CN116036318A

CN116036318A - PD-L1-targeted SPECT molecular imaging probe and preparation method and application thereof

Info

Publication number: CN116036318A
Application number: CN202211576037.XA
Authority: CN
Inventors: 张影影; 杨辉; 李文亮; 曲金荣
Original assignee: Henan Cancer Hospital
Current assignee: Henan Cancer Hospital
Priority date: 2022-12-08
Filing date: 2022-12-08
Publication date: 2023-05-02

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

PD-L1-targeted SPECT molecular imaging probe and preparation method and application thereof

Technical Field

The invention belongs to the fields of nuclear medicine molecular imaging and radiopharmaceuticals, and particularly relates to a SPECT molecular imaging probe targeting PD-L1, and a preparation method and application thereof.

Background

Programmed death receptor 1 and ligand (PD-1/PD-L1) checkpoint inhibitors are one of the hot spots of recent tumor immunotherapy research, and enhance the immune killing capacity of an organism on tumors by blocking a PD-1/PD-L1 signal path to reactivate T lymphocytes, have obtained good curative effects in different types of tumors, and the curative effects are closely related to PD-L1 expression. Studies show that the overall effective rate of PD-1/PD-L1 inhibition therapy is 20-40%, and the effective rate in PD-1/PD-L1 positive patients is as high as 90%. Therefore, PD-L1 positive is considered as a biological target effective in PD-1/PD-L1 inhibition treatment, and has important clinical significance for monitoring tumor PD-L1 expression conditions.

The most commonly used method at present is to detect the expression level of tumor PD-L1 by invasive puncture biopsy pathology Immunohistochemistry (IHC), which is limited in clinical application mainly because: (1) Tumor PD-L1 expression is temporally and spatially heterogeneous, i.e., there is a difference in expression of different tumor portions of the same patient, and there is dynamic variation during treatment. IHC based on biopsy or surgical specimens can only provide PD-L1 expression conditions of a sampling area, puncture evaluation cannot be carried out on all focuses, and false negative results can occur. Studies have shown that IHC tests for patients negative for PD-L1 expression respond well to PD-1/PD-L1 inhibitor treatment by about 10%. (2) IHC can not repeatedly and dynamically detect the PD-L1 expression level in the tumor progress process; (3) IHC examination belongs to invasive operation, and is difficult to be implemented in patients with multiple metastasis and poor constitution; (4) Different IHC kits detect inconsistent antibodies and platforms, and different PD-L1 positive judgment standards. The nuclear medicine molecular imaging of the targeted PD-L1 can carry out in-vivo detection of the expression level of the PD-L1 of the systemic tumor (primary tumor, metastasis and the like) in a noninvasive and real-time manner, so that patients suitable for immunotherapy are screened, treatment schemes are optimized, treatment response is monitored, and treatment effect is estimated.

Although various nuclides such as ⁶⁴ Cu， ⁸⁹ Zr、 ¹¹¹ In、 ¹²⁴ I、 ¹²⁵ I、 ⁶⁸ Ga、 ¹⁸ F and the like are used for PD-L1 imaging, but have some disadvantages. Such as nuclide-labeled intact monoclonal antibodies, due to molecular weightThe large (about 150 kDa) results in poor tumor penetration, long biological half-life, long imaging time (typically 2-7 days). And the intact monoclonal antibodies require long half-life nuclides for labeling, increasing unnecessary irradiation of normal and high blood flow perfused tissue. PET (positron emission computed tomography) imaging has higher sensitivity and resolution, but positron radionuclides required for PET imaging require cyclotron production, have limited sources, are expensive, and have strict requirements on PET probe synthesis conditions. Furthermore, many hospital nuclear medicine departments do not have PET and accelerator equipment, resulting in the inability of PET imaging to be widely developed and popularized in clinical settings. And SPECT (single photon emission computed tomography) imaging has the characteristics of wide application range, much more installed quantity than PET and relatively lower imaging cost. The common radionuclides for SPECT imaging are ^99m Tc, which is composed of ⁹⁹ Mo/ ^99m The Tc generator is convenient to obtain, low in cost and ^99m the Tc half-life and the radiation energy are moderate, and unnecessary irradiation to an inspector is avoided while a high-quality developed image is obtained. The SPCECT imaging probe has the characteristics of simple preparation, mild reaction conditions and high yield. With the upgrade of hardware, the SPECT equipment is advanced and the reconstruction algorithm is continuously improved, so that the SPECT can perform quantitative research like PET, and the method is beneficial to ^99m Clinical popularization and application of Tc-marked SPCECT imaging probes. Thus (2) ^99m Tc-labeled PD-L1-targeted small molecular weight antibody probes have very important social significance and potential economic value.

KN035 (Kangning JieJieJie Biotechnology Co., ltd.) is a subcutaneously injected PD-L1 single domain antibody Fc fusion protein which has been developed and approved for marketing for the first time worldwide and has a structure as shown in FIG. 1, a relatively small molecular weight (about 79.6 kDa), good solubility and stability, a strong tumor penetrating power, and a high affinity with PD-L1 (K) _D =3.0 nM), IC blocking PD-1 binding to PD-L1 ₅₀ 5.25 and nM. For this purpose, the invention constructs a single photon nuclide based on KN035 ^99m Tc-marked SPECT molecular imaging probe targeting PD-L1 ^99m Tc-KN035。

Disclosure of Invention

Aiming at solving the technical problems in the prior art, the invention aims to provide a SPECT molecular imaging probe targeting PD-L1, and a preparation method and application thereof.

For this purpose, the invention constructs a single photon nuclide based on KN035 ^99m Tc-marked SPECT molecular imaging probe targeting PD-L1 ^99m Tc- KN035。

The invention provides a SPECT molecular imaging probe targeting PD-L1 ^99m Tc-marked PD-L1 targeted SPECT molecular imaging probe ^99m Tc-KN 035), the specific steps are as follows.

(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 by using a centrifuge at 1500 Xg, discarding the buffer, and repeating for 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 by using a centrifuge at 1500 Xg, discarding the buffer, and repeating for 3 times to obtain another 1 activated Zeba desalting column; the 10X Conjugating Buffer buffer was a buffer containing 1.0M phosphate, 1.5M NaCl, pH 6.0.

(2) Purification of KN035

The purified KN035 was obtained by diluting KN035 having a concentration of 200. Mu.g/. Mu.L of 2 uL with ultrapure water to a volume of 70 uL, adding to the activated Zeba desalting column of the above step 1.2, and centrifuging at 4℃with 1500 Xg for 2 minutes with a centrifuge.

（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。

^99m Tc is low in price and easy to obtain ⁹⁹ Mo- ^99m Tc generator), half-life and radiation energy are moderate, and high quality display is obtainedThe image is imaged while avoiding unnecessary illumination of the inspector. KN035 has the advantages of convenient acquisition, relatively small molecular weight, good solubility and stability, strong tumor penetrating power and high affinity with PD-L1. ^99m Tc marked KN035 has simple preparation, mild reaction condition, high yield, good probe stability, high affinity with PD-L1 and strong targeting property. With the upgrade of hardware, the SPECT equipment is advanced and the reconstruction algorithm is continuously improved, so that the SPECT can perform quantitative research like PET, and the quantitative research is beneficial to the future ^99m Tc-KN035 SPECT imaging is clinically popularized and applied. Preclinical validation was performed by SPECT/CT in vivo animal imaging. The probe shows good tumor specificity, is beneficial to screening the population with the dominant immunotherapy, individuates and optimizes the therapy, and finally realizes clinical transformation.

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

1. synthesized according to the invention ^99m Tc-KN035 probe, the synthetic material is easy to obtain, KN035 is commercialized and is easy to purchase; ^99m tc is composed of ⁹⁹ Mo- ^99m The Tc generator is produced, the price is low, the acquisition is convenient, and any hospital nuclear medicine department can be equipped. Compared with positron labeling and a molecular imaging probe based on an intact monoclonal antibody, the probe has the advantages of simple preparation, mild reaction conditions, low cost and high yield, and can be prepared in any level of nuclear medicine department. In addition, the SPECT equipment has high popularization rate, and is favorable for popularization and application of the probe;

2. synthesized according to the invention ^99m KN035 in Tc-KN035 probe belongs to single domain antibody Fc fusion protein, the molecular weight is obviously reduced compared with the complete monoclonal antibody, the probe has good pharmacokinetics in vivo, the diagnosable image can be obtained by 4h after injection, the 8-18 h imaging effect is good, and 24h can still be clearly developed. Whereas prior art PD-L1 probes based on intact monoclonal antibodies often do not obtain clear diagnostic images until days after injection.

Drawings

FIG. 1 shows the sequence and structure of KN035.

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

FIG. 3 is a schematic view of ^99m Identification and stability of Tc-KN 035. Wherein A is ^99m Purifying Tc-KN035, using PBS as developing agent, B is ^99m Purifying Tc-KN035, using methanol and 1M ammonium acetate with volume ratio of 1:1 as developing agent, C is ^99m Stability of Tc-KN035 in PBS and FBS at different time points.

FIG. 4 shows the detection of PD-L1 on the surface of H1975 and A549 cells. Wherein A is immune cell fluorescence, red fluorescence represents the expression of PD-L1 on the cell surface, and blue fluorescence is cell nucleus; b is Western blot to detect the expression of PD-L1 in H1975 and A549 cells.

FIG. 5 is a schematic view of a display ^99m In vitro cell experiments with Tc-KN 035. Wherein A is ^99m Saturation experiment of Tc-KN035 in H1975 cell, B is H1975 and A549 cell uptake experiment, C is ^99m Blocking experiments of Tc-KN035 in H1975 cells. *p＜0.05, **p＜0.01, ***p＜0.001, ns, p＞0.05。

FIG. 6 shows the tail intravenous injection of tumor-bearing mice H1975, A549 and H1975 blocking group ^99m SPECT/CT imaging at different time points after Tc-KN035 probe, the arrow shows tumor site.

FIG. 7 is a bar graph of tumor uptake in tumor-bearing mice H1975, A549, and H1975 blocking group biodistribution. Wherein A is H1975 tumor-bearing mice; b is A549 tumor-bearing mice; c is H1975 tumor-bearing mouse blocking group; d is tumor uptake in different tumor-bearing mice; e is the tumor muscle ratio of different tumor-bearing mice. *p＜0.05, **p＜0.01, ns, p＞0.05。

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more clear, 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 apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.

Example 1

Single photon nuclide constructed based on KN035 ^99m Tc-marked SPECT molecular imaging probe targeting PD-L1 ^99m The preparation method of Tc-KN035 comprises the following specific steps.

(1) Activation of Zeba desalting columns

(1.2) adding 300. Mu.L of 10X Modification Buffer buffer (1.0M phosphate, 1.5M NaCl, pH 8.0) 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 for 3 times to obtain 1 activated Zeba desalting column;

(1.4) 300. Mu.L of 10X Conjugating Buffer buffer (1.0M phosphate, 1.5M NaCl, pH 6.0) was added to the Zeba desalting column of step 1.3, centrifuged at 1500 Xg for 1min at 4℃with a centrifuge, and the buffer was discarded and repeated 3 times to obtain another 1 activated Zeba desalting column.

(2) Purification of KN035

（3） ^99m Preparation of Tc-KN035

(3.1) weighing and dissolving 0.2 mg chelating agent SHNH (6-hydrazinonicotinic acid succinimidyl ester hydrochloride) powder in 20 uL anhydrous DMSO (dimethyl sulfoxide) to prepare a SHNH solution with the concentration of 10 mug/mu L;

(3.2) taking 10 mu g/mu L of SHNH solution with the concentration of 10 uL, uniformly mixing the solution with the purified KN035, and vibrating the solution at the temperature of 4 ℃ to react in a dark place for 12 h;

(3.4) mixing the above HYNIC-KN035 with 50. Mu.L Tricine (N-tris (hydroxymethyl) methylglycine) solution having a concentration of 200 mg/mL, 2. Mu.L SnCl having a concentration of 14 mg/mL ₂ (stannous chloride) 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 ℃;

Example 2

^99m And (3) detecting the labeling rate, specific activity, radiochemical purity and stability of Tc-KN 035.

（1） ^99m Tc-KN035 labeling rate was 75.87.+ -. 4.60% and specific activity was 2.68 MBq/. Mu.g.

(2) Radiochemical purity: the radiochemical purity of the markers was determined by iTLC-SG chromatography paper. Take 4. Mu.L ^99m Tc-KN035 was spotted at 1cm of the edge of the chromatographic paper using PBS and methanol and 1M ammonium acetate (volume ratio 1:1) as developing agents, respectively, and the result was shown in FIG. 3A as developing agent after purification using PBS, FIG. 3B is purified by using methanol and 1M ammonium acetate according to the volume ratio of 1:1 as developing agent, and shows that the purified product has no free radical ^99m Tc and ^99m the presence of Tc-colloid has a radiochemical purity of 99.40.+ -. 0.11%.

(3) Stability: purified is subjected to ^99m Tc-KN035 was incubated with PBS and Fetal Bovine Serum (FBS) at a volume ratio of 1:1, respectively, at 37 ℃. The radiochemical purity was measured at 2 h, 4h, 6 h, 12 h, 24h, respectively, and repeated 3 times at each time point. The results showed that 24h in both PBS and FBS had a radiochemical purity of greater than 95% (shown as C in fig. 3).

Example 3

Probe with a probe tip ^99m Tc-KN035 affinity and in vitro targeting verification.

(1) Expression of PD-L1 on tumor cell surface

The cell lines H1975 and A549 were analyzed for PD-L1 expression by cellular immunofluorescence and cellular immunohistochemistry. As a result, as shown in FIG. 4, the cell immunofluorescence showed that H1975 cells had stronger fluorescence, while A549 cells had weaker fluorescence. The cell immunohistochemical experiment shows that the ratio of H1975 cells is obviously higher than that of A549 cells (11.30 percent) through semi-quantitative analysis of the ratio of PD-L1 to the gray value of the internal reference beta-action bandvs96.26%). Thus, H1975 cells highly expressed PD-L1 and A549 cells lowly expressed PD-L1.

(2) Cell saturation experiment

(2.1) inoculating H1975 cell line which highly expresses PD-L1 and A549 cell line which lowly expresses PD-L1 into 24-well plates, respectively, each well being inoculated with 1.5X10 ⁵ Individual cells, at 37 ℃, 5% CO ₂ Culturing in an incubator until the cells adhere to the wall. Discarding the old culture medium, washing with PBS for 2 times, and performing the following experimental operation;

(2.2) 5 groups of 4 multiple wells/group were set, each group containing a different dose per well ^99m Tc-KN035 (0.37-185 KBq) serum-free RPMI 1640 medium at 37 ℃ with 5% CO ₂ Incubating in an incubator for 1 hour;

(2.3) collecting supernatant and cells, respectively: collecting cell culture solution, and washing cells with PBS for 2 times as supernatant; naOH digestion of 1N to obtain a cell suspension, and washing the cells with PBS 2 times as a cell suspension;

(2.4) measurement of supernatant and cell suspension counts by gamma counter, respectively, and calculation of cell saturation and specific uptake rate.

The results are shown as A in FIG. 5, following ^99m Tc-KN035 probe concentration was increased, cell uptake was increased, saturation was reached at 3.7 kBq/well, K _D The value was 31.04 nM.

(3) Cell binding assay

(3.1) inoculating H1975 cell line highly expressing PD-L1 and A549 cell line lowly expressing PD-L1 into 24-well plates, respectively, each well being inoculated with 1.5X10 ⁵ Individual cells, at 37 ℃, 5% CO ₂ Culturing in an incubator until the cells adhere to the wall. Discarding the old culture medium, washing with PBS for 2 times, and performing the following experimental operation;

(3.2) 3.7 KBq per well in cell binding experiments ^99m Tc-KN035, 5% CO at 37 ℃C ₂ Supernatant and cells were collected after incubation of 0.5 h, 1h, 2 h, 4h, 6 h and 8 h, respectively, in an incubator, and the supernatant and cell suspension counts were measured by gamma counter, respectively, to calculate the cell uptake rate.

The results are shown in figure 5B and table 1, ^99m the uptake rate of Tc-KN035 increased with time in PD-L1 highly expressed H1975 cells, reaching plateau at 6H. Compared with the A549 cells with low PD-L1 expression, the uptake rate of H1975 cells at each time point is obviously higher than that of the A549 cells, and the difference is statistically significant (x)p＜0.05, **p＜0.01, ***p＜0.001）。

Table 1. Cell uptake (%, n=4) of H1975 (high expression of PD-L1) and a549 (low expression of PD-L1) cell lines at different time points.

(4) Cell blocking assay

(4.1) inoculating H1975 cell line highly expressing PD-L1 into 24-well plates with 1.5X10 cells per well ⁵ Individual cells, at 37 ℃, 5% CO ₂ Culturing in an incubator until the cells adhere to the wall. Discard old medium, PBS wash2 times of the following experimental operation;

(4.2) setting 5 groups, each control group (unlabeled KN 035); different blocking groups, namely 25-fold, 50-fold, 100-fold and 500-fold unlabeled KN035. The 5 groups of cells were pretreated by 1h and then 3.7 KBq of the same were added ^99m Tc-KN035, after incubation of 1h, the supernatant and the cells were collected, respectively, and the supernatant and the cell suspension were counted by a gamma counter, respectively, to calculate the cell uptake rate.

The results are shown in figure 5C and table 2, ^99m Tc-KN035 was specifically blocked by 25-fold unlabeled KN035p< 0.01). 100 times and 500 times unlabeled KN035 blocking effect is not differentp> 0.05). The 100-fold unlabeled KN035 blocking group had a decrease in cell uptake of about 97.03% compared to the control group. Description of the above Experimental results ^99m Tc-KN035 was specifically taken up in PD-L1 positive cells.

Table 2. Uptake rates of H1975 cell lines at different fold blockages (%, n=4).

Example 4

Probe with a probe tip ^99m And (5) performing in vivo targeting verification on Tc-KN 035.

(1) Establishing a tumor-bearing mouse model

Will be 1X 10 ⁶ The H1975 and A549 cells are respectively suspended in PBS of 150 uL, are respectively inoculated on the armpit of the upper limb of a female nude mouse of 5 weeks old by subcutaneous injection, and are subjected to SPECT/CT imaging and biodistribution when the tumor volume is as long as 0.6-0.8 cm.

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

(2.1) H1975 and A549 tumor-bearing mice were injected into the tail vein in a volume of 150. Mu.L and at a radioactive dose of 29.6-37 MBq, respectively ^99m Tc-KN 035. After 100 times unlabeled KN035 was injected into the tail vein 1H in advance in the H1975 blocking group, the volume of the tail vein was 150. Mu.L, and the radioactive dose was 29.6-37 MBq ^99m Tc-KN035；

(2.2) SPECT/CT imaging of small animals at

lines

4h, 8 h, 12 h, 18 h and 24h post-injection (Yongxin small animal PET/SPECT/CT multimodal imaging system); 1.5% isoflurane-oxygen induced anesthesia, 1.0% isoflurane-oxygen maintenance anesthesia; SPECT parameters: acquisition time, 4, 8 h is 10 seconds/frame, 12, 18 h is 12 seconds/frame, 24h is 15 seconds/frame; matrix 256×512; CT parameters: bulb voltage 45 kVp, current 0.15 mA, exposure time 300 ms/frame;

(2.3) after the development is completed, the data is processed at a post-processing workstation (NMSOft-AIWS Version 1.7) to obtain SPECT/CT images.

As shown in FIG. 6, the arrow shows the tumor site, the H1975 tumor 4H tumor with high expression of PD-L1 was clearly developed, then the tumor site imaging agent was further concentrated, the radioactivity of other tissues and organs was gradually reduced, and the tumor was clearly seen until 24H. While the a549 tumor-bearing mice and the H1975-blocked group tumors were light in development at each time point. The probe can be specifically gathered on tumors with high expression of PD-L1, and has excellent targeting property.

(3) Tumor-bearing mouse biodistribution

(3.1) H1975 and A549 tumor-bearing rat tail intravenous volumes of 150. Mu.L with a radioactive dose of 29.6-37 MBq ^99m Tc-KN 035. H1975 blocking group advanced 1H tail vein with 100 times unlabeled KN035, and then tail vein with 150 μl volume and 29.6-37 MBq radioactive dose ^99m Tc-KN035；

(3.2) mice were sacrificed at 4h, 12 h and 24h post-injection, respectively, blocking groups to 24h post-injection. Taking the tissue of interest, such as blood, brain, heart, lung, liver, spleen, kidney, pancreas, stomach, small intestine, large intestine, muscle, bone and tumor, washing with ultrapure water, airing and weighing;

(3.3) gamma counter measures radioactivity counts and calculates the percent injected dose ratio per gram of tissue (% ID/g) and tumor/muscle ratio after radioactive decay correction.

As shown in FIG. 7 and Table 3, the uptake of H1975 tumor at 4H, 12H and 24H after injection was as high as 9.68.+ -. 0.91% ID/g, 11.97.+ -. 2.05% ID/g and 13.31.+ -. 2.23% ID/g, respectively, significantly higher than that of A549 tumor at the corresponding time points, 4.59.+ -. 0.76% ID/g, 5.43.+ -. 0.58% ID/g and 5.54.+ -. Respectively 0.28 %ID/g（**p< 0.01); h1975 tumor-blocked group showed significantly lower 24H tumor uptake than non-blocked group (5.64.+ -. 1.11% ID/g)vs 13.31 ± 2.23% ID/g, **p< 0.01). Has good consistency with SPECT imaging results. The tumor to muscle ratios showed that the H1975 tumor had excellent target ratios, the H1975 tumor to muscle ratios at 4.38+ -1.54,7.11 + -0.66,8.85 + -0.20 at 4H, 12H and 24H, respectively, were significantly higher than the ratio of the H1975 blocking group to the A549 tumor to muscle at the corresponding time points, and the differences were statistically significant (p＜0.05）。

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

SPECT molecular imaging probe of the invention ^99m The synthesis method of Tc-KN035 is simple, the reaction condition is mild, the yield is high, the cost is low, the affinity of the probe and PD-L1 is high, and the method can be developed in any level of nuclear medicine department. The probe has excellent affinity with PD-L1 and a smaller single-domain antibody Fc fusion protein structure, so that the probe has excellent in-vivo pharmacokinetics in vivo, the development of a tumor is clear after 4h of probe injection, the development is further enhanced along with time, the tumor is still clear and visible after 24h, and the target/cost ratio is high, thereby being beneficial to the detection of tumor metastasis of high-expression PD-L1.

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.

Claims

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

(1.2) 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;

(1.4) 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