CN111840585B - A drug combination for tumor immunotherapy - Google Patents

Abstract

The present invention is a pharmaceutical combination for tumor immunotherapy, which provides a new use of radionuclide or its marker, which is the application of radionuclide or its marker in remodeling tumor immune microenvironment. The present invention also provides a drug combination for tumor immunotherapy in humans or mice, which is composed of radionuclides or their markers and immune checkpoint inhibitors in specific proportions. The present invention also provides a kit for tumor immunotherapy, comprising the drug combination for tumor immunotherapy in humans or mice. The drug combination provided by the present invention can reshape the tumor immune microenvironment while detecting the tumor through radionuclide or its marker, and then significantly slow down the tumor growth rate and improve the survival period through the immune checkpoint inhibitor. The pharmaceutical combination of the present invention has good synergistic effect, and the kit of the present invention is convenient to use and easy to realize clinical promotion.

Description

A drug combination for tumor immunotherapy

technical field

The invention belongs to the field of chemical drugs, in particular to a drug combination for tumor immunotherapy.

Background technique

In recent years, malignant tumors have become one of the major public health problems that seriously threaten human health. In my country, both the incidence and mortality of cancer rank first in the world! Therefore, reducing the morbidity and mortality of malignant tumors in my country is still an arduous task.

A large number of clinical practices have proved that the combination of early diagnosis and early treatment can reduce the mortality rate of cancer patients and significantly prolong the survival period of cancer patients. Early malignant tumors usually lack obvious clinical signs, and imaging examination is the main means of early diagnosis. High-resolution molecular imaging technologies PET and SPECT combined with highly sensitive and specific radionuclide-labeled probes are expected to reflect the status of malignant tumor lesions at the molecular level. Compared with detection methods based on anatomical morphological changes, it can non-invasively display tumor invasion and metastasis in advance. biological behavior. In non-invasive, comprehensive, real-time and dynamic tracking of treatment effects and guidance of treatment, it has the advantages that biopsy and biomarker detection cannot match. In terms of tumor targets, a large number of basic researches have established a series of nuclide-labeled probes based on small molecules, macromolecules and nano-platforms through continuous screening, which provides a strong guarantee for the accurate diagnosis of tumors and the precise localization of lesions. Various radionuclide-labeled tracers have been reported so far, including iodine ( ¹²⁴ I, ¹²⁵ I, ¹³¹ I), technetium-99m ( ^99m Tc), fluorine-18 ( ¹⁸ F), copper- 64 ( ⁶⁴ Cu), gallium-68 ( ⁶⁸ Ga), zirconium-89 ( ⁸⁹ Zr), indium-111 ( ¹¹¹ In), etc. The commonly used clinical PET imaging agent [ ¹⁸ F]FDG is better than CT or MRI in imaging a variety of tumors (especially metastases).

So far, cancer treatment is mainly through surgery, chemotherapy, radiotherapy and immunotherapy which has emerged in recent years. Among them, surgical treatment is invasive and cannot be surgically removed for small occult lesions. However, chemotherapy and radiotherapy alone have great side effects on surrounding healthy tissues or the whole body of the patient, and are prone to drug resistance. Even if the tumor lesions can be accurately located in the early stage, it is difficult to solve the poor targeting in the subsequent treatment process. pain, etc. Immunotherapy has become the fourth major cancer treatment method after surgery, radiotherapy, and chemotherapy. A series of immune checkpoint inhibitors, such as CTLA-4 inhibitors, PD-1/PD-L1 inhibitors, have been introduced into the clinic. As a new type of cancer treatment, tumor immunotherapy has remarkable curative effect for some patients. Unfortunately, most patients cannot respond to immunotherapy alone. The main problem stems from the heterogeneity of the tumor immune microenvironment. Therefore, in order to improve the effect of immunotherapy and expand the proportion of the beneficiary population, on the one hand, it is necessary to identify the defects in the tumor immune process and realize personalized "precise immunotherapy" for patients; on the other hand, it is necessary to combine other means to reshape the tumor immune microenvironment , activate the activity of immune cells, make the tumor tissue from "cold" to "hot", so as to achieve the expected potential of immunotherapy. To date, reported approaches to remodel the tumor microenvironment include stimulatory changes induced by viral attack, laser photothermal activation of the immune system, vaccines for cancer therapy to accelerate immune responses, nano-drug-loaded immunotherapy, reprogramming of the microenvironment, and small molecule drugs to enhance immunity reaction etc. Various stimulation methods have their own advantages and disadvantages, and the combined use of immunotherapy can overcome the low immune response rate of some tumors to a certain extent, and improve the effect of immunotherapy.

SUMMARY OF THE INVENTION

The purpose of the first aspect of the present invention is to provide the application of radionuclides or their markers in remodeling tumor immune microenvironment.

The purpose of the second aspect of the present invention is to provide a drug combination for tumor immunotherapy, which can effectively inhibit tumor growth by remodeling the tumor immune microenvironment and based on the remodeled tumor immune microenvironment, with significant synergy treatment effect.

The purpose of the third aspect of the present invention is to provide a kit for tumor immunotherapy, which can be more conveniently applied to the clinical treatment of tumors.

The above-mentioned purpose of the present invention can be realized by following technical scheme:

First, a novel use of a radionuclide or its marker is provided, which is the application of the radionuclide or its marker in remodeling the tumor immune microenvironment.

For a long time, radiolabeled probes have been used for radionuclide imaging (PET and SPECT) and radionuclide-targeted therapy. Taking [ ¹⁸ F]FDG and Na ¹³¹ I, which are widely used clinically, as examples, the former is generally used for nuclear medicine PET imaging of tumors, and the latter is more used for the treatment of thyroid cancer. The inventors have found that different types of radionuclides can reshape the tumor immune microenvironment, such as upregulating the expression of PD-L1 in tumors, enhancing T cell infiltration, and so on.

In the application scheme of the present invention, the radionuclide labels can be either diagnostic radiopharmaceuticals (imaging agents) or therapeutic radiopharmaceuticals that are currently in clinical use, or can be used in preclinical research for different tumors. Individually designed probes.

Commonly used clinical and preclinical radionuclides or their labeled compounds that can be used in the present invention, including but not limited to ¹⁸ F, ^99m Tc, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ¹⁷⁷ Lu, ¹³¹ I, ¹²⁵ I, ¹²⁴ I , ⁶⁷ Ga, ⁶⁸ Ga, ¹¹¹ In, ⁸⁶ Y or ⁸⁹ Zr and other radionuclides or any compound labeled therewith.

Preferred radionuclides or their markers in the application scheme of the present invention are selected from:

(a) ¹⁸ F nuclide or ¹⁸ F-labeled probe, such as [ ¹⁸ F]FDG, ¹⁸ F- ^{Alfatide, [ 18 F]FLT or Na 18 F ,} etc.;

(b) ¹³¹ I nuclide or ¹³¹ I labeled probe, described ¹³¹ I labeled probe such as ¹³¹ I-MIBG or Na ¹³¹ I, etc.;

(c) ^99m Tc nuclide or ^99m Tc labeled probe, described ^99m Tc labeled probe such as Na ^99m TcO ₄ , ^99m Tc-MIBI,

^99m Tc-MAA or ^99m Tc-MDP, etc.;

(d) ⁶⁴ Cu nuclide or ⁶⁴ Cu labeled probe, the ⁶⁴ Cu labeled probe such as ⁶⁴ CuCl ₂ , ⁶⁴ Cu-EB-RGD,

⁶⁴ Cu-DOTATATE, etc.;

(e) ¹⁷⁷ Lu nuclide or ¹⁷⁷ Lu labeled probe, such as ¹⁷⁷ Lu-PSMA, ¹⁷⁷ Lu-EB- ^{TATE or 177 Lu} -EB-RGD, etc.

In the application scheme of the present invention, it is further preferred that the radionuclide or its marker is selected from any one of the following: Na ¹⁸ F, [ ¹⁸ F]FDG, ¹⁸ F-Alfatide, ^99m Tc-MDP, ^99m Tc - MAA, ^99m Tc-RGD, ⁶⁴ Cu-RGD or ¹⁷⁷ Lu-RGD; most preferably [ ¹⁸ F]FDG or ^99m Tc-RGD.

Based on the new use of the radionuclide or its marker according to the present invention, the present invention further provides a drug combination for human tumor immunotherapy, which is composed of a radionuclide or its marker and an immune checkpoint inhibitor with 20- 40MBq:1mg dose ratio composition.

At the same time, the present invention also provides a drug combination for mouse tumor immunotherapy, which is composed of radionuclide or its marker and immune checkpoint inhibitor in a dose ratio of 55.5-185 MBq:1 mg.

It should be noted that, in the drug combination for human or mouse tumor immunotherapy according to the present invention, the radionuclide or its marker and the immune checkpoint inhibitor play a synergistic effect through combined medication, and all of the two have a synergistic effect. The above-mentioned dose ratio (ie 20-40MBq:1mg and 55.5-185MBq:1mg) describes the relative proportional relationship range that the two can exert a synergistic therapeutic effect on the tumor, and the units "MBq" and "mg" only represent the two A certain dosage unit corresponding to the drug in the proportional relationship is not used to limit the absolute dosage of the two drugs in the drug combination of the present invention. For example, when the dose of the radionuclide or its label in the pharmaceutical combination for human of the present invention is 400MBq, the corresponding dose of the immune checkpoint inhibitor may be 10mg or 0.01g; When the dose of the radionuclide or its marker in the pharmaceutical combination for mice of the present invention is 555-1850 MBq, the corresponding dose of the immune checkpoint inhibitor can be 10 mg or 0.01 g. In a word, in the pharmaceutical combination of the present invention, the dose values of the two drugs can be expanded or reduced by the same multiple at the same time, and all the dose ratios in MBq and mg after conversion fall into the ratio range of the present invention, all belong to the present invention range of technical solutions.

In the combination of the present invention, the radionuclide or its marker can be either diagnostic radiopharmaceuticals (imaging agents) or therapeutic radiopharmaceuticals currently in clinical use, or can be used in preclinical research for different Probes for personalized design of tumors; specifically, can be selected from any one of ¹⁸ F, ^99m Tc, ⁶⁴ Cu, ¹⁷⁷ Lu, ¹³¹ I, ¹²⁵ I, ¹²⁴ I or ⁶⁸ Ga, or any compound labeled therewith; Preferably any one of ¹⁸ F, ^99m Tc, ⁶⁴ Cu, ¹⁷⁷ Lu or ¹³¹ I, or any compound labeled therewith; more preferably any one of ¹⁸ F, ^99m Tc, ⁶⁴ Cu or ¹⁷⁷ Lu, or its any compound labeled;

The ¹⁸ F-labeled compound can be selected from any one of [ ¹⁸ F]FDG, ¹⁸ F-Alfatide, [ ¹⁸ F]FLT or Na ¹⁸ F; the ¹³¹ I-labeled compound can be selected from ¹³¹ I -any one of MIBG or Na ¹³¹ I; the compound labeled with ^99m Tc can be selected from any one of Na ^99m TcO ₄ , ^99m Tc-MIBI, ^99m Tc-MAA or ^99m Tc-MDP; the The 64Cu-labeled compound can be selected from any one of ^64CuCl ₂ , ^64Cu -EB-RGD, or ^64Cu - ^DOTATATE ; the ^177Lu -labeled compound can be selected from ^177Lu -PSMA, ^177Lu- Either EB-TATE or ¹⁷⁷ Lu-EB-RGD.

In the pharmaceutical combination of the present invention, the radionuclide label is further preferably Na ¹⁸ F, [ ¹⁸ F]FDG, ¹⁸ F-Alfatide, ^99m Tc-MDP, ^99m Tc-MAA, ^99m Tc-RGD, ⁶⁴ Any of Cu-RGD or ¹⁷⁷ Lu-RGD; most preferably [ ¹⁸ F]FDG or ^99m Tc-RGD.

In the pharmaceutical combination of the present invention, the immune checkpoint inhibitor can be various types of antibodies or inhibitors used clinically or preclinically, including but not limited to any of the following:

(i) PD-L1 related inhibitors: including anti-mouse-PD-L1 antibody BP0101, anti-human-PD-L1 antibody SHR-1316, Atezolizumab (atezolizumab), Durvalumab (dulvalumab), Antibodies such as Avelumab, BMS-936559; or small molecule inhibitors such as JQ1, eFT508, Osimertinib, PlatycodinD, BMS-202, CA-170, TPP-1, DPPA-1, AUNP-12;

(ii) PD-1-related inhibitors: including antibodies such as Nivolumab, Pembrolizumab, Cemiplimab, Camrelizumab, Sintilimab or Toripalimab;

(iii) CTLA-4-related inhibitors: including antibodies such as Ipilimumab or Tremelimumab;

(iv) LAG3-related inhibitors: including IMP-321 or BMS-986016, etc.;

(v) STING-related inhibitors: including nitrofuran derivatives C-178 or C-176; nitrofatty acid inhibitors NO ₂ -FA or cyclic peptide Astin C, etc.;

(vi) Other immune checkpoint-related inhibitors: including V-region immunoglobulin inhibitor for T cell activation (VISTA), anti-KIR antibody lirilumab or A2aR adenosine receptor antagonist NIR178 (PBF-509), etc.

In the pharmaceutical combination of the present invention, the immune checkpoint inhibitor is preferably any one of PD-L1 inhibitor, PD-1 inhibitor or CTLA-4 inhibitor; more preferably anti-mouse PD-L1 antibody Any of BP0101, Atezolizumab, Avelumab, Nivolumab, Pembrolizumab, Cemiplimab, or Ipilimumab; most preferably anti-mouse PD -L1 antibody BP0101.

In the pharmaceutical combination for human tumor immunotherapy according to the present invention, the preferred dose ratio of the radionuclide or its marker and the immune checkpoint inhibitor is: 20MBq:1 mg.

In the drug combination for mouse tumor immunotherapy according to the present invention, the preferred dose ratio of the radionuclide or its marker and the immune checkpoint inhibitor is: 111-185MBq:1 mg; most preferably 111MBq : 1mg or 185MBq: 1mg.

In addition, based on the drug combination for human or mouse tumor immunotherapy according to the present invention, the present invention further provides a kit for tumor immunotherapy, wherein the kit contains the drug combination according to the present invention. Drug combinations for tumor immunotherapy in humans or mice.

In a preferred kit of the present invention, the radionuclide or its marker and the immune checkpoint inhibitor are individually packaged.

In a more preferred kit of the present invention, the dose of the radionuclide or its marker is 1-10 times the injection dose required for imaging in the same injection subject.

In a more preferred kit of the present invention, the immune checkpoint inhibitor is further divided into two equal doses.

The therapeutic principle of the clinical application of the drug combination and the kit of the present invention is as follows: firstly, the radionuclide or its marker is used to image the tumor and reshape the tumor immune microenvironment, so as to improve the immune response rate of the tumor, and then pass the immune checkpoint. Inhibitors for immunotherapy.

In practical applications of the drug combination of the present invention, a relatively uniform dosing standard, that is, the dosage per unit body weight, can usually be set for administration objects of the same species with different body weights. For example, when the drug combination of the present invention is used for human tumor immunotherapy, the administration standard of the radionuclide or its marker can be set to 40MBq/kg, and the administration standard of the corresponding immune checkpoint inhibitor can be 1 mg/kg, then the body weight is The injection dose of 70kg adult radionuclide or its marker is 2800MBq, and the injection dose of immune checkpoint inhibitor is 70mg; when used for mouse tumor immunotherapy, the administration of radionuclide or its marker can be set The standard is 1850MBq/kg, and the corresponding immune checkpoint inhibitor administration standard is 10mg/kg, then the injection dose of the radionuclide or its marker in a mouse weighing 20g is 37MBq, and the injection dose of the immune checkpoint inhibitor is is 200 μg.

The clinical application of the pharmaceutical combination and the kit of the present invention is as follows: firstly injecting the radionuclide or its marker, remodeling the tumor immune microenvironment, improving the immune response of the lesion, thereby enhancing immune checkpoints The effect of the inhibitor; and then inject the immune checkpoint inhibitor for tumor immunotherapy. In a preferred embodiment of the present invention, the immune checkpoint inhibitor is an anti-PD-L1 antibody, the nuclide is a positron nuclide ¹⁸ F, and the marker can be [ ¹⁸ F]FDG or Na ¹⁸ F. Within a certain time window (0-48h) after the injection of a certain dose (1-10 times the imaging dose) of radionuclide or radionuclide-labeled probes, immune checkpoint inhibitors are injected for treatment. In another preferred solution of the present invention, the time window is 4-8 hours. In yet another preferred embodiment of the present invention, the immune checkpoint inhibitor is administered in two divided doses within 4-8 hours of the day after the injection of the radiopharmaceutical and within the time window of the 4th day.

The beneficial effects of the present invention are:

The influence of radionuclides or nuclide markers on the tumor microenvironment, especially the remodeling of key tumor immune factors, has not been paid attention to. In the research of the present invention, we found that different types of nuclides ( ¹⁸ F, ^99m Tc, ⁶⁴ Cu, ¹³¹ I, ¹⁷⁷ Lu, etc.) can reshape the tumor immune microenvironment (such as up-regulating the expression of PD-L1 in tumors, Enhanced T cell infiltration, etc.). Generally speaking, the high expression of PD-L1 in tumor tissue is the basis for its immune checkpoint inhibitor therapy. Therefore, using the "nuclide tracing" and "immune remodeling" effects of radioactive probes, combined with immune checkpoint inhibitor therapy, is expected to significantly improve the effect of tumor immunotherapy. In particular, [ ¹⁸ F]FDG, as the most common tumor diagnostic probe in clinic, has unparalleled advantages compared with other radionuclides. If its therapeutic function can be expanded, it will have broad prospects for clinical application.

The invention utilizes the "nuclide tracing" and "immune remodeling" effects of radionuclides or their markers, and improves the effect of the immune checkpoint inhibitor on tumors by combining with the immune checkpoint inhibitor. The radionuclides and their labels that can be used in this combination are rich in variety and from a wide range of sources. It includes both diagnostic radiopharmaceuticals (imaging agents) and therapeutic radiopharmaceuticals currently in clinical use, as well as individually designed probes for different tumors in preclinical research. Immune checkpoint inhibitors can be related antibodies or various inhibitors used clinically or preclinically, so the drug combination has good clinical applicability. The invention expands the functions of commonly used clinical radionuclides and their labeled drugs, creatively develops the "one drug multi-purpose" function of imaging agents, and significantly enhances tumor immune checkpoint inhibition while achieving tumor imaging. Especially for tumors that are not sensitive to immunotherapy and heterogeneous tumors, it can significantly improve the effect of immune regulation and adjuvant immunotherapy.

Description of drawings

Figure 1 is an area % report of the TLC pattern for the determination of the radiochemical purity of [ ¹⁸ F]FDG in Example 1 of the present invention.

FIG. 2 is the area % report of the TLC chart of the radiochemical purity determination of ^99m Tc-RGD in Example 1 of the present invention.

Figure 3 shows the effect of different nuclides including Na ¹⁸ F, Na ^99m TcO ₄ , ⁶⁴ CuCl ₂ , ¹⁷⁷ LuCl ₃ , Na ¹³¹ I (all commercially available) on different cells (CT26, MC38, 4T1, The expression of PD-L1 on the cell surface was detected by flow cytometry after induction of B16F10).

Fig. 4 shows that CT26 and MC38 cells in Example 2 of the present invention were respectively induced by Na ¹⁸ F, Na ^99m TcO ₄ , ⁶⁴ CuCl ₂ , ¹⁷⁷ LuCl ₃ and then subjected to real-time fluorescence quantitative polymerase chain reaction to determine the change level of mRNA .

Figure 5 shows the detection of the expression level of PD-L1 on the cell surface by Western blotting after Na ¹⁸ F and Na ^99m TcO ₄ induction.

FIG. 6 is a PET imaging image of CT26, MC38, 4T1, and B16F10 tumor mice injected with [ ¹⁸ F]FDG in Example 2 of the present invention.

Figure 7 shows the treatment of CT26 and MC38 tumor mice with [ ¹⁸ F]FDG and ^99m Tc-RGD combined with PD-L1 antibody in Example 2 of the present invention, and the tumor growth rate and survival rate were monitored.

Detailed ways

The technical solutions of the present invention will be further illustrated and described below through specific embodiments in conjunction with the accompanying drawings.

Example 1

1. Synthesis of [ ¹⁸ F]FDG

[ ¹⁸ F]FDG was synthesized by the IBA automation module. Based on the premise of trifluoromannose, the method of chemical synthesis by alkali hydrolysis. The first step is the nucleophilic fluorination reaction: the cyclotron produces [ ¹⁸ F]F ^- , and under the transmission of helium, [ ¹⁸ F]F ^- is adsorbed on the QMASep-Pak anion exchange column, and under the action of the vacuum pump, A mixture of potassium carbonate (6 mg/mL) dissolved in water and phase transfer catalyst K2.2.2 (20 mg/mL) dissolved in acetonitrile was taken, and [ ¹⁸ F]F ^- was eluted into the reaction flask. The nucleophilic activity of [ ¹⁸ F]F ^-ion is one of the key factors affecting the reaction yield, so it is very important to remove water in the reaction system. Dry [ ¹⁸ F]F ^- was prepared by 2-3 evaporations of anhydrous acetonitrile to remove water, and the catalyst aminopolyether (K2.2.2) was used to chelate potassium ions in the system to expose [ ¹⁸ F]F ^- While increasing its nucleophilic activity (activation of fluoride ions), it undergoes a nucleophilic fluorination reaction with the reaction precursor trifluoromannose (15 mg/mL). The second step is the deprotection reaction, that is, the acetyl protecting group is removed under acidic or basic conditions. Purify by aluminum column and C18 column to remove free fluoride ions and intermediate products, and filter through sterile membrane to obtain the final preparation.

Physical and chemical properties: a colorless and clear liquid was observed behind the lead glass, and the pH value of the [ ¹⁸ F]FDG solution was measured to be 7.4.

Determination of radionuclear purity: The radionuclear purity measured by energy spectrometer is 0.511MeV, and the data is qualified.

Determination of radiochemical purity: Take an appropriate amount of injection solution and standard [ ¹⁸ F]FDG solution, respectively point them on a silica gel thin layer chromatography plate, and develop with 95% acetonitrile aqueous solution as the developing solvent, until the solution moves to 3 of the length of the chromatography plate. At /4, it was taken out and dried, and radio-TLC was used to measure the distribution of radioactivity. The results are shown in Figure 1. The radiochemical purity was greater than 99%, and the retention time was 0.728.

Determination of specific radioactivity: the specific activity of [ ¹⁸ F]FDG measured by an activity meter was 740MBq/mL, which was greater than the standard 370MBq/mL, and the test was qualified.

2. Synthesis of ^99m Tc-RGD

Before the reaction, the shaker was heated to 100 degrees Celsius, 1.85 GBq sodium pertechnetate (commercially available) was diluted with 2 ml of normal saline, and the solution was added to the reaction flask containing RGD polypeptide, reducing agent and co-ligand , and immediately put it into a heated shaker, and shake at 100 degrees Celsius for 30 minutes to obtain the desired injection. A colorless and clear liquid was observed behind the lead glass. The pH value of the solution was measured to be ^7.4 . The radioactivity distribution was determined by radio-TLC. 1.008, the specific activity of radioactivity measured by the activity meter is 800MBq/mL, and the test is qualified.

Example 2

The following is the determination of the effect of different nuclides on the expression of PD-L1 and the description of the in vivo distribution and therapeutic effect of the markers [ ¹⁸ F]FDG and ^99m Tc-RGD synthesized by the method of Example 1 above:

1. Flow cytometry to detect the effect of different nuclides on PD-L1 expression

CT26, MC38, 4T1 and B16F10 tumor cells were plated in six-well plates overnight, and 740kBq of radionuclide Na ¹⁸ F was added to each well, and an equal volume of normal saline was added to the cells of the control group. The cells of the group were cultured in different incubators to ensure that the control group was not affected. After incubation at different time points (0.5h, 2h, 4h, 8h and 24h), cells were harvested and washed twice with ice-cold PBS, then stained with anti-PD-L1 antibody (abcam, ab238697) and re-stained overnight Fluorescent secondary antibody, and washed with PBS to remove free antibody, and detected by flow cytometer. For other nuclides, including Na ^99m TcO ₄ , ⁶⁴ CuCl ₂ , ¹⁷⁷ LuCl ₃ , Na ¹³¹ I (the nuclides are all commercially available), the operation process is the same as the above. The final results are shown in Figure 3, demonstrating that different nuclides can induce increased PD-L1 expression in different cells, which is the first factor triggering immune checkpoint inhibitor therapy, which is a combination of radionuclides for immunotherapy. provided the foundation.

2. Real-time quantitative polymerase chain reaction (RT-qPCR) to determine the effect of different nuclides on PD-L1 expression CT26 and MC38 cells were plated in 12-well plates overnight, and 370kBq of radionuclide Na ¹⁸ F (or Na ^99m TcO ₄ , ⁶⁴ CuCl ₂ , ¹⁷⁷ LuCl ₃ ) were diluted in serum-free RPMI medium, then added to each well of cells and incubated at 37°C for different time points (2h, 6h, 24h), total RNA was removed from the cells was extracted, cDNA was obtained by reverse transcription, and gene expression analysis by RT-qPCR was performed. The results are shown in Figure 4, indicating that the stimulation of different nuclides can change the expression of PD-L1 at the gene level, so that the expression of PD-L1 gene doubles, and the stimulation of different nuclides to different cells is different. This provides a reference for different nuclides to select different cells for combined therapy.

3. Western blot analysis to verify the effect of different nuclides on PD-L1 expression

CT26 and MC38 cells were plated in 6-well plates overnight, and 740 kBq of the radionuclide Na ¹⁸ F or Na ^99m TcO ₄ was diluted in serum-free RPMI medium, then added to each well of cells and incubated at 37°C for different time points (2h, 4h, 8h and 24h), the control group was added with an equal volume of normal saline. After incubation at the corresponding time point, remove the culture medium, add pre-cooled PBS, shake gently for 1 minute, then completely remove the PBS, add PMSF-containing lysis buffer, lyse at 4°C for 30 minutes, and then use a clean scraper to remove the culture medium. Cells were scraped and cell debris and lysate were placed in a centrifuge tube. Finally, electrophoresis was performed to determine the protein content of PD-L1. The results are shown in Figure 5. Different imaging nuclides can change the expression of PD-L1 at the protein level, which lays the foundation for the research on the therapeutic function of imaging nuclides.

4. PET Imaging of Mice

The compound [ ¹⁸ F]FDG with a radiochemical purity greater than 95% was prepared according to Example 1, and 0.1 mL (about 3.7 MBq) was injected through the tail vein into CT26, MC38, 4T1 or B16F10 tumor mice (body weight about 18-20 g), and static PET image acquisition was performed. They were scanned at different time points after the injection of the radiotracer, and the imaging results are shown in Figure 6. [ ¹⁸ F]FDG can be significantly retained in the tumors of the four tumor-bearing mice, and clearer images can still be seen in the tumors as the time prolongs to 4 h, which is the "tracer" of the tumor. Triggered” treatment modality lays the foundation.

5. Radionuclide combined immunotherapy experiment

To investigate the therapeutic effect of [ ¹⁸ F]FDG combined with anti-PD-L1 antibody on tumor mice. CT26 and MC38 tumor cells were subcutaneously inoculated into the right lower limbs of female BALB/c mice (about 18-20 grams in weight) and female C57BL/6 mice (about 18-20 grams in weight), and the tumors grew to about 1 week later. Treatment started at 0.5 cm. 1110MBq/kg [ ¹⁸ F]FDG was injected into mice via tail vein, 200 μg PD-L1 antibody was injected after 0h, 6h, 24h intervals, and the dose was repeated 3 days after the first dose to boost curative effect. Mouse tumor size was measured by vernier calipers every other day, while changes in mouse body weight were monitored. Tumor volume V=length×width×width/2. The normal saline group and the simple anti-PD-L1 antibody group were set as the control group, and 100 μL of normal saline and 200 μg of anti-PD-L1 antibody were injected on the 0th day and the 4th day, respectively. In addition, a low-dose treatment group was set for comparison, that is, each mouse was given 555MBq/kg [ ¹⁸ F]FDG, and 200 micrograms of PD-L1 antibody was injected after an interval of 6 h, and the administration was repeated 3 days later. The treatment results are shown in Figure 7. Regardless of the high dose or the lower dose of [ ¹⁸ F]FDG, the growth trend of the tumor in the combination therapy group administered with anti-PD-L1 antibody at an interval of 6 hours was significantly slowed down and significantly prolonged. The survival time of mice was improved, and the tumor growth inhibitory effect of the combined treatment group with an interval of 0h and 24h was improved, but it was slightly inferior to that of the group of 6h interval. The tumor growth rate of the normal saline group, the pure [ ¹⁸ F]FDG group and the anti-PD-L1 antibody alone group was significantly higher than that of the combined treatment group. In all treatment groups, the body weight of the mice remained at normal levels, and no mice died during the treatment.

Similarly, the therapeutic effect of ^99m Tc-RGD combined with anti-PD-L1 antibody in the treatment of CT26 and MC38 tumor mice was investigated. The grouping situation was consistent with the above, and they were given high-dose ^99m Tc-RGD (1850MBq/kg) or low-dose ^99m Tc-RGD (925MBq/kg), combined with 200 micrograms of anti-PD-L1 antibody. And repeat the administration after 3 days to monitor the therapeutic effect. The results are shown in Figure 7. The combination therapy of high-dose or low-dose ^99m Tc-RGD and anti-PD-L1 antibody achieved obvious tumor inhibition effect in the 6-h interval administration group, and high-dose ^99m Tc-RGD and anti-PD-L1 antibody achieved obvious tumor inhibition effect. -PD-L1 antibody combined treatment group 2 mice with MC38 tumor were cured. The control group included the normal saline group, the pure ^99m Tc-RGD group and the anti-PD-L1 antibody group alone. The tumor growth rate was faster and the survival period was shorter.

The drug combination of the present invention uses an imaging agent to improve the tumor immune microenvironment, and has better universality than the current treatment method using X-rays. On the one hand, the current X-ray method is incapable of treating metastases or small hidden lesions. If the irradiation range is too large and the time is too long, it will cause irreversible damage to normal organs. The labeled compounds can not only actively target metastases, thereby reducing the irradiation of normal organs, but also maintain a low level of internal irradiation doses to non-target organs in the whole body with a faster excretion rate, which is safer. On the other hand, a large number of radiopharmaceuticals have been used in clinical or preclinical nuclear medicine imaging technology (PET/SPECT) for tumor diagnosis, identification, guidance and monitoring of treatment, and the combination of the drug combination method of the present invention can facilitate the diagnosis and treatment of tumors Integration, and ultimately achieve the purpose of precise tumor diagnosis and treatment.

The above are only the preferred embodiments of the present invention, so the scope of implementation of the present invention cannot be limited accordingly, that is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the description should still be covered by the present invention. In the range.

Claims (18)

1. A new use of a radionuclide or a marker thereof, the new use is the application of a radionuclide or a marker thereof in the preparation of a drug for up-regulating the level of PD-L1 in the tumor immune microenvironment; the radionuclide Or its label is any one selected from ¹⁸ F, ^99m Tc or ⁶⁴ Cu, or any compound of its label. 2. The use according to claim 1, characterized in that: the radionuclide label is selected from: ¹⁸ F-labeled probes, including [ ¹⁸ F]FDG, ¹⁸ F-Alfatide, [ ¹⁸ F]FLT or Na ¹⁸ F; ^99m Tc-labeled probes, including Na ^99m TcO ₄ , ^99m Tc-MIBI, ^99m Tc-MAA or ^99m Tc-MDP; ⁶⁴ Cu-labeled probes, including ⁶⁴ CuCl ₂ , ⁶⁴ Cu-EB-RGD or ⁶⁴ Cu-DOTATATE. 3. The use according to claim 1, wherein the radionuclide label is selected from any one of the following: Na ¹⁸ F, [ ¹⁸ F]FDG, ¹⁸ F-Alfatide, ^99m Tc-MDP, ^99m Tc-MAA, ^99mTc -RGD ^or64Cu -RGD. 4. The use according to claim 1, wherein the radionuclide label is selected from [ ¹⁸ F]FDG or ^99m Tc-RGD. 5. A drug combination for human tumor immunotherapy, comprising a radionuclide marker and an immune checkpoint inhibitor in a ratio of 20-40 MBq: 1 mg; the radionuclide marker is selected from ¹⁸ F. Any compound labeled with any one of ^99m Tc or ⁶⁴ Cu; the immune checkpoint inhibitor is selected from any of the following PD-L1 related inhibitors: anti-mouse-PD-L1 antibody BP0101, anti-human-PD-L1 antibody SHR-1316, Atezolizumab, Durvalumab, Avelumab, BMS-936559 antibody; or JQ1, eFT508, Osimertinib, PlatycodinD, BMS-202, CA -170, TPP-1, DPPA-1, AUNP-12 small molecule inhibitor. 6. A drug combination for human tumor immunotherapy, comprising a radionuclide marker and an immune checkpoint inhibitor in a ratio of 20 MBq: 1 mg; the radionuclide marker is selected from ¹⁸ F, Any compound labeled with either ^99m Tc or ⁶⁴ Cu; the immune checkpoint inhibitor is selected from any of the following PD-L1-related inhibitors: anti-mouse-PD-L1 antibody BP0101, anti- human-PD-L1 antibody SHR-1316, Atezolizumab, Durvalumab, Avelumab, BMS-936559 antibody; or JQ1, eFT508, Osimertinib, PlatycodinD, BMS-202, CA-170 , TPP-1, DPPA-1, AUNP-12 small molecule inhibitors. 7. A drug combination for mouse tumor immunotherapy, comprising a radionuclide marker and an immune checkpoint inhibitor in a ratio of 111 MBq: 1 mg or 185 MBq: 1 mg; the radionuclide marker is Any compound labeled with any one of ¹⁸ F, ^99m Tc, or ⁶⁴ Cu; the immune checkpoint inhibitor is selected from any of the following PD-L1-related inhibitors: anti-mouse-PD-L1 Antibody BP0101, anti-human-PD-L1 antibody SHR-1316, Atezolizumab, Durvalumab, Avelumab, BMS-936559 antibody; or JQ1, eFT508, Osimertinib, PlatycodinD, BMS- 202, CA-170, TPP-1, DPPA-1, AUNP-12 small molecule inhibitor. 8. The pharmaceutical combination according to any one of claims 5-7, wherein the ¹⁸ F-labeled compound is selected from [ ¹⁸ F]FDG, ¹⁸ F-Alfatide, [ ¹⁸ F]FLT or Na ¹⁸ F any of the . 9. the pharmaceutical combination described in any one of claim 5-7, it is characterised in that: described ^99m Tc labeled compound is selected from Na ^99m TcO , ^99m Tc-MIBI, ^99m Tc-MAA or ^99m Tc-MDP any kind. 10. The pharmaceutical combination according to any one of claims 5-7, wherein the 64Cu-labeled compound is selected from any one of ^64CuCl ₂ , ^64Cu -EB-RGD, or ^64Cu - ^DOTATATE kind. 11. The pharmaceutical combination according to any one of claims 5-7, wherein the radionuclide label is selected from Na ¹⁸ F, [ ¹⁸ F]FDG, ¹⁸ F-Alfatide, ^99m Tc-MDP, Any ^of99mTc -MAA, ^99mTc -RGD ^or64Cu -RGD. 12. The pharmaceutical combination according to any one of claims 5-7, wherein the radionuclide label is selected from [ ¹⁸ F]FDG or ^99m Tc-RGD. 13. The pharmaceutical combination according to any one of claims 5-7, wherein the immune checkpoint inhibitor is selected from any one of the following: anti-mouse PD-L1 antibody BP0101, Atezolizumab (atezolizumab) anti) or Avelumab. 14. The pharmaceutical combination according to any one of claims 5-7, wherein the immune checkpoint inhibitor is anti-mouse PD-L1 antibody BP0101. 15. A kit for tumor immunotherapy, wherein the kit contains the drug combination according to any one of claims 5-7. 16. The kit of claim 15, wherein the radionuclide or its marker and the immune checkpoint inhibitor are individually packaged. 17. The kit of claim 15, wherein the dose of the radionuclide or its marker is 1-10 times the injection dose required for imaging in the same injection subject. 18. The kit of claim 15, wherein the immune checkpoint inhibitor is further divided into two equal doses.

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