阅读说明：本技术 一种靶向pd-l1的pet分子探针及其制备方法与应用 (PET molecular probe targeting PD-L1 and preparation method and application thereof ) 是由 周明 胡硕 张现忠 王晓博 陈蓓 饶婉倩 于 2021-08-18 设计创作，主要内容包括：本发明提供了一种靶向PD-L1的PET分子探针及其制备方法与应用,属于核医学与放射性药物领域。本发明提供一种靶向PD-L1的PET分子探针及其制备方法和应用,并且实现了在多个动物肿瘤模型中的高特异性,高灵敏度显像。本发明制备获得的靶向PD-L1的PET分子探针在临床人体水平也得到了进一步验证,可以较好的对PD-L1高表达的肺癌患者中PD-L1的表达水平进行无创活体显像,有望为PD-L1患者的早期无创筛查已经免疫治疗过程中PD-L1表达水平的监测提供新的显像手段。(The invention provides a PET molecular probe targeting PD-L1, a preparation method and application thereof, and belongs to the field of nuclear medicine and radiopharmaceuticals. The invention provides a PET molecular probe targeting PD-L1, a preparation method and application thereof, and realizes high-specificity and high-sensitivity imaging in a plurality of animal tumor models. The PET molecular probe targeting PD-L1 prepared by the invention is further verified in clinical human body level, can better carry out noninvasive living body imaging on the expression level of PD-L1 in a lung cancer patient with high expression of PD-L1, and is expected to provide a new imaging means for monitoring the PD-L1 expression level in the early noninvasive screening of the PD-L1 patient in the immunotherapy process.)

1. A PET molecular probe targeting PD-L1, characterized in that: the structural formula of the molecular probe is as follows:

when in useThe PET molecular probe targeting PD-L1 is named as⁶⁸Ga-NOTA-NF12；

When in useThe PET molecular probe targeting PD-L1 is named as¹⁸F-AIF-NOTA-NF12。

2. The method for preparing the PET molecular probe targeting PD-L1 as claimed in claim 1, characterized in that: wherein, the⁶⁸The specific preparation steps of Ga-NOTA-NF12 are as follows:

(1) taking 25-30 mu g of labeled precursor compound NOTA-NF12, adding 1mL of 0.25M NaOAc aqueous solution, and uniformly mixing to obtain a NOTA-NF12 solution;

(2) 4mL of 0.05M HCl⁶⁸Leaching Ga into a NOTA-NF12 solution, wherein the radioactivity is about 10-50mCi, and reacting for 10min at 90 ℃;

(3) adding deionized water to quench the reaction to obtain a reaction system;

(4) c, passing the reaction system of the step (3) through₁₈Enrichment with Plus column and deionized water rinse C₁₈A Plus column;

(5) eluting the product into a product bottle with a filter membrane by using 1mL of ethanol and 10mL of normal saline in sequence to obtain the PET molecular probe targeting PD-L1⁶⁸Ga-NOTA-NF12。

3. The method for preparing the PET molecular probe targeting PD-L1 according to claim 1, characterized in that: wherein, the¹⁸The specific preparation steps of F-AIF-NOTA-NF12 are as follows:

1) production by cyclotron¹⁸F is transported to an anion exchange column QMA through a pipeline for capture, and N is used₂Drying the QMA; obtained by production thereof¹⁸The radioactivity of F is 250-300 mCi;

2) 300ug of AIF-NOTA-NF12,1.0mL of acetonitrile, 0.03mL of 0.4M A1C13 solution and 0.5mL of acetic acid buffer solution with pH 4.0 are added into a reaction tube in advance;

3)0.3mL of 0.9% NaCl the solution obtained in step 1)¹⁸F, leaching the mixture into a reaction tube to obtain 250mCi with the radioactivity of 200 and 110 ℃, and reacting for 10 min;

4) adding 5mL of deionized water, purifying by HPLC, and collecting the product peak with the radioactivity of 80-120 mci;

5) product solution passing through C₁₈Enriching the Plus column, then sequentially leaching with 2mL of ethanol and 10mL of normal saline, and passing the leaching solution through a sterile filtration membrane to obtain the PET molecular probe targeting PD-L1¹⁸F-AIF-NOTA-NF12。

4. The use of the PET molecular probe targeting PD-L1 according to claim 1 in a PD-L1 immunodiagnosis kit.

5. Use of the PET molecular probe targeting PD-L1 according to claim 1 in the preparation of a tumor PET imaging agent.

6. The use of the PD-L1-targeting PET molecular probe of claim 1 in tumor PET imaging.

Technical Field

The invention belongs to the field of nuclear medicine and radiopharmaceuticals, and particularly relates to a PD-L1-targeted PET molecular probe and a preparation method and application thereof.

Background

Tumor immunotherapy has become the fourth major tumor therapy after surgery, radiotherapy and chemotherapy drug therapy, and is the most popular tumor therapy at present. For example, immunotherapy directed to the PD-1/PD-L1 signaling pathway has had great success in the treatment of clinical tumor patients, and thus has received the 2018 nobel prize for medicine. However, not all tumor patients respond to this treatment; and due to different action mechanisms, compared with the conventional treatment means, the treatment response modes of the tumors are various, such as delayed response, pseudo progress, over progress, immune-related adverse events and the like, and difficulty and challenge are brought to clinical treatment decision and curative effect evaluation. Studies show that the immunotherapy effect of PD-1/PD-L1 is not only dependent on the expression level of PD-L1, but also closely related to the real-time dynamic change of PD-L1 in the treatment process. Therefore, it is important to accurately detect the expression and dynamic change of the immune checkpoint PD-L1 in real time. Traditional puncture pathological biopsy and immunohistochemistry are limited by the difficulty in accurately monitoring the dynamic change of the immune checkpoint PD-L1 in real time due to invasive operation, result distortion caused by tumor heterogeneity and the like; CT, MRI and ultrasound are structural imaging techniques based on changes in tumor morphology, and cannot reflect the expression and dynamic changes of PD-L1 at the molecular level. Therefore, there is an urgent clinical need for a novel detection method to achieve non-invasive, dynamic and accurate monitoring of PD-L1 expression.

Positron Emission Tomography (PET) can monitor and evaluate the expression level of the tumor immune check point in an overall intuitive and noninvasive manner due to the advantages of high resolution, high sensitivity and the like, provides more accurate, real-time and comprehensive information, avoids the influence of pathological sections and treatment means on the expression level of the organism immune check point, and provides accurate basis for screening patients with curative effect response of immune check point immune treatment, optimizing the treatment scheme of tumor anti-immune check point and evaluating prognosis. The molecular probe is a key for restricting PET molecular image, and the development of a radioactive molecular probe targeting PD-L1 is one of the key points of the current nuclear medicine and radiopharmaceutical field research, and is also a key technical problem to be solved urgently by researchers in the field.

Disclosure of Invention

The primary purpose of the embodiment of the invention is to provide a PET molecular probe targeting PD-L1, and to realize noninvasive, dynamic and accurate monitoring of PD-L1 expression.

The embodiment of the invention also aims to provide a preparation method of the PET molecular probe targeting PD-L1.

The embodiment of the invention further aims to provide the application of the molecular probe in PD-L1PET imaging.

The purpose of the invention is realized by the following technical scheme:

a PD-L1-targeted PET molecular probe, the molecular probe having the structural formula of formula I:

when in useThe PET molecular probe targeting PD-L1 is named as⁶⁸Ga-NOTA-NF12；

When in useThe PET molecular probe targeting PD-L1 is named as¹⁸F-AIF-NOTA-NF12。

Wherein, the⁶⁸The specific preparation steps of Ga-NOTA-NF12 are as follows:

(1) taking 25-30 mu g of labeled precursor compound NOTA-NF12, adding 1mL of 0.25M NaOAc aqueous solution, and uniformly mixing to obtain a NOTA-NF12 solution;

(2) 4mL of 0.05M HCl⁶⁸Leaching Ga into a NOTA-NF12 solution, wherein the radioactivity is about 10-50mCi, and reacting for 10min at 90 ℃;

(3) adding deionized water to quench the reaction to obtain a reaction system;

(4) c, passing the reaction system of the step (3) through₁₈Plus columnEnriching and washing C with deionized water₁₈A Plus column;

(5) eluting the product into a product bottle with a filter membrane by using 1mL of ethanol and 10mL of normal saline in sequence to obtain the PET molecular probe targeting PD-L1⁶⁸Ga-NOTA-NF12。

Further, obtained in step (5)⁶⁸Ga-NOTA-NF12 has a radioactivity of 20-26 mCi.

Further, the amount of deionized water used for quenching the reaction in step (3) was 10 mL.

Further, the amount of deionized water used for washing in step (4) was 10 mL.

Wherein, the¹⁸The specific preparation steps of F-AIF-NOTA-NF12 are as follows:

1) production by cyclotron¹⁸F is transported to an anion exchange column QMA through a pipeline for capture, and N is used₂Drying the QMA; obtained by production thereof¹⁸The radioactivity of F is 250-300 mCi;

2) 300ug of AIF-NOTA-NF12,1.0ml of acetonitrile, 0.03ml of 0.4M A1C13 solution and 0.5ml of acetic acid buffer solution with pH 4.0 are added into a reaction tube in advance;

3)0.3ml 0.9% NaCl the solution obtained in step 1) was washed with¹⁸F, leaching the mixture into a reaction tube to obtain 250mCi with the radioactivity of 200 and 110 ℃, and reacting for 10 min;

4) adding 5mL of deionized water, purifying by HPLC, and collecting the product peak with the radioactivity of 80-120 mci;

5) product solution passing through C₁₈Enriching the Plus column, then sequentially leaching with 2mL of ethanol and 10mL of normal saline, and passing the leaching solution through a sterile filtration membrane to obtain the PET molecular probe targeting PD-L1¹⁸F-AIF-NOTA-NF12。

Further, the cyclotron in step 1) is preferably a Qilin cyclotron, GE corporation.

Further, the anion exchange column QMA in step 1) is QMA ^ READI-CLING^TMAnd (4) forming the column.

Further, obtained in step 5)¹⁸The radioactivity of F-AIF-NOTA-NF12 was 50-60 mCi.

The PET molecular probe targeting PD-L1 is applied to a PD-L1 immune diagnosis and treatment kit.

The PET molecular probe targeting PD-L1 is applied to the preparation of a tumor PET imaging agent.

The application of the PET molecular probe targeting PD-L1 in tumor PET imaging.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

1. the technical problem to be solved by the invention is to provide⁶⁸Ga or¹⁸F-AIF labeled polypeptide targeting PD-L1PET molecular probe and preparation method thereof, and realizes high specificity and high sensitivity imaging in a plurality of animal tumor models (B16 melanoma, PANC02 pancreatic cancer, MC38 colon cancer, TNBC triple negative breast cancer tumor, H1975 lung adenocarcinoma and the like).

2. The PET molecular probe targeting PD-L1 prepared by the invention is further verified in clinical human body level, can better carry out noninvasive living body imaging on the expression level of PD-L1 in a lung cancer patient with high expression of PD-L1, and is expected to provide a new imaging means for monitoring the PD-L1 expression level in the early noninvasive screening of the PD-L1 patient in the immunotherapy process.

Drawings

FIG. 1 shows a flowchart of the present invention in example 1⁶⁸Graph of radiochemical purity HPLC results for Ga-NOTA-NF 12.

FIG. 2 shows a flowchart of example 2 of the present invention¹⁸F-AIF-NOTA-NF12 and standard substance¹⁹F-AIF-NOTA-NF12 radiochemical purity HPLC results.

FIG. 3 shows the rat tail vein injection of the B16 tumor model in detection example 1⁶⁸Ga-NOTA-NF12 and¹⁸F-AIF-NOTA-NF12B16 model visualization.

FIG. 4 shows the results of the detection of example 1 of the present invention¹⁸F-AIF-NOTA-NF12B16 showed patterns in PANC02 pancreatic cancer, MC38 colon cancer, TNBC triple negative breast cancer tumor, and H1975 lung adenocarcinoma tumor models.

FIG. 5 shows the results of the detection of example 2 of the present invention¹⁸The manifestation of F-AIF-NOTA-NF12 in patients with lung adenocarcinoma, wherein A is the tumorThe tumor site.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention provides a PET molecular probe targeting PD-L1, a preparation method and application, and aims to solve the problem of providing a detection probe for early noninvasive screening of a PD-L1 patient.

Example 1

This example provides a PD-L1-targeted PET molecular probe, which has the structural formula as follows:

in this example 1The PET molecular probe targeting PD-L1 is named as⁶⁸Ga-NOTA-NF12。

As described above⁶⁸The specific preparation steps of Ga-NOTA-NF12 are as follows:

(1) adding 1ml of 0.25MNaOAc aqueous solution into an EP tube containing a labeled precursor compound NOTA-NF12(25-30 mu g), and uniformly mixing to obtain a NOTA-NF12 solution;

(2) the NOTA-NF12 solution was transferred to a 20mL reaction tube; using 4mL0.05MHCl to react⁶⁸Leaching Ga into a reaction tube, wherein the radioactivity is about 10-50mCi, and reacting for 10min at 90 ℃;

(3) adding 10mL of deionized water to quench the reaction to obtain a reaction system;

(4) c, passing the reaction system of the step (3) through₁₈Enrichment with Plus column and washing C with 10mL deionized water₁₈PlA us column;

(5) sequentially eluting the product into a bottle filled with a filter membrane product by using 1mL of ethanol and 10mL of normal saline to form a PET molecular probe targeting PD-L1⁶⁸Ga-NOTA-NF12 having a radioactivity of about 20-26 mCi.

Obtained PET molecular probe targeting PD-L1⁶⁸Ga-NOTA-NF12 is product injection and is applied to PD-L1 immune diagnosis and treatment kits.

As shown in figure 1, the obtained PET molecular probe targeting PD-L1 is prepared⁶⁸And (3) carrying out HPLC purity analysis on Ga-NOTA-NF12 product injection: mobile phase a was distilled water containing 0.1% TFA, mobile phase B was acetonitrile containing 0.1% TFA, and the column was ZORBAXSB-C18; the elution mode is gradient elution (0-2 mim: 5% acetonitrile; 2-15 min: 90% acetonitrile), the product peak time is about 7.3min, and the purity is 95% -99%.

Example 2

This example provides a PD-L1-targeted PET molecular probe, which has the structural formula as follows:

in this example 2The PET molecular probe targeting PD-L1 is named as¹⁸F-AIF-NOTA-NF12。

The above¹⁸The specific preparation steps of F-AIF-NOTA-NF12 are as follows:

1) produced by using Qian cyclotron of GE company¹⁸F (250-300mCi) and transmitted to the anion exchange column QMA (QMA ^ READI-CLING) through the pipeline^TMShaped column) with N₂Drying the QMA;

2) 300ug of AIF-NOTA-NF12,1.0mL of acetonitrile, 0.03mL of 0.4M A1C13 solution and 0.5mL of acetic acid buffer solution with pH 4.0 are added into a reaction tube in advance;

3)0.3mL of 0.9% NaCl solution¹⁸F leaching the reaction tube to obtainThe emission activity is 200-250mCi, the reaction is carried out for 10min at 110 ℃;

4) 5mL of deionized water was added and the product peak (80-120mci) was collected by HPLC purification;

5) product solution passing through C₁₈Enriching by a Plus column, then sequentially leaching with 2mL of ethanol and 10mL of normal saline, and passing the leaching solution through a sterile filtration membrane to obtain the PET molecular probe (a) (PET molecular probe) targeting PD-L1¹⁸F-A1F-NF12) with an activity of 50-60 mCi.

Obtained PET molecular probe targeting PD-L1¹⁸F-AIF-NOTA-NF12 is a final imaging agent injection and is applied to a PD-L1 immune diagnosis and treatment kit.

Preparing the obtained PET molecular probe targeting PD-L1¹⁸Performing HPLC purity analysis on the F-AIF-NOTA-NF12 product injection; the analysis conditions were: high performance liquid chromatography (ZORBAX SB-C18,4.6X250mm,5.0 μm) with a mobile phase of 20% acetonitrile at a flow rate of 2.0mL/min and a radiation detector. Using standard products¹⁹F-AIF-NOTA-NF12 was used as a reference, and the results are shown in FIG. 2.

Detection example 1: constructing and detecting an animal model:

1. respectively constructing subcutaneous melanoma B16 models of PANC02 pancreatic cancer, MC38 colon cancer, TNBC triple negative breast cancer tumor and H1975 lung adenocarcinoma tumor of about 0.5cm by taking 50 mice as model mice for later use;

2. preparation of radiolabel by the methods of example 1 and example 2 above⁶⁸Ga-NOTA-NF12 and¹⁸F-AIF-NOTA-NF12；

mouse tail intravenous injection of B16 tumor model⁶⁸Ga-NOTA-NF12 and¹⁸F-AIF-NOTA-NF12(0.1-0.2mCi) (results are shown in FIG. 3), and the remaining models were injected¹⁸F-AIF-NOTA-NF12(0.1-0.2mCi) (results are shown in FIG. 4);

carrying out PET/CT imaging on the small animals after 4.30 min;

5. data analysis to obtain SUV value of tumor part, wherein⁶⁸The tumor uptake SUV value of the Ga-NOTA-NF12 probe is 7.1 +/-1.3,¹⁸the tumor uptake SUV value of the F-AIF-NOTA-NF12 probe was 7.3. + -. 1.1.

Detection example 2: imaging methods and results for patients with lung adenocarcinoma

The examinee lies on the back on the PET/CT examination bed, and is injected intravenously¹⁸After F-AIF-NOTA-NFET 124.44MBq/kg, respectively performing PET/CT static scanning at 5min, 15min, 30min, 45 min, 60 min, 75 min and 90min, firstly performing CT scanning, wherein the scanning range and sequence are brain-pelvic, then immediately performing PET acquisition, the acquisition range is the same as CT, the acquisition sequence is pelvic-brain, the total number of 8 beds is 15.7cm, each bed is overlapped by 3.7cm, the PET acquisition method adopts a 3D-TOF sequence, the voltage of a CT scanning tube is 120kV, the automatic mAs, the pitch is 0.984:1, the matrix 512 is 512, the PET matrix 256 is 256, and the thickness of the CT and PET layers is 3.75 mm. The PET image reconstruction uses an ordered subset maximum expectation value iteration (OSEM) method to obtain a three-dimensional image and transverse, coronal and sagittal sectional images. The PET and CT images are transmitted to a GEAW4.6 post-processing workstation, frame-to-frame image para-fusion is carried out, radioactive uptake of regions of interest (ROI) is respectively outlined at the brain, the lung, the heart, the liver, the spleen, the pancreas, the renal parenchyma, the bladder, the muscle, the bone marrow and the like of healthy volunteers, the average value of radioactive counts in the ROI region is taken, namely an average SUV value, all ROIs are measured for 3 times, a Standard Uptake Value (SUV) is calculated, and the radioactive concentration (kBq/ml)/injection dose (MBq)/weight (kg) of a focus is obtained according to a formula SUV, and the method is completed by the AW4.6 post-processing workstation. The imaging result is shown in fig. 5, which shows that about 30min after the injection of the drug, the tumor lesion has higher residual curve, the SUV value is about 3.3, and the target ratio is higher, wherein A is the tumor site.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

1. the technical problem to be solved by the invention is to provide⁶⁸Ga or¹⁸F-AIF labeled polypeptide targeting PD-L1PET molecular probe and preparation method thereof, and realizes high specificity and high sensitivity imaging in a plurality of animal tumor models (B16 melanoma, PANC02 pancreatic cancer, MC38 colon cancer, TNBC triple negative breast cancer tumor, H1975 lung adenocarcinoma and the like).

2. The PET molecular probe targeting PD-L1 prepared by the invention is further verified in clinical human body level, can better carry out noninvasive living body imaging on the expression level of PD-L1 in a lung cancer patient with high expression of PD-L1, and is expected to provide a new imaging means for monitoring the PD-L1 expression level in the early noninvasive screening of the PD-L1 patient in the immunotherapy process.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.