CN116036318A - A SPECT molecular imaging probe targeting PD-L1 and its preparation method and application - Google Patents

Abstract

The invention relates to the fields of nuclear medicine molecular imaging and radiopharmaceuticals, in particular to a SPECT molecular imaging probe targeting PD-L1, and a preparation method and application thereof. ^99m Tc-KN035 is a single photon nuclide ^99m Molecular imaging probes of Tc-labeled single domain antibody Fc fusion protein KN035. Compared with a positron labeling and a molecular imaging probe based on an intact monoclonal antibody, the probe has the advantages of easily obtained raw materials, low cost, simple labeling method, mild reaction conditions and the like in the aspect of synthesis, and is convenient to popularize. In the aspect of in vivo imaging, the method has good pharmacokinetics and good PD-L1 targeting, and can noninvasively and dynamically detect the expression of PD-L1 on the surface of tumor cells through SPECT imaging, guide the formulation of immune blocking treatment schemes, monitor the treatment effect and the like.

Description

A SPECT molecular imaging probe targeting PD-L1 and its preparation method and application

technical field

The invention belongs to the field of nuclear medicine molecular imaging and radiopharmaceuticals, and in particular relates to a PD-L1-targeted SPECT molecular imaging probe and its preparation method and application.

Background technique

Programmed death receptor 1 and its ligand (PD-1/PD-L1) checkpoint inhibitors are one of the hot spots in tumor immunotherapy research in recent years, which can reactivate by blocking the PD-1/PD-L1 signaling pathway T lymphocytes, which enhance the body's immune killing ability against tumors, have achieved good curative effects in different types of tumors, and the curative effects are closely related to the expression of PD-L1. Studies have shown that the overall effective rate of PD-1/PD-L1 inhibitory therapy is 20-40%, and the effective rate is as high as 90% in PD-1/PD-L1 positive patients. Therefore, PD-L1 positivity is considered to be an effective biological target for PD-1/PD-L1 inhibitory therapy, and it has important clinical significance for the monitoring of tumor PD-L1 expression.

At present, the most commonly used method is to detect the expression level of tumor PD-L1 by invasive biopsy and immunohistochemistry (IHC), which is limited in clinical application, mainly because: (1) tumor PD-L1 expression There is temporal and spatial heterogeneity, that is, there are differences in expression in different tumor parts of the same patient, and there are dynamic changes during treatment. However, IHC based on biopsy or surgical specimens can only provide the expression of PD-L1 in the sample area, and cannot perform puncture evaluation on all lesions, and false negative results may occur. Studies have shown that about 10% of patients with negative PD-L1 expression by IHC have a good response to PD-1/PD-L1 inhibitor therapy. (2) IHC cannot repeatedly and dynamically detect the expression level of PD-L1 during tumor progression; (3) IHC examination is an invasive operation, which is difficult to implement in patients with multiple metastases and poor physical fitness; (4) Different IHC reagents The box detection antibody and platform are inconsistent, and the criteria for judging PD-L1 positivity are different. Nuclear medicine molecular imaging targeting PD-L1 can detect PD-L1 expression levels of systemic tumors (primary tumors, metastases, etc.) Treatment response and assessment of treatment efficacy.

Although a variety of nuclides such as ⁶⁴ Cu, ⁸⁹ Zr, ¹¹¹ In, ¹²⁴ I, ¹²⁵ I, ⁶⁸ Ga, ¹⁸ F and other labeled antibodies have been developed for PD-L1 imaging, there are still some shortcomings. For example, nuclide-labeled intact monoclonal antibodies have poor tumor penetration, long biological half-life, and long imaging time (usually 2-7 days) due to their large molecular weight (about 150 kDa). Moreover, complete monoclonal antibodies need to be labeled with nuclides with long half-lives, which increases unnecessary irradiation of normal and hyperperfused tissues. Although PET (Positron Emission Tomography) imaging has high sensitivity and resolution, the positron radionuclides required for PET imaging need to be produced by cyclotrons, and its sources are limited and expensive. The synthesis conditions are harsh. In addition, the nuclear medicine departments of many hospitals do not have PET and accelerator equipment, resulting in the inability of PET imaging to be widely carried out and popularized clinically. SPECT (Single Photon Emission Computed Tomography) imaging has the characteristics of a wide range of applications, far more installed machines than PET, and relatively low imaging costs. The radionuclide commonly used in SPECT imaging is ^99m Tc, which is produced by a ⁹⁹ Mo/ ^99m Tc generator. It is easy to obtain, low in cost, and the half-life of ^99m Tc and the energy of the radiation are moderate. or cause unnecessary exposure. The SPCECT imaging probe has the characteristics of simple preparation, mild reaction conditions and high yield. And with the upgrading of hardware, the advancement of SPECT equipment and the continuous improvement of reconstruction algorithms, SPECT can conduct quantitative research like PET, all of which are conducive to the clinical application of ^99m Tc-labeled SPCECT imaging probes. Therefore, ^{the 99m} Tc-labeled small molecular weight antibody probe targeting PD-L1 has very important social significance and potential economic value.

KN035 (Corning Jierui Biopharmaceuticals, Suzhou/Suzhou Kangning Jierui Biotechnology Co., Ltd.) is currently the world's first domestically developed and approved subcutaneous injection of PD-L1 single domain antibody Fc fusion protein. Its structure is shown in Figure 1 , with relatively small molecular weight (about 79.6 kDa), good solubility and stability, strong tumor penetration, high affinity with PD-L1 (K _D =3.0 nM), blocking the IC of PD-1 binding to PD-L1 ₅₀ is 5.25 nM. To this end, the present invention constructs a single-photon nuclide ^99m Tc-labeled PD-L1-targeting SPECT molecular imaging probe ^99m Tc-KN035 based on KN035.

Contents of the invention

In order to solve the technical problems existing in the prior art, the object of the present invention is to provide a SPECT molecular imaging probe targeting PD-L1 and its preparation method and application.

To this end, the present invention constructs a single-photon nuclide ^99m Tc-labeled SPECT molecular imaging probe ^99m Tc-KN035 targeting PD-L1 based on KN035.

The present invention provides a method for preparing a PD-L1-targeting SPECT molecular imaging probe ( ^99m Tc-labeled PD-L1-targeting SPECT molecular imaging probe ^99m Tc-KN035), the specific steps of which are as follows.

(1) Activation of Zeba desalting column

(1.1) Take a Zeba desalting column, remove the top cap and bottom plug of the Zeba desalting column, put it into a 1.5 mL EP tube, and centrifuge at 4°C, 1500×g for 1 min to remove the Zeba desalting column storage solution;

(1.2) Add 300 μL of 10×Modification Buffer to the Zeba desalting column in step 1.1, centrifuge at 4°C, 1500×g for 1 min, discard the buffer, and repeat 3 times to obtain 1 tube Activated Zeba desalting column; the 10×Modification Buffer buffer is a buffer containing 1.0 M phosphate, 1.5 M NaCl, pH 8.0;

(1.3) Take another Zeba desalting column, remove the top cap and bottom plug of the Zeba desalting column, put it into a 1.5 mL EP tube, and centrifuge at 4°C, 1500×g for 1 min to remove the Zeba desalting column storage solution;

(1.4) Add 300 μL of 10×Conjugating Buffer to the Zeba desalting column in step 1.3, centrifuge at 4°C, 1500×g for 1 min, discard the buffer, and repeat 3 times to obtain another 1 An activated Zeba desalting column; the 10× Conjugating Buffer buffer is a buffer containing 1.0 M phosphate, 1.5 M NaCl, pH 6.0.

(2) Purification of KN035

Take 2 uL of KN035 with a concentration of 200 μg/μL, dilute it with ultrapure water to a volume of 70 uL, add it to the activated Zeba desalting column in step 1.2, and centrifuge at 4°C and 1500×g in a centrifuge 2 min, the purified KN035 was obtained.

(3) Preparation of ^99m Tc-KN035

(3.1) Weigh 0.2 mg of chelating agent SHNH powder and dissolve it in 20 uL of anhydrous DMSO to prepare a SHNH solution with a concentration of 10 μg/μL;

(3.2) Take 10 uL of SHNH solution with a concentration of 10 μg/μL and mix it with the purified KN035, shake at 4°C and react in the dark for 12 h;

(3.3) Add the reactant in the above step 3.2 to the activated Zeba desalting column in the step 1.4, centrifuge at 4°C, 1500×g for 2 min to remove excess SHNH, and obtain the reaction product of SHNH and KN035 Product HYNIC-KN035;

(3.4) Mix the above HYNIC-KN035 with 50 μL Tricine solution with a concentration of 200 mg/mL, 2 μL SnCl ₂ solution with a concentration of 14 mg/mL, and 600 μL Na ^99m TcO ₄ solution with a radioactivity of 25 mCi , 37°C shaking and dark reaction for 30 min;

(3.5) Take the PD-10 desalting column, remove the top cap and bottom plug, and remove the storage solution;

(3.6) Add 5 mL of PBS buffer to the PD-10 desalting column, and after the PBS buffer has run out, add 5 mL of PBS buffer again to the PD-10 desalting column to make the PBS buffer run out; Repeat the operation 4 times to obtain an activated PD-10 desalting column;

(3.7) Add the reactants in step 3.4 to the activated PD-10 desalting column, add PBS buffer to make up to a total volume of 2.5 mL, and connect the eluent with a 1.5 mL EP tube, each EP tube Take about 0.5 mL;

(3.8) After no liquid flows out of the PD-10 desalting column in the above step 3.7, add 0.5 mL of PBS buffer for rinsing, and at the same time connect the eluent with 1.5 mL EP tubes, each EP tube is connected with 0.5 mL; repeat the operation 15 times;

(3.9) Use a medical activity meter to measure the radioactivity of the eluate from each EP tube, and the one with the largest count is ^99m Tc-KN035.

^99m Tc is cheap, easy to obtain ( ⁹⁹ Mo- ^99m Tc generator), moderate half-life and ray energy, and avoids unnecessary exposure to the examiner while obtaining high-quality imaging images. KN035 is easy to obtain, relatively small molecular weight, good solubility and stability, strong tumor penetration, and high affinity with PD-L1. ^99m Tc-labeled KN035 is easy to prepare, with mild reaction conditions, high yield, good probe stability, high affinity with PD-L1, and strong targeting. And with the upgrading of hardware, the progress of SPECT equipment and the continuous improvement of reconstruction algorithms, SPECT can conduct quantitative research like PET, all of which are conducive to the promotion and application of ^99m Tc-KN035 SPECT imaging in the future. Preclinical validation was performed by SPECT/CT animal imaging in vivo. The probe shows good tumor specificity, which is helpful for screening immunotherapy advantage populations, making treatment individualized and optimized, and finally realizing clinical transformation.

Compared with the prior art, the present invention has the following beneficial effects:

1. ^{The 99m} Tc-KN035 probe synthesized by the present invention has easy-to-obtain synthetic raw materials, and KN035 has been commercialized and is easy to purchase; ^99m Tc is produced by a ^99Mo - ^99m Tc generator, which is cheap and easy to obtain. Available. Compared with positron labeling and molecular imaging probes based on complete monoclonal antibodies, the probe is simple to prepare, has mild reaction conditions, low cost and high yield, and can be prepared at any level of nuclear medicine. In addition, the high penetration rate of SPECT equipment is conducive to the promotion and application of this probe;

2. KN035 in the ^99m Tc-KN035 probe synthesized by the present invention belongs to the single-domain antibody Fc fusion protein, and its molecular weight is significantly lower than that of the complete monoclonal antibody. Diagnosable images can be obtained, and the imaging effect is good in 8–18 hours, and can still be clearly developed after 24 hours. However, the existing PD-L1 probes based on intact monoclonal antibodies often take several days after injection to obtain clear diagnostic images.

Description of drawings

Figure 1 shows the sequence and structure of KN035.

Figure 2 is a schematic diagram of the synthesis process and imaging of ^99m Tc-KN035.

Figure 3 shows the identification and stability of ^99m Tc-KN035. Among them, A is ^99m Tc-KN035 purified and then used PBS as a developing agent, B is ^99m Tc-KN035 purified and then used methanol and 1M ammonium acetate at a volume ratio of 1:1 as a developing agent, C is ^99m Tc-KN035 in PBS and Stability at different time points in FBS.

Figure 4 shows the detection of PD-L1 on the surface of H1975 and A549 cells. Among them, A is the fluorescence of immune cells, the red fluorescence indicates the expression of PD-L1 on the cell surface, and the blue fluorescence is the nucleus; B is the expression of PD-L1 in H1975 and A549 cells detected by Western blot.

Fig. 5 is the in vitro cell experiment of ^99m Tc-KN035. Among them, A is the saturation experiment of ^99m Tc-KN035 in H1975 cells, B is the uptake experiment of H1975 and A549 cells, and C is the blocking experiment of ^99m Tc-KN035 in H1975 cells. * p <0.05, ** p <0.01, *** p <0.001, ns, p >0.05.

Figure 6 is the SPECT/CT images at different time points after tail vein injection of ^99m Tc-KN035 probe in tumor-bearing mice H1975, A549 and H1975 blocking groups, and the arrows indicate the tumor sites.

Fig. 7 is a histogram of biodistribution and tumor uptake of H1975, A549 and H1975 blocking groups in tumor-bearing mice. Among them, A is the H1975 tumor-bearing mice; B is the A549 tumor-bearing mice; C is the blocking group of the H1975 tumor-bearing mice; D is the tumor uptake of different tumor-bearing mice; E is the ratio of tumor to muscle in different tumor-bearing mice. * p <0.05, ** p <0.01, ns, p >0.05.

Detailed ways

In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described The embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

The preparation method of the single-photon nuclide ^99m Tc-labeled PD-L1-targeting SPECT molecular imaging probe ^99m Tc-KN035 constructed based on KN035, the specific steps are as follows.

(1) Activation of Zeba desalting column

(1.1) Take a Zeba desalting column, remove the top cap and bottom plug of the Zeba desalting column, put it into a 1.5 mL EP tube, and centrifuge at 4°C, 1500×g for 1 min to remove the Zeba desalting column storage solution;

(1.2) Add 300 μL of 10×Modification Buffer (1.0 M phosphate, 1.5 MNaCl, pH 8.0) to the Zeba desalting column in step 1.1, centrifuge at 4°C, 1500×g for 1 min in a centrifuge, discard Remove the buffer and repeat 3 times to get an activated Zeba desalting column;

(1.3) Take another Zeba desalting column, remove the top cap and bottom plug of the Zeba desalting column, put it into a 1.5 mL EP tube, and centrifuge at 4°C, 1500×g for 1 min to remove the Zeba desalting column storage solution;

(1.4) Add 300 μL of 10×Conjugating Buffer (1.0 M phosphate, 1.5 MNaCl, pH 6.0) to the Zeba desalting column in step 1.3, centrifuge at 4°C, 1500×g for 1 min in a centrifuge, discard Remove the buffer and repeat 3 times to get another activated Zeba desalting column.

(2) Purification of KN035

Take 2 uL of KN035 with a concentration of 200 μg/μL, dilute it with ultrapure water to a volume of 70 uL, add it to the activated Zeba desalting column in step 1.2, and centrifuge at 4°C and 1500×g in a centrifuge 2 min, the purified KN035 was obtained.

(3) Preparation of ^99m Tc-KN035

(3.1) Weigh 0.2 mg of chelating agent SHNH (6-hydrazinonicotinic acid succinimidyl hydrochloride) powder and dissolve it in 20 uL of anhydrous DMSO (dimethyl sulfoxide) to prepare a concentration of 10 μg/ μL of SHNH solution;

(3.2) Mix 10 uL of SHNH solution with a concentration of 10 μg/μL with the purified KN035, shake at 4°C and react in the dark for 12 h;

(3.3) Add the reactants in the above step 3.2 to the activated Zeba desalting column in the above step 1.4, and use a centrifuge at 4°C, 1500×g for 2 minutes to remove excess SHNH to obtain the reaction product of SHNH and KN035 Product HYNIC-KN035;

(3.4) Mix the above-mentioned HYNIC-KN035 with 50 μL of Tricine (N-tris(hydroxymethyl)methylglycine) solution with a concentration of 200 mg/mL, 2 μL of SnCl ₂ (stannous chloride) with a concentration of 14 mg/mL ) solution and 600 μL of Na ^99m TcO ₄ solution with a radioactivity of 25 mCi were mixed evenly, and reacted in the dark at 37°C for 30 min;

(3.5) Take the PD-10 desalting column, remove the top cap and bottom plug, and remove the storage solution;

(3.6) Add 5 mL of PBS buffer to the PD-10 desalting column, and after the PBS buffer has run out, add 5 mL of PBS buffer again to the PD-10 desalting column to make the PBS buffer run out; Repeat the operation 4 times to obtain an activated PD-10 desalting column;

(3.7) Add the reactant in step 3.4 to the activated PD-10 desalting column, add PBS buffer to make up to a total volume of 2.5 mL, and connect the eluent with a 1.5 mL EP tube, each EP tube Take about 0.5 mL;

(3.8) After no liquid flows out of the PD-10 desalting column in the above step 3.7, add 0.5 mL of PBS buffer for rinsing, and at the same time connect the eluent with 1.5 mL EP tubes, each EP tube is connected with 0.5 mL; repeat the operation 15 times;

(3.9) Use a medical activity meter to measure the radioactivity of the eluate from each EP tube, and the one with the largest count is ^99m Tc-KN035.

Example 2

Detection of labeling rate, specific activity, radiochemical purity and stability of ^99m Tc-KN035.

(1) The labeling rate of ^99m Tc-KN035 was 75.87 ± 4.60 %, and the specific activity was 2.68 MBq/μg.

(2) Radiochemical purity: iTLC-SG chromatographic paper was used to determine the radiochemical purity of the marker. Take 4 μL ^{of 99m} Tc-KN035 and spot 1cm from the edge of the chromatography paper, use PBS, methanol and 1M ammonium acetate (volume ratio 1:1) as developing solvents respectively, the results are as shown in Figure 3. A is used as PBS after purification. Developing agent, B in Fig. 3 is that methanol and 1M ammonium acetate are used as the developing agent in a volume ratio of 1:1 after purification, showing that there is no free ^99m Tc and ^99m Tc-colloid in the purified product, and the radiochemical purity is 99.40 ± 0.11%.

(3) Stability: The purified ^99m Tc-KN035 was mixed with PBS and fetal bovine serum (FBS) at a volume ratio of 1:1, and incubated at 37°C. The radiochemical purity was measured at 2 h, 4 h, 6 h, 12 h, and 24 h, and each time point was repeated 3 times. The results showed that the 24 h radiochemical purity was greater than 95% in both PBS and FBS (shown as C in Figure 3).

Example 3

Probe ^99m Tc-KN035 affinity and in vitro targeting verification.

(1) Expression of PD-L1 on the surface of tumor cells

The expression of PD-L1 in cell lines H1975 and A549 was analyzed by immunofluorescence and immunohistochemistry. The results are shown in Figure 4. Cell immunofluorescence showed that H1975 cells had strong fluorescence, while A549 cells had weak fluorescence. In cell immunohistochemical experiments, semi-quantitative analysis was performed by the ratio of the gray value of PD-L1 to the internal reference β-action band. The results showed that the ratio of H1975 cells was significantly higher than that of A549 cells (11.30% vs. 96.26%). Therefore, H1975 cells highly express PD-L1, and A549 cells express low PD-L1.

(2) Cell saturation experiment

(2.1) Inoculate the H1975 cell line with high expression of PD-L1 and the A549 cell line with low expression of PD-L1 in a 24-well plate, inoculate 1.5×10 ⁵ cells per well, and inoculate at 37°C, 5% CO ₂ Cultured in an incubator until the cells adhered to the wall. Discard the old medium, wash 2 times with PBS, and perform the following experimental operations;

(2.2) Set up 5 groups, 4 duplicate wells/group, add serum-free RPMI 1640 medium containing different doses of ^99m Tc-KN035 (0.37-185kBq) to each well in each group, and incubate at 37°C and 5% CO ₂ Incubate in medium for 1 hour;

(2.3) Collect the supernatant and cells separately: collect the cell culture medium, wash the cells twice with PBS as the supernatant; digest with 1 N NaOH to obtain the cell suspension, and wash the cells twice with PBS as the cell suspension;

(2.4) Measure the supernatant and cell suspension counts with a gamma counter, and calculate cell saturation and specific uptake rates.

The results are shown in A in Figure 5. As the concentration of ^99m Tc-KN035 probe increased, the cellular uptake increased and reached saturation at 3.7 kBq/well, with a K _D value of 31.04 nM.

(3) Cell binding experiment

(3.1) Inoculate the H1975 cell line with high expression of PD-L1 and the A549 cell line with low expression of PD-L1 in a 24-well plate, inoculate 1.5×10 ⁵ cells per well, and incubate at 37°C and 5% CO ₂ Cultured in an incubator until the cells adhered to the wall. Discard the old medium, wash 2 times with PBS, and perform the following experimental operations;

(3.2) In the cell binding experiment, add 3.7 kBq ^99m Tc-KN035 to each well, incubate at 37°C, 5% CO ₂ incubator for 0.5 h, 1 h, 2 h, 4 h, 6 h and 8 h, then collect For the supernatant and cells, use a gamma counter to measure the counts of the supernatant and cell suspension, and calculate the cell uptake rate.

Results As shown in Figure 5 B and Table 1, the uptake rate of ^99m Tc-KN035 in H1975 cells with high expression of PD-L1 increased with time and reached a plateau at 6 h. Compared with A549 cells with low expression of PD-L1, the uptake rate of H1975 cells at each time point was significantly higher than that of A549 cells, and the difference was statistically significant (* p <0.05, ** p <0.01, *** p <0.001 ).

Table 1. Cell uptake rates of H1975 (high expression of PD-L1) and A549 (low expression of PD-L1) cell lines at different time points (%, n = 4).

(4) Cell blocking experiment

(4.1) Inoculate the H1975 cell line that highly expresses PD-L1 in a 24-well plate, inoculate 1.5×10 ⁵ cells per well, and culture in a 37°C, 5% CO ₂ incubator until the cells adhere to the wall. Discard the old medium, wash 2 times with PBS, and perform the following experimental operations;

(4.2) Set up 5 groups, which are control group (without unlabeled KN035); different blocking groups, namely 25 times, 50 times, 100 times and 500 times of unlabeled KN035. The above five groups of cells were pretreated for 1 hour, then 3.7 kBq of ^99m Tc-KN035 was added, and the supernatant and cells were collected after incubation for 1 hour. The supernatant and cell suspension were counted with a γ counter, and the cell uptake rate was calculated.

The results are shown in C in Fig. 5 and Table 2, ^99m Tc-KN035 can be specifically blocked by 25-fold unlabeled KN035 (** p <0.01). There was no difference in the blocking effect of 100-fold and 500-fold unlabeled KN035 ( p > 0.05). The cell uptake of the 100-fold unlabeled KN035 blocking group decreased by about 97.03% compared with the control group. The above experimental results indicated that ^99m Tc-KN035 was specifically uptake in PD-L1 positive cells.

Table 2. Uptake rate of H1975 cell line at different blocking folds (%, n=4).

Example 4

In vivo targeting verification of probe ^99m Tc-KN035.

(1) Establish a tumor-bearing mouse model

Suspend 1×10 ⁶ H1975 and A549 cells in 150 uL of PBS, respectively, and inoculate them into the armpits of 5-week-old female nude mice by subcutaneous injection, and perform SPECT/CT when the tumor volume grows to 0.6-0.8 cm Imaging and biodistribution.

(2) SPECT/CT imaging of tumor-bearing mice

(2.1) H1975 and A549 tumor-bearing mice were respectively injected with ^99m Tc-KN035 with a volume of 150 μL and a radioactive dose of 29.6-37MBq through the tail vein. In the H1975 blocking group, 100 times unlabeled KN035 was injected into the tail vein 1 hour in advance, and then ^99m Tc-KN035 with a volume of 150 μL and a radioactive dose of 29.6-37 MBq was injected into the tail vein;

(2.2) Small animal SPECT/CT imaging (Yongxin small animal PET/SPECT/CT multimodal imaging system) was performed at 4 h, 8 h, 12 h, 18 h and 24 h after injection; 1.5% isoflurane Anesthesia was induced by alkane-oxygen, anesthesia was maintained by 1.0% isoflurane-oxygen; SPECT parameters: acquisition time, 10 seconds/frame for 4 and 8 hours, 12 seconds/frame for 12 and 18 hours, 15 seconds/frame for 24 hours ; Matrix 256×512; CT parameters: tube voltage 45 kVp, current 0.15 mA, exposure time 300 ms/frame;

(2.3) After imaging, the data was processed on the post-processing workstation (NMSoft-AIWS Version 1.7) to obtain SPECT/CT images.

The results are shown in Figure 6. The arrow indicates the tumor site. The H1975 tumor with high expression of PD-L1 was clearly visualized at 4 hours, and then the imaging agent at the tumor site was further concentrated, and the radioactivity of other tissues and organs gradually decreased. At 24 hours, the tumor Still clearly visible. However, the tumors in A549 tumor-bearing mice and H1975 blocking group were light at each time point. It shows that the probe can be specifically aggregated in tumors with high expression of PD-L1, and has excellent targeting.

(3) Biodistribution of tumor-bearing mice

(3.1) ^99m Tc-KN035 with a volume of 150 μL and a radioactive dose of 29.6-37 MBq was injected into the tail vein of H1975 and A549 tumor-bearing mice. In the H1975 blocking group, 100-fold unlabeled KN035 was injected into the tail vein 1 hour in advance, and then ^99m Tc-KN035 with a volume of 150 μL and a radioactive dose of 29.6-37 MBq was injected into the tail vein;

(3.2) The mice were sacrificed at 4 h, 12 h and 24 h after injection, and the mice in the blocking group were sacrificed at 24 h after injection. Take the tissues of interest, such as blood, brain, heart, lung, liver, spleen, kidney, pancreas, stomach, small intestine, large intestine, muscle, bone, tumor, rinse with ultrapure water, dry and weigh;

(3.3) The gamma counter measures the radioactive count, and calculates the percent injected dose ratio per gram of tissue (%ID/g) and tumor/muscle ratio after radioactive attenuation correction.

The results are shown in Figure 7 and Table 3. The H1975 tumor uptake was as high as 9.68 ± 0.91%ID/g, 11.97 ± 2.05%ID/g and 13.31 ± 2.23%ID/g at 4 hours, 12 hours and 24 hours after injection, respectively. Significantly higher than the uptake of A549 tumor at the corresponding time point, respectively 4.59 ± 0.76 %ID/g, 5.43 ± 0.58 %ID/g and 5.54 ± 0.28 %ID/g (** p <0.01); H1975 tumor blocking group 24h Tumor uptake was significantly lower than in the non-blocking group (5.64 ± 1.11% ID/g vs 13.31 ± 2.23% ID/g, ** p < 0.01). There is a good agreement with the SPECT imaging results. The tumor-to-muscle ratios showed that H1975 tumors had excellent target-to-body ratios. The H1975 tumor-to-muscle ratios were 6.38±1.54, 7.11±0.66, and 8.85±0.20 at 4 h, 12 h, and 24 h after injection, which were significantly higher than those of H1975. The ratio of A549 tumor to muscle in the cut-off group and the corresponding time point, and the difference was statistically significant (* p <0.05).

Table 3. Biodistribution of ^99m Tc-KN035 in tumor-bearing mice (%ID/g ± SD, n = 4).

The SPECT molecular imaging probe ^99m Tc-KN035 of the present invention has a simple synthesis method, mild reaction conditions, high yield, low cost, high affinity between the probe and PD-L1, and can be carried out in any level of nuclear medicine. The excellent affinity of the probe to PD-L1 and its small single-domain antibody Fc fusion protein structure make it have good in vivo pharmacokinetics in vivo. The tumor was clearly visualized 4 hours after probe injection, and the development was further enhanced over time. , the tumor is still clearly visible at 24 hours, and the target/object ratio is high, which is conducive to the detection of tumor metastases with high expression of PD-L1.

The above are only preferred embodiments of the present invention, and do not limit the present invention in any way. All simple modifications, changes and equivalent transformations made to the above embodiments according to the technical essence of the present invention still belong to the technical solution of the present invention. scope of protection.

Claims (2)

1. The SPECT molecular imaging probe for targeting PD-L1 is characterized in that the probe is a single photon nuclide ^99m Tc marked KN035 ^99m Tc-KN035。

2. Preparation method of PD-L1 targeted SPECT molecular imaging probeThe probe is a single photon nuclide ^99m Tc marked KN035 ^99m Tc-KN035, comprising the steps of:

(1) Activation of Zeba desalting columns

(1.1) taking 1 Zeba desalting column, removing top cap and bottom plug of the Zeba desalting column, placing into an EP tube with specification of 1.5 ml, centrifuging at 4deg.C and 1500 Xg for 1min by using a centrifuge to remove the storage liquid in the Zeba desalting column;

(1.2) adding 300. Mu.l of 10X Modification Buffer buffer to the Zeba desalting column in the step 1.1, centrifuging at 4 ℃ for 1min with a centrifuge at 1500 Xg, discarding the buffer, and repeating 3 times to obtain 1 activated Zeba desalting column; the 10X Modification Buffer buffer is a buffer containing 1.0M phosphate, 1.5M NaCl and having a pH of 8.0;

(1.3) taking another 1 Zeba desalting column, removing top caps and bottom plugs of the Zeba desalting column, putting the Zeba desalting column into an EP pipe with the specification of 1.5 ml, and centrifuging at 4 ℃ for 1min by using a centrifuge at 1500 Xg to remove the storage liquid in the Zeba desalting column;

(1.4) adding 300. Mu.l of 10X Conjugating Buffer buffer to the Zeba desalting column in the step 1.3, centrifuging at 4 ℃ for 1min with a centrifuge at 1500 Xg, discarding the buffer, and repeating 3 times to obtain another 1 activated Zeba desalting column; the 10X Conjugating Buffer buffer is a buffer containing 1.0M phosphate, 1.5M NaCl and having a pH of 6.0;

(2) Purification of KN035

Diluting KN035 with concentration of 2 uL being 200 μg/μl with ultrapure water to volume of 70 uL, adding into the activated Zeba desalting column in step 1.2, centrifuging at 4deg.C and 1500×g for 2 min with a centrifuge to obtain purified KN035;

（3） ^99m preparation of Tc-KN035

(3.1) weighing the chelating agent SHNH powder of 0.2 mg, dissolving in anhydrous DMSO of 20 uL, and preparing into SHNH solution with the concentration of 10 mug/mu L;

(3.2) taking 10 uL of SHNH solution with the concentration of 10 mug/mu L, uniformly mixing the solution with the purified KN035, and vibrating and light-shielding reaction at the temperature of 4 ℃ for 12 h;

(3.3) adding the reactant in the step 3.2 into the activated Zeba desalting column in the step 1.4, and centrifuging at 4 ℃ and 1500 Xg for 2 min by using a centrifuge to remove excessive SHNH, so as to obtain a product HYNIC-KN035 after the reaction of SHNH and KN035;

(3.4) mixing the above HYNIC-KN035 with 50. Mu.L Tricine solution with a concentration of 200 mg/mL and 2. Mu.L SnCl with a concentration of 14 mg/mL ₂ Solution and 600. Mu.L of Na with a radioactivity of 25 mCi ^99m TcO ₄ Mixing the solution uniformly, and vibrating and light-shielding for reaction for 30 min at 37 ℃;

(3.5) taking a PD-10 desalting column, removing a top cap and a bottom plug, and removing storage liquid;

(3.6) adding 5 mL PBS buffer to the PD-10 desalting column, and after the PBS buffer is completely drained, adding 5 mL PBS buffer to the PD-10 desalting column again to completely drain the PBS buffer; repeating the operation for 4 times to obtain an activated PD-10 desalting column;

(3.7) adding the reactant of step 3.4 to the activated PD-10 desalting column, adding PBS buffer to make up to a total volume of 2.5 mL, and simultaneously bridging the eluent with an EP of 1.5 mL, each EP bridging about 0.5 mL;

(3.8) after no liquid flows out of the PD-10 desalting column in the step 3.7, adding 0.5 mL PBS buffer solution for leaching, and simultaneously connecting an eluent with the specification of 1.5 mL by using an EP pipe, wherein each EP pipe is connected with 0.5 mL; repeating the operation 15 times;

(3.9) measuring the radioactivity of the eluent in each EP tube by using a medical activity meter, wherein the maximum counting is ^99m Tc-KN035。

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