CN111592584B - HER2 affinity body and diagnosis and treatment nuclide marker as well as preparation method and application thereof - Google Patents
HER2 affinity body and diagnosis and treatment nuclide marker as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of clinical nuclear medicine, and relates to a HER2 affinity body and a nuclide marker for diagnosis and treatment, and a preparation method and application thereof. The sequence of the HER2affibody is shown in SEQ ID NO: 1 is shown. The HER2affibody sequence designed and synthesized by the invention has good in vivo stability and pharmacokinetic property, and the molecular probe synthesized by the molecular probe and labeled by diagnostic or therapeutic radionuclides has good affinity and functional activity for HER2 molecules, so that the HER2affibody sequence is expected to become a radiopharmaceutical for targeted HER2 imaging and tumor therapy with good application prospect.
Description
Technical Field
The invention belongs to the technical field of organic chemistry, radiochemistry labeled by radionuclide and clinical nuclear medicine, and particularly relates to a HER2 affinity (HER2 affibody) molecular structure, a modified HER2affibody, a nuclide marker for diagnosis and treatment, and a preparation method and application thereof.
Background
With the deep development and integration of nuclear medicine and molecular biology, medical imaging technology has advanced to the age of molecular imaging, in which Positron Emission Tomography (PET) is used for functional imaging, so that people really know and diagnose diseases from the molecular level, and particularly, the advantages of the PET are highlighted in the aspect of tumor diagnosis and treatment. By tracing the receptor changes, cell signals, of diseased tissueThe transduction abnormity overcomes the defects in the modern diagnosis technology, provides basis for early diagnosis, clinical staging and curative effect evaluation of the tumor, evaluates prognosis and can be applied to targeted therapy of the tumor. The tumor radioimmunoassay is that specific antibody or its fragment against tumor associated antigen is labeled with radioactive nuclide and injected into human body, and reaches tumor tissue along with blood flow, and is combined with tumor associated antigen to make local radioactive concentration of tumor tissue exceed that of normal tissue, then the nuclear medicine imaging equipment is used to qualitatively and positionally display primary focus and systemic metastasis focus, in particular, it can find hidden focus which is difficult to define by other diagnostic techniques. Alpha or beta radioactive nuclide can release alpha rays or beta rays and the like in the decay process, has stronger ionizing radiation effect and stronger killing effect on tumor cells or abnormal proliferation tissues and the like. Nuclide therapy means the use of radionuclides with therapeutic action, e.g.131I，90Y，177Lu，225Ac，213Bi and other nuclides or labeled drugs can kill diseased cells and tissues accurately in a close range, thereby achieving the purpose of treatment.
Human epidermal growth factor receptor 2 (HER 2), one of the Epidermal Growth Factor Receptor (EGFR) family members, is expressed to varying degrees in various tumor tissues. HER2 amplification or overexpression is closely related to high invasiveness, relapse susceptibility and increased mortality of tumors. Research shows that HER2 overexpression exists in different degrees in breast cancer (20-30 percent), gastric cancer (7-34 percent), ovarian cancer, lung cancer and prostate cancer, and the HER2 positive rate of domestic gastric cancer patients is 12-13 percent. HER2 overexpression is often predictive of poor patient prognosis. Patients with HER2 overexpression could benefit from HER2 targeted therapy. Several drugs targeting HER2 were presented sequentially, the most representative HER2 therapeutic drug trastuzumab (Herceptin ) selectively blocks HER 2-related signaling pathways upon binding to the extracellular domain of HER 2. Trastuzumab combined with chemotherapy can significantly prolong the overall survival of HER 2-overexpressed gastric cancer in the late stage compared with chemotherapy alone.
Has already provided muchThe research shows that the HER 2-targeted PET/CT molecular imaging can be used for focus imaging of breast cancer and gastric cancer, can show the heterogeneity of tumor HER2 expression, can be used for screening the positive focus of a primary focus HER2 expression negative patient, and can accurately predict the curative effect of targeted therapy.64Cu or89Clinical studies with Zr-labeled whole antibody (Trastuzumab, Pertuzumab) were in series and showed important values in HER2 positive patient screening, HER2 targeted therapeutic efficacy prediction, HER2 heterogeneity monitoring, etc. However, intact antibodies have slower blood pharmacokinetics, require nuclide labeling with relatively long half-lives, have high blood pool and liver background, and require imaging at longer time points for better image contrast, wherein,64the optimal imaging time of the Cu-trastuzumab is 1 to 2 days,89the optimal development time of the Zr-trastuzumab is 4-6 days of marking.
Affibody (Affibody) is an artificial protein molecule derived from the Z domain of staphylococcal protein a, is a single-chain structure consisting of 58 amino acids with a relative molecular mass of about 6.5kDa, and was the first Affibody used in molecular imaging with specificity of HER 2. The first generation HER2 targeted affibodies were predominantly ZHER 2: 4, second generation affibody ZHER 2: 342 were screened by site directed mutagenesis of the mature affibody series, with affinity comparable to that of ZHER 2: 4 was significantly increased (22pM VS 50 nM). In recent years, radionuclide-labeled ZHER 2: 342 and the analogues thereof (ZHER 2: 2891, ZHER 2: 2395, etc.) are beginning to receive wide attention in tumor molecular imaging. The Affinibody has small molecular weight and fast biological distribution, can be imaged in a short time (1-4h), and has unique advantages in the imaging of targeted HER2 due to the fact that the radiation is lower than a long half-life molecular probe.
After the Affinibody is modified by bifunctional coupling agents NOTA, HBED-CC and the like, diagnostic radionuclides can be carried out68Ga、18The mark F is marked with a mark F,68ga is positron radioactive nuclide and nuclide with wider application68Ga is composed of68Ge-68The Ga generator is prepared, the source is convenient,68ga is easy to coordinate with a compound, and the marking operation method is simple; drip washing target in contrast to cyclotronsRecord68The Ga radioactivity is relatively small, so that the quality control is convenient to carry out at any time; and is68The half-life period of Ga is short, diseases can be diagnosed in a noninvasive and real-time manner, and a whole body metabolic image can be obtained. As an ideal radionuclide of PET, with68Ga(T1/268min) of the two-phase flow,18f has a longer half-life (T)1/2109 min). And is18The F is prepared by an accelerator, so that the method is more suitable for batch preparation of a plurality of patients and is also more suitable for developing an automatic synthetic preparation method.
90Y and177lu is a beta-radionuclide and,90the energy of Y is high (2,280keV), the penetrating power is strong (12mm), and the Y has strong killing effect on tumor cells or abnormal proliferation tissues and the like. And90compared with the Y phase, the Y phase is,177lu emits beta-rays (0.5MeV) in a small ion range, which not only ensures the transmission of ray energy to the tumor region, but also reduces the damage to surrounding normal tissues.225Ac and213bi is an alpha radionuclide, which, in contrast to beta radionuclides,225ac (6000-213Bi(>6000keV) stronger ionization and weaker penetration (225Maximum Ac Range: 0.06-0.09 mm;213maximum range of Bi: 84 μm) so that the alpha radionuclide has a more potent cytotoxic effect than the beta radionuclide.
Therefore, the radioactive molecular probe for HER2affibody diagnosis is used for improving the diagnosis efficiency of HER2 state, and provides a noninvasive and effective molecular imaging means for screening HER2 positive breast cancer and gastric cancer patients, monitoring HER2 expression conditions and predicting and evaluating targeted therapy effect; the radioactive molecular probe for HER2 Affinibody treatment provides a new treatment means for HER2 positive breast cancer and gastric cancer patients after targeted treatment drug resistance; meanwhile, a new idea is provided for treating HER2 positive breast cancer and gastric cancer patients.
Disclosure of Invention
The invention aims to provide a molecular structure of an HER2affibody, a modified substance of the molecular structure of the HER2affibody, a further obtained diagnostic nuclide marker, a preparation method and application.
To achieve the above object, a first aspect of the present invention provides a HER2affibody, the sequence of the HER2affibody is (N-terminal-C-terminal):
(AEEA) -VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK [ (Acp) - (Acp) ], wherein Acp is 6-aminocaproic acid, attached to the C-terminus of K; the middle contiguous amino acid sequence (V to K) is as set forth in SEQ ID NO: 1 is shown in the specification;
the structure of the HER2affibody is shown as formula I:
formula I.
The structure of HER2affibody in the present invention is based on the second generation affibody ZHER 2: 342, in the case of ZHER 2: 342 is added with two Acp amino acid structures at the C end and one AEEA linker structure at the N end, thereby improving the stability of the Affinibody molecule in a human body and avoiding the radioactive self-decomposition of the synthesized radioactive molecular probe.
The HER2affibody can be prepared by various polypeptide synthesis/modification methods in the field, for example, an amino acid sequence is synthesized firstly, then the amino acid sequence reacts with Fmoc-AEEA (CAS No.:166108-71-0), and the product obtained by the reaction is subjected to Fmoc removal, so that the HER2affibody can be obtained, or can be obtained by whole commercial purchase.
In a second aspect the invention provides a modified HER2affibody, which is a HER2affibody according to the first aspect of the invention modified with a bifunctional coupling agent which is NOTA (1,4, 7-triazacyclononane-1, 4, 7-triacetic acid), HBED-CC (N, N "-bis [ 2-hydroxy-5- (carboxyethyl) -benzyl ] ethylenediamine-N, N" -diacetic acid), DOTA (1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid) or 3pC-NETA-NCS ({4- [2- (bis-carboxymethylamino) -5- (4-nitrophenyl) pentyl ] -7-carbonyl-dimethyl- [1,4,7] triazaaran-1-yl } acetic acid-N-chlorosuccinimide).
According to the present invention, preferably said bifunctional coupling agent is modified at the N-terminus of said HER2 affibody.
In order to further solve the problems of spatial structure of the affinity body, in vivo stability, lipid solubility and the like, the C-terminal of the HER2 affinity body is preferably modified with a maleimide (Mal) structure.
The method for modifying HER2affibody with bifunctional coupling agent can adopt various methods which are conventional in the art and are well known to those skilled in the art, and the present invention is not particularly limited thereto. For example, NaHCO at pH 8.5-9.03In a buffer system, HER2affibody and NOTA are mixed according to the ratio of 5-10: 1, and reacting at normal temperature for 30min-2 h.
In a third aspect, the invention provides a diagnostic nuclide marker that is a radionuclide-labeled modified HER2affibody according to the second aspect of the invention.
According to one embodiment of the invention, the radionuclide may be a diagnostic radionuclide, preferably the diagnostic radionuclide is68Ga and/or18F。
When the radionuclide is68Ga, the method for preparing a radionuclide-labeled HER2affibody may comprise the steps of:
1) by using68Ge-68Ga Generator preparation68Ga；
2) Leaching with 0.04-0.06M HCl solution68Ga is added into 0.8-1.2M NaAc solution;
3) adding the modified HER2affibody to the mixture obtained in step 2)68Uniformly mixing in a Ga NaAc solution system, and reacting for 5-20min at 90-100 ℃;
4) eluting the radioactive impurities in the reaction system in the step 3) by using normal saline, and then eluting by using ethanol to obtain a product68Ga-labeled HER2 affibody.
Specifically, taking NOTA modification of the bifunctional chelating agent as an example,68the labeling of modified HER2affibody by Ga can be performed by the following method:
after the HER2affibody sequence is modified by a bifunctional coupling agent NOTA, the sequence (NOTA) - (AEEA) -VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK [ (Acp) - (Acp) - (3-Mal) is obtained](NOTA-HER2 Affiniody); the prepared NOTA-HER 2Affibody is subjected to68Ga(T1/2＝68min；β+89 percent; e511 keV) a nuclide,68for Ga68Ge-68The Ga generator was prepared. Leaching 1mL of 0.05M HCl solution68Ga to 65 μ L in 1M NaAc; adding 0.1mL (100 μ g) of HER2affibody precursor into the above system, mixing, reacting at 95 deg.C for 10min, eluting radioactive impurities with 3mL of normal saline, and eluting the target compound with 0.8mL of 80% ethanol68Ga-HER2 Affinibody, the labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC. Obtained68Ga-HER2 Affinibody has radioactive chemical purity of more than 99%. When the labeling rate is less than 90%, the mixture is separated and purified by Sep-pak C18 Column, wherein the Sep-pak Column needs to be activated by 5mL of absolute ethyl alcohol and 5mL of high-purity water for standby. And (4) taking a proper amount of the product preparation subjected to sterile filtration for quality control inspection, and performing later-stage research after all the items are qualified.
When the radionuclide is18F, the method for preparing a radionuclide-labeled HER2affibody may comprise the steps of:
1) preparation by using a cyclotron18F；
2) Produced by a cyclotron and containing18F-H of (A) to (B)2 18O is passed through QMA ion exchange column18F-Adsorbing on a QMA column; washing the QMA column with 0.45-0.55mL of physiological saline, and eluting18F-；
3) Taking 0.09-0.11mL of the solution obtained in the step 2) containing18F-The normal saline with KHP and AlCl3Mixing the solutions, shaking, standing at room temperature for 4-6min, adding the modified HER2affibody, reacting at 100-120 deg.C for 10-20min, cooling the reaction solution, loading the product onto C18 separation column, washing with normal saline, and eluting with ethanol to obtain product18F-Labelled HER2 affibody.
Specifically, taking NOTA modification of the bifunctional chelating agent as an example,18the following methods can be used for F on modified HER2affibody markers:
HER2 affafter the ibody sequence is modified by a bifunctional coupling agent NOTA, the sequence (NOTA) - (AEEA) -VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK [ (Acp) - (Acp) - (3-Mal) is obtained](ii) a The prepared NOTA-HER 2Affibody is subjected to18F(T1/2＝109.8min；β+96.7 percent; e511 keV) a nuclide,18f is prepared by using a cyclotron. Produced by an accelerator containing18F-H of (A) to (B)2 18O is passed through QMA ion exchange column18F-Adsorbing on a QMA column; washing the QMA column with 0.5mL of physiological saline solution18F-Eluting into a reaction tube; taking 0.1mL of a solution containing18F-The reaction tube was then filled with 11. mu.L of 10-fold KHP and 6. mu.L of 2mM AlCl3Shaking the solution, standing at room temperature for 5 min; then adding 14 mu L of 10mg/mL labeled precursor NOTA-HER 2affibody, and reacting for 15min at 110 ℃; after the reaction solution was cooled to room temperature, the product was loaded onto a C18 separation column, washed with 4mL of physiological saline, and then washed out with 0.7mL of 80% ethanol and passed through a 0.22 μm sterile filter; n is a radical of2Drying the solvent, and preparing a product preparation by using normal saline; the labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC. The above-mentioned18The radiochemical purity of F-HER2 Affinibody after separation and purification is more than 99 percent. When the labeling rate is less than 90%, the mixture is separated and purified by Sep-pak C18 Column, wherein the Sep-pak Column needs to be activated by 5mL of absolute ethyl alcohol and 5mL of high-purity water for standby. And (4) taking a proper amount of the product preparation subjected to sterile filtration for quality control inspection, and performing later-stage research after all the items are qualified.
According to the invention, the radionuclide can also be a therapeutic radionuclide, so as to achieve the aim of HER2 high-expression tumor targeting molecule image therapy. The therapeutic radionuclide is preferably90Y、177Lu、225Ac and213at least one of Bi.
After the HER2 affinity body sequence is modified by a bifunctional coupling agent DOTA, a bifunctional coupling agent DTPA and a bimodal bifunctional ligand 3pC-NETA-NCS, therapeutic radionuclide90Y，177Lu，225Ac or213Marking with Bi to obtainTo90Y-HER2 Affibody、177Lu-HER2 Affibody、225Ac-HER2 Affibody、213Bi-HER2 Affinibody molecular probe for treatment.
Labeling of therapeutic radionuclides may be accomplished by any of a variety of methods conventional in the art, in accordance with a preferred embodiment of the present invention90Y，177Lu，225Ac or213The method for labeling HER2affibody by Bi can adopt the following methods: to be provided with177Lu for example, HER2affibody, after modification with the bifunctional chelating agents DOTA, DTPA, 3pC-NETA-NCS, gives the corresponding labeled precursor. Preparing a marking buffer solution according to 1mL of 0.05M HCl corresponding to 65 muL of 1M NaAc for later use; 100. mu.L (100. mu.g) of labeled precursor (DOTA-HER 2Affibody, DTPA-HER 2Affibody or 3pC-NETA-HER2 Affibody) was added with 100. mu.L of labeling buffer, followed by addition of 100. mu.L of labeling buffer177Lu; adjusting pH to 5.5, 70-95 deg.C, reacting for 10-15min, and separating and purifying with Sep-pak C18 Column when the labeling rate is less than 90% to obtain177Lu-HER2 Affibody. The labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC. Prepared by177The radiochemical purity of Lu-HER2 Affinibody after separation and purification is more than 99 percent.
Using diagnostic radionuclides68Ga、18F marks the modified HER2affibody of the invention to obtain68Ga/18The F-HER2 Affinibody molecular probe has good in-vivo stability, is safe and nontoxic, can be specifically combined with HER2 molecules, accurately positions HER2 high-expression tumor tissues, and shows good targeting property and specificity. Therefore, the HER2affibody of the invention also has good in vivo stability, pharmacokinetic property, targeting property and specificity.
The fourth aspect of the invention provides the application of the diagnostic nuclide marker (diagnostic radionuclide-labeled HER2 affibody) in the preparation of a HER2 targeted tumor PET imaging reagent.
The fifth aspect of the invention provides the application of the diagnostic nuclide marker (therapeutic radionuclide-labeled HER2 affibody) in the preparation of HER2 targeted tumor nuclide therapeutic drugs.
The invention provides an affibody structure targeting HER2, and labels and evaluations of diagnostic and therapeutic radionuclides. The synthesized HER2affibody molecular structure and the radionuclide-labeled HER2affibody molecular probe designed by the invention have the following advantages and beneficial effects:
(1) the HER2affibody structure has better in vivo stability, pharmacokinetic properties, affinity and specificity.
(2) Diagnostic radioactive molecular probes are useful68Ga/18PET imaging of F-HER2 Affinibody;68Ga/18the F-HER2 Affinibody can be used for screening HER2 positive breast cancer, gastric cancer and other tumor patients, treatment prediction and curative effect monitoring.
(3)68Ga/18F-HER2 Affinibody PET imaging can judge the curative effect response of patients to HER2 targeted therapy and judge whether the patients are suitable for using therapeutic nuclide90Y/177Lu/225Ac/213Bi substitution68Ga/18And F, carrying out nuclide targeted therapy.
(4)90Y/177Lu/225Ac/213Bi-HER2 Affinibody and other nuclide targeted therapy can provide a new treatment means for drug-resistant HER2 positive breast cancer and gastric cancer patients.
(5) According to the invention68Ga/18The preparation method of F-HER2 Affinimody has high marking rate,68the Ga marking rate can reach more than 80 percent,18the marking rate of F can reach more than 30%.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 is an HPLC representation of the HER2affibody sequence of the invention.
FIGS. 2a-2b are a structural diagram and a HPLC representation, respectively, of the HER2affibody sequence of the invention after NOTA modification.
FIG. 3 shows an embodiment of the present invention18F-HER 2affibody in vitro stability assay.
FIG. 4 shows an embodiment of the present invention18F-HER 2affibody was analyzed for pharmacokinetics in normal KM mice.
FIG. 5 shows an embodiment of the present invention18F-HER 2affibody micro-PET/CT distribution image within 1h in HER2 positive N-87 gastric cancer model.
FIG. 6 shows an embodiment of the present invention18F-HER 2affibody micro-PET/CT images in HER2 positive N-87 gastric cancer model and blocking group micro-PET/CT images.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
Example 1
This example serves to illustrate the synthesis and characterization of HER2affibody sequences of the present invention.
The sequence of the HER2affibody is:
(AEEA) -VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK [ (Acp) - (Acp) ], the structure is shown in formula I. The HER2affibody was obtained commercially and was verified to be correct in amino acid sequence.
Performing HPLC characterization of the molecular structure of the HER2affibody sequence; conditions for HPLC analysis: kromasil 100-5C18 gel filtration/size exclusion chromatography column, flow rate 1 mL/min; mobile phase a was acetonitrile containing 0.1% trifluoroacetic acid TFA; mobile phase B was water containing 0.1% trifluoroacetic acid TFA. Mobile phase gradient setup: 0.0min 25% A and 75% B; 20min 90% of A and 10% of B; 20.1min 100% A and 0% B. The HPLC results are shown in FIG. 1.
Example 2
This example serves to illustrate the preparation and characterization of the modified HER2affibody of the invention.
NOTA modification of the HER2affibody sequence with NaHCO at pH 8.83HER2Affibody was mixed with NOTA (10mg/mL) in a buffer system according to 8: 1, and reacting at room temperature for 1h to obtain modified HER2affibody, the structure of which is shown in FIG. 2 a. Performing HPLC characterization on the modified molecular structure; the HPLC analysis conditions were as in example 1, and the HPLC results are shown in FIG. 2 b.
Example 3
This example is for the purpose of illustration18Preparation of F-labeled HER2 affibody.
Produced by an accelerator containing18F-H of (A) to (B)2 18O is passed through QMA ion exchange column18F-Adsorbing on a QMA column; washing the QMA column with 0.5mL of physiological saline solution18F-Eluting into a reaction tube; taking 0.1mL of a solution containing18F-The reaction tube was then filled with 11. mu.L of 10-fold KHP and 6. mu.L of 2mM AlCl3Shaking the solution, standing at room temperature for 5 min; then adding 14 mu L of 10mg/mL labeled precursor NOTA-HER2 Affiniody, and reacting for 15min at 110 ℃; after the reaction solution was cooled to room temperature, the product was loaded onto a C18 separation column, washed with 4mL of physiological saline, and then washed out with 0.7mL of 80% ethanol and passed through a 0.22 μm sterile filter; n is a radical of2Drying the solvent, and preparing a product preparation by using normal saline; the labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC.
Through the determination, the method has the advantages that,18the labeling rate of F-HER2 Affinibody is 30%, and the radiochemical purity is more than 99%.
Example 4
This example illustrates the purification18In vitro stability assay of F-HER2 Affiniody.
Take 10. mu.L of purified product containing 1.11MBq (30. mu. Ci)18F-HER2 Affinibody was added to 200. mu.L of physiological saline (or 5% HSA solution) and incubated at 4 ℃; respectively incubating for 0h, 2h, 12h,Samples of 37-74kBq (1-2. mu. Ci) were taken at 24h, 36h and 60h for radio-TLC analysis; the analysis method comprises the following steps: taking 2 μ L of the extract containing 37-74kBq (1-2 μ Ci) radioactivity18F-HER2 Affinibody physiological saline solution or18F-HER2 Affinibody 5% HSA solution was added to 20. mu.L of saturated EDTA and mixed well for radio-TLC analysis. Dripping 2 μ L of sample at a distance of 1cm from the bottom end of Xinhua No. one filter paper, placing in a normal saline development system, taking out the filter paper after completely developing, air drying, performing radio-TLC detection, dissociating18F and18rf values of F-HER 2affibody are 0.9-1, 0-0.1, respectively; results display18F-HER 2affibody physiological saline solution or 5% HSA solution has good stability within 4 h. The specific results are shown in FIG. 3.
The above experiments show that the HER2affibody structure has good stability.
Example 5
This example illustrates the purification18F-HER2 Affiniody was analyzed for pharmacokinetics in normal KM mice.
5 KM mice (female, 5-6 weeks old, 18-20g) were prepared, and the purified KM mice were used18F-HER2 Affinibody was diluted to 18.5MBq/mL (0.5mCi/mL) with saline and each mouse was injected with 3.7MBq (0.1mCi, 200. mu.L) of the labeled product via the tail vein; at corresponding time points (1min, 3min, 5min, 10min, 15min, 30min, 45min and 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h, 36h) after injection of the labeled product, respectively, blood was drawn through the periocular venous plexus of the mice using capillaries and placed in a radioimmunoassay; administered intravenously from each tail18F-HER2 Affinibody activity 1% of the activity, namely 0.037MBq (1. mu. Ci, 2. mu.L), was used as the assay reference activity. The reference activity was measured with the collected blood samples using a Gamma counter, and after attenuation correction data analysis was performed using prism6.0 software, calculating the percent injection dose rate per gram of blood sample, and the results are expressed as% ID/g ± SD. In particular, referring to fig. 4, as can be seen from fig. 4,18F-HER2 Affinibody has good pharmacokinetic properties.
Example 6
This example is for the purpose of illustration18F-HER2 Affinibody was distributed within 1h of micro-PET/CT in HER2 positive N-87 gastric cancer model.
5 HER2 positive N87 gastric cancer animal models (female, 5-6 weeks old, 18-20g, tumor diameter of 0.8-1.0cm) were prepared. Mice were anesthetized with 3L/min isoflurane gas, the animal model was fixed in the center of a Micro-PET scanning bed in prone position, and 7.4MBq (0.2mCi, 200 μ L) was injected into the tail vein18Immediately after F-HER2 Affiniody, the mouse is scanned in a dynamic scanning mode with an energy window of 350 keV and 700keV, the scanning time is 1h, the anesthetized mouse is maintained by isoflurane gas of 1L/min in the scanning process, image reconstruction is carried out by Ordered Subsets Expectation Maximization (OSEM) software, and image analysis processing is carried out by MMWKS software after attenuation correction. The specific results are shown in figure 5 which shows,18F-HER2 Affinibody shows micro-PET/CT distribution image result within 1h in HER2 positive N-87 gastric cancer model18The F-HER2 Affiniody has better in-vivo stability and pharmacokinetic property in animals, the molecular probe is mainly metabolized through the kidney, and the non-specific uptake of the whole body tissues is lower; with systemic metabolism, specific radioactive accumulation occurs in the tumor tissue at 20-30min, and the radioactive uptake by the tumor tissue gradually increases.
Example 7
This example is for the purpose of illustration18F-HER2 Affinibody was distributed within 1h of micro-PET/CT in HER2 positive N-87 gastric cancer model.
10 HER2 positive N87 gastric cancer animal models (female, 5-6 weeks old, 18-20g, tumor diameter of 0.8-1cm) were prepared and randomly divided into experimental and blocking groups of 5 each. Experimental group 7.4MBq (0.2mCi, 200 μ L) was injected via tail vein for each18F-HER2 Affibody; blocking group Co-injection of 0.5mg affibody +7.4MBq (0.2mCi, 200. mu.L)18F-HER2 Affibody; micro-PET imaging is carried out on each animal model in each group 1h and 2h after injection; before micro-PET imaging is carried out at corresponding time, 3L/min isoflurane gas is used for anaesthetizing a mouse, a PDX model is fixed at the center of a micro-PET scanning bed in a prone position, a static scanning mode is adopted for scanning the mouse, an energy window is 350 keV and 700keV are scannedThe time is 15min, the scanning time is prolonged along with the metabolism and decay of the probe, the anesthesia mouse is maintained by isoflurane gas of 1L/min in the scanning process, the ordered subsets maximum expectation maximization (OSEM) software is adopted for image reconstruction, and the MMWKS software is adopted for image analysis processing after attenuation correction. The specific results are shown in FIG. 6.18F-HER2 Affinibody shows micro-PET/CT image results in a HER2 positive N-87 gastric cancer model,18F-HER2 Affiniody has higher radioactive uptake at the tumor position at 1h and 2 h; the fraction of tumor radioactivity uptake was significantly reduced after cold affibody blocking. Description of the invention18F-HER2 Affinibody has high specificity targeting on HER2 positive tumors.
The experiments prove that the HER2affibody sequence designed and synthesized by the invention has good in-vivo stability and pharmacokinetic properties, has good affinity and functional activity with molecular probes synthesized by diagnostic or therapeutic radionuclide labeling on HER2 molecules, and is expected to become a radiopharmaceutical for targeted HER2 imaging and tumor therapy with good application prospect.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Sequence listing
<110> research institute for tumor prevention and treatment in Beijing
<120> HER2 affinity body and diagnostic nuclide marker, and preparation method and application thereof
<130> BJI2000735BJZL
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 58
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Val Asp Asn Lys Phe Asn Lys Glu Met Arg Asn Ala Tyr Trp Glu Ile
1 5 10 15
Ala Leu Leu Pro Asn Leu Asn Asn Gln Gln Lys Arg Ala Phe Ile Arg
20 25 30
Ser Leu Tyr Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala
35 40 45
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys
50 55
Claims (10)
1. A modified HER2affibody, the modified HER2affibody being a HER2affibody modified by a bifunctional coupling agent, said bifunctional coupling agent being NOTA; the bifunctional coupling agent is modified at the N-terminus of the HER2 affibody; the C end of the HER2affibody is modified with a maleimide (Mal) structure;
the sequence of the HER2affibody is:
(AEEA) -VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK [ (Acp) - (Acp) ], wherein Acp is 6-aminocaproic acid;
the structure of the HER2affibody is shown as formula I:
2. a diagnostic nuclide marker that is a radionuclide-labeled modified HER2affibody according to claim 1.
3. The diagnostic radionuclide marker according to claim 2, wherein the radionuclide is a diagnostic radionuclide.
4. According to claimThe nuclide marker for diagnosis and treatment according to claim 3, wherein the diagnostic radionuclide is68Ga and/or18F。
5. The diagnostic nuclide marker as claimed in claim 2 wherein said radionuclide is a therapeutic radionuclide.
6. The diagnostic nuclide marker as claimed in claim 5 wherein said therapeutic radionuclide is90Y、177Lu、225Ac and213at least one of Bi.
7. Use of the nuclide marker for diagnosis and treatment according to claim 3 or 4 in the preparation of a HER 2-targeted tumor PET imaging agent.
8. Use of the nuclide marker for diagnosis and treatment according to claim 5 or 6 for the preparation of a HER2 targeted nuclide therapeutic drug for tumors.
9. The method for preparing a nuclide marker for diagnosis and treatment according to claim 3 or 4, wherein the radionuclide is68Ga, comprising the steps of:
1) by using68Ge-68Ga Generator preparation68Ga；
2) Leaching with 0.04-0.06M HCl solution68Ga is added into 0.8-1.2M NaAc solution;
3) adding the modified HER2affibody to the mixture obtained in step 2)68Uniformly mixing Ga in NaAc solution, and reacting for 5-20min at 90-100 ℃;
4) eluting the radioactive impurities in the reaction system in the step 3) by using normal saline, and then eluting by using ethanol to obtain a product68Ga-labeled HER2 affibody.
10. The method for preparing a nuclide marker for diagnosis and treatment according to claim 3 or 4, wherein the radionuclide is18F, comprising the following steps:
1) preparation by using a cyclotron18F-；
2) Produced by a cyclotron and containing18F-H of (A) to (B)2 18O is passed through QMA ion exchange column18F-Adsorbing on a QMA column; washing the QMA column with 0.45-0.55mL of physiological saline, and eluting18F-；
3) Taking 0.09-0.11mL of the solution obtained in the step 2) containing18F-The normal saline with KHP and AlCl3Mixing the solutions, shaking uniformly, standing at room temperature for 4-6min, adding the modified HER2affibody, reacting at 100-120 deg.C for 10-20min, cooling the reaction solution, loading the product on a C18 separation column, washing with normal saline, and eluting with ethanol to obtain the product18F-Labelled HER2 affibody.
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| PCT/CN2021/095455 WO2021238842A1 (en) | 2020-05-27 | 2021-05-24 | Her2 affibody, diagnostic or therapeutic nuclide-labeled substance, and preparation method for nuclide-labeled substance and application of nuclide-labeled substance |
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| CN111848746A (en) * | 2020-08-08 | 2020-10-30 | 四川大学华西医院 | Binding protein for targeted binding to HER2, and preparation method and application thereof |
| CN112999369B (en) * | 2021-03-03 | 2022-02-25 | 江苏元本生物科技有限公司 | HER2 affinity radionuclide marker composition and application thereof |
| CN114317385B (en) * | 2021-11-11 | 2024-02-13 | 元本(珠海横琴)生物科技有限公司 | Fermentation medium and fermentation process for promoting secretion and expression of HER2 affibody protein |
| CN114075268A (en) * | 2022-01-19 | 2022-02-22 | 北京肿瘤医院(北京大学肿瘤医院) | Affinity body targeting HER2 and application thereof |
| CN114874308B (en) * | 2022-05-26 | 2023-02-17 | 苏州大学 | Nuclide-labeled inhibitory peptide and preparation method and application thereof |
| CN115028710A (en) * | 2022-05-27 | 2022-09-09 | 北京肿瘤医院(北京大学肿瘤医院) | Radionuclide-labeled neutralizing antibody targeting new coronavirus, and preparation method and application thereof |
| CN116077687A (en) * | 2022-11-08 | 2023-05-09 | 苏州大学 | Nanometer nuclear medicine, preparation method and application thereof |
| CN116751258B (en) * | 2023-08-18 | 2023-11-17 | 北京肿瘤医院(北京大学肿瘤医院) | MDM2/MDMX targeting polypeptide and application thereof |
| CN117777296A (en) * | 2024-02-28 | 2024-03-29 | 北京肿瘤医院(北京大学肿瘤医院) | Preparation method and application of B7H3 affibody and diagnosis and treatment nuclide marker thereof |
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