HER2 (human epidermal growth factor receptor 2) receptor targeting diagnosis and treatment probe as well as labeled precursor, preparation method and application thereof

Abstract

The invention discloses a diagnosis and treatment probe of a targeted HER2 receptor and a labeled precursor, a preparation method and application of the diagnosis and treatment probe, and belongs to the technical field of radiopharmaceuticals. According to a biological orthogonal click chemical strategy, diagnosis and treatment are organically combined, HER2 is used as a target spot to design the nuclear medicine diagnosis and treatment integrated probe, the probe can achieve efficient and high-specificity tumor marking and treatment, and the accuracy and effect of clinical diagnosis and treatment are improved; meanwhile, by reducing accumulation of non-specific radioactive substances in the body, radiation damage to healthy tissue is remarkably reduced, a safe and effective treatment scheme is provided for patients, and potential clinical transformation prospects are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of radiopharmaceuticals, and in particular relates to a nuclear medicine diagnostic probe targeting HER2 receptors, a labeled precursor thereof, and a probe composition based on pre-positioning, as well as a preparation method and use thereof. Background Art

[0002] According to data from the National Cancer Center in 2024, the number of new cancer cases and deaths in China increased in 2022. The top five malignant tumors in terms of incidence are lung cancer, colorectal cancer, thyroid cancer, liver cancer, and gastric cancer; the top five malignant tumors in terms of mortality are lung cancer, liver cancer, gastric cancer, colorectal cancer, and esophageal cancer. Cancer prevention and control remains a major public health issue in China.

[0003] Human epidermal growth factor receptor 2 (HER2), also known as ERBB2, is a receptor tyrosine kinase. Compared with normal tissues / organs, HER2 shows significant overexpression in a variety of malignant tumors. For example, HER2 gene amplification or HER2 protein overexpression has been detected in breast cancer, gastric cancer, lung cancer, colorectal cancer, and ovarian cancer. HER2 is characterized by high-density expression on its cell membrane surface. This specificity makes HER2 an ideal pre-target, which helps to achieve precise positioning and targeted treatment of tumor tissues. How to accurately, holistically and non-invasively evaluate HER2 expression has become an urgent problem to be solved.

[0004] In recent years, radiotherapy and chemotherapy combined with targeted and immunotherapy have enriched the treatment options for advanced malignant tumors, and the median survival has also increased significantly. Traditional radioimmunoassay / therapy uses radionuclides to directly label large molecular monoclonal antibodies, giving full play to the excellent specificity and affinity of monoclonal antibodies. After a long period of metabolism, a very high target-to-substance ratio can usually be obtained, thereby achieving excellent quality images or significant therapeutic effects. However, too long a circulation time in the body will result in a large cumulative radiation dose, which may cause serious radiation damage to organs with rich blood supply and drug metabolism organs such as the liver and kidneys.

[0005] In order to improve this shortcoming, the modified antibody macromolecule is introduced in advance through bioorthogonal click chemistry. After a certain period of metabolism, the radionuclide-labeled small molecule probe is introduced into the body. Through the "click" reaction, the radionuclide-labeled small molecule binds to the modified antibody and traces or kills the target. This method combines the advantages of high affinity and high specificity of large molecule monoclonal antibodies with the advantages of rapid metabolism and low radiation cumulative dose of small molecules.

[0006] The present invention combines diagnosis and treatment organically according to the bio-orthogonal click chemistry strategy, and designs a nuclear medicine diagnosis and treatment integrated probe with HER2 as the target. The probe can use the same precursor to label different halogen-related radionuclides such as iodine-131, iodine-123, iodine-124, iodine-125, astatine-211, etc., and can achieve the following functions: 1. Guiding targeted medication: For patients who are difficult to sample pathologically, nuclear medicine imaging can be performed using the probe provided by the present invention, which can non-invasively, dynamically, and in real time evaluate the HER2 expression level to guide clinical medication. At the same time, whole-body PET imaging can also avoid the spatial heterogeneity and temporal heterogeneity of tumor HER2 expression; 2. Nuclide targeted therapy: Nuclide targeted therapy is performed for patients who progress after resistance to multiple cycles and multiple chemotherapy regimens. The probe provided by the present invention is integrated with diagnosis and treatment, and provides a new way for non-invasive evaluation of HER2 receptor expression levels and PRRT treatment of tumors with high HER2 receptor expression, which has important medical value. Summary of the invention

[0007] One object of the present invention is to provide a labeled precursor of an integrated diagnostic and therapeutic probe and a preparation method thereof for tumors with high HER2 expression; a second object of the present invention is to provide an integrated diagnostic and therapeutic probe labeled with a radionuclide; a third object of the present invention is to provide a probe composition based on a bioorthogonal click chemistry strategy; and a further object of the present invention is to provide a use of the probe and the probe composition.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] In a first aspect, the present invention provides a nuclear medicine diagnostic probe labeling precursor targeting HER2 receptor, having a structure as shown in Formula I:

[0010]

[0011] Wherein, "-" in the formula represents a connecting peptide or a peptide bond.

[0012] n is selected from an integer between 1 and 20; in a preferred embodiment of the present invention, n is 11.

[0013] Peptide has the following structure:

[0014]

[0015] The chelating ligand is selected from the chelating agents commonly used in the art that can chelate with radionuclides.

[0016] In some embodiments of the present invention, the probe labeling precursor has a structure shown in Formula II:

[0017]

[0018] The chelating ligand is selected from tyrosine (Tyr), HYNIC, DTPA, DOTA, NOTA and derivatives thereof. Those skilled in the art can modify the above chelating ligand onto the polypeptide structure by conventional techniques.

[0019] In a non-limiting manner, the chelating ligand of the present invention is selected from at least one of the following:

[0020] a1) Tyr;

[0021] a2) HYNIC;

[0022] a3)DOTA and its derivatives: DOTA-Mal, Butyne-DOTA-tris(t-Bu ester), Mono-amide-DOTA, Bn-SCN-DOTA, N3-DOTA-tBu, DOTA(tBu)-COOH;

[0023] a4) DTPA and its derivatives: DTPA-Mal, p-NH2-Bn-DTPA, p-NH2-Bn-DTPA-penta, p-SCN-Bn-DTPA, p-SCN-Bn-CHX-A-DTPA, DTPA-tetra (t-Bu ester);

[0024] a5) DOTA and its derivatives: DOTA, p-NH2-Bn-DOTA, p-SCN-Bn-DOTA, DOTA-bis(t-Buester), Maleimido-mono-amide-NOTA.

[0025] In a specific embodiment of the present invention, the probe labeling precursor has the following structure:

[0026]

[0027] The present invention is named as: Tyr-D-peptide-PEG 11 -Tz.

[0028] In a second aspect, the present invention provides a method for preparing a nuclear medicine diagnostic and therapeutic probe labeling precursor targeting HER2 receptor, comprising:

[0029] Step 1: Compound 3 was obtained by peptide solid phase synthesis;

[0030]

[0031] Step 2: removing the Boc protecting group of the Tz compound and coupling it with compound 3 to form compound 4;

[0032]

[0033] Step 3: After removing the Boc protecting group of compound 4, the compound 8 is reacted with activated Boc-D-Tyr(tBu)-OH;

[0034]

[0035] Step 4: removing the protecting group of compound 8 to obtain the probe labeling precursor.

[0036] In a third aspect, the present invention provides a nuclear medicine diagnostic probe targeting HER2 receptor, wherein the probe is a chelate of the probe labeling precursor described in the first aspect of the present invention and a radioactive nuclide.

[0037] The radioactive nuclide ions described in the present invention are nuclide ions well known to those skilled in the art and can be used for imaging, tracing and / or treatment.

[0038] Preferably, the radionuclides include but are not limited to 211 At 99 Tc, 99m Tc, 117 Sn, 111 In, 97 Such as 67 Ga, 68 Ga, 89 Zr, 177 Lu, 47 Sc, 105 Rh, 188 Re, 60 Cu, 62 Cu, 64 Cu, 67 Cu, 90 Y. 159 Gd, 149 Pr, 166 Ho, 131 I. 125 I. 124 I or 123 I.

[0039] In the most preferred embodiment of the present invention, the nuclear medicine diagnostic probe targeting HER2 receptor has the following structure:

[0040]

[0041] Name it: 131 I-Tyr-D-peptide-PEG 11 -Tz.

[0042] In a fourth aspect, the present invention provides a method for preparing a nuclear medicine diagnostic probe targeting HER2 receptor, the method comprising: under appropriate conditions, allowing a radioactive nuclide to undergo a chelation reaction with the probe labeling precursor described in the first aspect of the present invention, and purifying to obtain a target probe.

[0043] The appropriate conditions described in the present invention are the reaction conditions routinely used by those skilled in the art for labeling nuclides.

[0044] In a specific embodiment of the present invention, the method for preparing the probe comprises the following steps:

[0045] Step 1: Prepare Iodogen tube;

[0046] Step 2: Add the probe labeling precursor and Na 131 I, room temperature reaction;

[0047] Step 3: Purification to obtain the probe.

[0048] In a fifth aspect, the present invention provides a HER2 receptor pre-localization diagnostic and therapeutic probe composition based on AD click chemistry reaction, wherein the composition comprises the probe described in the third aspect of the present invention and an anti-HER2 antibody-TCO conjugate.

[0049] The anti-HER2 antibody is an antibody with HER2 receptor targeting function known to those skilled in the art. In a specific embodiment of the present invention, the anti-HER2 antibody is selected from Pertuzumab and / or Trastuzumab.

[0050] In a specific embodiment of the present invention, the anti-HER2 antibody is Pertuzumab, and the conjugate formed by the anti-HER2 antibody and TCO-NHS ester is Pertuzumab-TCO.

[0051] The Pertuzumab-TCO is prepared by the following method:

[0052] The pH of the Pertuzumab solution was adjusted to 7-9, TCO-NHS ester was added, and the mixture was reacted at 4° C. in the dark for 10-12 h, then the mixture was returned to room temperature and purified to prepare Pertuzumab-TCO.

[0053] In the most preferred embodiment of the present invention, the HER2 receptor pre-localization diagnostic and therapeutic probe composition based on AD click chemistry reaction is composed of 131 I-Tyr-D-peptide-PEG 11 -Tz and Pertuzumab-TCO.

[0054] The present invention is named as: 131 I-Tyr-D-peptide-PEG 11 -Tz / Pertuzumab-TCO.

[0055] Using the probe provided by the present invention 131 I-Tyr-D-peptide-PEG 11 -Tz / Pertuzumab-TCO can be used to diagnose and treat patients with tumors that overexpress HER2. The specific application method is: within a predetermined time, Pertuzumab-TCO is first injected into the body, and after it fully binds to the HER2 antigen on the surface of tumor cells and the unbound antibody conjugate is slowly excreted from the body, it is then injected intravenously. 131 I-Tyr-D-peptide-PEG 11 -Tz for imaging and / or treatment. At a predetermined time point, single photon emission computed tomography (SPECT) is used to non-invasively evaluate the status of HER2-overexpressing tumors. This process not only clearly shows the location and expression of the tumor, but also uses the beta rays emitted by iodine-131 to accurately kill tumor cells, thereby effectively reducing or inhibiting tumor growth.

[0056] In a sixth aspect, the present invention provides a use of the probe according to the third aspect of the present invention and / or the probe composition according to the fifth aspect of the present invention in at least one of the following:

[0057] 1) Use in the preparation of a reagent for assessing the HER2 expression level in a tumor;

[0058] 2) Use in the preparation of nuclear medicine imaging products for HER2-overexpressing tumors;

[0059] 3) Use in the preparation of nuclear medicines for treating HER2-overexpressing tumors.

[0060] The technical solution provided by the present invention has the following technical advantages:

[0061] 1) Probe structure stability: The present invention designs a new integrated diagnostic and therapeutic probe targeting HER2 receptor based on the bio-orthogonal click chemistry pre-targeting method. In view of the general problem of polypeptide stability, L-type amino acids are replaced with D-type amino acids, which significantly improves the stability of the probe.

[0062] 2) Flexibility of labeling with multiple radioactive isotopes: The probe-labeled precursors provided by the present invention can be radioactively labeled with more than just iodine-131. Those skilled in the art can select different radioactive isotopes according to different diagnostic and therapeutic needs, flexibly respond to different clinical applications, and improve the accuracy and efficacy of diagnosis and treatment.

[0063] 3) Complete separation of precursors and products: Since the polarity of the product changes significantly compared to the precursor after radioactive iodine labeling, the precursor and product can be completely separated by semi-preparative high performance liquid chromatography (HPLC), effectively eliminating the interference of unlabeled precursors and significantly reducing the chemical equivalents entering the body, thereby reducing side effects and improving the safety and effectiveness of treatment.

[0064] 4) Significantly improved stability: According to existing literature reports, the pre-targeting probes connected to polypeptides will be significantly decomposed within 2 hours, and the stability is poor. 131 I-Tyr-D-peptide-PEG11-Tz has significantly improved stability, which not only prolongs the effective time of the probe in the body and improves diagnostic efficiency, but also brings better expected results for subsequent treatments, which is much better than the current pre-targeting probes.

[0065] 5) Economical and efficient cost advantage: As we all know, 177 The price of Lu for radionuclide therapy is very expensive. The probe of the present invention is labeled with iodine-131, which is relatively cheap, greatly reducing the cost of treatment, making it affordable for more patients and improving the popularity and accessibility of nuclear medicine treatment.

[0066] The probe designed by the present invention realizes efficient and highly specific tumor labeling and treatment, greatly improving the accuracy and effect of clinical diagnosis and treatment. At the same time, by reducing the accumulation of non-specific radioactive substances in the body, the radiation damage to healthy tissues is significantly reduced, providing patients with a safe and effective treatment plan, and laying a good research foundation for the accurate diagnosis and effective treatment of HER2-overexpressing tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Schematic diagram of anti-HER2 antibody-TCO conjugates.

[0068] Figure 2 Compound Tyr-D-peptide-PEG 11 -Tz mass spectrum.

[0069] Figure 3 131 I-Tyr-D-peptide-PEG 11 -Tz semi-preparative HPLC purification diagram.

[0070] Figure 4 131 I-Tyr-D-peptide-PEG 11 -In vitro stability test diagram of Tz.

[0071] Figure 5 131 I-Tyr-D-peptide-PEG 11 -Pharmacokinetic profile of Tz.

[0072] Figure 6 131 I-Tyr-D-peptide-PEG 11 -Saturation experimental diagram of Tz.

[0073] Figure 7 Western blots and flow cytometry of SKOV3 cells that highly express HER2.

[0074] Figure 8 131 I-Tyr-D-peptide-PEG 11 -SPECT images of Tz / Pertuzumab-TCO in the SKOV3 animal model.

[0075] Fig. 9 131 I-Tyr-D-peptide-PEG 11 -SPECT images of Tz / Pertuzumab-TCO in the blocking group and MGC803-negative animal model. DETAILED DESCRIPTION

[0076] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0077] Explanation of terms

[0078] The term "Diels-Alder click chemistry reaction" refers to a classic reaction in which a conjugated diene and a dienophile construct a cyclohexene skeleton. The reaction has rich stereochemical presentations, and has stereoselectivity, stereospecificity, and regioselectivity. The reaction is a reversible reaction, and the reaction temperature for the forward ring formation is relatively low, while the reverse ring-opening reaction requires a higher temperature. This reaction can synthesize a six-membered ring with very little energy, and is one of the very important cyclization methods for preparing unsaturated six-membered rings.

[0079] The term "Tz" is an abbreviation for 1,2,4,5-tetrazine.

[0080] The term "TCO-NHS ester" has the structural formula

[0081] The term "FBS" refers to fetal bovine serum.

[0082] The term "1H-Pyrazole-1-Carboxamidine Hydrochloride" is the English name of the compound 1H-pyrazole-1-carboxamidine hydrochloride.

[0083] 1. Preparation and characterization of compounds

[0084] (1) Synthesis of Pertuzumab-TCO

[0085] Synthesis route such as Figure 1 As shown, the synthesis method is as follows:

[0086] S1) dissolving 10 mg of TCO-NHS ester in 1 mL of DMSO to prepare a 10 mg / mL TCO-NHS ester solution for later use;

[0087] S2) taking a 5.0 mg / mL pertuzumab solution and adjusting the pH value to 7-9 for later use;

[0088] S3) dissolving TCO-NHS ester in 3 volumes of 0.2 M sodium carbonate buffer and adding Pertuzumab solution so that the molar ratio of TCO-NHS ester to Pertuzumab in the reaction solution is 50:1, reacting at 4° C. in the dark for 12 hours, and then returning to room temperature;

[0089] S4) Purification was performed using a PD-10 desalting column to obtain the final antibody conjugated product Pertuzumab-TCO, which was stored in a PBS (pH ~ 7.4) buffer.

[0090] (2) Synthesis of the compound Tyr-D-peptide-PEG11-Tz 11 -Tz synthesis route is as follows:

[0091]

[0092] The specific synthesis method is:

[0093] Step a:

[0094]

[0095] TFA (7.68 g, 67.3 mmol, 5 mL, 81.1 eq) was added to a solution of compound 1 (250 mg, 829 μmol) dissolved in 20 mL DCM, and the reaction solution was stirred at 25 ° C for 2 h. LCMS was used for quality control, and the raw material compound 1 was completely reacted. The reaction solution was adjusted to pH 7-8 with NaHCO3 solution, and the reactant was washed 3 times with 1N HCl (250 mL), H2O (250 mL), and brine (250 mL), and dried over Na2SO4. The filtered liquid was lyophilized to obtain a red solid compound 2 (208 mg), which was confirmed by LCMS, HPLC and HNMR.

[0096] Step b:

[0097]

[0098] Peptide solid phase synthesis method:

[0099] 1) Dissolve 2-CTC Resin (0.50 mmol, Sub = 1.10 mmol / g) and Fmoc-PEG in 10 mL DCM. 11 -OH (0.39g, 1.00eq) solution was added dropwise with DIEA (4.00eq) and reacted for 2h under nitrogen blowing. Then 1.0mL methanol was added to the resin and nitrogen blowing was performed for 30 minutes. The resin was washed with DMF (10mL) 5 times and treated with DMF (5ml) containing 20% ​​piperidine at 20°C with nitrogen blowing for 30min to remove Fmoc. After ultrafiltration of the reaction solution, the resin was washed with DMF (10mL) 5 times for later use.

[0100] 2) Add the resin to a solution of Fmoc-D-isoGln(Trt)-OH (3.00 eq), HOAt (3.00 eq) and DIC (3.00 eq) dissolved in DMF (10 mL), and react for 30 min under nitrogen bubbling at 20°C. Monitor the coupling reaction by the ninhydrin test. If it is colorless, the coupling is complete. Then wash the resin 5 times with DMF (10 mL).

[0101] 3) DMF (10 ml) containing 20% ​​piperidine was treated with nitrogen gas at 20°C for 30 min, and the resin was washed 5 times with DMF (10 mL). The reaction was monitored by ninhydrin test, and if blue or other brownish red color was shown, the reaction was complete.

[0102] 4) Repeat steps 2-3 for the following amino acid extensions: numbered 3-9.

[0103] Table 1 Raw materials for solid phase synthesis of peptides

[0104]

[0105] 5) After the reaction of the last amino acid was completed, the resin was washed five times with DMF (10 mL) and MeOH (10 mL) and dried in vacuo.

[0106] 6) Add the lysate (HFIP / DCM, v / v, 20 / 80, 20 mL) to the flask containing the side chain protected peptide and react with nitrogen for 10 min at room temperature.

[0107] 7) The reaction solution was filtered and the filtrate was concentrated under pressure.

[0108] 8) After freeze-drying, crude compound 3 (385 mg) was obtained as a yellow solid, which was quality controlled by LCMS.

[0109] Step c:

[0110]

[0111] EDCI (53.0 mg, 276 μmol, 1.20 eq) and HOBt (37.3 mg, 276 μmol, 1.20 eq) were added to a solution of compound 2 (60.2 mg, 299 μmol, 1.30 eq) and compound 3 (365 mg, 230 μmol, 1.00 eq) containing 4 mL of DMF, and the reaction solution was stirred at 0 ° C for 2 h, and LCMS was used for quality control. The reaction solution was precipitated with cold isopropyl ether (10 mL) and centrifuged (3000 rpm, 2 min). The product was vacuum dried for 2 h to obtain a red solid crude compound 4 (458 mg).

[0112] Step d:

[0113]

[0114] Compound 4 (458 mg, 259 μmol) was added to a solution of TFA (3.84 g, 33.6 mmol, 2.50 mL) containing 2.50 mL of DCM, and the reaction solution was stirred at 25 ° C for 2 h. LCMS was used for quality control. The reaction solution was precipitated with cold isopropyl ether (50 mL) and centrifuged (at 3000 rpm, 2 min), and then washed twice with isopropyl ether (50 mL). The product was vacuum dried for 2 h and then purified by preparative HPLC. After LCMS and HPLC quality control, a white solid compound 5 (255 mg, 160 μmol, yield 61.9%, purity 97.8%) was obtained.

[0115] Step e:

[0116]

[0117] HOSu (1.36 g, 11.86 mmol, 4.00 eq) and EDCI (1.14 g, 5.93 mmol, 2.00 eq) were added to a solution of compound 6 (1.00 g, 2.96 mmol, 1.00 eq) dissolved in 10 mL DMF at 20°C. The reaction solution was stirred at 20°C for 2 h. LCMS quality control showed that the raw material compound 6 reacted completely. The product was separated and purified by pre-HPLC to obtain a white solid compound 7 (778 mg, yield 60.21%, purity 99.65%).

[0118] Step f:

[0119]

[0120] Compound 7 (11.9 mg, 27.4 μmol, 1.10 eq) and DIEA (16.1 mg, 124 μmol, 21.73 μL, 5.00 eq) were added to a solution of compound 5 (30 mg, 24.9 μmol, 1.00 eq) dissolved in 0.05 mL DMF at 20°C. The reaction solution was stirred at 20°C for 1 h. LCMS quality control was performed, and the raw material compound 5 was completely reacted. The product was separated and purified by pre-HPLC to obtain a red solid compound 7 (16.5 mg, yield 45.6%, purity 99.36%).

[0121] Step g:

[0122]

[0123] Compound 8 (16.5 mg) dissolved in 0.5 mL TFA / DCM (volume ratio of 1:1) was stirred for 2 h at 20 °C. The mixture was added with isopropyl ether and centrifuged at 3000 rpm for 3 min to obtain a precipitate, and the product was washed twice with isopropyl ether. The product was dried under reduced pressure for 2 h. LCMS was used for quality control, and the raw material compound 8 was completely reacted. The product was separated and purified by pre-HPLC to obtain a red solid Tyr-D-peptide-PEG 11 -Tz (8.6 mg, yield 56.8%, purity 99.52%).

[0124] (3) 131 I-Tyr-D-peptide-PEG 11 Synthesis of -Tz

[0125] Dissolve Iodogen in dichloromethane to a concentration of 1 mg / mL, add 30 μL of Iodogen solution to the bottom of the glass tube, blow nitrogen until the dichloromethane is completely evaporated and Iodogen forms a thin film at the bottom of the tube to obtain an Iodogen tube. Add 60 μg of Tyr-D-peptide-PEG to the Iodogen tube in turn. 11 -Tz and Na 131 I (37 MBq) was reacted at room temperature, and the test tube was shaken every 5 minutes. The reaction was completed after 15 minutes. After semi-preparative HPLC purification, 131 I-Tyr-D-peptide-PEG 11 -Tz.

[0126] (4) Experimental results

[0127] 1) Radioactive compound labeled precursor mass spectrometry results such as Figure 2 shown.

[0128] 2) 131 I-Tyr-D-peptide-PEG 11 -Tz was purified by semi-preparative HPLC as Figure 3 The following mobile phase and gradient were used at a rate of 3 ml / min: 0-25 min, 80%-65% solution A (0.1% TFA dissolved in H2O) and 20%-35% solution B (0.1% TFA dissolved in ACN). The compound with a retention time of 22.814 minutes was Tyr-D-peptide-PEG 11 -Tz, the product with retention time of 25.205 minutes is 131 I-Tyr-D-peptide-PEG 11 -Tz. After adding an iodine atom, the polarity of the target product and the precursor changes dramatically. In this way, the precursor and the product are completely separated to avoid competition with the precursor for the target.

[0129] 3) The product obtained by semi-preparative HPLC purification was diluted and solvent replaced with an activated C-18 extraction column, and eluted with a prepared eluent (70% ethanol, 10% 0.1M hydrochloric acid, 20% pure water). The eluted product was collected into an EP tube, 0.2 mL per tube, and the eluent with the highest radioactivity was detected as the target product. The ethanol concentration was diluted to less than 10% with PBS to obtain the final target product.

[0130] two, 131 I-Tyr-D-peptide-PEG 11 -Tz in vitro stability test

[0131] Will131 I-Tyr-D-peptide-PEG 11 -Tz molecular probes were placed in PBS and 10% FBS, respectively, and Radio-HPLC quality control was performed after the predetermined time. Among them, the PBS group was directly subjected to Radio-HPLC quality control, and the 10% FBS group was added with an equal volume of acetonitrile before Radio-HPLC quality control, mixed thoroughly, and centrifuged at high speed, and then the supernatant was taken for quality control. The results are shown in Figure 4 As shown in the figure, the molecular probe has a stability of more than 80% in both 10% FBS and PBS within 120 hours, indicating that the molecular probe prepared by the present invention has a stable 131 I-Tyr-D-peptide-PEG 11 -The stability of Tz molecular probe has been significantly improved.

[0132] three, 131 I-Tyr-D-peptide-PEG 11 - Pharmacokinetics and saturation experiments of Tz

[0133] 1) 131 I-Tyr-D-peptide-PEG 11 -The pharmacokinetics of Tz Figure 5 An appropriate amount of probe was injected into the tail vein of mice, and blood was collected at a predetermined time point. The blood sample mass was calculated by the difference method, and then its %IA / g was calculated. Its distribution half-life and elimination half-life were calculated, T 1 / 2α =3.378min, T 1 / 2β =95.36min.

[0134] 2) 131 I-Tyr-D-peptide-PEG 11 -Tz saturation experiment Figure 6 As shown. Gradient concentrations of 131 I-Tyr-D-peptide-PEG 11 -Tz, after incubation at room temperature for 1 h, measure its CPS, draw a saturation curve, and obtain the target K d Value and B max , K d =47.15nM, B max =634.1CPS.

[0135] HER2 Animal Model 131 I-Tyr-D-peptide-PEG 11 -Tz micro-SPECT imaging

[0136] The cell line SKOV3 with high expression of HER2 was screened by Western blots and flow cytometry. Figure 7 As shown. SKOV3 cells and MGC803 cells were used to construct animal models, and SKOV3 tumor model mice and MGC803 tumor model mice were obtained.

[0137] The SKOV3 tumor model mice were randomly divided into 3 groups, named group 1, group 2, and group 3. Group 1 was given pertuzumab-TCO in the tail vein at a dose of 0.1 mg. 48 hours after the administration, the probe was given in the tail vein. 131 I-Tyr-D-peptide-PEG 11 -Tz. Group 2 was given Pertuzumab-TCO by tail vein, the dosage was 0.2mg, and probe was given by tail vein 48h after administration. 131 I-Tyr-D-peptide-PEG 11 -Tz. Group 3 was given Pertuzumab-TCO by tail vein, the dosage was 0.1mg, and probe was given by tail vein 24h after administration. 131 I-Tyr-D-peptide-PEG 11 -Tz. The probe injection volume was 500μCi / mouse. Micro-SPECT imaging was performed 6h, 24h, 48h, and 120h after probe injection. The results are shown in Fig. 9 As shown. It can be seen that the probe 131 I-Tyr-D-peptide-PEG 11 -Tz successfully clicked Pertuzumab-TCO pre-located in the tumor site in vivo, and the tumor site had obvious uptake at 6h imaging, and the probe had a fast blood clearance rate. At the same time, the probe at the tumor site had high stability, and uptake could still be observed at the tumor site at 120h. The above results prove that the probe prepared by the present invention has therapeutic potential, which can be verified by subsequent experiments.

[0138] The MGC803 tumor model mice were named the negative group, and the SKOV3 tumor model mice were named the blocking group. The blocking group mice were given HER2 antibodies to block the receptor. Pertuzumab-TCO was administered to the tail vein of the negative group and the blocking group mice at a dose of 0.2 mg. The probe was administered to the tail vein 48 hours after administration. 131 I-Tyr-D-peptide-PEG 11-Tz. The probe injection amount was 500 μCi / mouse, and micro-SPECT imaging was performed at 6h, 24h, 48h, and 120h after probe injection, respectively. The results are shown in Figure 10. As can be seen from the figure, there was no obvious uptake in the tumor site in the blocking group and the negative group mice at 6h imaging, indicating that the probe prepared by the present invention has HER2 receptor specificity.

[0139] The embodiment of the present invention provides 131 I-Tyr-D-peptide-PEG 11 The probe combination composed of -Tz and Pertuzumab-TCO is only a technical solution using HER2 receptor as an example target, and is not intended to limit the scope of the present technical solution. Those skilled in the art should know that when Pertuzumab is replaced by an antibody (mAb) targeting any other target, 131 I-Tyr-D-peptide-PEG 11 The probe combination composed of -Tz and mAb-TCO can be used for pre-targeting diagnosis and treatment of the receptor based on AD click chemistry reaction. 131 I-Tyr-D-peptide-PEG 11 The probe composition composed of -Tz and mAb-TCO is also included in the technical solution described in the present invention.

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nuclear medicine diagnostic and therapeutic probe marker precursor targeting HER2 receptor, characterized in that: Having the structure shown in Formula I: Wherein, "-" in the formula represents a connecting peptide or peptide bond; n is selected from an integer between 1 and 20; Peptide has the following structure: The chelating ligand is selected from the chelating agents commonly used in the art that can chelate with radionuclides.

2. The probe labeling precursor according to claim 1, characterized in that It has the structure shown in Formula II: The chelating ligand is selected from tyrosine (Tyr), HYNIC, DTPA, DOTA, NOTA and derivatives thereof.

3. The probe labeling precursor according to claim 2, characterized in that It has the following structure: Name: Tyr-D-peptide-PEG 11 -Tz.

4. A method for preparing a nuclear medicine diagnostic and therapeutic probe labeling precursor targeting HER2 receptor according to claim 3, characterized in that: The method comprises: Step 1: Compound 3 was obtained by peptide solid phase synthesis; Step 2: remove the Boc protecting group of the Tz compound and couple it with compound 3 to form compound 4; Step 3: After removing the Boc protecting group of compound 4, the compound 8 is reacted with activated Boc-D-Tyr(tBu)-OH; Step 4: removing the protecting group of compound 8 to obtain the probe labeling precursor.

5. A nuclear medicine diagnostic probe targeting HER2 receptor, characterized in that: The probe is a chelate of the probe labeling precursor according to any one of claims 1 to 3 and a radioactive nuclide, and the radioactive nuclide ions are nuclide ions well known to those skilled in the art and can be used for imaging, tracing and / or treatment.

6. The probe according to claim 5, characterized in that It has the following structure: Named: 131 I-Tyr-D-peptide-PEG 11 -Tz.

7. A HER2 receptor pre-localization diagnostic and therapeutic probe composition based on AD click chemistry reaction, characterized in that: The composition comprises the probe according to any one of claims 5 to 6 and an anti-HER2 antibody-TCO conjugate, wherein the anti-HER2 antibody is an antibody known to those skilled in the art that has the function of targeting HER2 receptors.

8. The probe composition according to claim 7, characterized in that The anti-HER2 antibody is selected from Pertuzumab and / or Trastuzumab.

9. The probe composition according to claim 8, characterized in that The anti-HER2 antibody is Pertuzumab, and the conjugate formed by Pertuzumab and TCO-NHS ester is Pertuzumab-TCO; the probe composition is 131 I-Tyr-D-peptide-PEG 11 -Tz and Pertuzumab-TCO.

10. Use of the probe according to any one of claims 5 to 6 and / or the probe composition according to any one of claims 7 to 9 in at least one of the following: 1) Use in the preparation of a reagent for assessing the HER2 expression level in a tumor; 2) Use in the preparation of nuclear medicine imaging products for HER2-overexpressing tumors; 3) Use in the preparation of nuclear medicines for treating HER2-overexpressing tumors.

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