HER2 AFFIBODY 99MTC MARKER COMPOSITION AND APPLICATION THEREOF

Abstract

An HER2 affibody .sup.99mTc marker composition and application thereof are provided, which relates to the field of radiopharmaceuticals and nuclear medicine. The HER2 affibody .sup.99mTc marker composition contains HEPES, sodium glucoheptonate, vitamin C, cysteine, stannous chloride and an HER2 affibody.

Claims

1. An HER2 affibody .sup.99mTc labelling composition, comprising: HEPES, sodium glucoheptonate, vitamin C, cysteine, stannous chloride and an HER2 affibody.

2. The composition according to claim 1, wherein the HEPES has a concentration of 2-30 mmol/L, the sodium glucoheptonate has a concentration of 5-40 mmol/L, the vitamin C has a concentration of 2-30 mmol/L, the cysteine has a concentration of 5-40 mmol/L, the stannous chloride has a concentration of 0.1-5 mmol/L, and the HER2 affibody has a concentration of 0.01-0.6 mmol/L, and the composition has a pH of 5.0-7.5.

3. The composition according to claim 2, wherein the HEPES has a concentration of 5-20 mmol/L, the sodium glucoheptonate has a concentration of 10-30 mmol/L, the vitamin C has a concentration of 5-20 mmol/L, the cysteine has a concentration of 10-30 mmol/L, the stannous chloride has a concentration of 0.2-2 mmol/L, and the HER2 affibody has a concentration of 0.03-0.3 mmol/L, and the composition has a pH of 6.0-7.0.

4. The composition according to claim 2, wherein the HEPES has a concentration of 10 mmol/L, the sodium glucoheptonate has a concentration of 20 mmol/L, the vitamin C has a concentration of 10 mmol/L, the cysteine has a concentration of 20 mmol/L, the stannous chloride has a concentration of 0.4 mmol/L, and the HER2 affibody has a concentration of 0.05 mmol/L, and the composition has a pH of 6.6.

5. The composition according to claim 4, the HER2 affibody has GGGC at carboxyl terminus and HEHEHE at amino terminus.

6. An imaging agent comprising the HER2 affinity .sup.99mTc labelling composition according to claim 1.

7. An imaging agent product, comprising the HER2 affibody .sup.99mTc labelling composition according to claim 1.

8. The imaging agent product according to claim 7, wherein the imaging agent is a PET or SPECT imaging agent and the product is a separate agent or a kit.

9. A method for preparing an HER2 affibody .sup.99mTc labelling composition, comprising the following steps: adding 500-20000 ?Ci of .sup.99mTc into the HER2 affibody .sup.99mTc labelling composition, and reacting at room temperature for 5-60 min under sealed conditions.

10. The method according to claim 9, wherein the .sup.99mTc is added in an amount of 2000-10000 ?Ci, preferably 5000 ?Ci; reaction time is 10-15 min.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is a graph showing the results of dimer identification by SDS-PAGE after 60 days of preservation at a low temperature and at room temperature;

[0021] FIG. 2 is a graph showing the results of time limits of preservation of HER2 affibody in different formulas;

[0022] FIG. 3 is a graph showing the results of the stability tests of the HER2 affibody-expressing Escherichia coli supernatant and the HER2 affibody-expressing Escherichia coli bacterial cells;

[0023] FIG. 4 is a group of graphs showing the ITLC detection results of .sup.99mTc-ABH2 in Experimental Examples 2-10;

DETAILED DESCRIPTION

[0024] The present disclosure will be described in further detail with reference to specific examples, which are not intended to limit the present disclosure but only to illustrate the present disclosure. Unless otherwise stated, the experimental methods used in the following examples, and the experimental methods without specific conditions indicated in the examples are generally performed under conventional conditions. Unless otherwise stated, the materials, reagents and the like used in the following examples are commercially available.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those of skill in the art.

Example 1. Preparation of HER2 Affibody (ABH2)

[0026] The carboxyl terminus of the affibody was designed with a GGGC sequence for isotope labeling, and the amino terminus was designed with an HEHEHE sequence for affinity column purification. An HER2 affibody gene (SEQ ID NO: 1) with a preference for Escherichia coli expression was designed by using DNAstar software according to a triple code, the HER2 affibody gene was then synthesized by the oligonucleotide synthesis and the overlap PCR method, the enzyme digestion by HindIII was carried out to identify the length of the synthesized gene, and the sequence of the synthesized gene was determined by gene sequencing.

[0027] The HER2 affibody gene was digested with enzymes Ncol and EcoRI, inserted into a pET22b(+) vector subjected to enzyme digestion in the same way, and then transformed into DH5a competent Escherichia coli. The bacterial liquid was sent for gene sequencing identification after screening with antibiotics, and a plasmid was extracted and transformed into BL21(DE3) competent Escherichia coli. The expression was induced with IPTG, and bacterial clones expressing the nascent protein were identified by SDS-PAGE.

[0028] The Escherichia coli expressing the HER2 affibody was centrifuged, and the bacterial cells were washed with a washing buffer (50 mM Na.sub.2HPO.sub.4, 300 mM NaCl, pH 7.0), resuspended in a purification buffer (20 mM Tris HCl, 500 mM NaCl, pH 7.9) and crushed by ultrasonication. Cell debris was removed by centrifugation and the supernatant was treated at 60? ? C. for 10 min. Then the supernatant was centrifuged, filtered through a filter membrane and applied to a nickel column equilibrated in advance with a crushing buffer (20 mM Tris HCl, 500 mM NaCl, 1 mM EDTA, pH 7.9). After washing with a purification buffer containing 5 mM imidazole, purification was performed by eluting with a purification buffer containing 60 mM imidazole. Then the ion exchange purification was performed through a Q column, and the purification was carried out under the condition of 0.5 M NaCl. The product obtained after purification was subjected to ultrafiltration to remove the buffer and replace it with an aqueous solution, thus obtaining the HER2 affibody-expressing Escherichia coli bacterial cells.

Example 2

[0029] The difference from Example 1 is that after the Escherichia coli expressing the HER2 affibody was centrifuged, the supernatant was directly dialyzed against a purification buffer (20 mM Tris HCl, 500 mM NaCl, pH 7.9) overnight and then centrifuged, and the supernatant was filtered through a filter membrane and applied to a nickel column equilibrated in advance with a crushing buffer. After washing with a purification buffer containing 5 mM imidazole, purification was performed by eluting with a purification buffer containing 60 mM imidazole. Then the ion exchange purification was performed through a Q column, and the purification was carried out under the condition of 0.5 M NaCl. The product obtained after purification was subjected to ultrafiltration to remove the buffer and replace it with an aqueous solution, thus obtaining the HER2 affibody-expressing Escherichia coli supernatant.

Example 3

[0030] HER2 Affibody .sup.99mTc Labelling .sup.99mTc was used to label the HER2 affibody, and the specific steps are as follows: vitamin C was added to the affibody prepared in Example 1 or 2, and the concentration of the affibody was adjusted to 1 mg/mL. 100 ?L of the affibody after the concentration adjustment was added into a labeling bottle, 200 ?L of deoxidized labeling buffer (10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 20 mmol/L of cysteine, 0.4 mmol/L of stannous chloride) was added, and then 5000 ?Ci of .sup.99mTc was added. The mixture was reacted at room temperature for 10 min to complete the reaction.

[0031] The ITLC detection showed that the .sup.99mTc-ABH2 was significantly different from the .sup.99mTc, the Rf of the .sup.99mTc-ABH2 was 0, and the Rf of the .sup.99mTc was 1.

Experimental Example 1: Stability Test

[0032] 1. The purified HER2 affibody in Example 1 was added to different composition formulas and preserved at room temperature and a low temperature of 4? C. separately, and dimer production was identified by SDS-PAGE every 2-3 days. The composition formulas are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Composition formulas No. Conditions Composition formula 1 Low temperature 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate (LT) 2 Low temperature 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 0.4 mmol/L (LT) of stannous chloride 3 Low temperature 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, argon (LT) 4 Low temperature 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 0.4 mmol/L (LT) of stannous chloride, argon 5 Low temperature 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 20 mmol/L (LT) of cysteine 6 Low temperature 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 10 mmol/L (LT) of vitamin C 7 Low temperature 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 10 mmol/L (LT) of vitamin C, 20 mmol/L of cysteine 8 Low temperature 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 10 mmol/L (LT) of vitamin C, 20 mmol/L of cysteine, 0.4 mmol/L of stannous chloride 9 Room 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate temperature (RT) 10 Room 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 0.4 mmol/L temperature (RT) of stannous chloride 11 Room 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, argon temperature (RT) 12 Room 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 0.4 mmol/L temperature (RT) of stannous chloride, argon 13 Room 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 20 mmol/L temperature (RT) of cysteine 14 Room 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 10 mmol/L temperature (RT) of vitamin C 15 Room 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 10 mmol/L temperature (RT) of vitamin C, 20 mmol/L of cysteine 16 Room 10 mmol/L of HEPES, 20 mmol/L of sodium glucoheptonate, 10 mmol/L temperature (RT) of vitamin C, 20 mmol/L of cysteine, 0.4 mmol/L of stannous chloride

[0033] FIG. 1 is a graph showing the results of dimer identification by SDS-PAGE after 60 days of preservation at a low temperature and at room temperature, and FIG. 2 is a graph showing the time limit of storage of HER2 affibody in different formulas.

[0034] As can be seen from FIG. 1, the HER2 affibody had poor stability and was easy to generate dimers when vitamin C and cysteine were not added; the stability when cysteine was added was poorer than that when vitamin C was added; the stability when both vitamin C and cysteine were added was better than that when vitamin C or cysteine was added alone.

[0035] As can be seen from FIG. 2, the formulas showing better preservation conditions when preserved at a low temperature involved addition of vitamin C and SnCl.sub.2, introduction of argon and the like; HER2 affibody could be preserved for more than 60 days at room temperature when cysteine, vitamin C and SnCl.sub.2 were all added.

[0036] 2. The purified HER2 affibody in Example 2 was preserved at room temperature, at a low temperature of 4? C. and at a freezing condition of ?20? C. separately, and the results are shown in FIG. 3. As can be seen from FIG. 3, the stability of the HER2 affibody-expressing Escherichia coli supernatant was lower than that of the HER2 affibody-expressing Escherichia coli bacterial cells.

Experimental Example 2

[0037] Composition formula: 10 mmol/L of HEPES; 20 mmol/L of sodium glucoheptonate; 10 mmol/L of vitamin C; 20 mmol/L of cysteine; 0.4 mmol/L of stannous chloride; 0.05 mmol/L of HER2 affibody; pH 6.6.