CN116212058A - Immune PET molecular imaging probe for targeting apoptosis - Google Patents
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Abstract
The invention discloses an immune PET molecular imaging probe for targeting apoptosis, which is a cPARP antibody modified by a bifunctional chelating agent NOTA modified tetrazine small molecule ligand combined with gallium-68 positron nuclide and targeted connection TCO. The synthesis path of the probe comprises: by TCO-PEG 4 -NHS modified resultant cprp antibody; obtaining NOTA-Tz through NOTA modification of the tetrazine small molecule ligand; reacting gallium-68 nuclide with NOTA-Tz to obtain nuclide-labeled [ gallium-68 ]]-NOTA-Tz; finally, the probe of the invention is obtained through click chemistry-biological orthogonal reaction. The probe can be used for detecting the apoptosis level of cancer cells after radiation, and has the advantages of simple preparation process, high specificity, high in vivo stability, short imaging period, low radiation dose, high biological safety and easy transformation to clinical application.
Description
Technical field:
the invention relates to an antibody probe, in particular to an immune PET molecular imaging probe for targeting apoptosis.
The background technology is as follows:
the current detection technology for apoptosis is mainly in vitro experiments, such as morphological detection of nuclear atrophy by microscopy, detection of phosphatidylserine eversion for apoptosis markers (Annexin V method), detection of chromosome DNA double-strand break or single-strand break in apoptosis by TUNEL method, analysis of activated Caspase-3 cleavage fragment of substrate poly (ADP-ribose) polymerase (PARP), etc. (Drug resistance update.2010; 13 (6): 172-9). The methods are mainly used for scientific research, the samples are processed through a series of complex processes after the samples are acquired, and then the detection of related signals is carried out, so that the clinical application value is low. Because apoptosis is a dynamic change process, apoptosis processes and dynamic changes caused by different cell types and treatment modes have larger differences, so that in-vitro experiments are required to be sampled for multiple times to determine apoptosis response of cells, and clinical diagnosis is not facilitated.
Molecular imaging techniques such as SPECT and PET can perform dynamic, continuous, noninvasive in vivo imaging of different molecular targets, thus having better clinical conversion value. Annexin V (Annexin V) is a Ca-dependent phospholipid binding protein with a molecular weight of 35-36kD, has high affinity with phosphatidylserine, and has been developed for detecting apoptosis by molecular imaging techniques. The technetium-99 marked Annexin V has been tested in the prior art for apoptosis detection and preclinical research, however, the resolution of SPECT molecular imaging technology of technetium-99 is not high, and the scientific research application value is lacked (Eur J Nucl Med Mol imaging.2015;42 (13): 2083-97). Fluorine-18 labeled Annexin V has also been tested for the basic study of apoptosis (nucleic Med biol.2004;31 (6): 747-52). However, the nuclide probes based on Annexin V have poor in vivo stability and pharmacokinetics, are not suitable for observation and measurement of dynamic changes of apoptosis over a long period of time, and thus have not been clinically employed.
The shortcomings of the existing apoptosis detection technology are concentrated in: in vitro diagnosis of cancer cell apoptosis requires tissue sample biopsy, thus being an invasive detection method, while apoptosis is a dynamic process, and multiple sampling is required to extract valuable diagnostic information, thus the acceptance of patients is low. The mode of detecting apoptosis by using a medical imaging technology is still immature, and the apoptosis marker phosphatidylserine is detected by using Annexin V, so that the image resolution and the in-vivo stability are poor, and the capability of dynamically detecting apoptosis is lacking. The apoptosis diagnostic image obtained by SPECT imaging of technetium-99 labeled Annexin V is of lower resolution. The nuclide probe based on Annexin V has poor in vivo stability and pharmacokinetics and is not suitable for clinical transformation.
The therapeutic effect of cancer is difficult to predict due to the complex molecular pathology mechanism. At present, methods such as radiotherapy and chemotherapy, molecular targeted therapy and the like clinically adopted are all aimed at eliminating cancer cells, and apoptosis is an important mode of cancer cell death. Therefore, the detection of cancer apoptosis can predict the effect of a treatment scheme, and the corresponding treatment scheme is designed according to the sensitivity of individual tumors. The current in vitro experimental method can not meet the clinical problems of dynamic tracking and real-time diagnosis of the treatment effect. Furthermore, apoptosis is affected by the tissue microenvironment, and therefore detection by in vivo experiments is required, excluding deviations from in vitro experiments. Positron emission computed tomography (PET) is an advanced molecular medical imaging technology, and can be used for dynamic and living imaging aiming at specific apoptosis targets by utilizing a positron nuclide probe tracer, so that the PET has high clinical acceptance. PARP1 is released by cleavage by Caspase3 protease during cell death, especially apoptosis, thereby degrading full length PARP1 (116 Kda) into two sub-fragment proteins of 85Kda and 31Kda, called cprp. Thus, expression of cancer cell apoptosis before and after tumor treatment can be specifically detected through the cPARP antibody probe, and sensitivity of individual tumors to specific treatment modes can be determined.
In view of the above, basic research and clinical diagnosis and treatment have urgent demands for in vivo imaging and quantitative tracking of apoptosis, which are not satisfied at present. Therefore, researchers have been working on developing novel in vivo apoptosis detection probes. The PET molecular imaging technology has the advantages of high sensitivity, high molecular specificity and noninvasive diagnosis, and is very suitable for developing apoptosis detection technology. The cPARP is an apoptosis-specific fragment, and a PET imaging technology for detecting the molecule is not established yet, but is expected to have higher molecular specificity and clinical transformation value.
The invention comprises the following steps:
the invention aims to solve the defects in the prior art and provides a novel PET molecular medical imaging probe based on a positron nuclide tracer and a gallium-68 marking method. The probe combines the target specificity of a monoclonal antibody and the short half-life (68 minutes) of gallium-68 nuclide, and can detect the sensitivity of a tumor to a specific treatment mode by forming the probe from methyl tetrazine (Tz) carrying gallium-68 through Tz-TCO click chemistry and targeting cPARP in vivo through a biological orthogonal click chemistry technology of trans-cyclooctene (TCO) and detecting positron signals emitted by the probe through a PET imaging technology.
The invention provides an immune PET molecular imaging probe for targeting apoptosis, and the design of the probe is based on that a large number of cPARP molecules are generated during apoptosis, so that the cPARP can be used as a biomarker for apoptosis with strong specificity for detection. The vectors employed herein are immunopet probes that can specifically target the expression of large fragments of PARP1 (89 kDa) without recognizing full length PARP1 or other PARP isoform molecules, thus allowing for high signal-to-noise ratios.
The immune PET molecular image probe for targeting apoptosis is a cPARP antibody modified by a bifunctional chelating agent NOTA modified tetrazine small molecule ligand and combined with positron nuclides such as gallium-68 and the like and connected with TCO in a targeting way.
Furthermore, the immune PET molecular imaging probe for targeting apoptosis is a [ gallium-68 ] -NOTA-Tz-TCO-cPARP antibody probe, and has the following structure:
the invention also provides a method for preparing the immune PET molecular imaging probe for targeting apoptosis, which comprises the following steps:
step 1, preparing a TCO-cPARP antibody by modifying the cPARP antibody with TCO-PEG 4-NHS;
step 2, obtaining NOTA-Tz by modifying tetrazine micromolecule Tz through NOTA;
step 3, reacting the gallium-68 nuclide with NOTA-Tz to obtain [ gallium-68 ] -NOTA-Tz;
step 4, PET imaging is performed by co-incubating the TCO-cPARP antibody and the [ gallium-68 ] -NOTA-Tz ligand to cause bioorthogonal reaction to generate a cPARP-targeted [ gallium-68 ] -NOTA-Tz-TCO-cPARP antibody probe.
The TCO-cPARP antibody (cPARP mAb-TCO) product produced by the preparation method has the following structure:
further, the preparation route of the cprp antibody modified by TCO is:
firstly, dissolving a cPARP antibody (clear PARP (Asp 214) mAb, # 5625) in a phosphate buffer solution, and then adjusting the pH value of the solution to 8.8-9.0 by using a saturated potassium carbonate solution; adding a proper amount of a DMSO solution (10 mg/mL) of trans-cyclooctene-tetra-polyethylene glycol-active ester (TCO-PEG 4-NHS); the reaction system was incubated at room temperature for 30 minutes with gentle shaking; the mixture solution was purified using a PD-10 desalting column to obtain a high purity TCO-cPARP antibody.
Further, a chemical synthesis path of the [ gallium-68 ] -NOTA-Tz ligand is obtained by modifying the tetrazine small molecule Tz by NOTA and then labeling by gallium-68 ions:
removing tert-butoxycarbonyl (Boc) protected amino group by using Ethyl Acetate (EA) acidified by hydrochloric acid (HCk); mixing with bifunctional chelating agent NOTA (1, 4, 7-triazacyclonane-1, 4, 7-triacid), heating, dehydrating, and performing condensation reaction to generate NOTA-Tz; adjusting the pH value of a gallium ion solution to 4 through sodium acetate, and adding 5-10nM of a pre-prepared tetrazine small molecule ligand NOTA-Tz of the modified bifunctional chelating agent; fully and uniformly mixing, and reacting at 100 ℃ for 10 minutes to obtain a crude product of the tetrazine small molecule ligand marked with gallium-68 nuclide; adsorbing the product on a C18 column, eluting the C18 column twice with 20mL of water, and removing residual gallium-68 ions in the C18 column; eluting the product in the C18 column with 2mL ethanol/water mixture (V/V1:1) into a transfer bottle containing 10mL normal saline, and performing sterile filtration to obtain the tetrazine small molecule ligand [ gallium-68 ] -NOTA-Tz marked by gallium-68 nuclide.
Further, the labeled product [ gallium-68 ] -NOTA-Tz (37 MBq,1 nmol) and TCO-cPARP antibody (150 micrograms, 1 nmol) were mixed uniformly and then subjected to continuous shaking mixing reaction at room temperature for 20 minutes, after the reaction was completed, the labeled mixture was purified by a PD-10 desalting column, and the final product [ gallium-68 ] -NOTA-Tz-TCO-cPARP antibody probe was obtained by bio-orthogonal-click chemistry reaction. Finally, the analysis and purification are carried out by HPLC connected with a radioactive detection module. The identification of the antibody probe is carried out by HPLC, 40 microliters of antibody probe is sampled, the mobile phase is acetonitrile water solution, the antibody probe is subjected to analysis by a C18 semi-preparative column, the peak time of the antibody probe is 7.9 minutes, the peak time of [ gallium-68 ] -NOTA-Tz is 7.3 minutes, and the success of the gallium-68 labeling of the antibody probe can be determined when the peak time of a specific radioactivity detector, such as the peak of the antibody probe and the peak of a radioactivity detector, occurs simultaneously. The structure of the final product antibody probe is as follows:
the immune PET molecular imaging probe provided by the invention can be applied to detecting the apoptosis level in a subject.
Apoptosis detection is a research direction of significant clinical conversion value for treatment response assessment of tumors. The detection of the expression of cprp by Western immunoblotting (Western blot) is one of the important means to determine apoptosis. The method is mainly used for scientific research, and a series of complex processes are needed to process the sample after the sample is acquired, and then the detection of the related signals is carried out. Since apoptosis is a dynamic process, the method requires multiple samples to determine the apoptotic response of the cells, which causes great trauma and is not clinically adopted. Molecular imaging techniques such as SPECT and PET can perform dynamic, continuous, noninvasive in vivo imaging of different molecular targets, thus having better clinical conversion value. Currently, many studies are conducted to develop apoptosis detection techniques based on Annexin V. However, due to the defects of the in vivo stability of Annexin V, the related apoptosis detection technology has not been widely applied to clinical diagnosis. Unfortunately, no molecular imaging probes targeting cprp have been developed. Aiming at the cPARP specific molecular imaging probe, the invention is expected to realize accurate evaluation of tumor treatment effect.
At present, no molecular imaging probe for targeting the cPARP exists at home and abroad. The preparation process of the apoptosis probe based on zirconium-89 and fluorine-18 is complex and expensive. These probes also have a longer half-life (zirconium-89 up to 78 hours, fluorine-18 110 minutes) and thus the patient is more exposed to radiation. These limitations limit their clinical application value.
The invention realizes noninvasive living body imaging of apoptosis by using TCO-Tz click chemistry technology and [ gallium-68 ] nuclide marked cPARP antibody as an immune PET probe.
The invention has the advantages that:
the novel probe [ gallium-68 ] -NOTA-Tz-TCO-cPARP antibody has the advantages of simple preparation process (the NOTA-Tz and the TCO-cPARP antibody can be prepared in a large quantity for batch use, only gallium-68 is marked by a one-step method before PET imaging), high molecular specificity (the cPARP large fragment is only expressed in apoptosis, thus the signal to noise ratio is extremely high), high in vivo stability (the antibody can be recycled in the body for 3 days after one injection, and small molecules and polypeptides are only maintained for a plurality of hours), short imaging period (short time injection and rapid imaging are realized through a pretargeting technology), low radiation dose (the half life of gallium-68 is only 68 minutes, the radiation rays disappear soon after the probe is injected and imaged), high biosafety (the radiation dose is lower than 10mCi, the probe concentration is low, and no toxic or side effect) and easy conversion to clinical application.
1. The current technology does not diagnose the sensitivity of an individual's cancer to a particular mode of treatment. The biological characteristics of individual cancers are greatly different, and the responses to radiotherapy, chemotherapy and targeted therapies are different. In order to achieve the aim of accurately treating cancer, reduce the probability of toxic and side effects or ineffective treatment, and design a treatment scheme according to the sensitivity of individual tumors is a necessary route. The invention provides a novel apoptosis detection mode which can detect the sensitivity of tumors to various treatment modes such as radiation and the like.
2. Basic and clinical studies are in urgent need for methods for detecting apoptosis in living cells. Apoptosis is a concern in both basic research in molecular biology and clinical research in cancer treatment. Apoptosis is affected by the surrounding tissue environment, so that a living body detection method is important for the development of the field. The technology provides a new way for researching and detecting apoptosis by using the gallium-68 nuclide marked cPARP monoclonal antibody as a probe for detecting apoptosis in living body.
3. Apoptosis can be dynamically tracked and quantitatively detected. Apoptosis is a dynamically changing process, and therefore, there is a need to develop techniques for dynamically detecting apoptosis levels at different time periods. The PET molecular imaging technology is an advanced medical imaging means capable of dynamically imaging and quantitatively analyzing target molecules, and can dynamically image at a plurality of time points through the bio-orthogonal-click chemistry technology, quantitatively analyze apoptosis levels and be beneficial to accurately detecting apoptosis states of cancer cells.
Drawings
FIG. 1 is a synthetic route for [ gallium-68 ] -NOTA-Tz-TCO-cPARP monoclonal antibody probes; a is the route for the cprp antibody to modify TCO; b is the path of Tz modified NOTA; c is the Tz-NOTA-labeled gallium-68, and binds to the cPARP antibody-TCO.
FIG. 2 shows the results of detecting apoptosis of cancer cells with a cPARP monoclonal antibody and a TCO modified product.
FIG. 3 is a schematic representation of apoptosis levels after detection of mouse tumor irradiation by cPARP-TCO antibody click chemistry techniques.
FIG. 4 is an imaging result of a mouse tumor model of [ gallium-68 ] -NOTA-Tz-TCO-cPARP monoclonal antibody probe.
Detailed Description
The detailed description is presented in terms of a number of preferred embodiments.
Example 1
As shown in FIG. 1, the synthesis path of the [ gallium-68 ] -NOTA-Tz-TCO-cPARP monoclonal antibody probe is divided into three steps:
1. 200 mu.1 of the cPARP antibody (about 50mg;Cleaved PARP (Asp 214) mAb, # 5625) was dissolved in 5ml of phosphate buffer and the pH of the solution was adjusted to 8.8-9.0 with saturated potassium carbonate solution; adding a proper amount of a DMSO solution of trans-cyclooctene-tetra-polyethylene glycol-active lipid (TC 0-PEG 4-NHS) according to the calculation of 10 mg/mL; incubating the mixture at room temperature with gentle shaking for 30 minutes; purifying the mixed solution by using a PD-10 desalting column to obtain 30mg of high-purity TCO-cPARP monoclonal antibody; the concentration of TC0-cPARP was measured using a NanoDrop and placed in a 4℃refrigerator for use.
2. 1g of 4- (1, 2,4, 5-tetrazine-3) tert-butylbenzyl formate (Tz-Boc) was weighed accurately; removing tert-butoxycarbonyl (Boc) protected amino group by using Ethyl Acetate (EA) acidified by 1M hydrochloric acid (HCl); the product was mixed with a bifunctional chelating agent NOTA (1, 4, 7-triazacyclonane-1, 4, 7-triacylic acid) and dehydrated by heating at 75deg.C for 2 minutes to produce NOTA-Tz.
3. Adjusting the pH value of the gallium ion solution to 4 through 2mol/L sodium acetate, and adding 5-10nM NOTA-Tz of the pre-prepared modified bifunctional chelating agent; fully and uniformly mixing, and reacting at 100 ℃ for 10 minutes to obtain a crude product of the tetrazine small molecule ligand marked with gallium-68 nuclide; adsorbing the product on a C18 column, eluting the C18 column twice with 20mL of water, and removing residual gallium-68 ions in the C18 column; eluting the product in the C18 column with 2mL of ethanol/water mixed solution (V/V1:1) into a transfer bottle containing 10mL of physiological saline, and filtering with a sterile filter membrane to obtain the tetrazine small molecule ligand [ gallium-68 ] -NOTA-Tz marked by gallium-68 nuclide. Continuously and uniformly oscillating and mixing the [ gallium-68 ] -NOTA-Tz and the [ gallium-68 ] -NOTA-Tz solutions in the ratio of 1:1 at room temperature for reaction for 20min, purifying the labeled mixture after the reaction is finished by a PD-10 desalting column, and clicking a final product [ gallium-68 ] -NOTA-Tz-TCO-cPARP antibody probe obtained by chemical-biological orthogonal reaction.
Example 2
Quality control of the [ gallium-68 ] -NOTA-Tz-TCO-cPARP monoclonal antibody probe was carried out by thin layer chromatography and High Performance Liquid Chromatography (HPLC). Spotting 2 μl of [ gallium-68 ] -NOTA-Tz-TCO-cprp antibody probe solution on a silica gel plate by a micropipette; the radiochemical purity of the gallium-68 labeled probe was determined on a radiothin layer chromatograph (Eckert & Ziegler) using a 0.1M sodium citrate solution (ph=5). Further determining the radiochemical purity, integrity and immunoreactivity of the antibody probe using HPLC coupled with a radioactive detection module and an ultraviolet detector; using 0.1M phosphate-0.1M sodium sulfate as a mobile phase, and diluting the antibody to 5.0mg/mL by using the mobile phase solution before detection; sample 10. Mu.L (Agilent 1260) was introduced after filtration using a 0.22 μm filter; the column (7.8 mm. Times.30 cm,5 μm) was analyzed by TSKgel G3000SWXL chromatography at 25℃with a flow rate of 1.0mL/min and isocratic for 20min.
Example 3
As shown in fig. 2, the effect of the cprp-TCO antibody on apoptosis in cancer cells in vitro was detected. Inducing apoptosis of head and neck cancer cells by radiation or cisplatin drugs; 2x10 6 Collecting cell proteins after 10Gy radiotherapy of cancer cells is irradiated by an X-ray biological irradiation instrument (Rad Source RS 2000) or after 8 mu M cisplatin treatment for 24 hours; after measuring the protein concentration by Nanodrop, 50. Mu.g of the sample was collected; the expression level of the large fragment of cPARP (89 kDa) was detected by Western blotting. The cPARP antibody determines that untreated cells express the intact PARP1 protein (116 kDa), but not cPARP; radiation or cisplatin can significantly increase the expression of cprp, meaning that apoptosis occurs in large amounts, and thus apoptosis can be detected specifically by the cprp antibody; TCO modified cprp antibodies can also detect radiation or cisplatin-induced apoptosis signals in cancer cells in vitro.
Example 4
As shown in fig. 3, apoptosis levels after tumor irradiation in mice were detected by click chemistry techniques with a cprp-TCO antibody. The mouse tumor model generates apoptosis after radiation treatment; the mice are anesthetized by 0.3 percent sodium pentobarbital, and the purpose of only irradiating tumor parts is achieved by lead protection during radiation; 24-48 hours after irradiation, each mouse was injected with 100-200. Mu. Ci of Tz-NOTA- [ 68 Ga]Ligand and 10mg of cPARP-TCO antibody for targeting cPARP-TCCarrying out click chemistry reaction on the O antibody; PET imaging (Mediso nanoScan) of the mouse tumor model was performed after 10 minutes.
Effect experiment:
as shown in FIG. 4, the [ gallium-68 ] -NOTA-Tz-TCO-cPARP monoclonal antibody probe can detect obvious apoptosis signals 24 hours after irradiation, and has excellent PET imaging quality of a mouse tumor model. In this experiment, mouse tumors were divided into 3 groups; the no antibody control group was not injected with the cPARP antibody-TCO, but only with Tz-NOTA- [ gallium-68 ]; the no-radiation control group was not treated with radiation, but was injected with the cPARP antibodies-TCO and Tz-NOTA- [ gallium-68 ] 2 hours prior to radiation; the radiation group was subjected to radiation treatment, and was injected with the cPARP antibodies-TCO and Tz-NOTA- [ gallium-68 ]; a is the cross-section PET/CT imaging result of three groups of mouse tumor models; b is the longitudinal PET/CT imaging result of three groups of mouse tumor models; the circular white line area is a tumor part, and probe signal quantification and tissue distribution analysis are performed through Amide software. As shown, when no cPARP antibody-TCO is injected, tumor sites are not visualized after Tz-NOTA- [ gallium-68 ] injection; when the mice are not irradiated by X-rays, the tumor part is not developed; when the tumor is subjected to radiotherapy, the tumor part has higher signal during PET imaging.
The invention confirms through the above examples that the [ gallium-68 ] -NOTA-Tz-TCO-cPARP antibody probe can detect apoptosis signals after the radiation of a tumor model of a living mouse through a PET imaging technology.
Claims (5)
1. An immune PET molecular imaging probe for targeting apoptosis is characterized in that the probe is a cPARP antibody modified by a bifunctional chelating agent NOTA modified tetrazine small molecule ligand and combined with a gallium-68 positron nuclide and targeted and connected with TCO.
2. An immune PET molecular imaging probe targeting apoptosis in accordance with claim 1, wherein said probe is a [ gallium-68 ] -NOTA-Tz-TCO-cprp antibody probe.
3. An immunopet molecular imaging probe targeted to apoptosis according to claim 1 or 2, wherein said probe has the following structure:
4. an immune PET molecular imaging probe targeting apoptosis according to claim 1 or 2, wherein said probe is prepared by bio-orthogonal reaction of TCO-cprp antibody and [ gallium-68 ] -NOTA-Tz.
5. Preparation of an immune PET molecular imaging probe targeting apoptosis according to claim 1 or 2, characterized by comprising the steps of:
1) Preparing a TCO-cPARP antibody by modifying the cPARP antibody with TCO-PEG 4-NHS;
2) Obtaining NOTA-Tz through NOTA modification of tetrazine small molecules Tz;
3) Reacting the gallium-68 nuclide with NOTA-Tz to obtain [ gallium-68 ] -NOTA-Tz;
4) PET imaging was performed by co-incubation of TCO-cprp antibody with [ gallium-68 ] -NOTA-Tz ligand resulting in bioorthogonal reactions to generate a cprp-targeted [ gallium-68 ] -NOTA-Tz-TCO-cprp antibody probe.
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| US20150359913A1 (en) * | 2013-01-25 | 2015-12-17 | Memorial Sloan-Kettering Cancer Center | Tetrazines/trans-cyclooctenes in solid phase synthesis of labeled peptides |
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