CN100586483C - Medicine in use for diagnosing gastric cancer, and preparation method - Google Patents
Medicine in use for diagnosing gastric cancer, and preparation method Download PDFInfo
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Abstract
The present invention relates to a medicine for diagnosing carcinoma of stomach and its preparation method. Said medicine includes antibody and radioactive nuclide, the descried antibody is monoclonalantibody 3H11, and the described radioactive nuclide can utilize a bifunctional connecting agent to mark the described monoclonal antibody 3H11, and the monoclonal antibody marked by the described radioactive nuclide is a colourless transparent liquid injection. Because the tumor cell surface can express specific antigen, so that it can utilize the specific combination of antibody and antigen, and utilize said antibody to make the radioactive nuclide be loaded on the tumor region, and utilize nuclear medicine image technique to make diagnosis and location of tumor.
Description
Technical field
The present invention relates to a kind of medicine that is used for diagnosing gastric cancer and preparation method thereof, this medicine arrives monoclonal antibody 3H11 by a bifunctional linking reagent with the radionuclide indirect labelling, because tumor cell surface expression specific antigen, utilize antibody and antigenic specificity combination, make the antibody of labelling inject dense gathering of back in the body in tumor locus, by the single photon tomography technology of nuclear medicine, gastric cancer is diagnosed.
Background technology
Tumor is one of principal disease of harm humans life and health, and how carrying out early diagnosis and therapy is the target that biomedical worker unremitting effort is for a long time pursued.Tumor cell surface expression specific antigen, monoclonal antibody have the antigenic specificity of identification, thereby can utilize monoclonal antibody to diagnose the illness.Because radionuclide labelled monoclonal antibodies has specificity and high-affinity to tumor, simultaneously also because the fast development of antibody engineering technology, radio-immuno-image becomes a new ways and means of diagnosing tumor in recent years, and has obtained noticeable diagnosis effect clinically.
Chinese patent 97108618.4 discloses the agent of a kind of human liver cancer monoclonal antibody HAb18 radioimmunodiagnosis, and at home and abroad there is no the medicine that be used for diagnosing gastric cancer of radionuclide by the indirect method labelling.
3H11 is the anti-McAb against gastric carcinoma of China's development, and uses
99mTc,
188Re and
131The I labelling has carried out zoopery and clinical research.The labeling method that above-mentioned research is adopted is direct labelling method, owing in labeling process, added Reducing agent or oxidant, so the relatively large influence of the active generation of antagonist.
Summary of the invention
Order of the present invention is to provide a kind of medicine that is used for diagnosing gastric cancer and preparation method thereof, this medicine is by bifunctional linking reagent radionuclide indirect labelling monoclonal antibody 3H11, make the antibody of labelling inject dense gathering of back in the body in tumor locus, by the single photon tomography technology of nuclear medicine, gastric cancer is diagnosed; Medicine by the preparation of indirect labelling method both can reduce the active influence of antagonist, can strengthen stability of radioactive drugs again, improved tumor simultaneously to radiopharmaceutic picked-up.
The objective of the invention is to be achieved through the following technical solutions: a kind of medicine that is used for diagnosing gastric cancer, comprise antibody and radionuclide, described antibody is monoclonal antibody 3H11, described radionuclide is by the described monoclonal antibody 3H11 of bifunctional linking reagent labelling, and described radionuclide labelled monoclonal antibodies 3H11 is the colourless liquid injection.
A kind of preparation method that is used for the medicine of diagnosing gastric cancer, its step is as follows:
I, be that monoclonal antibody 3H11 solution and the 5 μ L concentration of 5.0mg/mL are 1.55 * 10 with 0.9mL concentration
-2The S-HYNIC solution of mg/mL adds in the reaction tube, reacts 5 hours under 4 ℃ of conditions of lucifuge; The mol ratio of S-HYNIC and monoclonal antibody 3H11 is 8: 1 in this reaction; After finishing, reaction obtains reaction solution;
II, with described reaction solution use that pH value is 7.4, concentration is the citrate buffer solution dialysis treatment of 20mM 2 times, each dialysis time is 8 hours, contains 100mM NaCl in the used citrate buffer solution; To use that pH value is 5.2 through the described reactant liquor after 2 dialysis treatment, concentration is the citrate buffer solution of 20mM dialysis treatment 2 times again, each dialysis time is 8 hours, contain 100mMNaCl in the used citrate buffer solution, after dialysis treatment is finished, obtain conjugate HYNIC-3H11 solution;
III, be that the described conjugate HYNIC-3H11 of 3.0mg/mL, Tricine, the 20.0 μ g concentration that 20.0mg concentration is 100.0mg/mL are the SnCl of 3.0mg/mL with 0.2mg concentration
2Na with 1110MBq
99mTcO
4Add in the reaction tube room temperature reaction 30 minutes successively; Reactant by the PD-10 column purification, is obtained radiochemical purity greater than 95% radiopharmaceutical
99mTc-HYNIC-3H11.
A kind of preparation method that is used for the medicine of diagnosing gastric cancer, its step is as follows:
I, be that the monoclonal antibody 3H11 solution of 5.0mg/mL and EDTA solution that 1mL concentration is 1.0mM join respectively in the ultra-filtration centrifuge tube with 1.2mL concentration, centrifugal 45 minutes of the speed of under 4 ℃ of conditions, changeing with per minute 5000;
II, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, the speed of changeing with per minute 5000 under 4 ℃ of conditions is centrifugal 45 minutes for the second time;
III, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, the speed of changeing with per minute 5000 under 4 ℃ of conditions is centrifugal 45 minutes for the third time;
IV, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, centrifugal 45 minutes of the speed of under 4 ℃ of conditions, changeing for the 4th time with per minute 5000;
V, the monoclonal antibody 3H11 solution of ultrafiltration centrifugal treating is taken out ultra-filtration centrifuge tube, in this monoclonal antibody 3H11 solution, add Na
2HPO
4Solution dilution is measured the concentration of monoclonal antibody 3H11 in this solution and is preserved data to 0.5mL, preserves this antibody-solutions under 4 ℃ of conditions; Described Na
2HPO
4The pH value of solution is 7.5, and concentration is 0.1M, described Na
2HPO
4Contain 0.15mM NaCl in the solution;
VI, dissolve DOTA-NHS, be mixed with the solution that concentration is 33.45g/L with DMF; Get this solution 10 μ L and join the above-mentioned in the monoclonal antibody 3H11 of ultrafiltration centrifugal treating solution of 0.5mL at twice, this mixed liquid reacted 12 hours under 4 ℃ of conditions of lucifuge, obtained reaction solution;
VII, described reaction solution is transferred in the dialysis card, at 1.0L Na
2HPO
4Dialysis treatment is 24 hours in the solution, and the dialysis treatment temperature is 4 ℃, described Na
2HPO
4The pH value of solution is 7.5, and concentration is 0.1M, described Na
2HPO
4Contain 0.15mM NaCl in the solution;
VIII, will use 1.0L NH through the described reaction solution of a dialysis treatment
4OAc solution carries out the dialysis treatment second time, and the dialysis treatment time is 18 hours, and the dialysis treatment temperature is 4 ℃; Repeat described dialysis procedure 3 times under the same conditions again; After dialysis treatment is finished, obtain DOTA-3H11 solution; In DOTA-3H11 solution, add NH
4OAc solution carries out concentration adjustment, and obtaining concentration is the conjugate DOTA-3H11 solution of 2.8mg/mL, described NH
4The pH value of OAc solution is 7.0, concentration is 0.05M; Data are measured and preserved to concentration to conjugate DOTA-3H11 in this solution, preserves this conjugate solution under 4 ℃ of conditions;
IX, described conjugate DOTA-3H11 solution and the 100 μ L sodium-acetate buffers of 50 μ L are joined in the reaction tube, the pH value of described sodium-acetate buffer is 5.2, concentration is 0.2M, adds 5 μ L again
111InCl
3Solution, described
111InCl
3Be dissolved in the 0.04M HCl solution; Should react 1 hour under 43 ℃ of conditions by mixed liquid, add 20 μ L EDTA then, the pH value of described EDTA is 5.0, concentration is 6.0mM, reacts at ambient temperature 10 minutes; Reactant by the PD-10 column purification, is obtained radiochemical purity greater than 99% radiopharmaceutical
111In-DOTA-3H11.
A kind of preparation method that is used for the medicine of diagnosing gastric cancer, its step is as follows:
I, be that the monoclonal antibody 3H11 solution of 5.0mg/mL and EDTA solution that 1mL concentration is 1.0mM join respectively in the ultra-filtration centrifuge tube with 1.2mL concentration, centrifugal 45 minutes of the speed of under 4 ℃ of conditions, changeing with per minute 5000;
II, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, the speed of changeing with per minute 5000 under 4 ℃ of conditions is centrifugal 45 minutes for the second time;
III, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, the speed of changeing with per minute 5000 under 4 ℃ of conditions is centrifugal 45 minutes for the third time;
IV, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, centrifugal 45 minutes of the speed of under 4 ℃ of conditions, changeing for the 4th time with per minute 5000;
V, the monoclonal antibody 3H11 solution of ultrafiltration centrifugal treating is taken out ultra-filtration centrifuge tube, in this monoclonal antibody 3H11 solution, add Na
2HPO
4Solution dilution is measured the concentration of monoclonal antibody 3H11 in this solution and is preserved data to 0.5mL, preserves this antibody-solutions, described Na under 4 ℃ of conditions
2HPO
4The pH value of solution is 7.5, and concentration is 0.1M, described Na
2HPO
4Contain 0.15mM NaCl in the solution;
VI, with DMF dissolving DTPA derivant, be mixed with the solution that concentration is 33.45g/L; Get this solution 10 μ L and join the above-mentioned in the monoclonal antibody 3H11 of ultrafiltration centrifugal treating solution of 0.5mL at twice, this mixed liquid reacted 12 hours under 4 ℃ of conditions of lucifuge, obtained reaction solution;
VII, described reaction solution is transferred in the dialysis card, at 1.0L Na
2HPO
4Dialysis treatment is 24 hours in the solution, and the dialysis treatment temperature is 4 ℃, described Na
2HPO
4The pH value of solution is 7.5, and concentration is 0.1M, described Na
2HPO
4Contain 0.15mM NaCl in the solution;
VIII, will use 1.0L NH through the described reaction solution of a dialysis treatment
4OAc solution carries out the dialysis treatment second time, and the dialysis treatment time is 18 hours, and the dialysis treatment temperature is 4 ℃; Repeat described dialysis procedure 3 times under the same conditions again.After dialysis treatment is finished, obtain DTPA-3H11 solution; In DTPA-3H11 solution, add NH
4OAc solution carries out concentration adjustment, and obtaining concentration is the conjugate DTPA-3H11 solution of 2.8mg/mL, described NH
4The pH value of OAc solution is 7.0, concentration is 0.05M; Data are measured and preserved to concentration to conjugate DTPA-3H11 in this solution, preserves this conjugate solution under 4 ℃ of conditions;
IX, described conjugate DTPA-3H11 solution and the 100 μ L sodium-acetate buffers of 50 μ L are joined in the reaction tube, the pH value of described sodium-acetate buffer is 5.2, concentration is 0.2M, adds 5 μ L again
111InCl
3Solution, described
111InCl
3Be dissolved in the 0.04M HCl solution; Should react at ambient temperature 30 minutes by mixed liquid, add 20 μ L EDTA then, the pH value of described EDTA is 5.0, concentration is 6.0mM, reacts at ambient temperature 10 minutes; Reactant by the PD-10 column purification, is obtained radiochemical purity greater than 99% radiopharmaceutical
111In-DTPA-3H11.
The present invention compared with prior art has following advantage: because the tumor cell surface expression specific antigen, utilize antibody and antigenic specificity combination, by antibody with the radionuclide carrier band to tumor locus, utilize the nucleus medical image technology, tumor is diagnosed the location.In prior art, the labeling method of monoclonal antibody 3H11 is direct labelling method, owing to added Reducing agent or oxidant in labeling process, so antagonist is active produces relatively large influence.The present invention adopts the indirect labelling method, promptly introduces a bifunctional linking reagent between monoclonal antibody and radionuclide.This both can reduce the active influence of antagonist, can strengthen stability of radioactive drugs again, improved tumor simultaneously to radiopharmaceutic picked-up.
Description of drawings
The invention will be further described below in conjunction with drawings and Examples.
Fig. 1 is the analysis chart of HYNIC coupling monoclonal antibody 3H11 of the present invention;
Fig. 2 compares sketch map for label stability of the present invention;
Fig. 3 is label stability of the present invention another sketch map relatively;
Fig. 4 is a label activity determination of radioimmunoassay sketch map of the present invention;
Fig. 5 is 4 hours γ video picture of the present invention figure as a result;
Fig. 6 is 24 hours γ video picture of the present invention figure as a result;
Fig. 7 is 4 hours γ video picture of direct labelling method figure as a result;
Fig. 8 is 24 hours γ video picture of direct labelling method figure as a result;
4 hours γ video picture of the negative contrast of Fig. 9 is figure as a result;
24 hours γ video picture of the negative contrast of Figure 10 is figure as a result;
Figure 11 removes relatively sketch map of result for the blood of label of the present invention.
The specific embodiment
Embodiment one:
A kind of medicine that is used for diagnosing gastric cancer, comprise antibody and radionuclide, described antibody is monoclonal antibody 3H11, described radionuclide is by the described monoclonal antibody 3H11 of bifunctional linking reagent labelling, and described radionuclide labelled monoclonal antibodies 3H11 is the colourless transparent liquid injection.
In the present invention, described monoclonal antibody 3H11 comprises Mus endogenous antibody and segment, single-chain antibody (ScFv), double-stranded antibody (Diabody), miniaturization antibody (Minibody) and the chimeric antibody (Chimeric) of genetic engineering preparation.
In the present invention, described radionuclide is
99m(technetium-99m), described bifunctional linking reagent is S-HYNIC (a butanimide hydrazino-nicotinamide, succinimidyl-hydrazinonicotinamide is abbreviated as S-HYNIC) to Tc, and the radiopharmaceutical that described radionuclide labelled monoclonal antibodies forms is
99mTc-HYNIC-3H11.
Described monoclonal antibody 3H11 coupling and purification adopt following method:
0.9mL monoclonal antibody 3H11 (5.0mg/mL) and 5 μ L S-HYNIC (are dissolved in the anhydrous dimethyl formamide 1.55 * 10
-2Mg/mL) under 4 ℃ of conditions of lucifuge, reacted 5 hours.The mol ratio of S-HYNIC and antibody 3H11 is 8: 1.After reaction is finished, be respectively 7.4 and 5.2 in pH, concentration is respectively to dialyse 2 times in the citrate buffer solution (containing 100mM NaCl) of 20mM, and each dialysis time is 8 hours, obtains conjugate HYNIC-3H11; Referring to Fig. 1, Fig. 1 is the HPLC analysis chart of HYNIC coupling monoclonal antibody 3H11 of the present invention before and after dialysis: wherein, (a) be the UV of conjugate before the dialysis purification
280nmAbsorption curve (b) is the UV of conjugate behind the dialysis purification
280nmAbsorption curve, HPLC are high performance liquid chromatography.
Further the conjugate HYNIC-3H11 that obtains is carried out radioisotope labeling, described monoclonal antibody 3H11's
99mTc labelling and purification adopt following method:
(concentration is 3.0mg/mL with 0.2mg HYNIC-3H11, being dissolved in pH value is 5.2, contain in the 0.02M citric acid solution of 0.1M NaCl), 20.0mg Tricine (concentration is 100.0mg/mL, and being dissolved in pH value is 6.0, in the succinic acid buffer solution of 0.1M), 20.0 μ g SnCl
2(concentration is 3.0mg/mL, is dissolved among the 0.1N HCl) and 1110MBq (30mCi) Na
99mTcO
4Add in the reaction tube room temperature reaction 30 minutes successively.((pH 7.5, are leacheate 0.1M), collect label behind the purification and measure radiochemical purity with PBS (phosphate buffer) for G-25 Sephadex, Pharmacia) purification by the PD-10 post with reactant.
In the present invention, described radiopharmaceutical
99mThe dosage of Tc-HYNIC-3H11 is 10-30mCi.
To described radiopharmaceutical
99mIt is as follows that the mark rate of Tc-HYNIC-3H11 and the radiochemical purity behind the purification are carried out method for measuring:
1.ITLC method
Get 1 μ L label point sample on ITLC-SG, then respectively at normal saline, butanone and water: ethanol: ammonia=5: 2: 1 (V: V: V) ascending development 10cm in the mixed liquid, wherein the technetium colloid (
99mTcO
2) and free
99mTcO
4-Rf value (Rf value) is 0.0 and 0.9-1.0 in three kinds of expansion systems,
99mTc-(Tricine)
2R in three kinds of expansion systems
fValue is respectively 0.9-1.0,0.0 and 0.9-1.0,
99mTc-HYNIC-3H11 is R in three kinds of expansion systems
fValue is respectively 0.0,0.0 and 0.9-1.0.
2.HPLC method
Sample HPLC on the 10 μ L labels, chromatographic column is that (7.8 * 300mm, TosoHaas), (pH 6.5, and 0.02M contains 0.9%NaCl and 0.05%NaN for phosphate buffer for TSK G3000 WXL
3) be mobile phase, flow velocity 1.0mL/min.
99mThe retention time of Tc-HYNIC-3H11 is 8.2min,
99mTc-(tricine)
2With free
99mTcO
4 -Retention time be 12.3min.
Adopt described method to carry out labelling and purification, its measurement result is:
99mThe mark rate of Tc-HYNIC-3H11 is greater than 90%, and by radiochemical purity is greater than 95% behind the PD-10 column purification, specific radioactivity is greater than 5000MBq/mg.
For
99mThe vitro stability of Tc-HYNIC-3H11 is measured, and can adopt following method:
A, with the label of direct labelling method preparation (
99mTc-3H11) with the label of indirect labelling method preparation (
99mTc-HYNIC-3H11) under room temperature and 37 ℃ of conditions, be stored in normal saline and the calf serum respectively, and behind incubation different time (0,1,2,4,8,14,24 and 48 hours) sampling and measuring radiochemical purity.Referring to Fig. 2, Fig. 2 compares sketch map for the vitro stability of label of the present invention in normal saline and calf serum, and experimental result shows that two kinds of labels all show good stable.
B, two kinds of labels (2.6 * 10
-10Mol/mL) with the DTPA and the L-cysteine incubated at room 4h of different mole multiples, the ITLC method is measured radiochemical purity.Referring to Fig. 3, Fig. 3 compares sketch map for the vitro stability of label of the present invention in DTPA and L-cysteine solution, and experimental result shows that two kinds of labels all show good stable in the DTPA of variable concentrations solution.In L-cysteine solution,
99mTc-HYNIC-3H11 has good stable, and
99mThe radiochemical purity of Tc-3H11 descends along with the raising of L-cysteine concentration.
For
99mThe activity determination of radioimmunoassay of Tc-HYNIC-3H11, adopt following method:
99mTc-HYNIC-3H11 reaches
99mThe immunocompetence of Tc-3H11 detects with reference to the Lindmo method, and promptly 7 * 10
7Centrifugal 5 minutes of the speed that individual BGC823 cell changes with per minute 1000, (pH 7.4,0.01M) wash twice, and reuse contains 5 titres of PBS doubling dilution of 1%BSA with the PBS that contains 1%BSA; Label is diluted to 40 μ g/L, cell and label mixed, and 37 ℃ of incubations 2 hours, the radiocounting (B) of measuring gross activity (T) and sedimentation cell, the T/B value of calculating variable concentrations cell number correspondence.With T/B value pair cell inverse concentration (1/F) mapping, the extended line of gained curve is the active mark of radioimmunity that label keeps in the inverse of the intercept of y axle.Referring to Fig. 4, Fig. 4 is a label activity determination of radioimmunoassay result schematic diagram of the present invention, the active mark of radioimmunity that the label of indirect labelling method and direct labelling method preparation is tried to achieve in calculating is respectively 68% and 41%, illustrates that the label of indirect labelling method preparation has kept better immunocompetence.
Utilize the antibody 3H11 of described radioisotope labeling to carry out zooperal method and the result as follows:
Distribute in a, the tumor bearing nude mice body: 16 BALB/C lotus people gastric cancer nude mices are divided into 2 groups, 8 every group at random.Two groups of nude mices are respectively through tail vein injection 100 μ L's
99mTc-HYNIC-3H11 and
99mTc-3H11 (～370KBq contains the 3H11 antibody of 5.6 μ g approximately).Put to death 4 at 4 hours and 24 hours disconnected necks respectively for every group, get main organs and blood measuring weight and radiocounting, behind decay correction, calculate every gram and organize percentage injection dose rate (%ID/g).Table one is listed
99mTc-HYNIC-3H11 and
99mTc-3H11 is distributed data in the tumor bearing nude mice body.It is right to inject back 24 hours tumors
99mThe picked-up of Tc-HYNIC-3H11 is right apparently higher than tumor
99mThe picked-up of Tc-3H11, explanation
99mTc-HYNIC-3H11 has better immunocompetence, and people's gastric cancer tumor cell is had good affinity.
Table one
99mTc-HYNIC-3H11 reaches
99mThe distribution of Tc-3H11 tumor bearing nude mice (n=4, %ID/g ± SD)
B, tumor bearing nude mice γ video picture: 3 BALB/C lotus people gastric cancer nude mices are injected 25.9MBq's respectively
99mTc-HYNIC-3H11,
99mTc-3H11 and
99mTc-IgG (negative control), and carried out the γ video picture in back 4 hours and 24 hours in injection.Referring to Fig. 5-Figure 10, Fig. 5 is lotus people gastric cancer nude mice injection the present invention
99m4 hours γ video picture figure as a result behind the Tc-HYNIC-3H11, Fig. 6 are lotus people gastric cancer nude mice injection the present invention
99m24 hours γ video picture figure as a result behind the Tc-HYNIC-3H11, Fig. 7 are the preparation of lotus people gastric cancer nude mice injection direct labelling method
99m4 hours γ video picture figure as a result behind the Tc-3H11, Fig. 8 are the preparation of lotus people gastric cancer nude mice injection direct labelling method
99m24 hours γ video picture figure as a result behind the Tc-3H11, Fig. 9 are the injection of lotus people gastric cancer nude mice
99m4 hours γ video picture figure (negative control) as a result behind the Tc-IgG, Figure 10 are the injection of lotus people gastric cancer nude mice
99m24 hours γ video picture figure (negative control) as a result behind the Tc-IgG.Injected back 24 hours,
99mTc-HYNIC-3H11 and
99mTc-3H11 has dense preferably poly-at tumor locus, tumor can clear video picture, and does not observe radioactive dense poly-in negative control group.Explanation
99mThe monoclonal antibody 3H11 of Tc labelling can be used as the video picture that diagnostic medicine is used for gastric cancer.With respect to
99mTc-3H11,
99mTc-HYNIC-3H11 is dense poly-more obvious tumor locus.
C, blood are removed: referring to Figure 11, Figure 11 is the present invention
99mTc-HYNIC-3H11 and direct labelling method preparation
99mThe blood of Tc-3H11 is removed relatively sketch map of result; Get 14 of the BABL/C mices of 18-22g, be divided into 2 groups, 7 every group, two groups respectively through tail vein injection 0.15mL's
99mTc-HYNIC-3H11 and
99mTc-3H11, and in injection back different time points, 0.5, cut tail in 1,2,3,4,5,6,8,10,12,15,19 and 24 hour and get blood, wound surface is handled with YUNNAN BAIYAO, on gamma-counter, measure the blood sample radiocounting then, calculate %ID/g, draw blood clearance curve, and calculate and remove parameter.
99mThe T of Tc-HYNIC-3H11
1/2 αAnd T
1/2 βBe respectively 1.30 hours and 22.70 hours,
99mThe T of Tc-3H11
1/2 αAnd T
1/2 βBe respectively 4.0 hours and 37.52 hours.
Embodiment two:
Present embodiment is that described radionuclide is with the described monoclonal antibody 3H11 of another kind of radioisotope labeling
111In (indium-111), described bifunctional linking reagent be DOTA (1,4,7,10-tetraazacyclododecanand-N ', N ", N " ', N " "-tetraacethyl, 1,4,7,10-tetraazacyclododecanetetraacetic acid), the radiopharmaceutical that described radionuclide labelled monoclonal antibodies forms is
111In-DOTA-3H11.
In the present invention, described bifunctional linking reagent comprises the derivant of DOTA.
In the present invention, described bifunctional linking reagent comprises DTPA (diethylene triamine pentacetic acid (DTPA), diethylenetriaminepenta-acetic acid).
In the present invention, described bifunctional linking reagent comprises the derivant of DTPA
111In is the good radionuclide of another physical characteristic, is suitable for nuclear medicine image.
111The In traget antibody still need pass through the indirect labelling method, promptly introduces bifunctional linking reagent between antibody and radionuclide, is generally DOTA and derivant thereof and DTPA and derivant thereof.With DOTA is example, sets forth its preparation process.
Described monoclonal antibody 3H11 coupling and purification adopt following method:
1.2mL antibody 3H11 (5.0mg/mL) and 1mL 1.0mM ethylenediaminetetraacetic acid (EDTA) are joined (Centricon-30 in the ultra-filtration centrifuge tube respectively, 10K, Amicon), centrifugal 45 minutes of the speed of under 4 ℃ of conditions, changeing, the surplus approximately 0.2mL of solution in the centrifuge tube with per minute 5000.Add Na to centrifuge tube then
2HPO
4Solution (pH7.5,0.1M contain 0.15mM NaCl) 2.0mL, centrifugal 45 minutes of the speed of changeing with per minute 5000 under 4 ℃ of conditions repeats this process 3 times.The antibody taking-up is added Na
2HPO
4(pH 7.5,0.1M) are diluted to 0.5mL, 4 ℃ of preservations after ultraviolet 280nm measures antibody concentration for solution.
The pretreated purpose of antibody is for regulating pH value and removing various impurity in the antibody-solutions.The optimal pH of reaction solution is 7.5 during with the DOTA-NHS coupling.Other metal ion in the complexation antibody-solutions of acting as that adds 1.0mM EDTA makes its removal by ultrafiltration is centrifugal.The pretreated yield of antibody is 83%, and antibody concentration is 10.0mg/mL.
With anhydrous dimethyl formamide (DMF) dissolving 1,4,7,10-tetraazacyclododecanand-N ', N "; N ' ", N " "-tetraacethyl-N-hydroxy thiosuccinimide ester (DOTA-NHS, N-Hydroxysulfosuccinimidyl-1,4,7,10-tetraazacyclododecanetetraacetic acid), compound concentration is the solution of 33.45g/L.(0.667 μ mol 0.3345mg) joins in the above-mentioned 3H11 antibody of 0.5mL (10.0mg/mL, 0.0333 μ mol) at twice, the following 4 ℃ of reactions of lucifuge condition 12 hours to get 10 μ L.Reaction finish back transfer reaction solution in the dialysis card (Slide-A-Lyzer, 10K, Pierce) in, at 1.0L Na
2HPO
4Dialysed 24 hours under 4 ℃ of conditions in the solution (pH 7.5, and 0.1M contains 0.15mM NaCl), use 1.0L NH then
4(pH 7.0,0.05M) 72 hours (wherein changing dialysis solution 5 times) of dialysis under 4 ℃ of conditions for OAc solution.3H11 conjugate (DOTA-3H11) after the dialysis is measured antibody concentration at ultraviolet 280nm, and estimate purity with the HPLC method.Use NH
4OAc solution (pH 7.0,0.05M) dilution DOTA-3H11 to 2.8mg/mL, and 4 ℃ of preservations are stand-by.
With
111In labelling DOTA-3H11 and purification thereof adopt following method:
Get 50 μ L DOTA-3H11 join 100 μ L sodium-acetate buffers (pH 5.2,0.2M) in, add 5.0 μ L again
111InCl
3(20mCi is dissolved in the 0.04M HCl solution), room temperature reaction 20-30 minute, add then 20 μ L EDTA (pH 5.0,6.0mM), room temperature reaction 10 minutes.((pH 7.5, are leacheate 0.1M), collect label behind the purification and measure radiochemical purity with PBS for G-25Sephadex, Pharmacia) purification by the PD-10 post with reactant.
In the present invention, described radiopharmaceutical
111The dosage of In-DOTA-3H11 is 5-15mCi.
Described radiopharmaceutical
111The mark rate of In-DOTA-3H11 and the mensuration of radiochemical purity thereof adopt following method
1.ITLC method:
With add in the 10 μ L labels 20 μ L EDTA (pH 5.0, and 6.0mM), room temperature reaction 10 minutes is got 1 μ L point sample on ITLC-SG, ascending development 10cm in 10.0mM EDTA developping solution then, wherein
111In-DOTA-3H11 is retained in initial point (R
f=0.0),
111The In-EDTA exhibition is to forward position (R
f=0.9-1.0).
2.HPLC method
Adding 20 μ L EDTA in the 10 μ L labels (pH 5.0,6.0mM), and room temperature reaction 10 minutes, get sample HPLC on the 20 μ L, chromatographic column be TSK G3000WXL (7.8 * 300mm, TosoHaas), (pH 6.5, and 0.02M contains 0.9%NaCl and 0.05%NaN for phosphate buffer
3) be mobile phase, flow velocity 1.0mL/min.
111The retention time of In-DOTA-3H11 is 8.2min,
111The retention time of In-EDTA is 12.3min.
For
111The vitro stability of In-DOTA-3H11 is measured, and adopts following method:
Label
111In-DOTA-3H11 is stored in normal saline and the calf serum under room temperature and 37 ℃ of conditions, and behind incubation different time (0,1,2,4,8,14,24 and 48 hours) sampling and measuring radiochemical purity.
For
111The activity determination of radioimmunoassay of In-DOTA-3H11, can adopt following method:
111The immunocompetence of In-DOTA-3H11 detects with reference to the Lindmo method, and promptly 7 * 10
7Centrifugal 5 minutes of the speed that individual BGC823 cell changes with per minute 1000, (pH 7.4,0.01M) wash twice, and reuse contains 5 titres of PBS doubling dilution of 1%BSA with the PBS that contains 1%BSA; Label is diluted to 40 μ g/L, cell and label mixed, and 37 ℃ of incubations 2 hours, the radiocounting (B) of measuring gross activity counting (T) and sedimentation cell, the T/B value of calculating variable concentrations cell number correspondence.With T/B value pair cell inverse concentration (1/F) mapping, the extended line of gained curve is the active mark of radioimmunity that label keeps in the inverse of the intercept of y axle.
Embodiment three:
A kind of preparation method that is used for the medicine of diagnosing gastric cancer, its step is as follows:
I, be that monoclonal antibody 3H11 solution and the 5 μ L concentration of 5.0mg/mL are 1.55 * 10 with 0.9mL concentration
-2The S-HYNIC solution of mg/mL adds in the reaction tube, reacts 5 hours under 4 ℃ of conditions of lucifuge; The mol ratio of S-HYNIC and monoclonal antibody 3H11 is 8: 1 in this reaction; After finishing, reaction obtains reaction solution;
II, with described reaction solution use that pH value is 7.4, concentration is the citrate buffer solution dialysis treatment of 20mM 2 times, each dialysis time is 8 hours, contains 100mM NaCl in the used citrate buffer solution; To use that pH value is 5.2 through the described reactant liquor after 2 dialysis treatment, concentration is the citrate buffer solution of 20mM dialysis treatment 2 times again, each dialysis time is 8 hours, contains 100mM in the used citrate buffer solution
NaCl after dialysis treatment is finished, obtains conjugate HYNIC-3H11 solution;
III, be that the described conjugate HYNIC-3H11 of 3.0mg/mL, Tricine, the 20.0 μ g concentration that 20.0mg concentration is 100.0mg/mL are the SnCl of 3.0mg/mL with 0.2mg concentration
2Na with 1110MBq
99mTcO
4Add in the reaction tube room temperature (20 ℃-25 ℃) reaction 30 minutes successively; Reactant by the PD-10 column purification, is obtained radiochemical purity greater than 95% radiopharmaceutical
99mTc-HYNIC-3H11.
In the present embodiment, employed monoclonal antibody 3H11 is the solution that is dissolved in PBS, and its concentration is 5.0mg/mL.
Employed S-HYNIC is dissolved in the anhydrous dimethyl formamide reagent, and its concentration is 1.55 * 10
-2Mg/mL.
It is 5.2 that described conjugate HYNIC-3H11 is dissolved in pH value, and concentration is in the citrate buffer solution of 20mM, contains 100mM NaCl in this citrate buffer solution, and the concentration of HYNIC-3H11 is 3.0mg/mL, and 0.2mg represents employed in the method chemistry amount.
Described Tricine is a kind of conventional reagent, and being dissolved in pH value is 6.0, and in the succinic acid buffer solution of 0.1M, its concentration is 100.0mg/mL, and 20.0mg represents employed in the method chemistry amount.Described SnCl
2Be stannous chloride, be dissolved among the 0.1N HCl (hydrochloric acid) that its concentration is 3.0mg/mL, 20.0 μ g represent employed in the method chemistry amount.Described Na
99mTcO4 is a sodium pertechnetate solution, and 1110MBq represents employed in the method radioactivity.
Described PD-10 post is the normal experiment material, buys from Pharmacia company.
Embodiment four: a kind of preparation method that is used for the medicine of diagnosing gastric cancer, and its step is as follows:
I, be that the monoclonal antibody 3H11 solution of 5.0mg/mL and EDTA solution that 1mL concentration is 1.0mM join respectively in the ultra-filtration centrifuge tube with 1.2mL concentration, centrifugal 45 minutes of the speed of under 4 ℃ of conditions, changeing with per minute 5000;
II, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, the speed of changeing with per minute 5000 under 4 ℃ of conditions is centrifugal 45 minutes for the second time;
III, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, the speed of changeing with per minute 5000 under 4 ℃ of conditions is centrifugal 45 minutes for the third time;
IV, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, centrifugal 45 minutes of the speed of under 4 ℃ of conditions, changeing for the 4th time with per minute 5000;
V, the monoclonal antibody 3H11 solution of ultrafiltration centrifugal treating is taken out ultra-filtration centrifuge tube, in this monoclonal antibody 3H11 solution, add Na
2HPO
4Solution dilution is measured the concentration of monoclonal antibody 3H11 in this solution and is preserved data to 0.5mL, preserves this antibody-solutions under 4 ℃ of conditions; Described Na
2HPO
4The pH value of solution is 7.5, and concentration is 0.1M, described Na
2HPO
4Contain 0.15mM NaCl in the solution;
VI, dissolve DOTA-NHS, be mixed with the solution that concentration is 33.45g/L with DMF; Get this solution 10 μ L and join the above-mentioned in the monoclonal antibody 3H11 of ultrafiltration centrifugal treating solution of 0.5mL at twice, this mixed liquid reacted 12 hours under 4 ℃ of conditions of lucifuge, obtained reaction solution;
VII, described reaction solution is transferred in the dialysis card, at 1.0L Na
2HPO
4Dialysis treatment is 24 hours in the solution, and the dialysis treatment temperature is 4 ℃, described Na
2HPO
4The pH value of solution is 7.5, and concentration is 0.1M, described Na
2HPO
4Contain 0.15mM NaCl in the solution;
VIII, will use 1.0LNH through the described reaction solution of a dialysis treatment
4OAc solution carries out the dialysis treatment second time, and the dialysis treatment time is 18 hours, and the dialysis treatment temperature is 4 ℃; Repeat described dialysis procedure 3 times under the same conditions again; After dialysis treatment is finished, obtain DOTA-3H11 solution; In DOTA-3H11 solution, add NH
4OAc solution carries out concentration adjustment, and obtaining concentration is the conjugate DOTA-3H11 solution of 2.8mg/mL, described NH
4The pH value of OAc solution is 7.0, concentration is 0.05M; Data are measured and preserved to concentration to conjugate DOTA-3H11 in this solution, preserves this conjugate solution under 4 ℃ of conditions;
IX, described conjugate DOTA-3H11 solution and the 100 μ L sodium-acetate buffers of 50 μ L are joined in the reaction tube, the pH value of described sodium-acetate buffer is 5.2, concentration is 0.2M, adds 5 μ L again
111InCl
3Solution, described
111InCl
3Be dissolved in the 0.04M HCl solution; Should react 1 hour under 43 ℃ of conditions by mixed liquid, add 20 μ L EDTA then, the pH value of described EDTA is 5.0, concentration is 6.0mM, reacts at ambient temperature 10 minutes; Reactant by the PD-10 column purification, is obtained radiochemical purity greater than 99% radiopharmaceutical
111In-DOTA-3H11.
In the present embodiment, employed monoclonal antibody 3H11 is dissolved in PBS solution, and its concentration is 5.0mg/mL.Employed EDTA is an edta solution, and its pH value is 5.0, and concentration is 6.0mM, is conventional reagent.Employed DMF is an anhydrous dimethyl formamide solution, is conventional reagent.Employed DOTA-NHS is a bifunctional linking reagent, and its Chinese name is called 1,4,7,10-tetraazacyclododecanand-N ', N ", N " ', N " "-tetraacethyl-N-hydroxy thiosuccinimide ester, its English name is N-Hydroxysulfosuccinimidyl-1,4,7,10-tetraazacyclododecanetetraacetic acid purchases the Macrocyclic company in the U.S..Described PD-10 post is the normal experiment material, buys from Pharmacia company.Described ultra-filtration centrifuge tube is the normal experiment material, buys from Amicon company.
Embodiment five: a kind of preparation method that is used for the medicine of diagnosing gastric cancer, and its step is as follows:
I, be that the monoclonal antibody 3H11 solution of 5.0mg/mL and EDTA solution that 1mL concentration is 1.0mM join respectively in the ultra-filtration centrifuge tube with 1.2mL concentration, centrifugal 45 minutes of the speed of under 4 ℃ of conditions, changeing with per minute 5000;
II, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, the speed of changeing with per minute 5000 under 4 ℃ of conditions is centrifugal 45 minutes for the second time;
III, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, the speed of changeing with per minute 5000 under 4 ℃ of conditions is centrifugal 45 minutes for the third time;
IV, to add pH value in described ultra-filtration centrifuge tube be 7.5, and concentration is the Na of 0.1M
2HPO
4Solution 2.0mL, centrifugal 45 minutes of the speed of under 4 ℃ of conditions, changeing for the 4th time with per minute 5000;
V, the monoclonal antibody 3H11 solution of ultrafiltration centrifugal treating is taken out ultra-filtration centrifuge tube, in this monoclonal antibody 3H11 solution, add Na
2HPO
4Solution dilution is measured the concentration of monoclonal antibody 3H11 in this solution and is preserved data to 0.5mL, preserves this antibody-solutions, described Na under 4 ℃ of conditions
2HPO
4The pH value of solution is 7.5, and concentration is 0.1M, described Na
2HPO
4Contain 0.15mM NaCl in the solution;
VI, with DMF dissolving DTPA derivant, be mixed with the solution that concentration is 33.45g/L; Get this solution 10 μ L and join the above-mentioned in the monoclonal antibody 3H11 of ultrafiltration centrifugal treating solution of 0.5mL at twice, this mixed liquid reacted 12 hours under 4 ℃ of conditions of lucifuge, obtained reaction solution;
VII, described reaction solution is transferred in the dialysis card, at 1.0L Na
2HPO
4Dialysis treatment is 24 hours in the solution, and the dialysis treatment temperature is 4 ℃, described Na
2HPO
4The pH value of solution is 7.5, and concentration is 0.1M, described Na
2HPO
4Contain 0.15mM NaCl in the solution;
VIII, will use 1.0L NH through the described reaction solution of a dialysis treatment
4OAc solution carries out the dialysis treatment second time, and the dialysis treatment time is 18 hours, and the dialysis treatment temperature is 4 ℃; Repeat described dialysis procedure 3 times under the same conditions again.After dialysis treatment is finished, obtain DTPA-3H11 solution; In DTPA-3H11 solution, add NH
4OAc solution carries out concentration adjustment, and obtaining concentration is the conjugate DTPA-3H11 solution of 2.8mg/mL, described NH
4The pH value of OAc solution is 7.0, concentration is 0.05M; Data are measured and preserved to concentration to conjugate DTPA-3H11 in this solution, preserves this conjugate solution under 4 ℃ of conditions;
IX, described conjugate DTPA-3H11 solution and the 100 μ L sodium-acetate buffers of 50 μ L are joined in the reaction tube, the pH value of described sodium-acetate buffer is 5.2, concentration is 0.2M, adds 5 μ L again
111InCl
3Solution, described
111NCl
3Be dissolved in the 0.04M HCl solution; Should react at ambient temperature 30 minutes by mixed liquid, add 20 μ L EDTA then, the pH value of described EDTA is 5.0, concentration is 6.0mM, reacts at ambient temperature 10 minutes; Reactant by the PD-10 column purification, is obtained radiochemical purity greater than 99% radiopharmaceutical
111In-DTPA-3H11.
In the present embodiment, employed monoclonal antibody 3H11 is dissolved in PBS solution, and its concentration is 5.0mg/mL.Employed EDTA is an edta solution, and its pH value is 5.0, and concentration is 6.0mM, is conventional reagent.Employed DMF is an anhydrous dimethyl formamide solution, is conventional reagent.Employed DTPA derivant is purchased the Macrocyclic company in the U.S..Described PD-10 post is the normal experiment material, buys from Pharmacia company.Described ultra-filtration centrifuge tube is the normal experiment material, buys from Amicon company.
Claims (2)
1, a kind of medicine that is used for diagnosing gastric cancer comprises antibody and radionuclide, it is characterized in that: described antibody is monoclonal antibody 3H11, and described radionuclide is
99mTc, described radionuclide
99mTc is by the described monoclonal antibody 3H11 of bifunctional linking reagent S-HYNIC labelling, and described radionuclide labelled monoclonal antibodies is
99mTc-HYNIC-3H11, described radionuclide labelled monoclonal antibodies 3H11 are the colourless transparent liquid injection.
2, a kind of preparation method that is used for the medicine of diagnosing gastric cancer is characterized in that:
I, be that monoclonal antibody 3H11 solution and the 5 μ L concentration of 5.0mg/mL are 1.55 * 10 with 0.9mL concentration
-2The S-HYNIC solution of mg/mL adds in the reaction tube, reacts 5 hours under 4 ℃ of conditions of lucifuge; The mol ratio of S-HYNIC and monoclonal antibody 3H11 is 8: 1 in this reaction; After finishing, reaction obtains reaction solution;
II, with described reaction solution use that pH value is 7.4, concentration is the citrate buffer solution dialysis treatment of 20mM 2 times, each dialysis time is 8 hours, contains 100mM NaCl in the used citrate buffer solution; To use that pH value is 5.2 through the described reactant liquor after 2 dialysis treatment, concentration is the citrate buffer solution of 20mM dialysis treatment 2 times again, each dialysis time is 8 hours, contain 100mMNaCl in the used citrate buffer solution, after dialysis treatment is finished, obtain conjugate HYNIC-3H11 solution;
III, be that the described conjugate HYNIC-3H11 of 3.0mg/mL, Tricine, the 20.0 μ g concentration that 20.0mg concentration is 100.0mg/mL are the SnCl of 3.0mg/mL with 0.2mg concentration
2Na with 1110MBq
99mTcO
4Add in the reaction tube room temperature reaction 30 minutes successively; Reactant by the PD-10 column purification, is obtained radiochemical purity greater than 95% radiopharmaceutical
99mTc-HYNIC-3H11.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5053503A (en) * | 1989-02-17 | 1991-10-01 | Centocor | Chelating agents |
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Non-Patent Citations (6)
| Title |
|---|
| 多肽的放射性标记. 朱小华.放射学实践，第19卷第11期. 2004 |
| 多肽的放射性标记. 朱小华.放射学实践，第19卷第11期. 2004 * |
| 放射性核素标记单克隆抗体的化学研究进展. 刘斌.化学进展，第6卷第1期. 1994 |
| 放射性核素标记单克隆抗体的化学研究进展. 刘斌.化学进展，第6卷第1期. 1994 * |
| 用In-111通过1B4M-DTPA标记单克隆抗体的研究. 刘斌等.北京大学学报（自然科学版），第30卷第6期. 1994 |
| 用In-111通过1B4M-DTPA标记单克隆抗体的研究. 刘斌等.北京大学学报(自然科学版)，第30卷第6期. 1994 * |
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