CN117752824A - Pretargeted tumor immune probe, bioorthogonal preparation, kit and application - Google Patents
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Abstract
The invention provides a pretargeted tumor immune probe, a bio-orthogonal preparation, a kit and application thereof, belonging to the technical field of PET probes. The pre-targeting tumor immune probe provided by the invention is NOTA-Reppe anhydride marked with radionuclide, and the NOTA-Reppe anhydride has a structure shown in a formula I. The pre-targeting tumor immune probe provided by the invention can construct a PD-1/PD-L1 targeting PET probe based on a tetrazine-Reppe anhydride biological orthogonal reaction system, can realize specific imaging of tumors, and can evaluate the expression level of PD-1/PD-L1 in the tumors.Formula I.
Description
Technical Field
The invention relates to the technical field of PET probes, in particular to a pretargeted tumor immune probe, a biological orthogonal preparation, a kit and application.
Background
Tumor immunotherapy is one of the most promising tumor treatment schemes at present, and is different from the action mechanism of traditional chemotherapy, the immunotherapy acts on the immune system of human body, induces and regulates the immune response of the body to tumor, monitors and kills tumor cells. The immune check point formed by combining the programmed death factor 1 (programmed cell death-1, PD-1) and the ligand (programmed cell death ligand-1, PD-L1) thereof in the tumor tissue microenvironment can inhibit tumor immune response and generate immune tolerance, and promote tumor immune escape. Thus blocking the PD-1/PD-L1 signal pathway can activate effector T cells and inhibit tumor immune escape, thereby enhancing the anti-tumor immune response of the organism. PD-1/PD-L1 immune checkpoint inhibitors have been applied to various types of tumor treatment, but due to individual differences in the expression levels of PD-1/PD-L1, PD-1/PD-L1 immune checkpoint inhibitors are only effective in 10-30% of patients with solid tumors. Therefore, the method has important clinical significance for evaluating the PD-1/PD-L1 expression level in the tumor microenvironment of the patient, can predict the immunotherapy response of the tumor patient, and is used as a screening means for judging whether the patient is suitable for PD-1/PD-L1 immunotherapy.
At present, no standard detection means exists for evaluating the expression level of PD-1/PD-L1, and the standard detection means mainly comprises an immunohistochemical detection method, a circulating tumor cell or related biomarker blood detection method and other invasive methods. Because of factors such as tumor heterogeneity and difficulty in standardization of quantification of PD-1/PD-L1 expression levels, the evaluation of PD-1/PD-L1 at the present stage often needs to combine multiple detection means to realize comprehensive dynamic monitoring. The traditional curative effect prediction means based on tissue biopsy has the problems of poor prediction accuracy, strong invasiveness, incapability of sampling for multiple times and the like. The immune PET technology combines the high sensitivity of PET imaging and the high specificity of antibodies, is similar to performing accurate immunohistochemical staining in living bodies, reveals the expression level of tumor markers in tumor patients in a non-invasive mode, can realize long-term dynamic visual monitoring, and is beneficial to screening and formulation of immune treatment schemes of tumor patients. The immune treatment of tumors can also benefit from an immune PET imaging technology, the immune PET technology can visualize and quantitatively evaluate the expression of immune checkpoints in vivo, patients possibly benefiting from PD-1/PD-L1 immune treatment can be screened out in early stage, and real-time monitoring of treatment effect can be realized. At present, a targeting PET probe constructed by a Pd-1/PD-L1 targeting monoclonal antibody with equal-half-life nuclide markers such as Zr-89 and I-124 is used for evaluating the expression level of PD-1/PD-L1 in tumors. The long half-life nuclide labeled targeting PET probe is matched with the pharmacokinetic properties of the monoclonal antibody in vivo, and the optimal imaging time point is about one week after the probe is injected into the body. While obtaining images with high contrast and high specificity, the longer circulation half-life of the probe in the body causes higher radiation damage to non-target organs of the organism.
The bio-orthogonal chemistry (Bioorthogonal chemistry) system is widely applied to the fields of protein labeling, molecular imaging and the like, has the advantages of rapidness, high selectivity, high affinity and the like, can occur in living cells, does not influence the biochemical process of organisms, and has become an important means of pretargeting imaging. The bio-orthogonal chemical system is established by respectively labeling two functional molecules capable of being specifically combined on functional biological macromolecules (proteins, antibodies, nano particles and the like) and reporter molecules (fluorophores, radionuclides and the like) through a chemical modification means, and then monitoring the real-time state of the labeled functional biological macromolecules in cells or living bodies by utilizing the characteristics (fluorescence, radioactivity and the like) of the labeled reporter molecules. Researches on bioorthogonal chemistry-based immune PET imaging technology are widely focused, short half-life nuclides such as F-18, ga-68, cu-64 and the like are marked on bioorthogonal chemistry-based pre-targeting immune PET imaging probes, in-vivo marking and real-time functional monitoring of antibodies can be achieved, and radiation damage to organisms is obviously lower than that of the PET probes with long half-life nuclides directly marked with the antibodies.
The current bio-orthogonal chemical reaction system mainly comprises a tetrazine-bridged cycloolefin diels-Alder reaction (inverse electron demand Diels-Alder reaction, IED-DAR) with inverse electronic requirements, a metal-catalyzed azido-acetylenic click chemical system and the like, wherein the IED-DAR reaction based on tetrazine and trans-cyclooctene (TCO) is a bio-orthogonal chemical reaction which is widely applied at present. However, the steric conformation of trans-cyclooctene is unstable, the binding effect with tetrazine in vivo is affected, and the research of pre-targeting immune PET imaging on a bio-orthogonal chemical reaction system for evaluating the PD-1/PD-L1 expression level in tumors has not been reported at present.
Disclosure of Invention
The invention aims to provide a pretargeted tumor immunity probe, a biological orthogonal preparation, a kit and application, and the pretargeted tumor immunity probe provided by the invention can construct a PD-1/PD-L1 targeted PET probe based on a tetrazine-Reppe anhydride biological orthogonal reaction system, can realize specific imaging of tumors, and can evaluate the expression level of PD-1/PD-L1 in the tumors.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a pretargeted tumor immune probe, which is NOTA-Reppe anhydride marked with radionuclide, wherein the NOTA-Reppe anhydride has a structure shown in a formula I:
formula I.
The invention provides a biological orthogonal preparation, which comprises a split charging monoclonal antibody-tetrazine conjugate and a PET imaging probe; the monoclonal antibody in the monoclonal antibody-tetrazine conjugate is a PD-1 monoclonal antibody inhibitor or a PD-L1 monoclonal antibody inhibitor; the PET imaging probe is NOTA-Fabry-Perot anhydride marked with radionuclide, and the NOTA-Fabry-Perot anhydride has a structure shown in formula II:
a formula II;
in the formula II, R is，n=0~4。
Preferably, the PD-1 monoclonal antibody inhibitor is nano Wu Liyou mab or palboc Li Zhushan antibody.
Preferably, the PD-L1 monoclonal antibody inhibitor is alemtuzumab or devaluzumab.
Preferably, the radionuclide comprises 18 F、 68 Ga or 64 Cu。
The invention provides an application of the pretargeted tumor immune probe or the bioorthogonal preparation in preparing a PD-1 or PD-L1 expression level detection reagent in a tumor microenvironment.
The invention provides an application of the pretargeted tumor immune probe or the bioorthogonal preparation in preparation of PET imaging agent for detecting PD-1 or PD-L1 expression level in tumor microenvironment.
Preferably, the tumor comprises human brain glioma, colorectal cancer, ovarian cancer or non-small cell lung cancer.
The invention provides a kit for detecting the expression level of PD-1 or PD-L1 in a tumor microenvironment, which comprises a packaged monoclonal antibody-tetrazine conjugate and a PET imaging probe; the monoclonal antibody in the monoclonal antibody-tetrazine conjugate is a PD-1 monoclonal antibody inhibitor or a PD-L1 monoclonal antibody inhibitor; the PET imaging probe is NOTA-Fabry-Perot anhydride marked with radionuclide, and the NOTA-Fabry-Perot anhydride has a structure shown in formula II:
a formula II;
in the formula II, R is，n=0~4。
Preferably, the administration dosage of the monoclonal antibody-tetrazine conjugate is 0.5-2 mg/kg, and the administration dosage of the PET imaging probe is 37-74 MBq/kg.
The beneficial effects are that: the pre-targeting tumor immune probe provided by the invention can construct a PD-1/PD-L1 targeting PET probe based on a tetrazine-Reppe anhydride biological orthogonal reaction system, can realize specific imaging of tumors, and can evaluate the expression level of PD-1/PD-L1 in the tumors. Specifically, the Fabry-Perot anhydride is used as a novel bridged cycloolefin, has higher stereo conformational stability and higher reaction rate with tetrazine compared with a tetrazine-cyclooctene bio-orthogonal reaction system, and is a more ideal functional molecule for constructing bio-orthogonal chemical reaction. Probes incorporating long chain PEG (n=4) therein can achieve longer in vivo circulation half-lives while reducing the steric hindrance of the reaction with tetrazine coupled to the surface of the antibody, thereby better recognizing the antibody.
Drawings
FIG. 1 is a schematic illustration of the preparation of a monoclonal antibody-tetrazine conjugate with [ in example 1 ] 18 F]A reaction scheme of AlF-NOTA-Reppe anhydride;
FIG. 2 is a MALDI-TOF MS diagram of NOTA-Reppe anhydride;
FIG. 3 is [ 18 F]An AlF-NOTA-Reppe anhydride radiochemical purity test chart;
FIG. 4 is [ 18 F]Stability test result graphs of AlF-NOTA-Reppe anhydride in FBS and PBS;
FIG. 5 is [ 18 F]Cytotoxicity test result diagrams of AlF-NOTA-Reppe anhydride and NOTA-Reppe anhydride;
FIG. 6 is [ 18 F]Metabolic profile of AlF-NOTA-Reppe anhydride in BALB/c normal mice;
FIG. 7 is [ 18 F]Metabolic profile of AlF-NOTA-Reppe anhydride in BALB/c normal mice (PET/CT images);
FIG. 8 is a PET/CT image of pre-targeted immune PET imaging of Dewaruzumab in glioma PDX model;
FIG. 9 is a diagram of the preparation [ in example 2 ] 18 F]AlF-NOTA-PEG 4 -a reaction scheme of a reptile anhydride;
FIG. 10 is NOTA-PEG 4 MALDI-TOF MS map of Reppe's anhydride;
FIG. 11 is [ 18 F]AlF-NOTA-PEG 4 -a graph of stability test results of the rapier anhydride in HSA;
FIG. 12 is a PET/CT image of pre-targeting immune PET imaging of Dewaruzumab in an A549 non-small cell lung cancer model.
Detailed Description
The invention provides a pretargeted tumor immune probe, which is NOTA-Reppe anhydride marked with radionuclide, wherein the NOTA-Reppe anhydride has a structure shown in a formula I:
formula I.
In the present invention, the preparation method of the NOTA-Reppe anhydride and the method of preparing the target probe by further labeling the radionuclide will be described in detail later. In the present invention, the radionuclide preferably comprises 18 F、 68 Ga or 64 Cu is more preferably 18 F。
The invention provides a biological orthogonal preparation, which comprises a split charging monoclonal antibody-tetrazine conjugate and a PET imaging probe; the monoclonal antibody in the monoclonal antibody-tetrazine conjugate is a PD-1 monoclonal antibody inhibitor or a PD-L1 monoclonal antibody inhibitor; the PET imaging probe is NOTA-Fabry-Perot anhydride marked with radionuclide, and the NOTA-Fabry-Perot anhydride has a structure shown in formula II:
a formula II;
in the formula II, R is，n=0~4。
In the present invention, n=0, 1,2, 3 or 4, preferably 0 or 4. In the present invention, the PD-1 monoclonal antibody inhibitor is preferably nano Wu Liyou mab or palbociclib mab; the PD-L1 monoclonal antibody inhibitor is preferably alemtuzumab or Dewaruzumab, more preferably Dewaruzumab. In the present invention, the radionuclide preferably comprises 18 F、 68 Ga or 64 Cu is more preferably 18 F。
In the present invention, the preparation method of the mab-tetrazine conjugate preferably comprises the following steps:
mixing tetrazine-carboxylic acid derivative, 1-ethyl-3- [3- (dimethylamino) propyl ] carbodiimide and N-hydroxysuccinimide with a first organic solvent, and performing activation treatment to obtain a product system containing tetrazine-succinimide ester;
mixing the product system containing tetrazine-succinimidyl ester with a monoclonal antibody solution, and performing a coupling reaction to obtain the monoclonal antibody-tetrazine conjugate;
in the present invention, the structural formula of the tetrazine-carboxylic acid derivative is as follows:
。
the invention mixes tetrazine-carboxylic acid derivative, 1-ethyl-3- [3- (dimethylamino) propyl ] carbodiimide, N-hydroxysuccinimide and first organic solvent, and carries out activation treatment to obtain a product system containing tetrazine-succinimide ester. In the present invention, the molar ratio of the tetrazine-carboxylic acid derivative, 1-ethyl-3- [3- (dimethylamino) propyl ] carbodiimide, to N-hydroxysuccinimide is preferably 1: 1.8-2.2: 1.8 to 2.2, more preferably 1:2:2; the first organic solvent is preferably acetonitrile, and the dosage of the acetonitrile is not particularly limited, so that the activation treatment is ensured to be carried out smoothly. In the invention, the temperature of the activation treatment is preferably 15-35 ℃, more preferably room temperature, and in the embodiment of the invention, the room temperature is specifically 25 ℃; the activation treatment time is preferably 1.5 to 2.5 hours, more preferably 2 hours.
After the activation treatment, the method does not need any post treatment, and the obtained product system containing tetrazine-succinimidyl ester is directly mixed with a monoclonal antibody solution for coupling reaction to obtain the monoclonal antibody-tetrazine conjugate. In the invention, the molar ratio of the monoclonal antibody to the tetrazine-succinimidyl ester in the monoclonal antibody solution is preferably 1:8 to 12, more preferably 1:10; the solvent of the monoclonal antibody solution is preferably a second organic solvent, and the second organic solvent is preferably dimethyl sulfoxide (DMSO), and the dosage of the second organic solvent is not particularly limited in the invention, so that the coupling reaction can be ensured to be smoothly carried out. In the invention, the temperature of the coupling reaction is preferably 2-6 ℃, more preferably 4 ℃; the time is preferably 2.5 to 3.5 hours, more preferably 3 hours. After the coupling reaction, the obtained product system is preferably purified by a PD-10 column (the mobile phase is preferably PBS buffer solution) to obtain the monoclonal antibody-tetrazine conjugate.
In the invention, the preparation method of the NOTA-Reppe anhydride with the structure shown in the formula II preferably comprises the following steps:
mixing an N-Boc protected Reppe anhydride derivative and a trifluoroacetic acid aqueous solution with a third organic solvent, and carrying out deprotection reaction to obtain amino-functionalized Reppe anhydride trifluoroacetate;
mixing the amino-functionalized Reppe anhydride trifluoroacetate, a bifunctional chelating agent p-SCN-Bn-NOTA, an alkali reagent and a fourth organic solvent for substitution reaction to obtain NOTA-Reppe anhydride with a structure shown in a formula II;
the structural formula of the N-Boc protected Reppe anhydride derivative is shown as follows:
；
the structural formula of the N-Boc protected Reppe anhydride derivative is defined as formula II.
The invention mixes the N-Boc protected Reppe anhydride derivative, trifluoroacetic acid aqueous solution and a third organic solvent to carry out deprotection reaction, thus obtaining amino functional Reppe anhydride trifluoroacetate. In the invention, the concentration of the trifluoroacetic acid aqueous solution is preferably 15-25wt%, more preferably 20wt%; the dosage ratio of the N-Boc protected Reppe anhydride derivative to the trifluoroacetic acid aqueous solution is preferably 0.03mmol:0.4 to 0.6mL, more preferably 0.03mmol:0.5mL; the third organic solvent is preferably methanol, more preferably anhydrous methanol, and the amount of the third organic solvent is not particularly limited in the present invention, so that the reaction can be smoothly performed. In the invention, the temperature of the deprotection reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 3.5 to 4.5 hours, more preferably 4 hours. After the deprotection reaction, the obtained product system is preferably subjected to rotary evaporation to remove the solvent, so that the amino-functionalized Reppe anhydride trifluoroacetate is obtained.
After the amino-functionalized Reppe anhydride trifluoroacetate is obtained, the amino-functionalized Reppe anhydride trifluoroacetate, the bifunctional chelating agent p-SCN-Bn-NOTA, the alkali reagent and a fourth organic solvent are mixed for substitution reaction, so that the NOTA-Reppe anhydride with the structure shown in the formula II is obtained. In the invention, the molar ratio of the amino-functionalized Reppe anhydride trifluoroacetate to the bifunctional chelating agent p-SCN-Bn-NOTA is preferably 1:0.8 to 1.2, more preferably 1:1, a step of; the fourth organic solvent is preferably acetonitrile, the dosage of the fourth organic solvent is not particularly limited, and the reaction can be ensured to be carried out smoothly; the alkaline reagent is preferably triethylamine, and the amount of triethylamine is preferably based on the pH value of a system of 8.8-9.5, more preferably based on the pH value of the system of 9. In the invention, the temperature of the substitution reaction is preferably 15-35 ℃, more preferably room temperature; the time is preferably 0.5 to 1.5 hours, more preferably 1 hour. After the substitution reaction, the invention preferably carries out rotary evaporation on the obtained product system to remove the solvent so as to obtain a crude product; and purifying the crude product to obtain the NOTA-Reppe anhydride with the structure shown in the formula II. The present invention preferably selects an appropriate purification method according to the specific structure of the target product. Specifically, when n=0 in R, the crude product is preferably purified by a silica gel column, and the mobile phase used is preferably methanol: dichloromethane = 4: 1). When n=4 in R (i.e. NOTA-rep anhydride having the structure shown in formula I), the crude product is preferably purified by HPLC, and the mobile phase preferably comprises mobile phase a and mobile phase B; the mobile phase A is preferably TFA, CH 3 A mixture of CN and water, wherein the volume fraction of TFA in the mobile phase A is preferably 0.05%, CH 3 The volume fraction of CN is preferably 2%; the mobile phase B is preferably TFA, CH 3 A mixture of CN and water, wherein the volume fraction of TFA in the mobile phase B is preferably 0.05%, CH 3 The volume fraction of CN is preferably 90%.
In the present invention, radionuclides are used as 18 As an example, the method for labeling a radionuclide with a NOTA-reptile anhydride having a structure represented by formula II preferably comprises the steps of:
mixing NOTA-Reppe anhydride water solution and AlCl 3 Aqueous solution, ammonium acetate buffer solution 18 F - Mixing the ions for reaction, and purifying to obtain the labeled product 18 F - Is not a-rap anhydride.
In the invention, the concentration of the NOTA-Reppe anhydride aqueous solution is preferably 1mg/mL; the AlCl 3 The concentration of the aqueous solution is preferably 2mM; the concentration of the ammonium acetate buffer solution is preferably 0.1M, and the pH value is preferably 4.0; the NOTA-Reppe anhydride aqueous solution and AlCl 3 The volume ratio of the aqueous solution to the ammonium acetate buffer solution is preferably 100. Mu.L: 3 μL:1mL; the said 18 F - The radioactivity of the ions is preferably 185MBq, said 18 F - The ions are preferably prepared by a medical cyclotron and eluted into the reaction tube. In the invention, the reaction temperature is preferably 85-95 ℃, more preferably 90 ℃; the time is preferably 15 to 25 minutes, more preferably 20 minutes. The present invention preferably uses Oasis HLB 3-cc cartridge (Waters) column to purify the product system after the reaction is completed, specifically 1mL of 0.9% physiological saline is used for eluting to remove unreacted 18 F - Sum [ 18 F]AlF 2+ Then, 200 mu L of ethanol water solution with the volume fraction of 80% is adopted for flushing a column to obtain a target product.
The invention constructs the PD-1/PD-L1 targeted tumor diagnosis PET probe by adopting a tetrazine-Reppe anhydride biological orthogonal reaction system, can realize the specific imaging of tumors, can evaluate the expression level of PD-1/PD-L1 in the tumors, and can be used as an evaluation means for judging whether a tumor patient is suitable for PD-1/PD-L1 immunotherapy.
The invention provides an application of the pretargeted tumor immune probe according to the technical scheme or the biorthogonal preparation according to any one of the technical scheme in preparing a PD-1 or PD-L1 expression level detection reagent in a tumor microenvironment.
The invention provides an application of the pretargeted tumor immune probe or the bioorthogonal preparation in preparation of PET imaging agent for detecting PD-1 or PD-L1 expression level in tumor microenvironment.
In the present invention, the tumor preferably includes human glioma, colorectal cancer, ovarian cancer or non-small cell lung cancer, more preferably human glioma or non-small cell lung cancer.
The invention provides a kit for detecting the expression level of PD-1 or PD-L1 in a tumor microenvironment, which comprises a packaged monoclonal antibody-tetrazine conjugate and a PET imaging probe; the monoclonal antibody in the monoclonal antibody-tetrazine conjugate is a PD-1 monoclonal antibody inhibitor or a PD-L1 monoclonal antibody inhibitor; the PET imaging probe is NOTA-Fabry-Perot anhydride marked with radionuclide, and the NOTA-Fabry-Perot anhydride has a structure shown in formula II:
a formula II;
in the formula II, R is，n=0~4。
In the present invention, n=0, 1,2, 3 or 4, preferably 0 or 4. In the invention, the administration dosage of the monoclonal antibody-tetrazine conjugate is preferably 0.5-2 mg/kg (rodent-sized mice), and the administration dosage of the PET imaging probe is preferably 37-74 MBq/kg (rodent-sized mice).
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Tetrazine-carboxylic acid derivative (4- (6-methyl-1, 2,4, 5-tetrazin-3-yl) benzoic acid, abbreviated as Tz-COOH) and Rappet anhydride derivative (7, 8-N- [ tert-butyl (2-aminoethyl) carbamate)]succinimideendo-tricyclo-[4.2.2.0 2.5 ]deca-3, 9-diene) is purchased from south Beijing, entts biotechnology Co., ltd; the bifunctional chelator p-SCN-Bn-NOTA was purchased from american macrocyclic corporation (macrocirculations, inc.); 1-ethyl-3- [3- (dimethylamino) propyl group]Carbodiimide (English name 1-methyl-3- [3- (dimethyl imine) propyl)]carbodiimide abbreviated EDC), N-Hydroxysuccinimide (English name N-hydroxycicinimide abbreviated NHS) and aluminum chloride hexahydrate (AlCl) 3 ·6H 2 O) was purchased from Sigma Aldrich trade company (Shanghai).
Example 1
1.1 Synthesis of monoclonal antibody-tetrazine conjugate
B in fig. 1 is a reaction route for preparing a monoclonal antibody-tetrazine conjugate by chemically modifying a PD-L1 monoclonal antibody inhibitor (dewaruzumab) with a tetrazine-carboxylic acid derivative, and specifically comprises the following steps:
300. Mu.g (1.3. Mu. Mol) of a tetrazine-carboxylic acid derivative (Tz-COOH) was dissolved in acetonitrile (500. Mu.L), EDC and NHS were added, and the molar ratio of Tz-COOH, EDC and NHS was 1:2:2, performing activation treatment for 2 hours at room temperature (25 ℃) to obtain a product system containing tetrazine-succinimidyl ester; the product system containing tetrazine-succinimidyl ester was added to a DMSO (200 μl) solution of dewaruzumab (200 μg), and the molar ratio of tetrazine-succinimidyl ester to dewaruzumab was 10:1, reacting for 3 hours at the temperature of 4 ℃; after the reaction is finished, the obtained product system is purified by adopting a PD-10 column (the used mobile phase is PBS buffer solution) to obtain the monoclonal antibody-tetrazine conjugate.
Determining the coupling number of Tz-COOH and Dewaruzumab in the monoclonal antibody-tetrazine conjugate by adopting an ultraviolet-visible spectrophotometry, wherein the characteristic absorption peak of the Tz-COOH is 540nm, and the characteristic absorption peak of the Dewaruzumab is 280nm; the results show that each Devaluzumab binds approximately 2 Tz-COOH.
1.2 [ 18 F]Synthesis of AlF-NOTA-Reppe anhydride
A in FIG. 1 is preparation [ 18 F]The reaction route of AlF-NOTA-Reppe anhydride comprises the following specific steps:
(1) 0.03mmol of N-Boc protected Reppe anhydride derivative (n=0 in R) is dissolved in 1.0mL of absolute methanol, and then 0.5mL of aqueous trifluoroacetic acid solution with concentration of 20wt% is added for reaction for 4 hours at room temperature (25 ℃); and evaporating the obtained product system by adopting a rotary evaporator after the reaction is finished to obtain the amino-functionalized Reppe anhydride trifluoroacetate.
(2) Dissolving 0.03mmol of amino-functionalized Reppe anhydride trifluoroacetate in 1mL of acetonitrile, adding 0.1mmol of triethylamine (regulating the pH value of the system to 9), adding 0.03mmol of bifunctional chelating agent p-SCN-Bn-NOTA, and stirring at room temperature (25 ℃) for reaction for 1h; after the reaction is finished, performing rotary evaporation on the obtained product system to remove the solvent, so as to obtain a crude product; the crude product was purified using a silica gel column (methanol: dichloromethane=4:1 as mobile phase) to give NOTA-Reppe anhydride (NOTA-Reppe anhydride) as a pink product in a yield of 12mg and 55%.
FIG. 2 is a MALDI-TOF MS plot of NOTA-Reppe anhydride, wherein [ M+H ]] + m/z [C 34 H 42 N 6 O 8 SH] + Theoretical value: 695.280, measured value: 695.111.
(3) By [ using 18 F]AlF one-step chelation method for NOTA-Reppe anhydride 18 F labelling, in particular AlCl at a concentration of 2mM in 3. Mu.L 3 The aqueous solution and 100. Mu.L of 1mg/mL aqueous NOTA-Reppe anhydride solution were added to 1mL of 0.1M ammonium acetate buffer solution (pH=4.0), and 185MBq was added 18 F - Ions (prepared by a medical cyclotron and eluted into a reaction tube) are reacted for 20min at 90 ℃; after the reaction, the resulting product system was purified using an Oasis HLB 3-cc cartridge (Waters) column (specifically, 1mL of 0.9% physiological saline was used to remove unreacted 18 F - Sum [ 18 F]AlF 2+ Then, 200 mu L of ethanol water solution with the volume fraction of 80% is adopted for flushing to obtain the target product, thus obtaining the [ 18 F]AlF-NOTA-Reppe anhydride.
Determination of said [ using radiation thin layer chromatography ] 18 F]The result shows that the radiolabeling rate and radiochemical purity of AlF-NOTA-Reppe anhydride are 68+/-5%; FIG. 3 is [ 18 F]AlF-NOTA-Reppe anhydride radiochemical purity test chart, and the result shows that 18 F]Radiochemical purity of AlF-NOTA-Reppe anhydride>96%。
Test example 1
1.1 [ 18 F]In vitro stability test of AlF-NOTA-Reppe anhydride (abbreviated as PET Probe)
100. Mu.L of 3.7MBq PET probe was mixed with 900. Mu.L of Fetal Bovine Serum (FBS) and PBS buffer, respectively, sampled at different time points (0 h, 1h, 2h, 4h, 8h, 12h, 16 h), and time-radiochemical purity stability curves were plotted using paper chromatography Radio-TLC to test the stability of the PET probe in FBS and PBS.
FIG. 4 is [ 18 F]The stability test result diagram of AlF-NOTA-Reppe anhydride in FBS and PBS shows that the PET probe can maintain higher stability in both FBS and PBS for 12 hours.
1.2 Cytotoxicity experiment of PET Probe
The human brain glioma cell line U87MG is subjected to conventional resuscitation, culture and passage, and 1X 10 cells are paved in each well of a 96-well plate 3 The method comprises the steps of carrying out a first treatment on the surface of the Set 2 groups: a) A PET probe set, b) a label-free NOTA-reptile anhydride set; drug co-incubation with cells 24h, wherein drug concentration was set to 0 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, 400 μg/mL; the CCK8 method then measures PET probe toxicity, and the absorbance (OD value) was measured at 570 nm with an enzyme-labeled instrument, and cell viability was calculated.
FIG. 5 is [ 18 F]Cytotoxicity test result graph of AlF-NOTA-Reppe anhydride and NOTA-Reppe anhydride, wherein 18 F-RA correspondence [ 18 F]AlF-NOTA-Rayleigh anhydride, RA corresponds to the NOTA-Rayleigh anhydride; the results show that both drugs were non-toxic to the U87MG cell line.
1.3 In vivo biological distribution of PET probes in normal mice and PET imaging
(1) PET probes were injected into normal Balb/c mice via tail vein (37 KBq/10. Mu.L of 0.9% physiological saline), sacrificed and dissected at different time points by orbital exsanguination, the major organs (heart, lung, liver, spleen, kidney, stomach, small intestine, muscle, bone and other tissue organs) were weighed, their counts were measured by a gamma-counter, and the percent injection dose rate per gram of tissue (% ID/g) was calculated after decay correction: tissue injection dose distribution per gram (% ID/g) =radioactivity Count (CPM) of each tissue organ/mass (g) of each tissue organ/injection dose; the method comprises the steps of researching the behaviors of absorption, distribution, metabolism, elimination and the like in a human body, drawing pharmacokinetic curve parameters of a PET probe, and exploring the in-vivo distribution rule of the PET probe; and collecting blood from the eyebox at different time points, measuring the radioactivity count, drawing a time-activity curve, and calculating the half-clearance rate of blood.
FIG. 6 is [ 18 F]The result of the metabolism distribution diagram of AlF-NOTA-Reppe anhydride in BALB/c normal mice shows that the PET probe is mainly metabolized and rapidly cleared through the liver and the kidney, wherein the uptake of the liver (7.63% ID/g) and the kidney (10.22% ID/g) reaches a peak after tail vein injection for 1h, and the small intestine also presents higher uptake due to the hepatobiliary metabolism of the PET probe; after 2 hours, the PET probe can be rapidly cleared (0.84% ID/g of liver; 2.84% ID/g of kidney); non-metabolic organs such as lung, muscle and bone have low uptake of PET probes.
(2) Carrying out PET/CT imaging in a small animal body on a normal Balb/c mouse, and observing the biological distribution of a PET probe in a model body, wherein the specific steps are as follows: PET probes (14.9+ -0.5 MBq/100 μL of 0.9% physiological saline) were used to inject mice (5 mice/group) into tail vein, and were placed in a small animal PET/CT scanning device (Siemens Inveon MM, germany) for whole body PET/CT scanning, during which 2% isoflurane was used to mix with medical oxygen at a flow rate of 1mL/min to maintain the anesthetized state of the mice; after the scanning is finished, an image is reconstructed by adopting a filter back projection method (FBP, filtered back projection), the quantity (% ID/g) of the PET probe absorbed by each organ is calculated by taking CT as a template, and the optimal imaging time of the PET probe is determined by analyzing imaging results of different times (30 min, 1h and 2 h) after the PET probe is injected.
FIG. 7 is [ 18 F]The metabolic profile (PET/CT image) of AlF-NOTA-Reppe anhydride in BALB/c normal mice shows uptake and biology with the above organsThe results of the distribution experiment are consistent.
1.4 Pre-targeting immune PET imaging evaluation of PD-L1 expression level in human brain glioma PDX model
(1) Construction of human brain glioma PDX model: the tissue sample of the human brain glioma after the fresh surgery is inoculated onto a heavy immunodeficiency NSG mouse subcutaneously and is marked as generation P1; taking out tumor after P1 generation grows to about 1.5cm, cutting into small pieces, inoculating again into NSG mice, and marking as P2 generation; and (3) resuscitating the P2 generation tumor sample, inoculating the resuscitating sample into an NSG mouse, measuring the tumor volume every 5 days, and obtaining the human brain glioma PDX model when the tumor diameter reaches 0.5-1 cm.
(2) Pre-targeting animal immune PET/CT imaging is carried out on the constructed human brain glioma PDX model, the biological distribution of the PET probe in the model body is observed, and the diagnosis effect of the PET probe is evaluated, wherein the specific steps are as follows: dewaruzumab conjugated with tetrazine derivatives (i.e. the mab-tetrazine conjugate prepared in example 1) was injected into human brain glioma PDX model mice by tail vein (5, 200 μg mab-tetrazine conjugate per 200 μl of 0.9% physiological saline), then PET probes (14.9+ -0.5 MBq/100 μl of 0.9% physiological saline) were injected 48h later, scanned in a small animal PET/CT scanning device, and the optimal injection time and optimal imaging time of the PET probes were determined by analysis of imaging results at different times (1 h, 2h, 24 h) after injection of the PET probes to evaluate the accuracy and sensitivity of evaluation of PD-1/PD-L1 expression levels of the PET probes in the humanized PDX model.
FIG. 8 is a PET/CT image of pre-targeting immune PET imaging of Dewaruzumab in a glioma PDX model, where a is the in vivo profile 1h after PET probe injection, b is the in vivo profile 2h after PET probe injection, c is the in vivo profile 24h after PET probe injection, and white arrows are tumor areas; the results showed that the uptake of PET probe in tumors was 2.3% ID/g after 1h of tail vein injection of PET probe, 2.1% ID/g after 2h, and 1.7% ID/g after 24 h. This suggests that PET probes can achieve targeted imaging of tumors and assess the expression level of PD-L1 in tumors.
Example 2
FIG. 9 is a schematic diagram of preparation [ 18 F]AlF-NOTA-PEG 4 The reaction route of the Reppe anhydride comprises the following specific steps:
(1) 0.03mmol of N-Boc protected Reppe anhydride derivative (n=4 in R) is dissolved in 1.0mL of absolute methanol, and then 0.5mL of aqueous trifluoroacetic acid solution with concentration of 20wt% is added for reaction for 4 hours at room temperature (25 ℃); and evaporating the obtained product system by adopting a rotary evaporator after the reaction is finished to obtain the amino-functionalized Reppe anhydride trifluoroacetate.
(2) Dissolving 0.03mmol of amino-functionalized Reppe anhydride trifluoroacetate in 1mL of acetonitrile, adding 0.1mmol of triethylamine (regulating the pH value of the system to 9), adding 0.03mmol of bifunctional chelating agent p-SCN-Bn-NOTA, and stirring at room temperature (25 ℃) for reaction for 1h; after the reaction is finished, performing rotary evaporation on the obtained product system to remove the solvent, so as to obtain a crude product; the crude product was purified by HPLC (mobile phase a TFA, CH 3 A mixture of CN and water, wherein the volume fraction of TFA in the mobile phase A is 0.05%, CH 3 The volume fraction of CN was 2%; mobile phase B is TFA, CH 3 A mixture of CN and water, wherein the volume fraction of TFA in the mobile phase B is 0.05%, CH 3 CN with volume fraction of 90 percent) and freeze-drying to obtain NOTA-PEG 4 Reppe anhydride (NOTA-PEG) 4 Reppe anhydride) as a pale yellow product in a yield of 5.2mg and a yield of 21%.
FIG. 10 is NOTA-PEG 4 MALDI-TOF MS map of Reppe anhydride, wherein [ M+H ]] + m/z [C 42 H 58 N 6 O 12 SH] + Theoretical value: 872.020, measured value: 872.029.
(3) Operating as in example 1, the replacement of the NOTA-Reppe anhydride with NOTA-PEG 4 Reppe anhydride, finally prepared 18 F]AlF-NOTA-PEG 4 -a reptile anhydride.
Determination of said [ using radiation thin layer chromatography ] 18 F]AlF-NOTA-PEG 4 The radiolabelling rate of the Reppe anhydride and the radiochemical purity, which shows that the radiolabelling rate without attenuation correction is 30%, the radiochemical purity>95%。
Test example 2
1.1 [ 18 F]AlF-NOTA-PEG 4 Reppe anhydride (abbreviated as PEG) 4 -PET probe) in vitro stability test
100 μL of 3.7MBq PEG 4 PET probe was mixed with 900. Mu.L of Human Serum Albumin (HSA) solution, sampled at different time points (0 h, 0.5h, 1h, 3h, 6h, 12h, 24 h), and analyzed by chromatography on Radio-TLC, and the stability profile of time-radiochemical purity was plotted to test PEG 4 Stability of the PET probe in HSA.
FIG. 11 is [ 18 F]AlF-NOTA-PEG 4 Stability test result diagram of Reppe anhydride in HSA, and the result shows that PEG 4 The PET probe can maintain stability in HSA for a longer period of time.
1.2 In vivo pre-targeted PET/CT imaging of non-small cell lung cancer model based on bioorthogonal chemistry
(1) Construction of a non-small cell lung cancer model: the non-small cell lung cancer cell line A549 was taken at 1X 10 6 The cells are injected into the armpit of the right side of the BALB/c-nu nude mouse, the growth of the tumor is measured by observing every other day after injection, and the tumor is reserved when reaching 0.5-1 cm.
(2) Adopts a pre-targeting immune PET imaging strategy and uses a small animal PET/CT scanning device to inspect PEG 4 The PET probe is inoculated into the metabolism and biological distribution condition of a nude mouse model body under the skin of a non-small cell lung cancer cell line A549, and quantitative analysis is carried out, and the specific steps are as follows: the tail vein was pre-injected with the mab-tetrazine conjugate prepared in example 1 (4, each at a dose of 200 μg mab-tetrazine conjugate/200 μl of 0.9% physiological saline) and after 48h, PEG was injected again 4 -PET probe and by injection of PEG 4 Analysis of imaging results at different times (1 h, 2h, 24 h) after PET probe.
FIG. 12 is a PET/CT image of pre-targeting immune PET imaging of Dewaruzumab in A549 non-small cell lung cancer model, wherein a is PEG 4 -in vivo profile 1h after PET probe injection, b being PEG 4 -in vivo profile 2h after PET probe injection, c is PEG 4 -in vivo profile 24h after PET probe injection, white arrow is tumorA region; the results show that PEG 4 The uptake value of the PET probe in the tumor model after 1h injection is 1.6+/-0.5% ID/g, the highest value of the PET probe reaches 4.6+/-0.3% ID/g after 2h injection, and the uptake value of the PET probe in 24h is reduced to 0.5+/-0.2% ID/g. This illustrates the bio-orthogonal chemical PEG 4 The PET probes allow to visualize the expression level of PD-L1 in the tumor and to enable a quantitative assessment.
From the above examples and tests, the present invention constructs pre-targeting immune PET probes useful for assessing PD-1/PD-L1 expression levels in tumor microenvironments based on bio-orthogonal chemical reaction systems of tetrazine-Reppe anhydride derivatives. Specifically, the invention couples high affinity PD-1 monoclonal antibody inhibitors (such as nano Wu Liyou monoclonal antibody and palbociclib monoclonal antibody) or PD-L1 monoclonal antibody inhibitors (such as atteguzumab and Dewaruzumab) with tetrazine derivatives respectively, thereby constructing and obtaining 'bispecific' monoclonal antibody; meanwhile, the short half-life positron nuclides such as F-18, ga-68 and Cu-64 are subjected to radioactive labeling on the Reppe anhydride (Reppe anhydride) derivative, so that the PET probe capable of being specifically coupled with the monoclonal antibody is obtained. The in vivo PET imaging experiment of the tumor model proves that the PET probe can be applied to quantitatively evaluating the expression level of PD-1/PD-L1 in tumors, and can provide important basis for the formulation of PD-1/PD-L1 immunotherapy strategies of tumor patients and the evaluation of curative effects. The visual imaging detection of the expression level of PD-1/PD-L1 can overcome the problems of poor prediction accuracy, strong invasiveness, incapability of sampling for many times and the like of tissue biopsy, and meanwhile, the pre-targeting immune PET imaging strategy based on short half-life nuclides can definitely solve the problem of radiation damage, namely, firstly, the monoclonal antibody is injected into an organism, after the antibody is subjected to in vivo circulation for about 1 week, after the antibody and target tissues are subjected to specific binding, the PET probe marked by the short half-life nuclides is injected, and the PET probe is rapidly combined with the modified antibody which is injected first in the target tissues.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A pretargeted tumor immune probe, characterized by being a radionuclide-labeled NOTA-reptile anhydride having a structure according to formula I:
formula I.
2. A biorthogonal preparation, which is characterized by comprising a split charging monoclonal antibody-tetrazine conjugate and a PET imaging probe; the monoclonal antibody in the monoclonal antibody-tetrazine conjugate is a PD-1 monoclonal antibody inhibitor or a PD-L1 monoclonal antibody inhibitor; the PET imaging probe is NOTA-Fabry-Perot anhydride marked with radionuclide, and the NOTA-Fabry-Perot anhydride has a structure shown in formula II:
a formula II;
in the formula II, R is，n=0~4。
3. The biorthogonal formulation of claim 2, wherein the PD-1 monoclonal antibody inhibitor is nano Wu Liyou mab or palboc Li Zhushan antibody.
4. The biorthogonal formulation according to claim 2, wherein the PD-L1 monoclonal antibody inhibitor is alemtuzumab or devaluzumab.
5. The biorthogonal formulation according to any one of claims 2-4, wherein said radionuclide comprises 18 F、 68 Ga or 64 Cu。
6. The use of the pretargeted tumor immunity probe of claim 1 or the bioorthogonal preparation of any one of claims 2-5 in preparing a reagent for detecting the expression level of PD-1 or PD-L1 in tumor microenvironment.
7. Use of the pretargeted tumor immunity probe of claim 1 or the bioorthogonal preparation of any one of claims 2-5 in the preparation of a PET imaging agent for detecting the expression level of PD-1 or PD-L1 in a tumor microenvironment.
8. The use according to claim 6 or 7, wherein the tumour comprises human brain glioma, colorectal cancer, ovarian cancer or non-small cell lung cancer.
9. A kit for detecting the expression level of PD-1 or PD-L1 in a tumor microenvironment, comprising a packaged mab-tetrazine conjugate and a PET imaging probe; the monoclonal antibody in the monoclonal antibody-tetrazine conjugate is a PD-1 monoclonal antibody inhibitor or a PD-L1 monoclonal antibody inhibitor; the PET imaging probe is NOTA-Fabry-Perot anhydride marked with radionuclide, and the NOTA-Fabry-Perot anhydride has a structure shown in formula II:
a formula II;
in the formula II, R is，n=0~4。
10. The kit according to claim 9, wherein the dosage of the mab-tetrazine conjugate is 0.5-2 mg/kg and the dosage of the PET imaging probe is 37-74 mbq/kg.
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