specificity to corresponding antigen.Can prepare specificity bonded monoclonal antibody with it at specific molecular target.Therefore, monoclonal antibody has great potential and application prospect in the targeted therapy of tumour.Monoclonal antibody therapeutical agent or title monoclonal antibody medicine generally comprise two classes, and the one, antitumor monoclonal antibody; The 2nd, antitumor monoclonal antibody conjugate or title immune conjugate.The immune conjugate molecule is made of monoclonal antibody and " bullet " medicine two portions.Monoclonal antibody at molecular target be generally the tumor associated antigen of tumor cell surface or specific acceptor.The material that can be used as " bullet " mainly contains three classes, i.e. radionuclide, medicine and toxin are connected with monoclonal antibody and constitute radioimmunity conjugate, chemo-immunity conjugate and immunotoxin respectively.Since nineteen ninety-seven, Rituxan, Herceptin and Mylotarg are in the granted in succession clinical cancer therapy that is applied to of the U.S., and the research and development of monoclonal antibody medicine show the growth momentum that makes new advances, and become the new focus in biotech drug field.
That development monoclonal antibody targeted drug needs is clear and definite, with the molecular target of oncotherapy height correlation.To consider humanization, miniaturization and the high efficiency of monoclonal antibody medicine on this basis.Humanization be meant the antagonist molecule particularly its Fc fragment transform the human antimouse antibody reaction (HAMA) that produces when using to be reduced in human body.Miniaturization be to use antibody fragment (Fab, Fab ' or scFv) and " bullet " material make up conjugate.The monoclonal antibody medicine of miniaturization more easily by capillary endothelium layer and tumour cell external series gap, may arrive the solid tumor deep; And owing to removed the Fc fragment, the monoclonal antibody medicine of miniaturization also can reduce the HAMA reaction.High efficiency is to make up the monoclonal antibody medicine that tumour cell is had extremely strong lethality.Because use to show curative effect the low dose of and short course of treatment, the high efficiency monoclonal antibody medicine also may reduce the HAMA reaction.Complete monoclonal antibody molecule contains the Fc fragment, helps transferring the effector function that body immune system is attacked tumour cell; If utilize complete monoclonal antibody molecule and efficient " bullet " medicine to make up the conjugate of high efficiency, might not only reduce the HAMA reaction but also can keep the segmental effector function of Fc.Therefore, development is still significant in neoplasm targeted therapy based on the immune conjugate of complete monoclonal antibody.
Lidamycin (LDM) (LDM has another name called C-1027) is screening anti-tumor medicine method-spermatogonium method that Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences utilizes our unit to set up, and finds through a large amount of fermentation broth sample of screening.The generation bacterium of LDM is to separate to obtain from the soil in Qianjiang county, China Hubei Province, name for Streptomyces globisporus C-1027 (submit to before this " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " preservation, deposit number is CGMCC No.0704).The LDM molecule is by a chromophoric group (Chromophore) and the new compound that acidic protein constitutes.Chromophoric group contains a nonatomic ring enediyne core texture, and molecular weight is 843Da, and protein part is made up of 110 amino acid, and molecular weight is 10506Da.LDM has the intensive killing activity to cancer cells, and animal vivo test shows, to mice transplanted tumor and the growth of tumor such as people's liver cancer that nude mice is transplanted have remarkable restraining effect (Chinese microbiotic magazine 1994,19＜2 〉: 164).With LDM be " bullet " medicine respectively with the Fab fragment (Acta Pharmaceutica Sinica 1993 of anti-liver cancer monoclonal antibody 3A5,28＜4 〉: 260-265) or Fab ' fragment (Chinese Academy of Medical Sciences's journal 2001,23＜6 〉): 563) conjugate of Gou Chenging shows stronger antitumor action at animal experiment.Conjugate about LDM and complete monoclonal antibody structure does not still have report so far.More than Bao Dao 3A5 is with BEL-7402 liver cancer cell immune rat and through the rat monoclonal antibody of hybridoma technology preparation, is that the mouse monoclonal antibody 3G11 of antigen prepd is different fully with the present invention with IV Collagen Type VI enzyme.
Matrix metalloproteinase (MMPs) exists confidential relation with growth, invasion and attack, the transfer of malignant tumour.The contiguous mesenchymal cell of cancer cells or its is by secreting multiple matrix metalloproteinase degrade basilar membrane and extracellular matrix components, thereby helps the invasion and attack and the transfer process of tumour.Studies show that the expression of MMPs and active height are relevant with the grade malignancy of tumour, and MMPs also there is material impact in the vasculogenesis of tumour.Therefore, MMPs becomes the molecular target of noticeable oncotherapy.IV Collagen Type VI enzyme is called gelatinase again, comprises two components of MMP-2 and MMP-9, be the important member in the MMPs family, it can be degraded and make up the IV Collagen Type VI of support in the basilar membrane as other component, thereby helps tumor cell destruction and penetrate basilar membrane, attacks and shifts.It is reported, suppress the secretion of IV Collagen Type VI enzyme or other MMPs and active small-molecule drug thereof have in vivo the effect that suppresses tumor growth and transfer (J Natl Cancer Inst 2001,93:178).Inventor laboratory once utilized IV Collagen Type VI enzyme monoclonal antibody 3D6 and Zhengguangmycin A5 to make conjugate, and in animal body the evidence conjugate than Zhengguangmycin A5 have stronger tumor-inhibiting action (Chinese microbiotic magazine 2002,27＜8 〉: 496).Utilize IV Collagen Type VI enzyme monoclonal antibody and efficient " bullet " medicine lidamycin (LDM) to make conjugate, still do not have report so far.
The monoclonal antibody 3G11 that the present invention relates to (its hybridoma 3G11 cell strain on November 7th, 2002 submit to the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " preservation; deposit number is CGMCC No.0831), be to adopt the preparation of hybridoma technology.After IV Collagen Type VI enzyme immunity BALB/c mouse, get its splenocyte and mouse myeloma SP2/0 cytogamy, the hybridoma cell strain excretory antibody that behind colony screening, is obtained.This antibody molecule amount is about 150kDa, and its activity can combine and neutralize with IV Collagen Type VI enzyme spcificity.This laboratory study finds that 3G11 carries out immunohistochemical staining with the type-IV collagenase-resisting monoclonal antibody, and tumor tissues coloration results such as people's lung cancer, colorectal carcinoma, the esophageal carcinoma, cancer of the stomach and mammary cancer are positive.Human colon carcinoma tissue and normal adjacent tissue thereof compared when observing find, healthy tissues comprises that the immunohistochemical staining of enteraden cell and mesenchymal cell and monoclonal antibody 3G11 is negative, colon cancer tissue then presents positive staining in various degree, shows that IV Collagen Type VI enzyme is specific expressed at colon cancer cell.Show that through the ELISA detection monoclonal antibody 3G11 and people's lung cancer PG cell, human colon carcinoma HT-29 cell, rat liver cancer H22 cell and mouse junction cancer C26 cell are positive.At people's lung cancer PG tumor model that nude mice is transplanted, use
[3]The monoclonal antibody 3G11 intravenous injection of iodine labeling is tumor imaging clearly as seen.Research recently shows that also LDM has obvious restraining effect to transplanting in people's lung cancer PG of nude mice growth of tumor.The present invention be with Lidamycin as antineoplastic antibiotic (LDM) as the bullet medicine, make 3G11-LDM conjugate and Fab '-LDM conjugate with monoclonal antibody 3G11 and Fab ' fragment thereof respectively, and observed its antitumor action of testing in animal body.The result shows that compare with the free LDM of Isodose, the antitumor action of 3G11-LDM conjugate and Fab '-LDM conjugate significantly strengthens.Therefore, 3G11-LDM conjugate and Fab '-LDM conjugate might become the new antitumoral targeted drug of treatment tumour.
The objective of the invention is to prepare monoclonal antibody 3G11-LDM conjugate and monoantibody segment Fab '-LDM conjugate, and this conjugate is applied to tumor treatment with suitable method.
Summary of the invention:
1. monoclonal antibody 3G11-LDM conjugate
1.1 monoclonal antibody 3G11-LDM conjugate involved in the present invention is the complete antibody molecule of type-IV collagenase-resisting monoclonal antibody 3G11 and antitumor antibiotics LDM with 1: 1 molecular ratio, connects formed chemo-immunity conjugate by the small molecules linking agent.A kind of conjugate molecular structure wherein by link molecule 2-imino-tetramethylene sulfide (2-IT) and N-hydroxy-succinamide base--(N-dimaleoyl imino) benzoic ether (MBS) etc. is connected to form; The another kind of molecular structure of 3G11-LDM conjugate involved in the present invention can be to connect formed chemo-immunity conjugate by link molecule such as N-hydroxy-succinamide base-3-(2-pyridine disulfide group)-propionic ester (SPDP) etc.The 3G11-LDM conjugate is measured through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and molecular weight is about 160kDa.Wherein the molecular weight of monoclonal antibody 3G11 is about 150kDa, and the molecular weight of LDM is 11349Da, and the molecular weight of 3G11-LDM conjugate is equivalent to both sums, illustrates that the molecular ratio of monoclonal antibody 3G11 and LDM is 1: 1 in the conjugate.
1.2 utilize type-IV collagenase-resisting monoclonal antibody 3G11 as the oriented carrier that carries antitumor drug with 2-IT and MBS as the preparation of the monoclonal antibody 3G11-LDM conjugate of corsslinking molecular: (1) monoclonal antibody 3G11 behind sad ammonium sulfate precipitation and hydroxyapatite column purification, put in the dialysis tubing that molecular weight cut-off is 10kDa, (pH8.0) dialyses with the 0.05M borate buffer, and regulating its concentration is 5mg/ml.Get that to be dissolved in 0.05M borate buffer (pH8.0) concentration be that the 2-IT solution 10 μ l of 4mg/ml add in the 3G11 solution of above-mentioned 5mg/ml, under the condition of inflated with nitrogen in room temperature reaction 40min.After reaction finished, putting molecular weight cut-off was in the centrifugal evaporating pipe of 30kDa, (pH6.8) carried out buffer-exchanged with 0.05M phosphate buffered saline buffer (PB), and centrifugally removed unreacted 2-IT.(2) power taking reaches mycin and is dissolved in the concentration that is made into 5mg/ml among the 0.05MPB (pH6.8), stirs that to add the concentration that is dissolved in dimethyl formamide down be the MBS of 10mg/ml, room temperature reaction 40min.After reaction finishes, put in the dialysis tubing that molecular weight cut-off is 10kDa, with 0.05MPB (pH6.8) 16h that fully dialyses.After finishing, dialysis carries out linked reaction with the monoclonal antibody 3G11 that modifies through 2-IT immediately, room temperature reaction 6h or spend the night.Reaction solution is put in the centrifugal evaporating pipe of ultrafiltration that molecular weight cut-off is 30kDa, and centrifugal ultrafiltration 3-4 time is removed the lidamycin (LDM) that unreacted MBS modifies, and obtains the conjugate of 3G11-LDM.
1.3 with the preparation of N-hydroxy-succinamide base-3-(2-pyridine the disulfide group)-propionic ester (LC-SPDP) of SPDP or long-chain as the 3G11-LDM conjugate of corsslinking molecular: (1) get LDM 2mg be dissolved in phosphate buffered saline buffer (PBS) (pH7.4) in, be made into the concentration of 4mg/ml, adding mole number then is 10 times of excessive SPDP or LC-SPDP (15mg/ml, be dissolved in the dimethyl formamide), room temperature reaction 30min.It is fully dialysis in the 0.1mol/L acetate buffer solution (pH4.5) that reaction finishes rearmounted concentration.Get dialyzed solution, adding DTT (DTT) is 10mmol/L to final concentration, and room temperature reaction 30min puts fully dialysis among the PBS then.(2) get monoclonal antibody 3G11 2mg in addition and be dissolved in the 0.2mol/L phosphate buffered saline buffer (pH6.8), adding mole number is 10 times of excessive SPDP or LC-SPDP, room temperature reaction 30min, fully dialysis in PBS liquid.(3) dialyzed solution in the merging (1) and (2), room temperature reaction spends the night.Separate through the SephadexG-75 post then, collect first peak, obtain the conjugate of 3G11-LDM.
2. monoantibody segment Fab '-LDM conjugate
2.1 monoantibody segment Fab ' involved in the present invention-LDM conjugate is the Fab ' fragment of type-IV collagenase-resisting monoclonal antibody 3G11 and antitumor antibiotics LDM with 1: 1 molecular ratio, connects formed chemo-immunity conjugate by micromolecular linking agent.Fab ' the fragment of monoclonal antibody 3G11 can obtain Fab '-LDM conjugate by the sulfydryl and the LDM coupling of modifying through special-shaped bi-functional cross-linking agent MBS of its molecule hinge area.The SDS-PAGE electrophoresis result shows that the molecular weight of Fab '-LDM conjugate is about 65000Da, compares the lucky molecular weight (11345Da) that has increased a LDM molecule with Fab ' fragment, and Fab ' is 1: 1 with the molecular ratio of LDM in the supposition conjugate.
2.2 the monoclonal antibody 3G11 of the segmental preparation purifying of monoclonal antibody 3G11 Fab ' (pH7.4) fully dialyses with 0.05mol/LTrisHCl damping fluid (containing the EDTA that concentration is 2mmol/L), adjusting its concentration with same buffer is 4mg/ml.Behind 37 ℃ of pre-temperature 30min, add in advance with the same buffer dissolving and at the ficin (0.06U/mg antibody) of 37 ℃ of pre-temperature 30min, and add the halfcystine activating reaction that 1/9 volumetric concentration is 10mmol/L, put 37 ℃ of slow stirring reaction 5h.Then, with 1/10 volumetric concentration be the NEM termination reaction of 100mmol/L.Reaction solution is with Sephadex G-150 column chromatography purification, collects F (ab ')
2Fragment.The F of purifying (ab ')
2Fragment is dialysed with 0.1mol/L TrisHCl damping fluid (containing 10mmol/L EDTA) (pH 7.5), and becomes 6～10mg/ml through ultrafiltration and concentration.Then with the beta-mercaptoethanol of 10～15mmol/L, 37 ℃ of reduction 1h or 37 ℃ of reductase 12 h of 10mmol/L halfcystine, reduzate with 0.05mol/L phosphoric acid buffer (PB) (pH6.8) under the condition of inflated with nitrogen, fully dialysis obtains Fab ' fragment, is used for lidamycin (LDM) crosslinked immediately.
2.3 the preparation lidamycin (LDM) 20mg of monoantibody segment Fab '-LDM conjugate dissolves with 0.05mol/LPB (pH6.8), be mixed with the concentration of 5mg/ml, adding mole number then is 10 times of excessive MBS, room temperature lucifuge reaction 40min, reaction solution is put in the dialysis tubing that molecular weight cut-off is 10kDa, abundant dialysis 16h removes excessive MBS in 0.05mol/LPB (pH6.8).Dialysis finishes, and mixes with the Fab ' fragment of sulfhydrylation immediately, and room temperature reaction spends the night under the lucifuge condition, with the NEM termination reaction.Reaction solution is put in the centrifugal evaporating pipe of ultrafiltration that molecular weight cut-off is 30kDa, and centrifugal ultrafiltration 3-4 time is removed the LDM that unreacted MBS modifies, and obtains monoantibody segment Fab '-LDM conjugate.
3. the immunocompetence and the antitumous effect of monoclonal antibody 3G11-LDM conjugate and monoantibody segment Fab '-LDM conjugate
The said monoclonal antibody 3G11 of the present invention shows in the immunohistochemical staining experimental result of different human body tumor tissues, malignant tumor tissues such as colorectal carcinoma, lung cancer, mammary cancer, the esophageal carcinoma, cancer of the stomach present positive staining to monoclonal antibody 3G11, and the negative dyeing of healthy tissues around colorectal carcinoma illustrates that the expression of IV Collagen Type VI enzyme in multiple malignant tumor tissue has specificity.ELISA detects and shows, monoclonal antibody 3G11 and Fab ' fragment thereof show immunoreactivity to colorectal carcinoma, lung cancer, liver cancer cell.Experiment showed, that the present invention's said monoclonal antibody 3G11-LDM conjugate and monoantibody segment Fab '-LDM conjugate has kept the binding ability with IV Collagen Type VI enzyme and above-mentioned various tumour cells.The said 3G11-LDM conjugate of the present invention and Fab '-LDM conjugate show that to the lethal effect experiment of tumour cell conjugate is stronger to the cytotoxicity of tumour cell than using LDM separately, and become dependence with the concentration of conjugate.Animal vivo test proves, when said 3G11-LDM conjugate of the present invention and Fab '-LDM conjugate intravenous administration, rat liver cancer, colorectal carcinoma had notable therapeutic effect, and its tumour inhibiting rate is apparently higher than independent use LDM.
Effect of the present invention is, utilize the immunoreactivity of malignant tumor tissues such as type-IV collagenase-resisting monoclonal antibody 3G11 and colorectal carcinoma, lung cancer, mammary cancer, the esophageal carcinoma, cancer of the stomach and the characteristics of distribution of specific in vivo thereof, make 3G11-LDM conjugate and Fab '-LDM conjugate respectively with high vigor " bullet " medicine lidamycin (LDM); Determined the preparation method of 3G11-LDM conjugate and Fab '-LDM conjugate; In vitro tests shows that 3G11-LDM conjugate or Fab '-LDM conjugate is compared with free LDM, and tumour cell is shown stronger lethal effect; Animal vivo test proves that 3G11-LDM conjugate or Fab '-LDM conjugate has very strong restraining effect to tumor growth, and its antitumous effect is apparently higher than free LDM or antibody itself.Therefore, expection monoclonal antibody 3G11-LDM conjugate and fragment Fab '-LDM conjugate thereof are expected to develop and become novel antineoplastic target medicine and be used for clinical cancer therapy, and obtain good effect.
Description of drawings:
The immunohistochemical staining of Fig. 1 monoclonal antibody 3G11 and human colon carcinoma tissue
Wherein: the figure right side is a colon cancer tissue, has the dyeing positive particle to be distributed in the cell top in the cancer cells that visible body of gland sample is arranged; The figure left side is the body of gland of normal mucous membrane of colon.
The immunohistochemical staining of Fig. 2 monoclonal antibody 3G11 and people's cancerous lung tissue
Wherein: lung carcinoma cell is positive, and more lymphocyte is arranged on every side.
The immunohistochemical staining of a Fig. 3 monoclonal antibody 3G11 and a routine human colon carcinoma tissue
Wherein: have the dyeing positive particle to be distributed in the cell top in the cancer cells of figure right side for the arrangement of body of gland sample; The figure left side is the body of gland of normal mucous membrane of colon.
The immunoreactivity of Fig. 4 monoclonal antibody 3G11 and lung cancer PG cell, colorectal carcinoma HT-29 cell, liver cancer H22 cell, colorectal carcinoma C26 cell and oral squamous cell carcinomas KB cell
--PG ■--HT-29 △--H22 *--KB Θ--C-26 wherein: υ
Fig. 5 is inoculated in 96 hours the radio-immuno-image of human cytomegalovirus lung cancer PG tumour of nude mice.
The immunoreactivity of Fig. 6 monoclonal antibody 3G11-LDM conjugate and colorectal carcinoma HT-29 cell, liver cancer H22 cell, colorectal carcinoma C26 cell and oral squamous cell carcinomas KB cell
--HT-29 ■--KB ▲--C-26--H-22 wherein: υ
Fig. 7 monoclonal antibody 3G11-LDM conjugate is to the restraining effect (mtt assay) of H22 cell proliferation
Wherein: ▲--LDM ■--3G11-LDM
Fig. 8 monoantibody segment Fab '-LDM conjugate is to the restraining effect (mtt assay) of H22 cell proliferation
Wherein: ◆--LDM ■--Fab '-LDM
Fig. 9 monoclonal antibody 3G11-LDM conjugate is to the restraining effect of liver cancer H22 tumor growth
Wherein: zero--contrast ■--3G11 (0.75) △--LDM (0.05)
□---3G11+LDM(0.75+0.05) υ--3G11-LDM(0.025)
●--3G11-LDM(0.05) ▲--3G11LDM(0.10)
(bracket inner digital is represented used dosage mg/kg)
Figure 10 monoantibody segment Fab '-LDM conjugate is to the restraining effect of liver cancer H22 tumor growth
Wherein: zero--contrast ■--Fab ' (0.25) △--LDM (0.05)
●--Fab’-LDM(0.025)
□--Fab’-LDM(0.05) ▲--Fab’-LDM(0.10)
(bracket inner digital is represented used dosage mg/kg)
Embodiment:
The embodiment of following the anti-tumor activity for the 3G11-LDM conjugate, just illustrative for the purpose of the present invention, and nonrestrictive.
Embodiment 1
Monoclonal antibody 3G11 is at the conventional dewaxing of the immunohistochemical staining paraffin section entry of different people tumor tissues, 3%H
2O
2Deactivation endogenous peroxidase, immerse 0.01M citrate buffer (pH6.0) microwave antigen retrieval, I is anti-to be monoclonal antibody 3G11, incubated at room 1 hour, adopt strepto-avidin-vitamin H-enzyme connection mixture (SABC) method to dye, with DAB test kit color development at room temperature about 30 seconds to 5 minutes again, conventional Hematorylin was slightly redyed, and transparent mounting dewaters.
Coloration result is observed judging criterion: the dyeing of IV Collagen Type VI enzyme is painted for cell cytosol, has pale brown color depth to dye the positive cell of particle in the endochylema.The employing semiquantitative method is analyzed, and artificial grade scale is divided into 0 with the cell dyeing depth degree: negative (-), 1: the weak positive (+), 2: positive (++), 3: strong positive (+++), 4: the extremely strong positive (++ ++).
The image quantitative analysis stained deposits in the computer through microimaging scanning, and carries out quantitative analysis with LEICAQ500IW type image analysis system.20 visuals field are chosen in every section respectively, automatically (the gray-scale value scope is between 0-255 to detect and calculate the average positive percentage in each visual field, positive area percentage and positive staining intensity=255-average gray value, the most black place of 0 representative, 255 represent high light).
Monoclonal antibody 3G11 malignant tumor tissues such as human colorectal cancer, lung cancer, mammary cancer, the esophageal carcinoma, cancer of the stomach, kidney are shown high positive reactivity (table 1) (Fig. 1, Fig. 2).Tissue slice in the experiment is equipped with blank (substituting monoclonal antibody 3G11 with PBS), negative control (substituting monoclonal antibody 3G11 with irrelevant monoclonal antibody F9), and coloration result is all negative.The immunohistochemical staining of proof monoclonal antibody 3G11 and tumour has specificity.
The immunohistochemical staining of table 1 monoclonal antibody 3G11 in the different human body tumor tissues
| Tumor sample | Positive staining example number | Negative staining example number | Stained positive rate (%) |
| Large bowel cancer cancer of the stomach esophageal carcinoma mammary cancer lung cancer kidney bladder cancer | 30 7 4 7 2 3 0 | 8 5 2 9 0 0 3 | 78.9(30/38) 58.3(7/12) 66.7(4/6) 43.8(7/16) (2/2) (3/3) (0/3) |
In addition, observe the immunohistochemical staining of 5 routine colorectal carcinomas and normal adjacent tissue thereof, (dyeing that comprises enteraden cell and mesenchymal cell is negative, and cancer cells is the positive staining (table 2) of showed different then for healthy tissues.Brown engrain particle is positioned at the top of the tumour cell of body of gland sample arrangement in cell, matter interior (Fig. 3) between indivedual cases see on every side.The result has shown the specificity that IV Collagen Type VI enzyme is expressed in colon cancer tissue.
Table 2 monoclonal antibody 3G11 is at the immunohistochemical staining of colorectal carcinoma and normal adjacent tissue thereof
| The case sequence number | Cancer cells dyeing situation | Contiguous normal gland cell dyeing situation |
| 1 2 3 | ++++ +++ + | - - - |
| 4 5 | ++++ ++ | - - |
Embodiment 2:
The immunoreactivity of monoclonal antibody 3G11 and tumour cell detects with the ELISA method, and monoclonal antibody 3G11 presents good immunity with people's lung cancer PG cell, human colon carcinoma HT-29 cell, human oral squama cancer KB cell, rat liver cancer H22 cell one and mouse junction cancer 26 cells and combines active (Fig. 4).At nude mice armpit subcutaneous vaccination people lung cancer PG tumor tissues fritter (the about 1mm of diameter), when treating tumor growth to the about 1cm of diameter, the monoclonal antibody 3G11 of intravenous injection employing Iodogen method mark (
131I-3G11, about 200 μ Ci), carry out immune video picture in different time points.As seen the result has video picture clearly (Fig. 5) at the position of PG lung cancer.
Embodiment 3:
Detect with ELISA, monoclonal antibody 3G11 has kept and various tumour cell bonded abilities (Fig. 6) with conjugate 3G11-LDM after LDM is connected.
Measure with tetrazole indigo plant (MTT) method, 3G11-LDM conjugate or Fab '-LDM conjugate show the lethal effect stronger to tumour cell (Fig. 7, Fig. 8).3G11-LDM conjugate and free LDM are respectively 5.1 * 10 to the IC50 (50% inhibition concentration) of the liver cancer H22 cell of vitro culture
-12Mol/L and 6.2 * 10
-11Mol/L.The IC50 of Fab '-LDM conjugate is 9.3 * 10
-12Mol/L, the IC50 of free LDM is 2.5 * 10
-11Mol/L, Fab '-LDM conjugate is better than free LDM to the inhibited proliferation of H22 cell, and the ratio of both IC50 is 2.7.
Embodiment 4:
The intravenous injection of 3G11-LDM conjugate is got the mouse junction cancer C26 tumor tissue that goes down to posterity under the animal skin to the curative effect of mouse junction cancer C26, makes suspension with physiological saline by 1: 3 dilution proportion, and it is subcutaneous only to be inoculated in the BALB/c mouse armpit by 0.2ml/.Inoculate 72 hours posterior vein drug administration by injection, be administered once altogether.Control group gives physiological saline.Test and measured the gross tumor volume of each treated animal on the 10th day, and press gross tumor volume and calculate tumour inhibiting rate.The result shows (table 3), 3G11-LDM conjugate 0.025mg/kg, and the tumour inhibiting rate of 0.05mg/kg and 0.10mg/kg dosage is respectively 54.5%, 70.3% and 81.2%; And the tumour inhibiting rate of free LDM dosage 0.05mg/kg is 56.4%, and the tumour inhibiting rate of monoclonal antibody 3G11 dosage 0.75mg/kg is 25.7%, and the tumour inhibiting rate of conjugate and monoclonal antibody combined group (3G11+LDM) is 38.6%.The tumour inhibiting rate of 3G11-LDM conjugate is significantly higher than isodose free LDM.
Table 3 3G11-LDM conjugate and free LDM are to the growth-inhibiting effect of mouse transplantability colorectal carcinoma C26
| Medicine | Dosage (mg/kg) | Number of animals | Body weight (g) | Knurl heavy (mg) | Tumour inhibiting rate (%) |
| Beginning/end | Beginning/end | Xaver±SD | |||
| Contrast 3G11 LDM 3G11+LDM 3G11-LDM 3G11-LDM 3G11-LDM | 0.75 0.05 0.75+0.05 0.025 0.05 0.10 | 10/10 10/10 10/10 10/10 10/10 10/10 10/10 | 18.4/18.2 18.0/17.9 18.1/18.3 18.5/18.1 18.2/17.9 18.1/16.4 18.6/16.3 | 1.01±0.19 0.75±0.20 0.44±0.16 0.62±0.13 0.46±0.09 0.30±0.06 0.20±0.06 | 25.7 56.4* 38.6* 54.5* 70.3** 81.2** |
The subcutaneous vaccination tumour, the 3rd day intravenous administration is once;
* compare with control group P＜0.01, and compare with the lidamycin (LDM) group * * P＜0.01
Embodiment 5:
The intravenous injection of 3G11-LDM conjugate is 120 of the Kunming kind female mices of 18-22g to the efficacy experiment body weight of rat liver cancer H22, random packet, 10 every group.Get rat liver cancer H22 ascites, making cell count with the physiological saline dilution is 7.5 * 10
6The suspension of/ml, it is subcutaneous only to be inoculated in mouse armpit by 0.2ml/.The administration of inoculated tumour 24h posterior vein, a Zhou Houzai is administered once, totally 2 times.Control group intravenous injection physiological saline 0.2ml/ only.Experimental session was measured the major diameter a and the vertical with it minor axis b of a tumour in per 3～4 days, and is write down the weight of animals and animal dead situation.V=1/2ab by formula
2Calculate gross tumor volume, draw tumor growth curve, and calculate tumour inhibiting rate.Observe of the therapeutic action of conjugate different dosing regimes to mouse bearing liver cancer.The result shows (Fig. 9), and three dosage of 3G11-LDM conjugate 0.025mg/kg, 0.05mg/kg and 0.1mg/kg all can suppress the growth of mouse bearing liver cancer H22 significantly, and 3G11-LDM conjugate specific ionization LDM shows stronger tumor growth restraining effect.Compare by gross tumor volume, tested the 11st day, the tumour inhibiting rate of the 3G11-LDM conjugate of 0.025mg/kg, 0.05mg/kg and 0.1mg/kg dosage is respectively 66.3%, 87.8% and 97.2%; And the tumour inhibiting rate of the free LDM of 0.05mg/kg dosage is 67.1%.3G11-LDM conjugate 0.05mg/kg dosage is compared with the free LDM of Isodose, and tumor-inhibiting action significantly strengthens (P＜0.05).
Embodiment 6:
Fab '-LDM conjugate intravenous injection is 120 of the Kunming kind female mices of 18-22g to the efficacy experiment body weight of rat liver cancer H22, random packet, 10 every group.Get rat liver cancer H22 ascites, making cell count with the physiological saline dilution is 7.5 * 10
6The suspension of/ml, it is subcutaneous only to be inoculated in mouse armpit by 0.2ml/.The administration of inoculated tumour 24h posterior vein, a Zhou Houzai is administered once, totally 2 times.Control group intravenous injection physiological saline 0.2ml/ only.Experimental session was measured the major diameter a and the vertical with it minor axis b of a tumour in per 3～4 days, and is write down the weight of animals and animal dead situation.V=1/2ab by formula
2Calculate gross tumor volume, draw tumor growth curve, and calculate tumour inhibiting rate.Observe of the therapeutic action of conjugate different dosing regimes to mouse bearing liver cancer.The result shows (Figure 10), and the tumor growth of Fab '-LDM conjugate treatment group mouse is subjected to remarkable inhibition).Tested the 15th day, press gross tumor volume and calculate tumour inhibiting rate, the tumour inhibiting rate of Fab '-LDM conjugate 0.025mg/kg, 0.05mg/kg and 0.1mg/kg dosage group is respectively 84.5%, 93.9% and 96.0%, and the tumour inhibiting rate of free LDM dosage 0.05mg/kg is 78.3%.To testing the 21st day, Fab '-LDM conjugate 0.025mg/kg, the tumour inhibiting rate of 0.05mg/kg and three dosage groups of 0.1mg/kg is respectively 76.7%, 93.3% and 94.8%; And the tumour inhibiting rate of free LDM dosage 0.05mg/kg is 76.1%.Compare with the free LDM of Isodose, Fab '-LDM conjugate shows stronger tumor growth restraining effect (P＜0.01).
Sequence table
＜110〉Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences
＜120〉lidamycin (LDM) and monoclonal antibody and Fab ' thereof segmental conjugate and in neoplasm targeted therapies such as colorectal carcinoma
Use
<140>02153655.4
<141>2002-12-3
<160>1
<210>1
<211>110
<212>PRT
＜213〉organism: actinomycetes (Streptomyces globisporus C-1027)
<400>1
Ala Pro Ala Phe Ser Val Ser Pro Ala Ser Gly Leu Ser Asp Gly Gln
1 5 10 15
Ser Val Ser Val Ser Val Ser Gly Ala Ala Ala Gly Glu Thr Tyr Tyr
20 25 30
Ile Ala Gln Cys Ala Pro Val Gly Gly Gln Asp Ala Cys Asn Pro Ala
35 40 45
Thr Ala Thr Ser Phe Thr Thr Asp Ala Ser Gly Ala Ala Ser Phe_Ser
50 55 60
Phe Val Val Arg Lys Ser Tyr Thr Gly Ser Thr Pro Glu Gly Thr Pro
65 70 75 80
Val Gly Ser Val Asp Cys Ala Thr Ala Ala Cys Asn Leu Gly Ala Gly
85 90 95
Asn Ser Gly Leu Asp Leu Gly His Val Ala Leu Thr Phe Gly
100 105 110
Claims (6)
1, lidamycin (LDM) LDM and type-IV collagenase-resisting monoclonal antibody 3G11 immune conjugate 3G11-LDM is characterized in that said immune conjugate is made of monoclonal antibody 3G11 and lidamycin (LDM).
2, a kind of preparation method of immune conjugate according to claim 1, it is characterized in that with lidamycin (LDM) earlier with special-shaped bi-functional cross-linking agent N-hydroxy-succinamide base--(N-dimaleoyl imino) benzoic ether (MBS) modifies, add monoclonal antibody 3G11 then, the purified conjugate 3G11-LDM that obtains in reaction back through 2-imido grpup tetramethylene sulfide (2-IT) sulfhydrylation; Or lidamycin (LDM) carried out linked reaction with N-hydroxy-succinamide base-3-(2-pyridine the disulfide group)-propionic ester (LC-SPDP) of special-shaped bi-functional cross-linking agent N-hydroxy-succinamide base-3-(2-pyridine disulfide group)-propionic ester (SPDP) or long-chain as mesosome and monoclonal antibody 3G11, the purified conjugate 3G11-LDM that obtains in reaction back.
3, as the application of immune conjugate 3G11-LDM as described in the claim 1 in preparation resistive connection intestinal cancer and hepatoma-targeting medicine.
4, Fab ' fragment immune conjugate Fab '-LDM of lidamycin (LDM) LDM and type-IV collagenase-resisting monoclonal antibody 3G11 is characterized in that said immune conjugate is made of Fab ' fragment and the lidamycin (LDM) of monoclonal antibody 3G11.
5, a kind of preparation is as the method for immune conjugate Fab '-LDM as described in the claim 4, it is characterized in that with lidamycin (LDM) earlier with special-shaped bi-functional cross-linking agent N-hydroxy-succinamide base--(N-dimaleoyl imino) benzoic ether (MBS) modifies, Fab ' the fragment that adds sulfhydrylation then, purified immune conjugate the Fab '-LDM that obtains in reaction back.
6, as the application of immune conjugate Fab '-LDM as described in the claim 4 in preparation resistive connection intestinal cancer and hepatoma-targeting medicine.
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| CN1232537C (en) * | 2004-04-23 | 2005-12-21 | 中国医学科学院医药生物技术研究所 | Heavy chain variable region single domain antibody reinforced fusion protein VH-LDP-AE |
| WO2005117952A2 (en) * | 2004-05-14 | 2005-12-15 | Abraxis Bioscience, Inc. | Treatment methods utilizing albumin-binding proteins as targets |
| CN1307201C (en) * | 2005-04-27 | 2007-03-28 | 中国医学科学院医药生物技术研究所 | Coupled substance between Lidamycin and segments of monoclonal antibody 3G11, 3G11Fab' |
| AR059900A1 (en) * | 2006-03-17 | 2008-05-07 | Genentech Inc | ANTI-TAT226 ANTIBODIES AND IMMUNOCATE PLAYERS |
| CN102866052A (en) * | 2012-10-16 | 2013-01-09 | 百奥迈科生物技术有限公司 | Cell staining kit and use method thereof |
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