CN102830223A - Screening method for drugs used for treating neoplastic diseases in mammals - Google Patents
Screening method for drugs used for treating neoplastic diseases in mammals Download PDFInfo
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Abstract
The invention discloses a screening method for drugs used for treating neoplastic diseases in mammals. According to the invention, sample 1 and sample 2 are extracted from a cell population containing suspected tumor cells respectively before and after administration of a drug to a measured body; then an antibody containing an amino acid sequence recorded by SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8 is added into the sample 1 and the sample 2, and the antibody is used for labeling insulin-like growth factor I and insulin-like growth factor II to determine whether the sample 1 and the sample 2 contain tumor cells; if there are tumor cells, identification is carried out on the tumor cells, the amount of the tumor cells in the sample 1 and the sample 2 is compared, and if the amount of the tumor cells in the sample 2 is less than the amount of the tumor cells in the sample 1, it is proved that the drug has a therapeutic effect on neoplastic diseases.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method of screening treatment mammal tumor disease medicament.
Background technology
The screening mode of traditional treatment tumor disease medicine is: tumor cell line is implanted in the immunodeficiency mouse body; Treat to give again after tumour grows up to the screening compounds of various dose; Whether observation post's examination compound can suppress the growth of tumour cell then, even makes death of neoplastic cells or disappearance.Remake computational analysis then, draw indexs such as MID, meta inhibition dosage, LC50, with the antitumaous effect intensity of these indexs as the measurement test-compound.The advantage of this method is that the medicine that obtains can pass cell membrane and some physiologic barrier, and shortcoming is that resulting medicine is only better to tumour (like leukaemia or the lymthoma) curative effect of quick division, and far unsatisfactory to the treatment of solid tumor.And this screening mode can only be to confirming to suffer from the individuality of tumor disease, and can not judge whether tested individuality suffers from tumour, and whole screening institute time-consuming major part is used for the cultivation of tumour, causes this screening technique inefficiency.
Cell division and cell proliferation faster can cause the incidence probability of tumor disease to improve.Clinical research in recent years shows that the morbidity of IGF and multiple kinds of tumor disease (like breast cancer, prostate cancer, lung cancer and colon cancer etc.) has direct relation.IGF is brought into play in mitosis and is regulated cell proliferation, differentiation and effect of apoptosis, it not only the irritation cell proenzyme also suppress Apoptosis, this generation to tumour has direct influence.
So be necessary to develop and a kind ofly judge that through IGF whether tested individuality suffers from the method for tumor disease and screening treatment mammal tumor disease medicament, to shorten screening time, improves screening effeciency.
Summary of the invention
The technical matters that will solve required for the present invention provides a kind of method of screening treatment mammal tumor disease medicament.Antibody through having the specific amino acids sequence that the application puts down in writing acts on sample, but whether judgement sample contains tumour cell, thereby shortens screening time, improves screening effeciency.
It is following that the present invention solves the problems of the technologies described above the technical scheme that is adopted:
A kind of method of screening treatment mammal tumor disease medicament, it may further comprise the steps:
A. sampling in the blood of a doubtful individuality of suffering from tumour or tissue obtains sample one, and sample one comprises a doubtful cell mass that contains tumour cell;
B. drug candidate is administered to said individuality;
C. sampling in the blood of said individuality or tissue obtains sample two, and sample two comprises a doubtful cell mass that contains tumour cell;
D. in sample one and sample two, add and contain SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, the antibody of the amino acid sequence of putting down in writing among SEQ ID NO:7 or the SEQ ID NO:8;
Be attached to said antibody specificity on insulin-like growth factor I or insulin-like growth factor I and the insulin-like growth factor II mark insulin-like growth factor I and insulin-like growth factor II;
E. detect in sample one and the sample two and whether have insulin-like growth factor I label or insulin-like growth factor I label and insulin-like growth factor II label;
F. as containing insulin-like growth factor I label or insulin-like growth factor I label and insulin-like growth factor II label in sample one and the sample two; Then sample one and sample two are analyzed; Tumour cell in recognition sample one and the sample two; Reduce compared to sample one like the tumour cell quantity in the sample two, explain that described drug candidate has curative effect for the mammal tumor disease.
Described antibody is connected with the video picture composition.
Particularly, said video picture composition is fluorescent dye, radioactively labelled substance, microbubble contrast agent, enzyme or calorimetry label.
Further, described fluorescent dye is that thiocyanic acid luciferin, rhodamine or Texas are red.
Described radioactively labelled substance does
3H,
14C,
35S,
125I,
121I,
112In or
99MTc.
Described enzyme is horseradish peroxidase, alkaline phosphatase or hydrolytic enzyme.
Described calorimetry label is nanometer gold bead, coloured glass pearl or plastic bead.
Described tumour is metastatic tumo(u)r or commitment tumour.
The invention has the beneficial effects as follows: the antibody that the present invention will have the specific amino acids sequence is used for mark insulin-like growth factor I and insulin-like growth factor II; To judge whether comprise tumour cell in the said sample; Avoid the cultivation of tumour, thereby shortened screening time, improved screening effeciency.
Description of drawings
Fig. 1 has showed that MK35A, MK35B and MK35C are attached to the situation of insulin-like growth factor I
Fig. 2 has showed that MK35A, MK35B and MK35C are attached to the situation of insulin-like growth factor II
Fig. 3 has showed that the situation that MK35C and MK35C-1 are attached to insulin-like growth factor I and insulin-like growth factor II shows.
Embodiment
Below in conjunction with specific embodiment the present invention is described in further detail, protection scope of the present invention is not limited only to following embodiment.
Embodiment 1:
At present, most of available IGF antibody is not from the mankind, but from muroid.The inventor utilizes the human source Fab storehouse that comprises 1010 different bacteriophages displayings to develop the monoclonal antibody to IGF.The enzyme linked immunosorbent assay of being known by one of skill in the art utilizes insulin-like growth factor I as target antigen, through the three-wheel screening, obtains 200 random individual phage clones.Monoclonal antibody to obviously being attached to insulin-like growth factor I checks order, and wherein 3 monoclonal antibodies have unique sequence, and they are expressed with solubility Fab in bacteriophage.One of them monoclonal antibody MK35C is in above-mentioned enzyme linked immunosorbent assay detects; Insulin-like growth factor I and insulin-like growth factor II are all demonstrated stronger binding specificity; Two other monoclonal antibody, MK35A and MK35B then only demonstrate binding specificity to insulin-like growth factor I.
The human source Fab storehouse is as the source of upsetting the whole ingredients of specific antibody chain variable region gene VL in the storehouse.When the preparation of bacteriophage is carried out in the human source Fab storehouse, at first carry out enzyme and cut with Nco I and Spe I, carry out agarose gel electrophoresis then, to remove all specific antibody heavy chain variable region gene VH.Gene code to the VH district of monoclonal antibody MK35C increases, and introduces random mutation, merges mutually with the genetic fragment of CHI then.Merge fragment and carry out enzyme through Nco I and Spe I and cut, then its purifying from gel is come out, and be connected to the main body carrier of purifying, obtain upsetting whole ingredients of specific antibody chain variable region gene VL in the storehouse.From upset the storehouse, obtain 2 * 10
8Individual independently monoclonal antibody, said monoclonal antibody are the variant of MK35C.Through to the screening of the two-wheeled of insulin-like growth factor I conjugated beads, and carry out second through enzyme linked immunosorbent assay and obtain 200 monoclonal antibodies after taking turns screening.Monoclonal antibody to obviously being attached to insulin-like growth factor I and insulin-like growth factor II checks order; Wherein 5 monoclonal antibody MK35C-1 have unique sequence, and he demonstrates very strong binding specificity to insulin-like growth factor I and insulin-like growth factor II.
Wherein, the specific antibody heavy chain variable region gene VH of MK35A comprises the amino acid sequence of putting down in writing among the SEQ ID NO:1, and the specific antibody chain variable region gene VL of MK35A comprises the amino acid sequence of putting down in writing among the SEQ ID NO:2;
The specific antibody heavy chain variable region gene VH of MK35B comprises the amino acid sequence of putting down in writing among the SEQ ID NO:3, and the specific antibody chain variable region gene VL of MK35B comprises the amino acid sequence of putting down in writing among the SEQ ID NO:4;
The specific antibody heavy chain variable region gene VH of MK35C comprises the amino acid sequence of putting down in writing among the SEQ ID NO:5, and the specific antibody chain variable region gene VL of MK35C comprises the amino acid sequence of putting down in writing among the SEQ ID NO:6;
The specific antibody heavy chain variable region gene VH of MK35C-1 comprises the amino acid sequence of putting down in writing among the SEQ ID NO:7, and the specific antibody chain variable region gene VL of MK35C-1 comprises the amino acid sequence of putting down in writing among the SEQ ID NO:8.
Above-mentioned 4 kinds of antibody and insulin-like growth factor I or insulin-like growth factor I and insulin-like growth factor II specificity combine; There are competitive relation in antibody and IGF-1; Thereby stop IGF to combine, make the required signal mediation of cell proliferation be suppressed with IGF-1.
As depicted in figs. 1 and 2; Test through enzyme linked immunosorbent assay; MK35A and MK35B combine with the insulin-like growth factor I specificity; Do not combine with insulin-like growth factor II, MK35C combines with insulin-like growth factor I and insulin-like growth factor II specificity, and its binding ability is superior to MK35A and MK35B.
As shown in Figure 3; Through the enzyme linked immunosorbent assay test, along with the raising of AC, the binding capacity of antibody and insulin-like growth factor I and insulin-like growth factor II increases gradually; Under the identical situation of concentration, the binding ability of MK35C-1 is superior to the binding ability of MK35C.Can find out that therefrom MK35C-1 is a preferred embodiments of the present invention.
Embodiment 2:
Present embodiment selects MK35C-1 that the video picture composition is described on the basis of embodiment 1, and said video picture composition can not combine to produce significant impact by antagonist with the specificity of insulin-like growth factor I or insulin-like growth factor II.
MK35C-1 and fluorescent dye covalent bond; Selectable fluorescent dye has thiocyanic acid luciferin, rhodamine or Texas red, and above-mentioned fluorescent dye can not combine to produce significant impact to MK35C-1 with the specificity of insulin-like growth factor I and insulin-like growth factor II.MK35C-1 is with after insulin-like growth factor I and insulin-like growth factor II specificity combine, and fluorescent dye can be realized mark to insulin-like growth factor I and insulin-like growth factor II.The thiocyanic acid luciferin can send strong green fluorescence after being dissolved in alkaline solution, can observe through naked eyes.Rhodamine and Texas are red can be detected through sensitive film or detection of electrons device (like CCD, photomultiplier etc.).
MK35C-1 and radioactively labelled substance covalent bond, selectable radioactively labelled substance has
3H,
14C,
35S,
125I,
121I,
112In or
99MTc, above-mentioned radioactively labelled substance can not influence the specificity of MK35C-1 and insulin-like growth factor I and insulin-like growth factor II.MK35C-1 is with after insulin-like growth factor I and insulin-like growth factor II specificity combine, and radioactively labelled substance can be realized mark to insulin-like growth factor I and insulin-like growth factor II.The detection of radioactively labelled substance can be carried out through the autoradiographic technique that contains a scintillation counter or sensitive film, and this technology is well known to those skilled in the art, and repeats no more here.
MK35C-1 and enzyme covalent bond, selectable enzyme has horseradish peroxidase, alkaline phosphatase or hydrolytic enzyme.Above-mentioned enzyme can not influence the specificity of MK35C-1 and insulin-like growth factor I and insulin-like growth factor II.MK35C-1 is with after insulin-like growth factor I and insulin-like growth factor II specificity combine, and enzyme can be realized mark to insulin-like growth factor I and insulin-like growth factor II.Detection to enzyme can detect its reaction product and carry out through suitable substrate is provided to enzyme then, obstructed according to the kind of enzyme, and substrate and reaction product be difference to some extent also.Because this technology is well known to those skilled in the art, and repeats no more here.
MK35C-1 combines with the calorimetry label, and selectable calorimetry label has nanometer gold bead, coloured glass pearl or plastic bead.The calorimetry label can not influence the specificity of antibody and antigen.Detection for label can be carried out through observing the color relevant with label.
Can also add microbubble contrast agent among the MK35C-1, microbubble contrast agent can not impact the specificity of MK35C-1 and insulin-like growth factor I and insulin-like growth factor II equally.Ultrasonic imaging to the detection of microbubble contrast agent can be known is by one of skill in the art accomplished.
Embodiment 3:
Present embodiment is used to explain the actual effect of this method, so the employed tested individuality of present embodiment is the mouse of implanting the metastatic breast cancer cell line and becoming knurl.
Present embodiment carries out on the basis of embodiment 1 and embodiment 2.
A kind of method of screening treatment mammal tumor disease medicament, it may further comprise the steps:
A. sampling in the blood of said mouse or tissue obtains sample one, and sample one comprises a mouse cell crowd;
B. to said injected in mice endoxan 100mg, and cultivate 6h;
C. sampling in the blood of said mouse or tissue obtains sample two, and sample two comprises a mouse cell crowd;
D. in sample one and sample two, add MK35C-1, MK35C-1 is connected with any one development composition described in the embodiment 2;
The MK35C-1 specificity is attached on insulin-like growth factor I and the insulin-like growth factor II, mark insulin-like growth factor I and insulin-like growth factor II;
E. sample one and sample two are washed; Remove the MK35C-1 that does not wherein combine with insulin-like growth factor I and insulin-like growth factor II; Detect in sample one and the sample two whether have insulin-like growth factor I label and insulin-like growth factor II label then, detection method is confirmed according to selected development composition among the step D.Testing result is found all to have insulin-like growth factor I label and insulin-like growth factor II label in sample one and the sample two; Explain that this mouse body contains tumour cell; Its detection method is distinguished according to said video picture composition difference to some extent; But all be well-known to those skilled in the art, repeat no more here;
F. sample one and sample two are analyzed, the tumour cell in recognition sample one and the sample two, find tumour cell quantity in the sample two be in the sample one tumour cell quantity 60%, explain that endoxan has result of treatment to metastatic breast cancer.
Embodiment 4:
Present embodiment is the Comparative Examples of embodiment 2.The employed tested individuality of present embodiment is healthy mouse.
A kind of method of screening treatment mammal tumor disease medicament, it may further comprise the steps:
A. sampling in the blood of said mouse or tissue obtains sample one, and sample one comprises a mouse cell crowd;
B. to said injected in mice endoxan 100mg, and cultivate 6h;
C. sampling in the blood of said mouse or tissue obtains sample two, and sample two comprises a mouse cell crowd;
D. in sample one and sample two, add and contain embodiment 1 described MK35C-1, MK35C-1 is connected with any one development composition described in the embodiment 2; Because mouse is a healthy mice, do not contain insulin-like growth factor I and insulin-like growth factor II in sample one and the sample two;
E. sample one and sample two are washed; Remove all MK35C-1; Detect sample one and sample two then; In sample one and sample two, do not find the label of insulin-like growth factor I and the label of insulin-like growth factor II, explain that said mouse is a healthy mice, does not contain tumour cell in its body.
Embodiment 5:
Present embodiment is used to explain the actual effect of this method, so the employed tested individuality of present embodiment is the mouse of implanting the metastatic breast cancer cell line and becoming knurl.
A kind of method of screening treatment mammal tumor disease medicament, it may further comprise the steps:
A. sampling in the blood of said mouse or tissue obtains sample one, and sample one comprises a mouse cell crowd;
B. to said injected in mice penicillin 100mg, and cultivate 6h;
C. sampling in the blood of said mouse or tissue obtains sample two, and sample two comprises a mouse cell crowd;
D. in sample one and sample two, add and contain embodiment 1 described MK35C-1, MK35C-1 is connected with any one development composition described in the embodiment 2;
The MK35C-1 specificity is attached on insulin-like growth factor I and the insulin-like growth factor II, mark insulin-like growth factor I and insulin-like growth factor II;
E. sample one and sample two are washed; Remove the MK35C-1 that does not wherein combine with insulin-like growth factor I and insulin-like growth factor II; Detect in sample one and the sample two whether have insulin-like growth factor I label and insulin-like growth factor II label then, detection method is confirmed according to selected development composition among the step D.Testing result is found all to have insulin-like growth factor I label and insulin-like growth factor II label in sample one and the sample two, explains that this mouse body contains tumour cell;
F. sample one and sample two are analyzed, the tumour cell in recognition sample one and the sample two, tumour cell quantity in the discovery sample two and the tumour cell quantity in the sample one explain that penicillin is to the readily good therapeutic effect of metastatic breast cancer much at one.
A kind of method of screening treatment mammal tumor disease medicament of the present invention, its principle is not intended for a certain specific tumors disease or a certain specific medication.Use the method for the invention can realize screening to treatment mammal tumor disease medicament.Embodiment described in the instructions has been enough to explain effect of the present invention.To other tumor disease or medicine describe can not be regarded as of the present invention disclose insufficient.
SEQUENCE LISTING
< 110>Sichuan remittance space pharmaceutical Co. Ltd
< 120>a kind of method of treating tumor disease in the mammalian body
< 130>a kind of method of treating tumor disease in the mammalian body
<160> 8
<170> PatentIn version 3.3
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Claims (8)
1. one kind is screened the method for treating the mammal tumor disease medicament, it is characterized in that it may further comprise the steps:
A. sampling in the blood of a doubtful individuality of suffering from tumour or tissue obtains sample one, and sample one comprises a doubtful cell mass that contains tumour cell;
B. drug candidate is administered to said individuality;
C. sampling in the blood of said individuality or tissue obtains sample two, and sample two comprises a doubtful cell mass that contains tumour cell;
D. in sample one and sample two, add and contain SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, the antibody of the amino acid sequence of putting down in writing among SEQ ID NO:7 or the SEQ ID NO:8;
Be attached to said antibody specificity on insulin-like growth factor I or insulin-like growth factor I and the insulin-like growth factor II mark insulin-like growth factor I and insulin-like growth factor II;
E. detect in sample one and the sample two and whether have insulin-like growth factor I label or insulin-like growth factor I label and insulin-like growth factor II label;
F. as containing insulin-like growth factor I label or insulin-like growth factor I label and insulin-like growth factor II label in sample one and the sample two; Then sample one and sample two are analyzed; Tumour cell in recognition sample one and the sample two; Reduce compared to sample one like the tumour cell quantity in the sample two, explain that described drug candidate has curative effect for the mammal tumor disease.
2. a kind of method of screening treatment mammal tumor disease medicament according to claim 1, it is characterized in that: described antibody is connected with the video picture composition.
3. a kind of method of screening treatment mammal tumor disease medicament according to claim 2, it is characterized in that: described video picture composition is fluorescent dye, radioactively labelled substance, microbubble contrast agent, enzyme or calorimetry label.
4. a kind of method of screening treatment mammal tumor disease medicament according to claim 3, it is characterized in that: described fluorescent dye is that thiocyanic acid luciferin, rhodamine or Texas are red.
5. a kind of method of screening treatment mammal tumor disease medicament according to claim 3, it is characterized in that: described radioactively labelled substance does
3H,
14C,
35S,
125I,
121I,
112In or
99MTc.
6. a kind of method of screening treatment mammal tumor disease medicament according to claim 3, it is characterized in that: described enzyme is horseradish peroxidase, alkaline phosphatase or hydrolytic enzyme.
7. a kind of method of screening treatment mammal tumor disease medicament according to claim 3, it is characterized in that: described calorimetry label is nanometer gold bead, coloured glass pearl or plastic bead.
8. a kind of method of screening treatment mammal tumor disease medicament according to claim 1, it is characterized in that: described tumour is metastatic tumo(u)r or commitment tumour.
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| CN108114271A (en) * | 2016-11-29 | 2018-06-05 | 中国科学院上海生命科学研究院 | The pharmaceutical composition of insulin-containing like growth factor -2 and its application |
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| WO2007118214A2 (en) * | 2006-04-07 | 2007-10-18 | The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services | Antibody compositions and methods for treatment of neoplastic disease |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108114271A (en) * | 2016-11-29 | 2018-06-05 | 中国科学院上海生命科学研究院 | The pharmaceutical composition of insulin-containing like growth factor -2 and its application |
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