HER2 binding protein molecular imaging probe labeled by radionuclide as well as preparation method and application of HER2 binding protein molecular imaging probe

Abstract

The invention relates to the technical field of biomedicine, and particularly discloses a radionuclide-labeled HER2 binding protein molecular imaging probe as well as a preparation method and application thereof. The molecular imaging probe is labeled by radionuclides < 99m > Tc, < 68 > Ga and < 18 > F respectively so as to target HER2 binding protein. By optimizing the type of the buffer solution of the precursor protein, the ratio of the precursor protein to the chelating agent and the conditions of labeling reaction, the molecular imaging probe shows high radiation purity and good in-vitro stability, and in a tumor-bearing mouse model, the probe shows excellent uptake rate and good retention capacity at a tumor site, so that the molecular imaging probe has good application prospects. And the accuracy of the probe is obviously improved. Therefore, the probe has a wide application prospect in the aspect of preparing HER2 positive tumor imaging agents.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a radionuclide-labeled HER2-binding protein molecular imaging probe, a preparation method thereof, and an application thereof. Background Art

[0002] Breast cancer is one of the primary threats to women's health globally and is the most frequently occurring malignant tumor. Among them, up to 20%-30% of primary female breast cancer patients have overexpression or amplification of the human epidermal growth factor receptor 2 (HER2) gene. This type of breast cancer is usually highly invasive, leading to malignant tumor behavior and poor clinical outcomes. HER2 has become an important predictive and prognostic indicator for breast cancer and is also the core target of current immuno-molecular therapy. Therefore, accurately detecting and monitoring the expression level of HER2 in vivo is crucial for the diagnosis and treatment of breast cancer patients.

[0003] Currently, common detection methods for HER2 in breast cancer tissues are based on histopathological techniques such as immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). However, these methods require invasive biopsies, and the sampling is local, lacking comprehensiveness and real-time nature. In addition, for some tumor metastasis patients, especially those involving specific organ metastases (such as brain metastases), it is difficult to obtain samples. If a whole-body imaging method that can simultaneously detect the HER2 expression in the primary tumor and metastatic lesions can be developed, it will provide more accurate information for the comprehensive evaluation of HER2 expression. Therefore, developing a non-invasive diagnostic method in vivo to monitor the HER2 expression in tumor patients is crucial for the precise diagnosis and personalized treatment of breast cancer.

[0004] Clinically commonly used molecular diagnostic techniques are based on radionuclide molecular probe techniques. This technique can detect the HER2 expression in patients in real-time and non-invasively at the molecular level, with the advantages of high sensitivity, high specificity, and quantitative analysis, and has been widely used in the precise diagnosis and treatment evaluation of lesions in tumor patients. HER2-targeted therapy has significantly improved the survival rate of HER2-positive breast cancer patients.

[0005] To achieve individualized precision treatment, it is necessary to select appropriate targeted drugs according to the molecular characteristics of the patient's biomarkers. Therefore, it is necessary to develop a variety of novel targeted molecules. The emergence of molecular probe targeted diagnostic techniques has great significance in the field of "precision medicine". Currently, multiple clinical trials on radionuclide-labeled imaging probes of HER2 therapeutic antibodies have been carried out abroad, such as using 111 In, 124 I, 64 Cu, and 89Trastuzumab labeled with radionuclides such as Zr is used to detect whether there are HER2-positive metastatic foci in breast cancer with HER2-negative in situ cancer foci. However, the imaging probe labeled with "whole antibody" radionuclide has problems such as slow blood clearance rate, poor tissue permeability, easy non-specific binding caused by the Fc segment, poor thermal stability, complex preparation process and high production cost.

[0006] Therefore, there is an urgent need to develop a new type of HER2-targeted binding protein tumor imaging agent to optimize the diagnostic and therapeutic effects. Summary of the Invention

[0007] The present invention provides a radioactive nuclide-labeled HER2 binding protein molecular imaging probe, its preparation method and application, aiming to solve the deficiencies of the existing technology in HER2-positive tumor imaging. The following is a summary of the present invention:

[0008] A molecular imaging probe, wherein the molecular imaging probe is a radioactive nuclide-labeled HER2 binding protein molecular imaging probe, and the amino acid sequence of the HER2 binding protein is shown in SEQ ID NO.1.

[0009] Preferably, the radioactive nuclide is 99m Tc, and the structural formula of the molecular imaging probe is shown in Formula I;

[0010] Or, the radioactive nuclide is 68 Ga, and the structural formula of the molecular imaging probe is shown in Formula II;

[0011] Or, the radioactive nuclide is 18 F, and the structural formula of the molecular imaging probe is shown in Formula III;

[0012]

[0013] Wherein, BindHer is the HER2 binding protein.

[0014] The present invention also provides a method for preparing the above molecular imaging probe, and the method includes the following steps:

[0015] (a) Reacting the HER2 binding protein with a chelating agent in a solvent to obtain a precursor protein solution;

[0016] (b) Reacting the radioactive nuclide solution with the precursor protein solution to obtain the molecular imaging probe.

[0017] Preferably, the radioactive nuclide is 99mAt Tc, the preparation method of the precursor protein solution comprises the following steps: mixing a HER2 binding protein, buffer 1, stannous chloride, EDTA and sodium gluconate, followed by sterile filtration and pH adjustment to obtain the precursor protein solution; the pH is 5.5 - 6.5, and buffer 1 is selected from at least one of citric acid - sodium citrate buffer, sodium dihydrogen phosphate - disodium hydrogen phosphate buffer, and disodium hydrogen phosphate - citric acid buffer; in the precursor protein solution, the concentration of the HER2 binding protein is 0.5 - 1.5 mg / mL, the concentration of disodium hydrogen phosphate - citric acid is 15 - 25 mM, the concentration of stannous chloride is 5.0 - 6.0 mg / mL, the concentration of EDTA is 7.0 - 8.0 mg / mL, and the concentration of sodium gluconate is 260 - 300 mg / mL;

[0018] Or, the radionuclide is 68 At Ga, the preparation method of the precursor protein solution comprises the following steps: reacting the HER2 binding protein with the chelating agent MAL - NOTA to obtain NOTA - BindHer; adding NOTA - BindHer to buffer 2 to obtain the precursor protein solution; buffer 2 is a sodium acetate solution with a concentration of 0.05 - 0.2 M and a pH of 5.5 - 6.5, and the concentration of the precursor protein solution is 1 - 4 mg / mL;

[0019] Or, the radionuclide is 18 At F, the preparation method of the precursor protein solution comprises the following steps:

[0020] (1) Protein pretreatment: reacting the HER2 binding protein with a thiol reducing agent to obtain the pretreated protein;

[0021] (2) Coupling reaction: carrying out a coupling reaction between the pretreated protein and the chelating agent MAL - NOTA in buffer 3 to obtain the precursor protein solution; buffer 3 is PBS, and the molar ratio of the pretreated protein to the chelating agent is 1:(1 - 5); in the precursor protein solution, the concentration of the pretreated protein is 2.0 - 3.0 mg / mL.

[0022] Preferably, the radionuclide is 99m At Tc, buffer 1 is selected from disodium hydrogen phosphate - citric acid buffer; the pH is 6.0; in the precursor protein solution, the concentration of the HER2 binding protein is 1 mg / mL, the concentration of disodium hydrogen phosphate - citric acid is 20 mM, the concentration of stannous chloride is 5.6 mg / mL, the concentration of EDTA is 7.5 mg / mL, and the concentration of sodium gluconate is 280 mg / mL;

[0023] Or, the radionuclide is 68When the radionuclide is [[ID=]], the buffer solution 2 is a sodium acetate solution with a concentration of 0.1 M and a pH of 6.0, and the concentration of the precursor protein solution is 2 mg / mL;

[0024] Or, when the radionuclide is 18 F, the thiol reducing agent is TCEP, the molar ratio of the pretreated protein to the chelating agent is 1:3, and in the precursor protein solution, the concentration of the pretreated protein is 2.2 - 2.5 mg / mL.

[0025] Preferably, when the radionuclide is 99m Tc, the preparation method of the radionuclide solution includes the following steps: rinsing a 99 Mo - 99m Tc generator with physiological saline to obtain 99m TcO4 - solution, which is the radionuclide solution;

[0026] Or, when the radionuclide is 68 Ga, the preparation method of the radionuclide solution includes the following steps: rinsing a germanium - gallium generator with hydrochloric acid to obtain 68 Ga solution, mixing the 68 Ga solution with the sodium acetate solution to obtain a pre - buffer 68 Ga solution, which is the radionuclide solution;

[0027] Or, when the radionuclide is 18 F, the preparation method of the radionuclide solution includes the following steps: rinsing a fluorine accelerator with physiological saline to obtain 18 F ion solution, which is the radionuclide solution.

[0028] Preferably, when the radionuclide is 99m Tc, the radioactivity of the radionuclide solution is 20 - 50 MBq, preferably 37 MBq;

[0029] Or, when the radionuclide is 68 Ga, the radioactivity of the radionuclide solution is 20 - 40 MBq, preferably 37 MBq;

[0030] Or, when the radionuclide is 18 F, the radioactivity of the radionuclide solution is 0.5 - 2 mCi, preferably 1 mCi.

[0031] Preferably, when the radionuclide is 99m Tc, the volume ratio of the radionuclide solution to the precursor protein solution in step (b) is 1:(0.25 - 1); the reaction temperature is 20 - 30 °C, and the reaction time is 15 - 25 minutes;

[0032] Or, the radionuclide is 68 When it is 68 Ga, the volume ratio of the radionuclide solution to the precursor protein solution in step (b) is 1:(0.5 - 2); the temperature of the reaction is 65 - 85 °C, the reaction time is 10 - 20 minutes, and the pH of the reaction is 4.0 - 6.0;

[0033] Or, the radionuclide is 18 When it is 18 F, step (b) includes the following steps: mixing the radionuclide solution with the precursor protein solution and aluminum chloride solution, adding ethanol, and reacting to obtain; the molar ratio of the precursor protein to aluminum chloride is 1:(3 - 12), and the volume ratio of the radionuclide solution to the precursor protein solution is 1:(2 - 10); the reaction temperature is 80 - 120 °C, and the reaction time is 10 - 20 minutes.

[0034] Preferably, the radionuclide is 99m When it is 99m Tc, the volume ratio of the radionuclide solution to the precursor protein solution is 1:0.5; the temperature of the reaction is 25 °C, and the reaction time is 20 minutes;

[0035] Or, the radionuclide is 68 When it is 68 Ga, the volume ratio of the radionuclide solution to the precursor protein solution is 1:1; the temperature of the reaction is 75 °C, the reaction time is 15 minutes, and the pH of the reaction is 4.0 - 5.0;

[0036] Or, the radionuclide is 18 When it is 18 F, the molar ratio of the precursor protein to aluminum chloride is 1:6, and the volume ratio of the radionuclide solution to the precursor protein solution is 1:5; the reaction temperature is 100 °C, and the reaction time is 15 minutes.

[0037] The present invention also provides the use of the above molecular imaging probe in the preparation of a HER2 - positive tumor imaging agent.

[0038] The 99m 99m Tc - BindHer molecular imaging probe provided by the present invention has the advantages of high radiochemical purity, good in - vitro stability, excellent uptake and retention effects at the tumor site of HER2 - positive breast cancer - bearing mice, and low uptake values in non - target organs; 68 The 68 Ga - NOTA - BindHer molecular imaging probe, after structural optimization, has the advantages of strong hydrophilicity, increased detection rate of HER2 breast cancer and reduced liver absorption, improving the accuracy of the probe; 18 The 18 F - NOTA - BindHer molecular imaging probe has the advantages of specifically targeting HER2 - positive tumors, significantly increased tumor uptake rate, and significantly reduced liver uptake rate; In summary, through the optimization of the present invention, 99m Tc, 68 Ga,18 Molecular imaging probes of F-radionuclide-labeled targeting HER2-binding proteins show great potential in the characterization and treatment monitoring of HER2-positive tumors.

[0039] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0040] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Shows the trends of protein content, purity and labeling rate of three formulation prescriptions over time at 37°C. (A) is the graph of protein content change; (B) is the graph of protein purity change; (C) is the graph of labeling rate change. Formulation B1 - B3 represent three different formulation prescriptions; LCL represents the lowest detection limit of quality control.

[0042] Figure 2 For 99m Results of radio-purity determination of Tc-labeled molecular probes. (A) On the left is the migration map with PBS as the developing agent, and on the right is the thin-layer migration map with pyridine: glacial acetic acid: water = 10:6:3 as the developing agent. When using pyridine: glacial acetic acid: water = 10:6:3 as the developing agent, 99m TcO4 - and the Rf of technetium-labeled protein is 1, 99m the Rf of Tc colloid is 0; with PBS as the developing agent, 99m TcO4 - has an Rf of 1, 99m the Rf of Tc colloid and technetium-labeled protein is 0. (B) The upper part is the radio-purity display diagram of the labeling of Tc-BindHer, and the lower part is the radio-purity display diagram of the labeling of Tc-ABY-025. 99m Tc-BindHer 99m Tc-ABY-025

[0043] Figure 3 For 99m In vitro radiochemical stability assessment of Tc-BindHer (red curve) and 99m Tc-ABY-025 (blue curve) in PBS and serum at 37°C.

[0044] Figure 4 For 99mSPECT imaging results of Tc-BindHer in different tumor-bearing mice. (A) shows 99m the biodistribution of the Tc-BindHer molecular probe in different tumor-bearing mice; (B) shows 99m the comparison of the uptake of Tc-BindHer in the tumor sites of three groups of tumor-bearing mice. At each time point, there were extremely significant statistical differences (P < 0.0001) between the unblocked and blocked groups of SK-BR-3 and the HER2-negative tumor group at the tumor site (n = 3); (C) shows 99m the biodistribution of Tc-BindHer at different time points in SK-BR-3 tumor-bearing mice, MDA-MB-231 tumor-bearing mice, and SK-BR-3 tumor-bearing mice under blocking conditions 2 hours after injection. There were extremely significant statistical differences (P < 0.0001) between the unblocked and blocked groups of SK-BR-3 and the HER2-negative tumor group at the tumor site (n = 3).

[0045] Figure 5 shows 99m the comparison of the SPECT / CT imaging effects of Tc-BindHer and 99m Tc-ABY-025 in SK-BR-3 tumor-bearing mice. (A) The upper part shows the SPECT / CT three-dimensional reconstructed image (T: tumor site, K: kidney, L: liver), and the lower part shows the imaging of the cross-section of the tumor site and the liver site; (B) shows 99m the comparison of the uptake of Tc-BindHer and 99m Tc-ABY-025 at the tumor site and the comparison of the uptake at the liver site in HER2 tumor-bearing mice. There were statistical differences at the 1st hour, 2nd hour, and 4th hour (P < 0.01) (n = 3).

[0046] Figure 6 shows the HPLC analysis results of the reaction products of BindHer and MAL-NOTA at different chelation ratios.

[0047] Figure 7 shows the electrophoretic result analysis of the reaction products of BindHer and MAL-NOTA at different chelation ratios. Lanes 1-4 are in sequence: the original protein, the protein coupled with NOTA at a 1:1 molar ratio, the protein coupled with NOTA at a 1:3 molar ratio, and the protein coupled with NOTA at a 1:5 molar ratio.

[0048] Figure 8 shows the mass spectrometry molecular weight analysis results of the reaction product NOTA-BindHer under the condition that the reaction ratio of BindHer and MAL-NOTA is 1:3.

[0049] Figure 9Results of HPLC purity identification of the protein before and after conjugation. After conjugation, the free MAL-NOTA in the protein has been completely removed. HPLC analysis after mixing shows that the retention times of the protein before and after conjugation differ by only about 0.2 minutes.

[0050] Figure 10 Analysis of the conjugate stability of BindHer-NOTA after heating at 85 °C for different times. The results show that the intermediate formed by NOTA and the protein is very stable and there is basically no detachment.

[0051] Figure 11 Analysis of the conjugate stability of BindHer-NOTA after being placed at 25 °C for a certain time. The results show that the intermediate formed by NOTA and the protein is very stable and there is basically no detachment.

[0052] Figure 12 For radionuclide 68 Optimization of the conditions for labeling NOTA-BindHer with 68 Ga. (A) HPLC analysis of the labeling efficiency of NOTA-BindHer labeled with 68 Ga under different temperature conditions; (B) HPLC analysis of the labeling efficiency of NOTA-BindHer labeled with

[0053] Figure 13 Verified 68 The HPLC detection results of whether the reaction of the

[0054] Figure 14 For the labeling product 68 HPLC purity detection results of

[0055] Figure 15 For the result of detecting the purity of the 68 Ga-NOTA-BindHer molecular probe using thin-layer chromatography.

[0056] Figure 16 For 68 Results of the stability identification of the

[0057] Figure 17 For 68 PET / CT dynamic scanning imaging results of 68The imaging results of Ga-NOTA-BindHer in HER2-positive cell tumors, blocking the HER2 receptor, and in the case of HER2-negative cell tumors; (B) Statistical analysis of the tumor uptake trend graphs of the SK-BR-3 group, the SK-BR-3 blocking group, and the HER2-negative (MDA-MB-231) group; (C) When scanned to 90 minutes, 68 The absorption values of Ga-NOTA-BindHer in various tissues of HER2-positive tumor-bearing mice, and the statistical analysis of the comparison of the absorption values of the SK-BR-3 blocking group and the HER2-negative (MDA-MB-231) group with those of HER2-positive tumors.

[0058] Figure 18 For PET / CT imaging results. (A) During the dynamic scan 68 Ga-NOTA-BindHer and 68 Comparison of the in vivo targeted HER2-positive tumor and liver absorption values of Ga-NOTA-ABY-025; (B) Tumor and liver absorption value trend graphs.

[0059] Figure 19 For the results of radioactive ITLC analysis of the in vitro stability radioactive purity of fluorine-labeled proteins.

[0060] Figure 20 For the labeled product 18 HPLC purity detection results of 18F-NOTA-BindHer.

[0061] Figure 21 For 18 Comparison of the radioactive purity results of the 18F-NOTA-BindHer molecular probe before (A) and after (B) purification.

[0062] Figure 22 18 18F-NOTA-BindHer is for the tumor PET / CT dynamic scan imaging results. (A) 18 Imaging of the 18F-NOTA-BindHer molecular probe in HER2-positive cell tumors, blocking the HER2 receptor in advance, and in the case of HER2-negative cell tumors; (B) Statistical analysis of the tumor uptake trend graphs of the SK-BR-3 group, the SK-BR-3 blocking group, and the HER2-negative (MDA-MB-231) group; (C) When scanned to 90 minutes, 18 The absorption values of the 18F-NOTA-BindHer molecular probe in various tissues of HER2-positive tumor-bearing mice, and 18 Statistical analysis of the comparison of the absorption values of the 18F-NOTA-BindHer molecular probe in the tumors of Block and HER2-negative mice with those of HER2-positive tumors.

[0063] Figure 23 This is the PET / CT imaging result. (A) During the dynamic scan 18 F-NOTA-ABY-025 and 18 Comparison of in vivo targeting of HER2-positive tumors and liver uptake values of F-NOTA-BindHer; (B) Trend chart of tumor and liver uptake values. Detailed implementation manners

[0064] In the following examples and experimental examples, the reagents and raw materials not specifically described are all commercially available products.

[0065] In the examples and experimental examples, "normal temperature" is also called "room temperature", referring to 25 °C.

[0066] In the examples and experimental examples, the amino acid sequence of the BindHer protein is shown in SEQ ID NO.1: MAEAKYNEEMRITYWAIALMPNLDNDAKREYIRKLYDDPSQADDLLKAAEKEAQAN AQG

[0067] In the examples and experimental examples, BindHerNOTA, also known as NOTA-BindHer, also known as MAL-NOTA-BindHer. Unified: NOTA-BindHer.

[0068] Example 1 99m Preparation of the Tc-BindHer molecular imaging probe

[0069] 1. Preparation of the precursor protein

[0070] Under sterile conditions, according to formulation B3: 1 mg of BindHer protein, 20 mM disodium hydrogen phosphate-citric acid buffer, 280 mg of sodium gluconate, 7.5 mg of ethylenediaminetetraacetic acid (EDTA), 5.6 mg of stannous chloride, adjust the pH to 6.0, prepare the recombinant BindHer protein liquid preparation, after sterile filtration, fill it into a 2 mL middle borosilicate glass tube injection bottle, the injection solution is packaged with a halogenated butyl rubber stopper (brominated), and sealed with an aluminum-plastic combination cap for the antibiotic bottle, thus obtaining the precursor protein solution.

[0071] 2. 99m Synthesis of the Tc-BindHer molecular probe

[0072]

[0073] Wash with normal saline 99 Mo- 99m Tc generator (DRN4329, Curium Netherlands B.V.), obtain99m TcO4- solution. Then, using a syringe, 1 mL 99m TcO4- solution (with a radioactivity of 37 MBq) was injected into the vial containing the sterile-filtered recombinant BindHer protein liquid preparation in Step 1, mixed evenly, and reacted at 25 °C for 20 minutes to obtain 99m Tc-BindHer molecular imaging probe.

[0074] Example 2 68 Preparation of Ga-NOTA-BindHer Molecular Imaging Probe

[0075]

[0076] 1. Pre-buffering 68 Preparation of Ga solution

[0077] 100 μL of Ga eluted from a germanium-gallium generator with 0.05 M hydrochloric acid was mixed evenly with 100 μL of 0.1 M sodium acetate solution with a pH of 5.0 to obtain pre-buffered 68 Ga solution (with a radioactivity of 37 MBq). 68 Ga solution (with a radioactivity of 37 MBq).

[0078] 2. Preparation of NOTA-BindHer precursor protein

[0079] 1) Preparation of MAL-NOTA solution

[0080] Weighed 5 mg of MAL-NOTA (56491-86-2, Chematech) and dissolved it in 100 μL of ultrapure water to prepare a 50 mg / mL MAL-NOTA solution.

[0081] 2) Protein pretreatment

[0082] According to the ratio of adding 1 mM TCEP per 1 mg of BindHer protein, TCEP solution was added, mixed evenly, and left to stand at room temperature for 30 minutes. Subsequently, solution replacement was carried out through a NAP-5 desalting column to transfer the BindHer protein to PBS solution while removing TCEP, thereby obtaining the pretreated BindHer protein.

[0083] 3) Coupling reaction of BindHer protein with MAL-NOTA

[0084]

[0085] The coupling reaction was carried out according to the molar ratio of BindHer protein to the chelator MAL-NOTA of 1:3: Take 300 μL of BindHer protein with a concentration of 2.5 mg / mL, add 3.5 μL of MAL-NOTA solution with a concentration of 50 mg / mL to it, mix well and then place it at 25 °C for reaction for 16 hours.

[0086] 4) Solution replacement

[0087] Using a NAP-5 column, equilibrate with PBS for 10 column volumes, then pass NOTA-BindHer through the column. After complete breakthrough, elute with 250 μL of PBS each time, collect the eluate, and remove free MAL-NOTA.

[0088] 3. Preparation of precursor protein solution

[0089] Using 0.1 M sodium acetate solution with pH 6.0, adjust the concentration of NOTA-BindHer to 2 mg / mL to obtain the precursor protein solution.

[0090] 4. Labeling reaction

[0091] Mix the pre-buffered 68 Ga solution with the precursor protein solution in equal volume, react at pH 4.0 and 75 °C for 15 minutes to obtain 68 Ga-NOTA-BindHer molecular imaging probe.

[0092] Example 3 18 Preparation of 18F-NOTA-BindHer molecular imaging probe

[0093]

[0094] First, rinse the fluorine accelerator with physiological saline to obtain 18 18F ion solution; prepare the NOTA-BindHer precursor protein according to the steps of Example 2. Subsequently, mix 100 μL of NOTA-BindHer (concentration: 2 mg / ml, 25 nmol) and 7.5 μL of 2 mM aluminum chloride solution (the molar ratio of protein to aluminum chloride is 1:0.6), and then add 20 μL of 18 18F (radioactivity is 37 MBq). Finally, add an equal volume of absolute ethanol (127 μL) to make the volume ratio of ethanol to the previous reaction mixture 1:1 and react at 100 °C for 15 minutes.

[0095] After the reaction is completed, purify it using a NAP-5 column. Equilibrate with PBS for 10 column volumes, then pass the mixed reaction solution through the column. After it completely penetrates the column, elute with 250 μL of PBS each time, collect the eluate, and thus obtain 18F-NOTA-BindHer molecular imaging probe.

[0096] The beneficial effects of the present invention are demonstrated by the following experimental examples.

[0097] Experimental Example 1 99m Condition optimization and performance evaluation of the Tc-BindHer molecular probe

[0098] 1. 99m Condition optimization of the Tc-BindHer molecular probe

[0099] (1) Experimental samples

[0100] Formulation B3: It is the prescription preparation B3, that is, the preparation prescription of step 1 in Example 1;

[0101] Formulation B1: It is the prescription preparation B1, referring to the preparation prescription of step 1 in Example 1, with the difference being the buffer type. The specific composition of Formulation B1 is as follows: 1 mg of BindHer protein, 10 mM citric acid-sodium citrate buffer, 280 mg of sodium gluconate (BCBS0946, Sigma), 7.5 mg of EDTA (WXBC6464V, VETEC TM Company), 5.6 mg of stannous chloride (MKCD4836, Sigma), and the pH is adjusted to 6.0 (Table 1);

[0102] Formulation B2: It is the prescription preparation B2, referring to the preparation prescription of step 1 in Example 1, with the difference being the different buffer type. The composition of the prescription preparation B2 is: 1 mg of BindHer protein, 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, 280 mg of sodium gluconate, 7.5 mg of EDTA, 5.6 mg of stannous chloride, and the pH is adjusted to 6.0 (Table 1).

[0103] (2) Experimental method

[0104] Under a sterile environment, stannous chloride, sodium gluconate, EDTA, BindHer protein and buffer were sequentially added according to the formula sequence, the pH value was adjusted to 6.0, and then fully mixed for 20 minutes to obtain the recombinant BindHer protein liquid preparation. Subsequently, the preparation was dispensed into 2 mL vials, with each vial having a specification of 0.5 mL. After capping and labeling, medicine boxes were made. 100 bottles were prepared for each formula, and the batch numbers were 20210401-B1, 20210401-B2, and 20210401-B3 respectively. Subsequently, accelerated stability studies were carried out on the finished products at 37 °C.

[0105] In the stability test, samples were randomly taken from Formulations B1, B2, and B3 at time points of 0, 7, 14, 28, and 56 days for detection of protein content, purity, and radiolabeling rate. The lower limit of the minimum standard for the experiment was set as follows: the protein mass content was not less than 80% of the labeled amount, the protein purity was not less than 85%, and the labeling rate was not less than 85%. If the above standards were not met, the stability experiment would be terminated.

[0106] Table 1 Prescription of Recombinant BindHer Protein Liquid Preparation

[0107]

[0108] (3) Experimental Results

[0109] The experimental results are as Figure 1 shown in and Table 2:

[0110] 1) Protein content: As Figure 1 (A) shows, after storing the three prescription preparations at 37°C for 56 days, there was no significant change in their protein content compared with that at day 0.

[0111] 2) HPLC purity: As Figure 1 (B) shows, the purity of Formulation B1 dropped below 85% after being placed at 37°C for 14 days; the purity of Formulation B2 also dropped below 85% after storing for 28 days; while the purity of Formulation B3 was still higher than 85% even after being stored at 37°C for 56 days.

[0112] 3) 99m Tc radiolabeling rate: As Figure 1 (C) shows, for Formulation B1 at 37°C, the labeling rate gradually decreased with the extension of time, and the labeling rate dropped below 85% after 56 days; Formulation B2 showed a similar trend. In contrast, after storing Formulation B3 at 37°C for 56 days, the labeling rate was still higher than 95%.

[0113] Based on the above results, compared with Formulations B1 and B2, Formulation B3 prepared the 99m Tc-BindHer molecular probe with significant stability advantages. Therefore, the present invention will adopt Formulation B3 as the final prescription of the recombinant BindHer protein liquid preparation. Specifically, it is as follows (per 1 mL content): 1 mg of BindHer protein, 1.79 mg of disodium hydrogen phosphate, 0.71 mg of citric acid, 5.6 mg of stannous chloride, 7.5 mg of EDTA, 280 mg of sodium gluconate, add sterile injection water to 1 mL; adjust the pH value to 6.0.

[0114] Table 2 Protein Content, Purity and Labeling Rate of Three Formulations

[0115]

[0116] 2、 99m Radiochemical Purity Analysis of Tc-BindHer Molecular Probe

[0117] (1) Experimental Materials

[0118] 99m Tc-BindHer molecular probe: Prepared according to the method of Example 1.

[0119] 99m Tc-ABY-025 molecular probe: Prepared according to the method of Example 1, except that the added protein is ABY-025.

[0120] (2) Experimental Methods

[0121] Analysis was performed using an instant thin-layer chromatography silica gel plate (iTLC-SG) (Agilent Technologies, SG10001). Using PBS as the developing agent, 99m TcO4 - has an Rf value of 1, 99m Tc colloid and technetium-labeled protein have an Rf value of 0; using pyridine: glacial acetic acid: water (10:6:3) as the developing agent, 99m TcO4- and technetium-labeled protein have an Rf value of 1, 99m Tc colloid has an Rf value of 0.

[0122] Spotting: Spot 1 μL of the sample on the thin-layer plate, generally as a round dot. The spotting baseline is 1.0 - 1.5 cm from the bottom edge, and the diameter of the sample spot is generally not more than 2 mm.

[0123] Development: Place the thin-layer plate spotted with the sample into a small beaker. The immersion depth in the developing agent should be 5 mm from the origin. Seal the beaker with tin foil. Wait until the development reaches the specified distance (generally 8 - 15 cm), and then take out the thin-layer plate.

[0124] Detection: Use a γ scanner (BioScan, model 3201) for scanning to detect the labeling rate. 99m The radiochemical purity (%) of Tc-labeled protein is calculated as shown in Figure 2 A.

[0125] (3) Experimental Results

[0126] As shown in Figure 2 B, the obtained 99m Tc-BindHer and 99mThe radiochemical purities of the Tc-ABY-025 molecular probe were as high as 98.4% and 98.1% respectively. These data indicate that labeling can be achieved under normal temperature conditions, and the purity of the obtained product exceeds 95%, eliminating the need for further purification and meeting the requirements for direct use in subsequent experiments.

[0127] 3. 99m In vitro Stability Study of the Tc-BindHer Molecular Probe

[0128] (1) Experimental Samples

[0129] 99m Tc-BindHer molecular probe: Prepared according to the method of Example 1.

[0130] 99m Tc-ABY-025 molecular probe: Prepared according to the method of Example 1, except that the added protein is ABY-025.

[0131] (2) Experimental Method

[0132] The molecular probes were separately mixed evenly with an equal volume of serum and phosphate buffer (PBS), then incubated at 37 °C, and detected at time points of 1, 2, and 4 hours.

[0133] (3) Experimental Results

[0134] After the molecular probes were placed at 37 °C in PBS and serum for 4 hours, as Figure 3 shown, compared with 99m Tc-ABY-025, 99mTc-BindHer has relatively higher stability in PBS or serum. These results indicate that 99m the Tc-BindHer molecular probe exhibits good stability in the in vitro environment.

[0135] 4. 99m Biodistribution of the Tc-BindHer Molecular Probe

[0136] (1) Experimental Samples

[0137] 99m Tc-BindHer molecular probe: Prepared according to the method of Example 1.

[0138] (2) Experimental Method

[0139] Biodistribution studies were performed on healthy BALB / C mice. The Tc-BindHer molecular probe was injected via the tail vein, and the mice were sacrificed at time points of 1 hour, 4 hours, and 8 hours after injection. Each organ was isolated to measure the radioactivity count, and the percentage injected dose per gram of tissue (%ID / g) of each organ was calculated. 99m Tc-BindHer molecular probe, and the mice were sacrificed at 1 hour, 4 hours, and 8 hours after injection, and each organ was isolated to measure the radioactivity count and calculate the percentage injected dose per gram of tissue (%ID / g) of each organ.

[0140] (3) Experimental results

[0141] The results are shown in Table 3. After injecting the 99m Tc-BindHer molecular probe into healthy mice, radioactive distribution was mainly found in the kidneys, indicating that the molecular probe was metabolized through the kidneys and finally excreted from the body through the bladder.

[0142]

[0143]

[0144] 5. 99m SPECT / CT imaging of the

[0145] (1) Experimental samples

[0146] 1) 99m Specific binding of the

[0147] SK-BR-3 group: A nude mouse model bearing HER2-positive breast cancer was established using SK-BR-3 cells. The 99m Tc-BindHer molecular probe was injected via the tail vein, and SPECT / CT imaging was performed at 1 hour, 2 hours, and 4 hours respectively;

[0148] SK-BR-3 + Blocking group: Similarly, a nude mouse model bearing HER2-positive breast cancer was constructed using SK-BR-3 cells. First, an excessive amount of unlabeled BindHer protein was injected via the tail vein, and then the 99m Tc-BindHer molecular probe was injected, and SPECT / CT imaging was performed at the same time points;

[0149] MDA-MB-231 group: A nude mouse model bearing HER2-negative tumors was established using MDA-MB-231 cells. The 99m Tc-BindHer molecular probe was injected, and SPECT / CT imaging was performed at 1 hour, 2 hours, and 4 hours.

[0150] 2) 99m Comparison of the 99m hepatic uptake in vivo of the

[0151] 99m Tc-BindHer group: The 99mThe Tc-BindHer molecular probe was injected into nude mice with subcutaneous xenografts of SK-BR-3 breast cancer cells, and SPECT / CT imaging was performed 1 hour, 2 hours, and 4 hours later.

[0152] 99m Group Tc-ABY-025: 99m The Tc-ABY-025 molecular probe was injected into nude mice with subcutaneous xenografts of SK-BR-3 breast cancer cells, and SPECT / CT imaging was performed at the same time points.

[0153] (2) Experimental methods

[0154] Establishment of animal model: The human-derived breast cancer HER2-positive cell line SK-BR-3 was cultured in a 37 °C constant temperature incubator by the conventional method. It was rinsed with serum-free medium during the logarithmic growth phase, digestive fluid was added, and after standing for 1 - 2 minutes, complete culture medium was added to terminate digestion. It was repeatedly pipetted into a cell suspension, centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, washed three times with PBS, and the density of the cell suspension was adjusted to 1×10 7 cells / 0.2 mL, and 200 μL of the cell suspension was inoculated into the right axilla of each nude mouse. The diet and mental state of the nude mice were observed daily, and the tumor size (volume V = π / 6 × length × width 2 ) and the body weight of the nude mice were measured every three days until the tumor volume reached 80 - 100 mm 3 , and it was regarded as the establishment of a HER2-positive breast cancer-bearing nude mouse model. MDA-MB-231 cells were inoculated in the same way to establish a HER2-negative control breast cancer-bearing nude mouse model.

[0155] SPECT / CT imaging of animal tumor models: The 99m Tc-labeled molecular probes were respectively injected into the body of nude mice with HER2-positive breast cancer xenografts via the tail vein. One hour, 2 hours, and 4 hours after injecting the imaging agent, animal imaging was performed using a single photon emission computed tomography (SPECT) imaging device (anoScan-SPECT / CT, MILabs) equipped with a pinhole collimator. All tumor-bearing nude mice were anesthetized with isoflurane before imaging and placed prone on the examination bed. Acquisition parameters: magnification 3.2, acquisition matrix 256×256, acquisition time 10 minutes.

[0156] (3) Experimental results

[0157] 1) 99m The Tc-BindHer molecular probe specifically binds to HER2-positive tumors in mice

[0158] The 99mTc-BindHer was injected into HER2-positive breast cancer-bearing nude mice, and SPECT / CT imaging was performed after 1, 2, and 4 hours. Figure 4 As shown in A, there was obvious radioactivity accumulation in the tumor site of mice in the SK-BR-3 group, while there was no radioactivity accumulation in the tumor site of the SK-BR-3+Blocking and MDA-MB-231 groups; the tumor absorption of the SK-BR-3 group was significantly different from that of the other two groups (P<0.001)(n=3)( Figure 4 B). There was no significant statistical difference in the radioactivity absorption of other organs of mice among the three groups of experiments ( Figure 4 C) indicates 99m Tc-BindHer can specifically bind to HER2-positive breast cancer.

[0159] 2) 99m Reduced nonspecific uptake of Tc-BindHer molecular probe in the liver of mice

[0160] Will 99m Tc-BindHer and 99m Tc-ABY-025 was injected into SK-BR-3 breast cancer cell-bearing nude mice, and SPECT / CT imaging was performed after 1 hour, 2 hours, and 4 hours. Figure 5 A, 5B show a cross section through the tumor site. 99m Tc-BindHer group and 99m The difference between the Tc-ABY-025 group and the liver was significant at the 2nd and 4th hours (p<0.01). 99m The Tc-ABY-025 group nonspecifically bound to the liver, which was particularly evident at the 2nd and 4th hours ( Figure 5 AB). 99m Tc-BindHer 99m Tc-ABY-025 molecular probes can target HER2-positive tumors in tumor-bearing mice, but 99m Tc-BindHer showed better liver absorption.

[0161] Experimental Example 2: Condition Optimization and Performance Evaluation of NOTA-BindHer Precursor Protein

[0162] 1. Optimization of conditions for NOTA-BindHer precursor protein

[0163] (1) Experimental samples

[0164] BindHer group: prepared according to step 2 of Example 2, but without coupling reaction with MAL-NOTA;

[0165] BindHer:NOTA (1:1) group: Prepared according to step 2 of Example 2, with a molar ratio of BindHer protein to chelating agent of 1:1;

[0166] BindHer:NOTA (1:3) group: Prepared according to step 2 of Example 2;

[0167] BindHer:NOTA (1:5) group: Prepared with reference to step 2 of Example 2, with a molar ratio of BindHer protein to chelating agent of 1:5;

[0168] (2) Experimental method

[0169] 1) Electrophoresis

[0170] Preparation of polyacrylamide gel: Since the protein molecular weight is 8 kDa, we use the formula for small molecular weight electrophoresis gel. According to the number of samples, we prepare polyacrylamide gels of different volumes.

[0171] Sample treatment: Centrifuge all purified samples at 13000 g for 5 minutes, resuspend the precipitate with PBS, add 4x loading buffer to all samples to make the final concentration 1x. To fully denature the protein, boil the treated samples in a 100 °C metal bath or boiling water for 5 minutes.

[0172] Loading: Use a loading needle for loading. The loading volume of both the sample and the marker is 5 μl, and record the

[0173] sample loading order. After loading, cover the electrophoresis tank, connect the switch, manually adjust the voltage to 120 V, and run for about 15 - 20 minutes; when the sample band runs into the separation gel, adjust the voltage to 200 V, and wait until the band runs to the lower plastic pad, then turn off the electrophoresis and use a gel stripper to strip the gel.

[0174] Staining and decolorization of polyacrylamide gel: Put the peeled complete gel into a 0.25% Coomassie Brilliant Blue staining solution, place it on a shaker for 2 h, set an appropriate rotation speed to ensure staining and the integrity of the gel. Until the protein band is very clear, discard the staining solution, rinse it with water, and finally pour in the decolorizing solution for decolorization for 2 h.

[0175] Scanning: Place the decolorized gel on glass paper for scanning.

[0176] 2) HPLC purity analysis

[0177] Chromatographic column variety and specifications: The packing material is octadecylsilane chemically bonded silica gel, with a pore size of 300 Å, a particle size of 5 μm, a diameter of 4.6 mm, and a length of 150 mm;

[0178] Temperature: The column temperature is 30 ± 5 °C, and the storage temperature of the sample to be measured is 4 °C;

[0179] Mobile phase: Solution A is pure water (containing 0.1% trifluoroacetic acid), and solution B of the mobile phase is an acetonitrile solution (containing 0.1% trifluoroacetic acid).

[0180] Running conditions: Set the column flow rate to 1 ml / min; the detection wavelength is 214 nm; perform gradient elution with mobile phase B increasing from 0% to 75% within 40 minutes.

[0181] 3) Mass spectrometry molecular weight analysis

[0182] Separation by ultra-high performance liquid chromatography: Chromatographic column: ACQUITY UPLC Protein BEH C4 Column( 1.7 μm, 2.1 mm × 50 mm); Mobile phase A: 0.1% FA, H2O; Mobile phase B: 0.1% FA, ACN; Flow rate: 0.300 mL / min; Liquid chromatography gradient: 10 minutes;

[0183] Mass spectrometry conditions: Operated using a time-of-flight mass spectrometer (Waters Q-TOF Premier, Waters).

[0184] (3) Experimental results

[0185] 1) Determination of coupling conditions

[0186] The samples before and after chelation were simultaneously subjected to HPLC, electrophoresis, and mass spectrometry molecular weight analysis. Through RP-HPLC purity analysis, as Figure 6 , the retention time of the BindHer group was 16.75 minutes; the peak elution time on the HPLC chromatogram of NOTA-BindHer after chelation was 16.50 minutes, which was about 0.2 minutes earlier than that of the BindHer group. It was preliminarily judged that when the reaction ratio of BindHer protein to the bifunctional chelating agent was 1:3, it was completely chelated.

[0187] As Figure 7 shown, the small molecule electrophoresis results showed that the protein band after complete chelation had a slight upward shift compared to the pure protein band. Mass spectrometry analysis showed that ( Figure 8 ), when the reaction ratio of BindHer protein to the chelating agent was 1:3, the molecular weight of the product was approximately 8.5 kDa, which was about 500 Daltons larger than the 8 kDa of the BindHer group, confirming that BindHer protein had been successfully chelated with MAL-NOTA to form a labeled intermediate.

[0188] 2) Identify the purity of the product after chelation.

[0189] Figure 9The results showed that the retention time of pure MAL-NOTA was approximately 6 minutes, the peak time of BindHer protein was approximately 16.75 minutes, and the peak time of the chelated product NOTA-BindHer was 16.50 minutes. Moreover, two peaks appeared in the mixed HPLC, indicating that different substances existed before and after chelation, which further proved that the protein had indeed chelated with NOTA. There were deviations in the peak times of the original protein and the chelated protein. Additionally, there were no impurity peaks at a retention time of 6 minutes for the chelated protein, indicating that free MAL-NOTA had been completely removed, and the purity reached over 95%, making it suitable for radionuclide labeling studies.

[0190] 3) Thermal stability

[0191] First, the protein intermediate NOTA-BindHer (at a concentration of 1 mg / mL) was heated at 85 °C for 20 minutes, 40 minutes, and 60 minutes respectively. At each time point, 50 μL of the sample was taken after centrifugation at 13,000 rpm for 10 minutes, and then the electrophoresis sample was prepared with 4x loading buffer. The results of small molecule electrophoresis were analyzed, the gray values of the electrophoresis bands were analyzed using ImageJ software, and finally, a statistical graph was plotted using Graphpad software. Figure 10 The results showed that when NOTA-BindHer was incubated at 85 °C for up to 1 hour, the degradation of its main band was not obvious, and the gray value of the main band remained above 95%. This indicated that the protein BindHer had good thermal stability after chelating with NOTA, and NOTA would not easily fall off the protein, which was beneficial for subsequent radionuclide labeling experiments.

[0192] 4) Storage stability

[0193] The protein intermediate NOTA-BindHer was placed at room temperature for 1 week, 2 weeks, and 4 weeks. At each time point, 50 μL of the sample was taken after centrifugation at 13,000 rpm for 10 minutes, and then the electrophoresis sample was prepared with 4x loading buffer. The results were analyzed by SDS-PAGE, the gray scale of the electrophoresis bands was analyzed using ImageJ software, and finally, a statistical graph was plotted using Graphpad software.

[0194] Figure 11 As shown, after the protein labeling intermediate was stored at room temperature for a certain period of time, the experimental results showed that the main electrophoresis band of the chelated protein NOTA-BindHer did not degrade significantly even after being placed at room temperature for up to one month. Based on the statistical analysis of the gray value of its main band, the gray values of the main bands of the two protein labeling intermediates still remained above 95% after being placed at room temperature for up to one month, indicating that BindHer was stable after chelating with NOTA, and NOTA would not easily fall off the protein, which was of great significance for the production of subsequent radionuclide-labeled drugs.

[0195] Experimental Example 3 68Condition Optimization and Performance Evaluation of Ga-NOTA-BindHer Molecular Probe

[0196] 1、 68 Condition Screening for Labeling NOTA-BindHer with Ga

[0197] (1) Experimental Samples

[0198] Synthesize according to the method of Ga-NOTA-BindHer molecular probe in Step 4 of Example 2, and change the reaction temperature (65 °C, 75 °C, 85 °C) and pH value (4.0, 5.0, 6.0) respectively to obtain experimental samples. 68

[0199] (2) Experimental Methods

[0200] 1) Take 100 μL of precursor protein NOTA-BindHer in three portions (the buffer is 0.1 M sodium acetate solution with pH 6.0, and adjust the concentration of precursor protein to 2 mg / mL), add 100 μL of pre-buffered 68 Ga solution (~37 MBq), mix evenly, and incubate at 65 °C, 75 °C, and 85 °C for 15 minutes respectively. The pre-buffered 68 Ga solution is obtained by eluting 100 μL of Ga from the generator with 0.05 M hydrochloric acid 68 and mixing it evenly with 100 μL of 0.1 M sodium acetate solution with pH 7.0, so that the pH of the 68 Ga solution is consistent with the pH of the protein intermediate during the reaction.

[0201] The labeling rate calculation formula is (%) 68 Ga-NOTA-BindHer) = (activity labeled on the protein / total radioactivity) * 100. (Where the total radioactivity = activity labeled on the protein + free 68 Ga radioactivity).

[0202] 2) Take 100 μL of precursor protein NOTA-BindHer in three portions (the buffer is 0.1 M sodium acetate solution with pH 4.0, 5.0, 6.0, and adjust the concentration of precursor protein to 2 mg / mL), add 100 μL of pre-buffered 68 Ga solution (~37 MBq), mix evenly, and incubate at 75 °C for 15 minutes respectively. The pre-buffered 68 Ga solution is obtained by eluting 100 μL of Ga from the generator with 0.05 M hydrochloric acid 68 and mixing it evenly with 100 μL of the corresponding 0.1 M sodium acetate solution with pH 7.0, 6.0, 5.0, so that the pH of the 68 Ga solution is consistent with the pH of the protein intermediate during the reaction. ​

[0203] (3) Experimental results

[0204] The present invention first studied the labeling efficiency under different temperature conditions, and the results are as Figure 12 shown in Figure A. It was found that when the temperature increased from 65 °C to 75 °C, the labeling rate increased from about 50% to about 70%. However, when the temperature continued to rise to 85 °C, the labeling rate did not increase significantly anymore. Therefore, 75 °C was selected.

[0205] The present invention further explored the role of pH in the labeling process. The results are as Figure 12 shown in Figure B. Under the condition of 75 °C, it was found that when the pH was 6.0, the labeling rate was only 70%. When the pH was 4.0 or 5.0, the labeling rates were both as high as 90%, and the labeling rate increased significantly. Therefore, the preferred labeling conditions are: pH 4.0 - 5.0, incubation at 75 °C for 15 minutes.

[0206] 2. Determination 68 The reaction of

[0207] (1) Experimental samples

[0208] BindHer group: Prepared according to the method of protein pretreatment in step 2 of Example 2;

[0209] BindHer-NOTA group: Prepared according to the method of Example 2, except that the pre-buffered 68 Ga solution was not added;

[0210] 68 Ga-BindHer group: Prepared according to the method of Example 2, except that NOTA was not conjugated;

[0211] 68 Ga-NOTA-BindHer group: Prepared according to the method of Example 2.

[0212] (2) Experimental method

[0213] The present invention took the BindHer protein without conjugated NOTA and the intermediate NOTA-BindHer conjugated with NOTA, and reacted them respectively under the selected labeling conditions, and then detected the labeling situation using a UV-Radio-HPLC system. The HPLC column was selected as C-18, 300A, 5 μm, 4.6 * 150 mm, and the detection wavelength was 280 nm. The HPLC chromatographic conditions were set as follows: mobile phase A: 5% acetonitrile, 95% water; mobile phase B: 95% acetonitrile, 5% water; the elution gradient of the mobile phase was A 75% - 25%, B 25% - 75%, the time was 30 minutes, and the flow rate was set at 1 mL / min. Observe the two cases 68The differences in the radioactive peak times of Ga were analyzed.

[0214] (3) Experimental results

[0215] Figure 13 The UV peak retention time of BindHer protein without NOTA chelation was still consistent with the above experiments, and the corresponding radioactive peak time was 2.2 minutes, which was consistent with the free 68 The peak time of Ga is consistent, indicating that the protein labeling intermediate is not labeled 68 Ga, while the UV peak and radioactive peak of the BindHer protein labeled intermediate chelated with NOTA are both present together, and the peak time is consistent with the previous experimental results, indicating that the protein without NOTA mediation cannot be labeled with nuclides 68 Ga, this labeling process must be mediated by NOTA to be completed.

[0216] 3. 68 Labeling effect and radioactivity purity of Ga-NOTA-BindHer molecular probe

[0217] (1) Experimental samples

[0218] 68 Ga-acetate group: According to the preparation method of Example 2, 100 μL of pre-buffered 68 The Ga (37 MBq) solution is different in that only 100 μL of 0.1 M sodium acetate buffer (without NOTA-BindHer) with a pH of 4.0 is added, mixed evenly, and reacted at 75° C. for 15 minutes to obtain the solution.

[0219] 68 Ga-NOTA group: According to the preparation method of Example 2, 100 μL of pre-buffered 68 The Ga (37 MBq) solution is different in that 100 μL of 0.1 M sodium acetate buffer with a pH of 4.0 and 2 μL of 50 mg / mL MAL-NOTA buffer are added, mixed evenly, and reacted at 75° C. for 15 minutes to obtain the solution.

[0220] 68 Ga-NOTA-BindHer group: prepared according to the method of Example 2;

[0221] 68 Ga-NOTA-ABY-025 group: prepared according to the method of Example 2, except that the precursor protein added was NOTA-ABY-025.

[0222] (2) Experimental methods

[0223] The labeling results were detected using a UV-Radio-HPLC system. The HPLC column was selected as C-18, 300A, 5μm, 4.6*150mm, and the detection wavelength was 280nm. The HPLC chromatographic conditions were set as follows: mobile phase A: 5% acetonitrile, 95% water; mobile phase B: 95% acetonitrile, 5% water; the elution gradient of the mobile phase was 75%-25% for A and 25%-75% for B, the time was 30 minutes, and the flow rate was set at 1 mL / min. The radioactivity detector detected whether the peak emergence time of 68 Ga was consistent with the UV peak emergence time of the protein, so as to judge whether 68 Ga was labeled on the protein intermediate.

[0224] Radioactive purity Radio-ITLC: Using 0.2M citric acid with pH 2.0 as the developing agent, suck 2 μL of the mixture with a capillary tube, release the sample on the ITLC thin-layer chromatography paper, and then vertically place the thin-layer chromatography paper into a beaker containing an appropriate amount of the developing agent, taking care to avoid submerging the sample spot in the developing agent.

[0225] (3) Experimental results

[0226] For 68 the purity identification of the Figure 14 Ga-NOTA-BindHer labeling product, the results were as 68 shown. The radioactive activity peak emergence time of pure 68 Ga was approximately 2.20 minutes under the system set in the present invention and no UV peak appeared. After MAL-NOTA was incubated with 68 Ga for the same time under the same conditions, the UV-Radio-HPLC results showed that the UV peak emergence time of MAL-NOTA was about 6 minutes, which was consistent with the previous purity HPLC results. At the same time, the radioactive peak retention time was about 6.25 minutes, indicating that MAL-NOTA could also react with 68 Ga and had a good labeling effect. After 68 Ga-NOTA-BindHer was incubated at 75°C for 15 minutes, its UV peak retention time was about 16.50 minutes, and the corresponding radioactive peak emergence time was 16.75 minutes. No impurity peaks such as MAL-NOTA were found under A280 UV detection, while only a little free 68 Ga impurity peak was detected by Radio-HPLC at about 2 minutes, indicating that there was no interference from

[0227] the Figure 15 Ga-MAL-NOTA signal, which was beneficial for subsequent in vivo experiments. 68 Ga-NOTA-BindHer and 68Ga-NOTA-ABY-025 remains at the starting point of spotting due to its relatively low polarity, while the reference substance 68 Ga-acetate has a relatively high polarity and migrates to the front of the developing solvent with the developing solvent, indicating that the radiochemical purity of the molecular probe prepared under this condition is high and no subsequent purification is required.

[0228] 4. 68 In vitro stability study of Ga-NOTA-BindHer molecular probe

[0229] (1) Experimental samples

[0230] 68 Ga-NOTA-BindHer molecular probe: Prepared according to the method of Example 2.

[0231] (2) Experimental method

[0232] The labeled product 68 Ga-NOTA-BindHer was incubated with an equal volume of normal saline or serum at 37 °C for 1 hour, 2 hours, and 4 hours. Since 68 the half-life of Ga is relatively short, the radiochemical purity within 4 hours of incubation at 37 °C was only measured.

[0233] (3) Experimental results

[0234] The results are as Figure 16 shown. The labeled product 68 Ga-NOTA-BindHer is quite stable in human serum. When incubated in serum and normal saline for 4 hours, its labeling rate did not decrease significantly, which is extremely beneficial for in vivo imaging.

[0235] 5. 68 PET / CT imaging of Ga-NOTA-BindHer molecular probe in animal tumor models

[0236] (1) Experimental samples

[0237] SKBR-3 group: A nude mouse model bearing HER2-positive breast cancer was constructed using SK-BR-3 cells. 68 The Ga-NOTA-BindHer molecular probe was injected into the nude mice bearing tumors via the tail vein for PET / CT imaging;

[0238] SKBR-3+Blocking group: A nude mouse model bearing HER2-positive breast cancer was constructed using SK-BR-3 cells. First, an excessive amount of unlabeled 68 BindHer protein of Ga was injected into the nude mice bearing tumors via the tail vein, and then 68Ga-NOTA-BindHer molecular probe was used for PET / CT imaging;

[0239] MDA-MB-231 group: An HER2-negative tumor-bearing nude mouse model was constructed using MDA-MB-231 cells. 68 The Ga-NOTA-BindHer molecular probe was injected into the tumor-bearing nude mice via the tail vein for PET / CT imaging;

[0240] 68 Ga-NOTA-BindHer group: Prepared according to the method of Example 2;

[0241] 68 Ga-NOTA-ABY-025 group: Prepared according to the method of Example 2, except that the precursor protein was NOTA-ABY-025.

[0242] (2) Experimental method

[0243] Establishment of mouse tumor model: The same as the part of establishing the animal model in Experimental Example 1.

[0244] PET / CT tumor imaging: 68 10 μg of Ga-labeled protein (3.7 MBq / mouse, diluted to 100 μL with normal saline) was injected into the breast cancer tumor-bearing mice via the tail vein respectively. After injecting the imaging agent, dynamic imaging of animals was performed using a PET imaging instrument. All tumor-bearing mice were anesthetized with isoflurane before imaging and placed prone on the examination bed. Acquisition parameters: magnification factor 3, acquisition matrix 256×256, acquisition time 1.5 hours. Finally, GraphPad software was used for analysis of the absorption values of each tissue, and the results were summarized into a bar chart or table.

[0245] (3) Experimental results

[0246] In the present invention, the labeled 68 Ga-NOTA-BindHer was used for PET / CT imaging research in nude mice. As Figure 17 shown, the SKBR-3 group could successfully target HER2-positive tumors in tumor-bearing mice; in the SKBR-3 + Blocking group, after blocking the HER2 receptor with an excessive amount of unlabeled protein in advance, 68Ga-NOTA-BindHer could not target tumors; in the MDA-MB-231 group, BindHer could not target and bind to tumors either, verifying that BindHer could perform in vivo imaging by specifically binding to HER2-positive receptors; from the results of statistical analysis, it was known that when scanning for 90 minutes, the absorption value of BindHer in the kidneys of HER2-positive tumor-bearing mice was the highest, followed by the absorption value in tumors. At the same time, the absorption values of the SKBR-3 + Blocking group and the MDA-MB-231 group were much lower than those of the SK-BR-3 group, successfully verifying once again the 68 Ga-NOTA-BindHer molecular probe could successfully target and bind to HER2-positive cell receptors.

[0247] 68 The Ga-NOTA-BindHer group and 68 comparison results of the tumor absorption values of the Ga-NOTA-ABY-025 group showed that ( Figure 18 ) 68 both the Ga-NOTA-BindHer and 68 Ga-NOTA-ABY-025 molecular probes could target HER2-positive tumors in tumor-bearing mice. At the same time, it was found that 68 the absorption value of the Ga-NOTA-BindHer group on tumors was higher than that of 68 the Ga-NOTA-ABY-025 group, and the trend of this value gradually increasing was more significant as time extended. The present invention also found that as time extended, 68 the hepatic absorption in the Ga-NOTA-BindHer group decreased rapidly in vivo, while 68 the hepatic absorption in the Ga-NOTA-ABY-025 group became more and more serious. The difference between the two was very significant statistically, indicating that the present invention successfully obtained a protein with better targeting and stability through protein design methods.

[0248] Experimental Example 4: 18 Condition optimization and performance evaluation of the F-NOTA-BindHer molecular probe

[0249] 1. 18 Condition optimization for the preparation of the F-NOTA-BindHer molecular probe

[0250] (1) Experimental samples

[0251] Referring to the method for synthesizing the 18 F-NOTA-BindHer molecular probe in Example 3, the molar ratio of protein to aluminum chloride in the reaction was changed to 1:0.3, 1:0.5, 1:0.6, 1:1 to obtain different experimental samples.

[0252] AlF 18 Group: Prepared according to the preparation method of Example 3, except that NOTA-BindHer is not added;

[0253] 18 F-NOTA-BindHer group: Prepared according to the method of Example 3.

[0254] (2) Experimental method

[0255] 1) 18 F labeling reaction

[0256] First, rinse the fluorine accelerator with normal saline to obtain 18 F ion solution. Subsequently, according to the molar ratios of four proteins to aluminum chloride (1:0.3, 1:0.5, 1:0.6, 1:1), mix NOTA-BindHer and aluminum chloride solution, then add 18 F ion solution. Finally, add an equal volume of absolute ethanol so that the volume ratio of ethanol to the previous reaction mixture is 1:1 and react at 100 °C for 15 minutes. Conduct radioactive yield purity detection to determine the optimal reaction conditions.

[0257] 2) Detection of in vitro stability of molecular probe in the reaction mixture

[0258] Incubate the labeled product 18 F-NOTA-BindHer with an equal volume of normal saline or serum at 37 °C for 3 hours, and then conduct Radio-ITLC method to detect the radioactive purity.

[0259] 3) Further purification of the molecular probe

[0260] After the reaction is completed, purify it using a NAP-5 column, equilibrate with PBS for 10 column volumes, then pass the mixed reaction solution through the column. After it completely penetrates the column, elute with 250 μL PBS each time, collect the eluate, and detect the radioactive purity according to the Radio-ITLC method.

[0261] 4) Quality detection

[0262] Radio-ITLC detection: The stationary phase is a filter paper with a length of 10 cm and a width of 2 cm, and the mobile phase is 0.1 M sodium acetate solution. Use a micropipette to take 1 μL of the sample and spot it in the middle of the filter paper strip, generally as a round dot. The spotting distance from the bottom edge is 1.0 - 1.5 cm, and the diameter of the sample spot is generally not more than 2 mm. Hang the filter paper strip with the spotted sample on the hook of the flask cap, cover the flask and immerse it in the developing agent. Wait until the development reaches a certain distance (generally 8 - 15 cm), and terminate the chromatography and take out the paper strip. The Rf of the labeled protein is 0; the Rf of free aluminum fluoride is 1. The radioactive purity of the labeled protein (%) 18F-NOTA-BindHer) = Radioactivity on protein label / Total radioactivity * 100 (Total radioactivity = Radioactivity on protein label + Radioactivity of free aluminum fluoride);

[0263] UV-Radio-HPLC detection: Conditions for radioactive reverse high performance liquid chromatography analysis: 1. Chromatographic column variety and specifications: The packing material is octadecylsilyl bonded silica gel, with a pore size of 300 Å, a particle size of 5 μm, a diameter of 4.6 mm, and a length of 150 mm; Temperature: The column temperature is 30 ± 5 °C, and the storage temperature of the test sample is 2 - 8 °C; Mobile phase A is pure water (containing 0.1% trifluoroacetic acid), mobile phase B is an acetonitrile solution (containing 0.1% trifluoroacetic acid), the column flow rate is set to 1 ml / min; Detection wavelength is 214 nm; Mobile phase gradient: Mobile phase A is 78% - 22%, mobile phase B is 22% - 78%, time is 0 - 30 minutes, and the flow rate is 1 mL / min.

[0264] (3) Experimental results

[0265] Since AlCl3 is used to form Al 18 F, therefore, during the labeling process, the addition amount of AlCl3 is crucial. At a molar ratio of protein to aluminum chloride of 1:0.3, the radioactive labeling yield is 10.17% ± 1.56%; at 1:0.5, the yield increases to 46.96% ± 3.41%; at 1:0.6, at the highest concentration, the radioactive labeling yield is the best, at 57.25% ± 2.25%, and at the highest ratio of 1:1, the radioactive labeling yield is 55.23% ± 2.25%. Reducing the amount of Al 3+ will result in a decrease in the amount of 3+ bound to Al 18 F. On the contrary, if a larger amount is used, more unwanted proteins labeled with Al 3+ instead of Al 18 F will be obtained. Therefore, we will adopt a molar ratio of protein to aluminum chloride of 1:0.6.

[0266] As Figure 19 shown by the results, 18 the reaction mixture of 18 F-labeled NOTA-BindHer was incubated at 37 °C for up to 3 hours in human serum and PBS without dissociating into Al 18 F, and the radiochemical purity did not decrease significantly. It shows that

[0267] The radioactive labeling rate obtained by the one-step method for fluorine labeling in the present invention reaches over 50% ( Figure 21 A), and we further purified it by gel filtration and carried out quality detection.

[0268] As Figure 21 shown by the Radio-ITLC results of Figure 21 , the fluorine-labeled protein 18 F-NOTA-BindHer was both at the origin, and the free aluminum fluoride AlF 18 moved to the front edge of the paper strip with the mobile phase of sodium acetate solution. The radiochemical purity of the product before and after purification indicated that the free aluminum fluoride could be removed by purification through the NAP-5 column, and the radiochemical purity was increased to over 90% ( Figure 21 B), meeting the requirements for subsequent in vivo experiments.

[0269] 2. 18 PET / CT imaging of the F-NOTA-BindHer molecular probe in an animal tumor model

[0270] (1) Experimental samples

[0271] SK-BR-3 group: A HER2-positive breast cancer xenograft nude mouse model was constructed using SK-BR-3 cells, and 18 the F-NOTA-BindHer molecular probe was injected into the xenograft nude mice via the tail vein, followed by SPECT / CT imaging;

[0272] SK-BR-3 + Blocking group: A HER2-positive breast cancer xenograft nude mouse model was constructed using SK-BR-3 cells. First, an excessive amount of unlabeled BindHer protein was injected via the tail vein, and then 18 the F-NOTA-BindHer molecular probe was injected for SPECT / CT imaging;

[0273] MDA-MB-231 group: A HER2-negative xenograft nude mouse model was constructed using MDA-MB-231 cells, and 18 the F-NOTA-BindHer molecular probe was injected into the xenograft nude mice via the tail vein, followed by SPECT / CT imaging;

[0274] 18 F-NOTA-BindHer group: Prepared according to the method of Example 3;

[0275] 18 F-NOTA-ABY-025 group: Prepared according to the method of Example 3, except that the precursor protein was NOTA-ABY-025.

[0276] (2) Experimental methods

[0277] Establishment of animal model: The establishment of the mouse tumor model referred to the part of the animal model establishment in Experimental Example 1.

[0278] PET / CT imaging: The18 F-NOTA-BindHer and 18 F-NOTA-ABY (3.7 MBq, 10 μg) was injected into tumor-bearing mice with breast cancer via the tail vein respectively, and dynamic imaging was performed using a PET / CT imaging device within 60 minutes after the injection of the imaging agent.

[0279] (3) Experimental results

[0280] As Figure 22 shown, compared with the SK-BR-3 group, the SK-BR-3+Blocking group could effectively block the binding to HER2-positive tumors, resulting in no obvious uptake at the tumor site. In addition, the results of the MDA-MB-231 group showed that 18 the F-NOTA-BindHer molecular probe also had no obvious uptake at HER2-negative tumor sites, indicating that 18 the F-NOTA-BindHer molecular probe could specifically target and bind to HER2-positive tumors.

[0281] Figure 23 As shown, the fluorine-labeled molecular probe 18 F-NOTA-ABY.025 and 18 F-NOTA-BindHer had certain radioactive uptake at the tumor site at 10 minutes, 30 minutes, and 60 minutes after injection; Figure 23 Panel B showed that at 60 minutes after injection, 18 the uptake value of the F-NOTA-BindHer group in HER2-positive tumors was 14.22±1.15% ID / g, and the uptake value in the liver was 5.41±0.26 ID / g; while 18 the uptake value of the F-NOTA-ABY.025 group in tumors was 6.52±1.31% ID / g, and the uptake value in the liver was 25.32±0.71% ID / g. It showed that compared with the 18 F-NOTA-ABY.025 molecular probe, 18 the F-NOTA-BindHer molecular probe had a significantly increased tumor uptake and a significantly decreased liver uptake.

[0282] The above experimental results showed that the optimized 99m Tc-BindHer molecular imaging probe of the present invention had the following advantages: high radiochemical purity, good in vitro stability, excellent uptake and retention effects at the tumor sites of HER2-positive breast cancer-bearing mice, and low uptake values in non-target organs. 68 After structural optimization, the Ga-NOTA-BindHer molecular imaging probe showed stronger hydrophilicity, better serum stability, improved detection rate of HER2 breast cancer, and reduced liver absorption, thus enhancing the accuracy of the probe.18 The F-NOTA-BindHer molecular imaging probe specifically targets HER2-positive tumors, significantly increasing the tumor uptake rate while significantly reducing the liver uptake rate. Therefore, a new molecular imaging probe with excellent performance is obtained, which can be used for the imaging of HER2-positive breast cancer.

Claims

1. A molecular imaging probe, characterized in that, The molecular imaging probe is a radionuclide-labeled HER2-binding protein molecular imaging probe, and the amino acid sequence of the HER2-binding protein is as shown in SEQ ID NO.

1.

2. The molecular imaging probe according to claim 1, wherein The radionuclide is 99m Tc, and the structural formula of the molecular imaging probe is shown in Formula I; Or, the radionuclide is 68 Ga, and the structural formula of the molecular imaging probe is as shown in Formula II; Or, the radionuclide is 18 F, and the structural formula of the molecular imaging probe is as shown in Formula III; Among them, BindHer is the HER2-binding protein.

3. A method for preparing the molecular imaging probe according to claim 1 or 2, characterized in that, The method includes the following steps: (a) React the HER2-binding protein with a chelating agent in a solvent to obtain a precursor protein solution; (b) React the radionuclide solution with the precursor protein solution to obtain a molecular imaging probe.

4. The method according to claim 3, characterized in that, The radionuclide is 99m When it is 99m Tc, the preparation method of the precursor protein solution comprises the following steps: mixing a HER2 binding protein, buffer solution 1, stannous chloride, EDTA and sodium gluconate, performing sterile filtration, and adjusting the pH to obtain the precursor protein solution; the pH is 5.5-6.5, and the buffer solution 1 is selected from at least one of a citric acid-sodium citrate buffer solution, a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, and a disodium hydrogen phosphate-citric acid buffer solution; in the precursor protein solution, the concentration of the HER2 binding protein is 0.5-1.5 mg / mL, the concentration of the disodium hydrogen phosphate-citric acid buffer solution is 15-25 mM, the concentration of stannous chloride is 5.0-6.0 mg / mL, the concentration of EDTA is 7.0-8.0 mg / mL, and the concentration of sodium gluconate is 260-300 mg / mL; Or, when the radionuclide is 68 Ga, the preparation method of the precursor protein solution comprises the following steps: reacting a HER2-binding protein with a chelating agent MAL-NOTA to obtain NOTA-BindHer; adding NOTA-BindHer to buffer solution 2 to obtain a precursor protein solution; the buffer solution 2 is a sodium acetate solution with a concentration of 0.05-0.2 M and a pH of 5.5-6.5, and the concentration of the precursor protein solution is 1-4 mg / mL; Or, when the radionuclide is 18 F, the method for preparing the precursor protein solution comprises the following steps: (1) Protein pretreatment: React the HER2-binding protein with a thiol reducing agent to obtain a pretreated protein; (2) Coupling reaction: Perform a coupling reaction on the pretreated protein and the chelating agent MAL-NOTA in buffer 3 to obtain a precursor protein solution; the buffer 3 is PBS, and the molar ratio of the pretreated protein to the chelating agent is 1:(1-5); in the precursor protein solution, the concentration of the pretreated protein is 2.0-3.0 mg / mL.

5. The method according to claim 4, wherein When the radionuclide is 99m Tc, the buffer solution 1 is selected from disodium hydrogen phosphate-citric acid buffer solution; the pH is 6.0; in the precursor protein solution, the concentration of HER2-binding protein is 1 mg / mL, the concentration of disodium hydrogen phosphate-citric acid buffer solution is 20 mM, the concentration of stannous chloride is 5.6 mg / mL, the concentration of EDTA is 7.5 mg / mL, and the concentration of sodium gluconate is 280 mg / mL; Or, when the radionuclide is 68 Ga, the buffer solution 2 is a sodium acetate solution with a concentration of 0.1 M and a pH of 6.0, and the concentration of the precursor protein solution is 2 mg / mL; Or, when the radionuclide is 18 F, the thiol reducing agent is TCEP, the molar ratio of the pretreated protein to the chelating agent is 1:3, and in the precursor protein solution, the concentration of the pretreated protein is 2.2 - 2.5 mg / mL.

6. The method according to claim 3, wherein The radionuclide is 99m When Tc is used, the preparation method of the radionuclide solution comprises the following steps: washing with physiological saline 99 Mo- 99m Tc generator, get 99m TcO4- solution, i.e., radionuclide solution; Or, when the radionuclide is 68 Ga, the preparation method of the radionuclide solution comprises the following steps: leaching a germanium-gallium generator with hydrochloric acid to obtain 68 Ga solution, and mixing the 68 Ga solution with a sodium acetate solution to obtain a pre-buffered 68 Ga solution, i.e., the radionuclide solution; Or, the radionuclide is 18 When it is 18 F, the method for preparing the radionuclide solution comprises the following steps: rinsing a fluorine accelerator with physiological saline to obtain 18 F ion solution, i.e., the radionuclide solution.

7. The method according to claim 6, characterized in that, When the radionuclide is 99m Tc, the radioactivity of the radionuclide solution is 20 - 50 MBq, preferably 37 MBq; Or, when the radionuclide is 68 Ga, the radioactivity of the radionuclide solution is 20 - 40 MBq, preferably 37 MBq; Or, when the radionuclide is 18 F, the radioactivity of the radionuclide solution is 0.5 - 2 mCi, preferably 1 mCi.

8. The method according to claim 3, characterized in that, The radionuclide is 99m When it is 99m Tc, the volume ratio of the radionuclide solution to the precursor protein solution in step (b) is 1:(0.25-1); the temperature of the reaction is 20-30°C, and the reaction time is 15-25 minutes; Or, the radionuclide is 68 When it is 68 Ga, the volume ratio of the radionuclide solution to the precursor protein solution in step (b) is 1:(0.5 - 2); the temperature of the reaction is 65 - 85 °C, the reaction time is 10 - 20 minutes, and the pH of the reaction is 4.0 - 6.0; Or, the radionuclide is 18 When it is 18 F, step (b) includes the following steps: mixing a radionuclide solution with a precursor protein solution and an aluminum chloride solution, adding ethanol, and reacting to obtain the product; the molar ratio of the precursor protein to aluminum chloride is 1:(3-12), and the volume ratio of the radionuclide solution to the precursor protein solution is 1:(2-10); the reaction temperature is 80-120°C, and the reaction time is 10-20 minutes.

9. The method according to claim 8, wherein: When the radionuclide is 99m Tc, the volume ratio of the radionuclide solution to the precursor protein solution is 1:0.5; the temperature of the reaction is 25 °C, and the reaction time is 20 minutes; Or, the radionuclide is 68 When it is 68 Ga, the volume ratio of the radionuclide solution to the precursor protein solution is 1:1; the temperature of the reaction is 75 °C, the reaction time is 15 minutes, and the reaction pH is 4.0 - 5.0; Or, the radionuclide is 18 When it is 18 F, the molar ratio of the precursor protein to aluminum chloride is 1:6, and the volume ratio of the radionuclide solution to the precursor protein solution is 1:5; the reaction temperature is 100 °C and the reaction time is 15 minutes.

10. Use of the molecular imaging probe according to any one of claims 1-2 in the preparation of an imaging agent for HER2-positive tumors.

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