CN111840585B

CN111840585B - Pharmaceutical composition for tumor immunotherapy

Info

Publication number: CN111840585B
Application number: CN202010697679.XA
Authority: CN
Inventors: 张现忠; 文雪君; 郭志德; 石昌荣; 曾馨莹
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2020-07-20
Filing date: 2020-07-20
Publication date: 2022-05-03
Anticipated expiration: 2040-07-20
Also published as: CN111840585A

Abstract

The invention relates to a pharmaceutical composition for tumor immunotherapy, and provides a new application of a radionuclide or a marker thereof in remodeling a tumor immune microenvironment. The invention also provides a pharmaceutical composition for tumor immunotherapy of human body or mice, which is composed of the radionuclide or the marker thereof and the immune checkpoint inhibitor according to a specific ratio. The invention also provides a kit for tumor immunotherapy, which contains the pharmaceutical composition for tumor immunotherapy of human bodies or mice. The pharmaceutical composition provided by the invention can remodel a tumor immune microenvironment while detecting tumors through the radioactive nuclide or the marker thereof, so that the growth speed of the tumors is remarkably slowed down through the immune checkpoint inhibitor, and the survival period is improved. The medicine composition has good synergistic effect, and the kit is convenient to use and easy to realize clinical popularization.

Description

Pharmaceutical composition for tumor immunotherapy

Technical Field

The invention belongs to the field of chemical medicines, and particularly relates to a pharmaceutical composition for tumor immunotherapy.

Background

In recent years, malignant tumors have become one of the major public health problems that seriously threaten human health. In China, the incidence and mortality of cancer account for the first! Therefore, reducing the morbidity and mortality of malignant tumors in China is still a difficult task.

A great deal of clinical practice proves that the combination of early diagnosis and early treatment can reduce the death rate of cancer patients and remarkably prolong the life span of the cancer patients. Early stage malignancies often lack significant clinical signs and imaging examinations are the primary means of early diagnosis. The high-resolution molecular imaging technology PET and SPECT are combined with a high-sensitivity high-specificity radionuclide labeled probe, which is expected to reflect the pathological change condition of malignant tumor from the molecular level, compared with the probe based on the high-resolution molecular imaging technology PET and SPECTThe detection method of the anatomical morphology change can noninvasively display the infiltrating and transferring biological behavior of the tumor in advance. Has the advantages that the biopsy and the biomarker detection cannot achieve in the aspects of non-invasive, comprehensive, real-time and dynamic tracking of the treatment effect and guidance of the treatment. In the aspect of tumor targets, a large number of basic researches establish a series of nuclide marking probes based on small molecules, macromolecules and nano platforms through continuous screening, and provide powerful guarantee for accurate diagnosis of tumors and accurate positioning of focuses. There are many radionuclides labeled with tracers reported so far, and the nuclides used include iodine (I), (II), (III), and (III)¹²⁴I、¹²⁵I、¹³¹I) Technetium-99 m (^99mTc), fluorine-18 (¹⁸F) Copper-64 (⁶⁴Cu), gallium-68 (⁶⁸Ga), zirconium-89 (⁸⁹Zr), indium-111 (¹¹¹In), and the like. PET imaging agent commonly used in clinic¹⁸F]FDG, imaging of a variety of tumors (particularly metastases) is superior to CT or MRI.

Cancer has been treated so far mainly by surgery, chemotherapy, radiotherapy and recently emerging immunotherapy. Among them, surgical treatment is invasive and surgical excision of small hidden lesions is impossible. The side effects of the individual chemotherapy and radiotherapy on surrounding healthy tissues or the whole body of a patient are large, the drug resistance is easy to generate, and even if the tumor focus can be accurately positioned in the early stage, the problems of poor targeting property, pain in the process and the like are difficult to solve in the subsequent treatment process. Immunotherapy is the fourth cancer treatment mode after surgery, radiotherapy and chemotherapy, and a series of immune checkpoint inhibitors such as CTLA-4 inhibitor, PD-1/PD-L1 inhibitor are continuously advanced to clinic. Tumor immunotherapy, a novel cancer treatment modality, is significantly effective in a subset of patients, unfortunately the majority of patients fail to respond clinically to immunotherapy alone. The main problem arises from the heterogeneity of the tumor immune microenvironment. Therefore, in order to improve the immunotherapy effect and expand the proportion of beneficial people, on one hand, the defects in the tumor immunization process need to be determined, and the personalized 'precise immunotherapy' for patients is realized; on the other hand, the tumor immune microenvironment is required to be remodeled by combining other means, the activity of immune cells is activated, and the tumor tissues are changed from cold to hot, so that the expected potential of immunotherapy is achieved. Today, reported methods of remodeling tumor microenvironment include virus challenge-induced stimulation changes, laser photothermal activation of the immune system, vaccine-accelerated immune responses in cancer therapy, drug-loaded nano-immunotherapy, reprogramming of microenvironment, and small molecule drug-enhanced immune responses. Various stimulation means have advantages and disadvantages respectively, and the combined immunotherapy can overcome the defect of low immune response rate of certain tumors to a certain extent and improve the immunotherapy effect.

Disclosure of Invention

The first aspect of the invention aims at: provides the application of the radionuclide or the marker thereof in remodeling tumor immune microenvironment.

A second aspect of the invention is directed to: the medicine composition for tumor immunotherapy can effectively inhibit tumor growth by remodeling a tumor immune microenvironment and based on the remodeled tumor immune microenvironment, and has a remarkable synergistic treatment effect.

A third aspect of the invention is directed to: provides a kit for tumor immunotherapy, which can be more conveniently applied to clinical treatment of tumors.

The above object of the present invention can be achieved by the following technical solutions:

firstly, a new use of the radionuclide or the marker thereof is provided, and the new use is the application of the radionuclide or the marker thereof in remodeling tumor immune microenvironment.

Radiolabeled probes have long played a role in nuclear imaging (PET and SPECT) and nuclear targeted therapies. Is widely used clinically¹⁸F]FDG and Na¹³¹For example, the former is generally used for nuclear medicine PET imaging of tumors, and the latter is more used for treatment of thyroid cancer. The inventor finds that different types of radionuclides can reshape the tumor immune microenvironment, such as up-regulating the expression of PD-L1 in the tumor, enhancing T cell infiltration and the like.

In the application scheme of the invention, the radionuclide marker can be a diagnostic radiopharmaceutical (imaging agent) or a therapeutic radiopharmaceutical which is already clinically used at present, and can also be a probe which is individually designed for different tumors in preclinical research.

Clinically and preclinically useful radionuclides or labeled compounds thereof that may be used in the present invention include, but are not limited to¹⁸F、^99mTc、⁶²Cu、⁶⁴Cu、⁶⁷Cu、¹⁷⁷Lu、¹³¹I、¹²⁵I、¹²⁴I、⁶⁷Ga、⁶⁸Ga、¹¹¹In、⁸⁶Y is or⁸⁹Radionuclides such as Zr, or any of the compounds labeled with Zr.

Preferred radionuclides or markers thereof for use in accordance with the present invention are selected from the group consisting of:

(a)¹⁸f nuclide or¹⁸F-labeled probe, said¹⁸F label probe such as [ 2 ]¹⁸F]FDG、¹⁸F-Alfatide、[¹⁸F]FLT or Na¹⁸F, etc.;

(b)¹³¹i nuclide or¹³¹I labeled probe, said¹³¹I labeling of the probes¹³¹I-MIBG or Na¹³¹I, etc.;

(c)^99mtc nuclide or^99mTc-labeled probe, said^99mTc-labeled probes, e.g. Na^99mTcO₄、^99mTc-MIBI、

^99mTc-MAA or^99mTc-MDP, etc.;

(d)⁶⁴cu nuclide or⁶⁴Cu-labeled probe, the⁶⁴Cu-labelled probes such as⁶⁴CuCl₂、⁶⁴Cu-EB-RGD、

⁶⁴Cu-DOTATATE, and the like;

(e)¹⁷⁷lu nuclide or¹⁷⁷Lu-labeled probe, the¹⁷⁷Lu-labeled probes such as¹⁷⁷Lu-PSMA、¹⁷⁷Lu-EB-TATE or¹⁷⁷Lu-EB-RGD, etc.

In the application embodiment of the present invention, it is further preferred that the radionuclide or the marker thereof is selected from any one of the following: na (Na)¹⁸F、[¹⁸F]FDG、¹⁸F-Alfatide、^99mTc-MDP、^99mTc-MAA、^99mTc-RGD、⁶⁴Cu-RGD or¹⁷⁷Lu-RGD; most preferably [ 2 ]¹⁸F]FDG or^99mTc-RGD。

Based on the new application of the radionuclide or the marker thereof, the invention further provides a pharmaceutical composition for human tumor immunotherapy, which is prepared from the radionuclide or the marker thereof and an immune checkpoint inhibitor in a dosage ratio of 20-40 MBq:1 mg.

Meanwhile, the invention also provides a pharmaceutical composition for immunotherapy of mouse tumors, which consists of the radionuclide or the marker thereof and the immune checkpoint inhibitor in a dosage proportion of 55.5-185 MBq:1 mg.

It should be noted that, in the pharmaceutical combination for immunotherapy of human or mouse tumor according to the present invention, the radionuclide or its marker and the immune checkpoint inhibitor exert synergistic effect by combination, and the dosage ratio of the two (i.e. 20-40 MBq:1mg and 55.5-185 MBq:1mg) describes the relative ratio range of the two with respect to tumor synergistic therapeutic effect, wherein the unit "MBq" and "mg" only represents a corresponding dosage unit of the two drugs in the ratio relationship, and is not used to limit the absolute dosage of the two drugs in the pharmaceutical combination according to the present invention. For example, when the dose of the radionuclide or its marker in the pharmaceutical combination for human of the present invention is 400MBq, the corresponding dose of the immune checkpoint inhibitor may be 10mg or 0.01 g; and when the dose of the radionuclide or the marker thereof in the pharmaceutical combination for mice of the present invention is 555 to 1850MBq, the corresponding dose of the immune checkpoint inhibitor may be 10mg or 0.01 g. In conclusion, in the pharmaceutical composition of the present invention, the dosage values of the two drugs can be simultaneously enlarged or reduced by the same factor, and all the dosage ratios converted by using MBq and mg as units fall within the range of the ratio of the present invention, which belongs to the technical scheme of the present invention.

In the combination of the present invention, the radionuclide or the label thereof may beIs a diagnostic radiopharmaceutical (imaging agent) or a therapeutic radiopharmaceutical which is currently used in clinic, and can also be a probe which is individually designed aiming at different tumors in preclinical research; may in particular be selected from¹⁸F、^99mTc、⁶⁴Cu、¹⁷⁷Lu、¹³¹I、¹²⁵I、¹²⁴I or⁶⁸Any one of Ga, or any compound labeled therewith; preference is given to¹⁸F、^99mTc、⁶⁴Cu、¹⁷⁷Lu or¹³¹Any one of I, or any compound labelled therewith; more preferably¹⁸F、^99mTc、⁶⁴Cu or¹⁷⁷Any one of Lu, or any compound labeled therewith;

said¹⁸The F-labeled compound may be selected from the group consisting of [ [ 2 ] ]¹⁸F]FDG、¹⁸F-Alfatide、[¹⁸F]FLT or Na¹⁸Any one of F; said¹³¹The I-labelled compound may be selected from¹³¹I-MIBG or Na¹³¹Any one of I; said^99mTc-labelled compounds may be selected from Na^99mTcO₄、^99mTc-MIBI、^99mTc-MAA or^99mAny one of Tc-MDP; said⁶⁴The Cu-labelled compound may be selected from⁶⁴CuCl₂、⁶⁴Cu-EB-RGD, or⁶⁴Any one of Cu-DOTATATE; said¹⁷⁷The Lu-labelled compound may be selected from¹⁷⁷Lu-PSMA、¹⁷⁷Lu-EB-TATE or¹⁷⁷Any one of Lu-EB-RGD.

In the pharmaceutical composition of the present invention, the radionuclide label is preferably Na¹⁸F、[¹⁸F]FDG、¹⁸F-Alfatide、^99mTc-MDP、^99mTc-MAA、^99mTc-RGD、⁶⁴Cu-RGD or¹⁷⁷Any one of Lu-RGD; most preferably [ 2 ]¹⁸F]FDG or^99mTc-RGD。

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

(i) PD-L1-related inhibitors: including anti-mouse-PD-L1 antibody BP0101, anti-human-PD-L1 antibody SHR-1316, Atezolizumab (Attuzumab), Durvalumab (Durvalizumab), Avelumab, BMS-936559 and other antibodies; or small molecule inhibitors such as JQ1, eFT508, Osimetinib, platycodinD, BMS-202, CA-170, TPP-1, DPPA-1, AUNP-12, etc.;

(ii) PD-1 related inhibitors: antibodies including Nivolumab (Wunacumab), Pembrolizumab (Pembrolizumab), Cemiplimab, Camrelizumab, Sintilimab or Tolipalimab;

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

(iv) LAG 3-related inhibitors: including IMP-321 or BMS-986016, etc.;

(v) STING-related inhibitors: including nitrofuran derivatives C-178 or C-176; nitro fatty acid inhibitor NO₂-FA or cyclic peptide Astin C, etc.;

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

In the pharmaceutical combination of the invention, the immune checkpoint inhibitor is preferably any one of a PD-L1 inhibitor, a PD-1 inhibitor or a CTLA-4 inhibitor; further preferably any one of anti-mouse PD-L1 antibody BP0101, Atezolizumab (Attuzumab), Avelumab, Nivolumab (Wunacumab), Pembrolizumab (Pembrolizumab), Cemiplimab or Iipilimumab (Epipilimumab); the anti-mouse PD-L1 antibody BP0101 is most preferred.

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

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

In addition, based on the pharmaceutical composition for tumor immunotherapy of human body or mouse of the present invention, the present invention further provides a kit for tumor immunotherapy, wherein the kit contains the pharmaceutical composition for tumor immunotherapy of human body or mouse of the present invention.

In a preferred kit of the invention, said radionuclide or marker thereof and said immune checkpoint inhibitor are packaged separately.

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

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

The treatment principle of the pharmaceutical composition and the kit in clinical application is as follows: firstly, the tumor is imaged by using the radioactive nuclide or the marker thereof, the tumor immune microenvironment is remodeled, the immune response rate of the tumor is improved, and then the immunotherapy is carried out by the immune checkpoint inhibitor.

In practical application, a relatively uniform administration standard, namely the administration amount per body weight, can be set for the administration subjects with different body weights in the same species. For example, when the pharmaceutical composition of the present invention is used for immunotherapy of human tumor, the standard for administration of the radionuclide or its marker can be set to 40MBq/kg, the standard for administration of the corresponding immune checkpoint inhibitor can be set to 1mg/kg, and then the injection dosage of the radionuclide or its marker for adult human with a weight of 70kg is 2800MBq, and the injection dosage of the immune checkpoint inhibitor is 70 mg; when the radionuclide or the marker thereof is used for the immunotherapy of the tumor of the mouse, the standard of administration of the radionuclide or the marker thereof is 1850MBq/kg, the standard of administration of the corresponding immune checkpoint inhibitor is 10mg/kg, and then the injection dosage of the radionuclide or the marker thereof of the mouse with the weight of 20g is 37MBq, and the injection dosage of the immune checkpoint inhibitor is 200 mug.

The application method of the pharmaceutical composition and the kit in clinical application comprises the following steps: firstly, the methodThe radionuclide or the marker thereof is injected to remodel a tumor immune microenvironment and improve the immune response of a focus part, so that the action effect of the immune checkpoint inhibitor is improved; and then the immune checkpoint inhibitor is injected for tumor immunotherapy. In a preferred embodiment of the invention, the immune checkpoint inhibitor is anti-PD-L1 antibody and the nuclide is a positron nuclide¹⁸F, the label thereof may be [ 2 ]¹⁸F]FDG or Na¹⁸F. The immune check point inhibitor is injected for treatment within a certain time window (0-48h) after the injection of a certain dose (1-10 times of the imaging dose) of nuclide or nuclide-labeled probe. In another preferred embodiment of the invention, the time window is 4 to 8 hours. In a further preferred embodiment of the invention, the immune checkpoint inhibitor is administered in two divided doses within the time window 4-8 hours and 4 days after the injection of the radiopharmaceutical.

The invention has the beneficial effects that:

the effects of radionuclide or nuclide markers on the tumor microenvironment, particularly on the remodeling action of key tumor immune factors, have not been of interest. In the present study we found that different types of nuclides: (¹⁸F,^99mTc,⁶⁴Cu,¹³¹I,¹⁷⁷Lu, etc.) can remodel the tumor immune microenvironment (e.g., up-regulate the expression of PD-L1 in tumors, enhance T cell infiltration, etc.). In general, high expression of PD-L1 in tumor tissue is the basis for immune checkpoint inhibitor therapy. Therefore, the 'nuclide tracing' and 'immune remodeling' functions of the radioactive probe are combined with the treatment of the immune checkpoint inhibitor, and the obvious improvement of the tumor immunotherapy effect is expected. In particular [ alpha ], [ alpha ]¹⁸F]FDG has incomparable advantages compared with other nucleic acids as the most common tumor diagnosis probe in clinic. If the treatment function can be expanded, the compound has wide clinical application prospect.

The invention utilizes the 'nuclide tracing' and 'immune remodeling' functions of the radionuclide or the marker thereof, and improves the effect of the immune checkpoint inhibitor on the tumor by combining with the immune checkpoint inhibitor. The radionuclide and the labeled species thereof which can be used for the combination are rich and have wide source range. Including both diagnostic (imaging) and therapeutic radiopharmaceuticals that are currently in clinical use, and probes individually designed for different tumors in preclinical studies. The immune checkpoint inhibitor can be related antibodies or various inhibitors used clinically or preclinically, so the pharmaceutical composition has better clinical universality. The invention expands the functions of the radionuclide and the labeled drug thereof which are commonly used clinically, creatively develops the 'one-drug-multiple-use' function of the imaging agent, realizes the tumor imaging and obviously improves the inhibition effect of the tumor immune check point. Especially for tumors insensitive to immunotherapy and heterogeneous tumors, the effects of immune regulation and adjuvant immunotherapy can be remarkably improved.

Drawings

FIG. 1 is a schematic view of the term "in example 1 of the present invention¹⁸F]Area% of TLC map of FDG radiochemical purity determination is reported.

FIG. 2 shows the results of example 1 of the present invention^99mRadioactive chemical purity determination of Tc-RGD area% report on TLC pattern.

FIG. 3 shows that different species include Na in example 2 of the present invention¹⁸F、Na^99mTcO₄、⁶⁴CuCl₂、¹⁷⁷LuCl₃、Na¹³¹I (all commercially available) cell surface expression of PD-L1 was measured by flow cytometry after induction of different cells (CT26, MC38, 4T1, B16F10) for different periods of time.

FIG. 4 shows the Na-passage of CT26 and MC38 cells in example 2 of the present invention¹⁸F、Na^99mTcO₄、⁶⁴CuCl₂、¹⁷⁷LuCl₃The variation level of mRNA is determined by real-time fluorescent quantitative polymerase chain reaction after the induction of several compounds respectively.

FIG. 5 shows Na¹⁸F and Na^99mTcO₄After induction, the expression level of PD-L1 on the cell surface is detected by Western blotting.

FIG. 6 shows the tumor mouse injections of CT26, MC38, 4T1 and B16F10 in example 2 of the present invention¹⁸F]PET visualization post FDG.

FIG. 7 is a schematic view of the term "in example 2 of the present invention¹⁸F]FDG and^99mthe Tc-RGD in combination with the PD-L1 antibody was used to treat CT26 and MC38 tumor mice and to monitor tumor growth rate and survival.

Detailed Description

The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.

Example 1

1.[¹⁸F]Synthesis of FDG

[¹⁸F]FDG was synthesized by IBA automation modules. The method is a chemical synthesis method by alkaline hydrolysis on the premise of mannose triflate. The step 1 is nucleophilic fluorination: production of [ 2 ] by cyclotron¹⁸F]F^-Under helium transmission¹⁸F]F^-Adsorbed on QMA Sep-Pak anion exchange column, and mixed solution of potassium carbonate (6mg/mL) dissolved in water and phase transfer catalyst K2.2.2(20mg/mL) dissolved in acetonitrile is taken and mixed under the action of vacuum pump¹⁸F]F^-And eluting into a reaction bottle. [¹⁸F]F^-The nucleophilic activity of the ion is one of the key factors affecting the reaction yield, and thus the removal of water from the reaction system is important. Preparation of dried [ alpha ], [ alpha ] or a mixture thereof by 2-3 times of evaporative water removal with anhydrous acetonitrile¹⁸F]F^-The catalyst aminopolyether (K2.2.2) is used to chelate potassium ion in the system to make the solution¹⁸F]F^-Exposed to increase its nucleophilic activity (activation of fluoride ion) followed by nucleophilic fluorination with the reaction precursor mannose triflate (15 mg/mL). Step 2 is deprotection reaction, namely, deacetylation protecting group is removed under acidic or basic conditions. Purifying with aluminum column and C18 column sequentially to remove free fluoride ion and intermediate product, and filtering with sterile filter membrane to obtain final preparation.

Physical and chemical properties: a colorless clear liquid was observed after the lead glass was observed, and [ 2 ]¹⁸F]The FDG solution had a pH of 7.4.

And (3) radioactive nucleus purity determination: the purity of the radioactive nucleus is measured to be 0.511MeV by an energy spectrometer, and the data is qualified.

Radiochemical purity determination: taking a proper amount of injection andstandard [ 2 ]¹⁸F]FDG solution was spotted on silica gel thin layer chromatography plate, and developed with 95% acetonitrile water solution as developing agent until the solution moved to 3/4 of the length of the chromatography plate, and then taken out for drying, and the radioactivity distribution was measured by radio-TLC, as shown in FIG. 1, the radiochemical purity was more than 99%, and the retention time was 0.728.

Specific activity determination: the value of [ 2 ] is measured by a liveness meter¹⁸F]The specific activity of FDG is 740MBq/mL which is more than 370MBq/mL specified by the standard, and the detection is qualified.

2.^99mSynthesis of Tc-RGD

Before the reaction, the shaker is heated to 100 ℃, 1.85GBq sodium pertechnetate (commercially available) is diluted with 2 ml of physiological saline, the solution is added into a reaction bottle containing RGD polypeptide, a reducing agent and a co-ligand, and the reaction bottle is immediately placed into the heated shaker, and the shaking reaction is carried out at 100 ℃ for 30 minutes to obtain the required injection. After the lead glass was observed to be colorless and clear liquid, pH of the solution was measured to be 7.4, and radioactive distribution was measured by radio-TLC, and the results are shown in FIG. 2 to obtain^99mThe radioactive chemical purity of Tc-RGD is more than 99%, the retention time is 1.008, the specific activity of the radioactivity is 800MBq/mL measured by an activity meter, and the detection is qualified.

Example 2

The following are the measurement of the influence of different nuclides on the expression of PD-L1 and the marker [ 2 ] synthesized by the method of example 1 described above¹⁸F]FDG and^99mdescription of in vivo distribution and therapeutic Effect of Tc-RGD:

1. flow cytometry to detect the effects of different nuclides on PD-L1 expression

CT26, MC38, 4T1 and B16F10 tumor cells were plated in six well plates overnight, respectively, and 740kBq of radionuclide Na was added to each well¹⁸And F, adding the same volume of normal saline into the cells of the control group, and culturing the cells of the experimental group and the cells of the control group in different culture boxes to ensure that the cells of the control group are not influenced. After incubation at different time points (0.5h, 2h, 4h, 8h and 24h), cells were harvested and washed twice with ice-cold PBS, stained with anti-PD-L1 antibody (abcam, ab238697), and restained overnight with fluorescent bis-fluorescent dyeAntibodies were raised and free antibodies were washed with PBS and detected by flow cytometry. For other nuclear species, including Na^99mTcO₄、⁶⁴CuCl₂、¹⁷⁷LuCl₃、Na¹³¹I (all the nuclides are commercially available), the procedure is as described above. The final results are shown in fig. 3, demonstrating that different nuclides can induce increased PD-L1 expression in different cells, which is the first factor to trigger immune checkpoint inhibitor therapy, providing the basis for radionuclide-combination immunotherapy.

2. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) determination of Effect of different nuclides on PD-L1 expression CT26 and MC38 cells were plated overnight in 12-well plates, and radionuclide Na was 370kBq¹⁸F (or Na)^99mTcO₄、⁶⁴CuCl₂、¹⁷⁷LuCl₃) The total RNA was extracted from the cells, cDNA was obtained by reverse transcription, and gene expression analysis by RT-qPCR was performed by dilution in serum-free RPMI medium and then added to each well of cells and incubated at 37 deg.C for various time points (2h, 6h, 24 h). The results are shown in FIG. 4, which indicates that stimulation by different nuclides can change the expression of PD-L1 at the gene level, so that the expression of PD-L1 gene is increased in a doubling way, and the stimulation degrees of different nuclides on different cells are different, thereby providing reference for the selection of different cells for combined treatment by different nuclides.

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

CT26 and MC38 cells were plated overnight in 6-well plates with 740kBq of radionuclide Na¹⁸F or Na^99mTcO₄Diluted in serum-free RPMI medium and added to each well of cells incubated at 37 degrees celsius for various time points (2h, 4h, 8h and 24h), and an equal volume of saline was added to the control group. After incubation for the corresponding time point, the culture solution was removed, precooled PBS was added and gently shaken for 1 minute, PBS was then completely aspirated, lysate containing PMSF was added and lysed at 4 ℃ for 30 minutes, then the cells were scraped off with a clean scraper and cell debris and lysate were placed in a centrifuge tube. Finally, electrophoresis is carried out, and PD-L1 eggs are measuredWhite content. As shown in FIG. 5, the expression of PD-L1 can be changed at protein level by different imaging nuclides, which lays a foundation for the research on the therapeutic function of the imaging nuclide.

4. PET imaging of mice

A compound having a radiochemical purity of greater than 95% was prepared as in example 1¹⁸F]FDG, 0.1mL (about 3.7MBq) was injected via tail vein into CT26, MC38, 4T1 or B16F10 tumor mice (approximately 18-20 g in body weight) and static PET image acquisition was performed. The radiotracer was scanned separately at different time points after injection and the imaging results are shown in figure 6. [¹⁸F]FDG can be obviously retained in the tumors of the 4 tumor-bearing mice, and can still see a clear image in the tumor after the time is prolonged to 4 hours, thereby laying a foundation for a treatment mode from 'tracing' to 'triggering' of the tumor.

5. Radionuclide-linked immunotherapy experiments

Para 2¹⁸F]The therapeutic effect of FDG in combination with anti-PD-L1 antibody on tumor mice was examined. Female BALB/C mice (weighing about 18-20 grams) and female C57BL/6 mice (weighing about 18-20 grams) were inoculated subcutaneously in the right lower limbs with CT26 and MC38 tumor cells, respectively, and treatment was initiated when the tumor diameter reached about 0.5cm after one week. Mixing 1110MBq/kg [, [ solution ]¹⁸F]FDG was injected into mice via the tail vein, and 200 micrograms of PD-L1 antibody was injected after intervals of 0h, 6h, and 24h, respectively, and the administration was repeated again 3 days after the first administration to enhance the therapeutic effect. Mouse tumor size was measured by vernier caliper every other day while monitoring the change in body weight of the mice. Tumor volume V ═ length × width/2. And a normal saline group and a single anti-PD-L1 antibody group are set as control groups, and 100 mu L normal saline and 200 mu g anti-PD-L1 antibodies are injected on the 0 th day and the 4 th day respectively. In addition, a low dose treatment group was set for comparison, i.e., 555 MBq/kg/mouse¹⁸F]FDG, and 200. mu.g of PD-L1 antibody was injected after 6h intervals, and the administration was repeated 3 days later. The therapeutic results are shown in FIG. 7, in both high and lower doses¹⁸F]FDG, the growth trend of tumors in the combined treatment group of anti-PD-L1 antibody administered at intervals of 6h is obviously slowed down, and the growth trend is obviously prolongedThe survival time of mice was improved in the combination treatment group with interval of 0h and 24h, but slightly after the 6h interval. The normal saline solution group, simple¹⁸F]The tumor growth rate of the FDG group and the anti-PD-L1 antibody group alone is obviously higher than that of the combination treatment group. The body weight of the mice was maintained at a normal level in all treatment groups, and there was no death of the mice during the treatment.

Likewise, for^99mThe therapeutic effect of Tc-RGD in combination with anti-PD-L1 antibody on CT26 and MC38 tumor mice was examined. The grouping was in accordance with the above, and high doses were given separately^99mTc-RGD (1850MBq/kg) or low dose^99mTc-RGD (925MBq/kg), in combination with 200. mu.g of anti-PD-L1 antibody. And repeat dosing after 3 days to monitor the effect of treatment. The results are shown in FIG. 7, high dose or low dose^99mThe Tc-RGD and anti-PD-L1 antibody combined treatment has obvious tumor inhibition effect in the administration group with 6h interval, and the high dose^99mThe Tc-RGD and anti-PD-L1 antibody combined treatment group 2 MC38 tumor mice were cured. While the control group included the normal saline group, alone^99mThe Tc-RGD group and the single anti-PD-L1 antibody group have higher tumor growth speed and shorter survival time.

The medicine composition of the invention improves the tumor immune microenvironment by using the imaging agent, and has better universality compared with the prior method for treating by using X-rays. On one hand, the existing X-ray method has no way to treat the metastasis tumor or the tiny hidden focus, and if the irradiation range is too large and the irradiation range is too long, irreversible damage can be caused to normal organs. On the other hand, a large number of radiopharmaceuticals are used for tumor diagnosis, identification, guidance and monitoring treatment through a nuclear medicine imaging technology (PET/SPECT) in clinic or before clinic, and the integration of tumor diagnosis and treatment can be conveniently realized by combining the drug combination method, so that the aim of accurate tumor diagnosis and treatment is finally fulfilled.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Claims

1. A new use of a radionuclide or a marker thereof in the preparation of a medicament for up-regulating the level of tumor immune microenvironment PD-L1; the radionuclide or the marker thereof is selected from¹⁸F、^99mTc or⁶⁴Any one of Cu, or any compound labeled therewith.

2. The use according to claim 1, characterized in that: the radionuclide label is selected from:

¹⁸a F-labeled probe comprising¹⁸F]FDG、¹⁸F-Alfatide、[¹⁸F]FLT or Na¹⁸F；

^99mTc-labeled probe comprising Na^99mTcO₄、^99mTc-MIBI、^99mTc-MAA or^99mTc-MDP；

⁶⁴A Cu-labeled probe comprising⁶⁴CuCl₂、⁶⁴Cu-EB-RGD or⁶⁴Cu-DOTATATE。

3. The use according to claim 1, characterized in that: the radionuclide marker is selected from any one of the following: na (Na)¹⁸F、[¹⁸F]FDG、¹⁸F-Alfatide、^99mTc-MDP、^99mTc-MAA、^99mTc-RGD or⁶⁴Cu-RGD。

4. The use according to claim 1, characterized in that: the radionuclide marker is selected from the group consisting of¹⁸F]FDG or^99mTc-RGD。

5. A pharmaceutical composition for immunotherapy of human tumor comprises a radionuclide marker and an immune checkpoint inhibitor at a ratio of 20-40 MBq:1 mg; the radionuclide label is selected from¹⁸F、^99mTc, or⁶⁴Any compound labeled with any one of Cu; the immune checkpoint inhibitor is selected from any one of the following PD-L1 related inhibitors: the anti-mouse-PD-L1 antibody BP0101, the anti-human-PD-L1 antibody SHR-1316, Atezolizumab (Attuzumab), Durvalumab (Durvalizumab), Avelumab, BMS-936559 antibody; or JQ1, eFT508, Osimetinib, platycodinD, BMS-202, CA-170, TPP-1, DPPA-1, AUNP-12 small molecule inhibitors.

6. A pharmaceutical composition for immunotherapy of human tumors comprises a radionuclide marker and an immune checkpoint inhibitor at a ratio of 20MBq to 1 mg; the radionuclide label is selected from¹⁸F、^99mTc, or⁶⁴Any compound labeled with any one of Cu; the immune checkpoint inhibitor is selected from any one of the following PD-L1 related inhibitors: the anti-mouse-PD-L1 antibody BP0101, the anti-human-PD-L1 antibody SHR-1316, Atezolizumab (Attuzumab), Durvalumab (Durvalizumab), Avelumab, BMS-936559 antibody; or JQ1, eFT508, Osimetinib, platycodinD, BMS-202, CA-170, TPP-1, DPPA-1, AUNP-12 small molecule inhibitors.

7. A pharmaceutical composition for immunotherapy of mouse tumor comprises radionuclide marker and immune checkpoint inhibitor at ratio of 111MBq:1mg or 185MBq:1 mg; the radionuclide label is selected from¹⁸F、^99mTc, or⁶⁴Any compound labeled with any one of Cu; the immune checkpoint inhibitor is selected from any one of the following PD-L1-related inhibitors: the anti-mouse-PD-L1 antibody BP0101, the anti-human-PD-L1 antibody SHR-1316, Atezolizumab (Attuzumab), Durvalumab (Durvalizumab), Avelumab, BMS-936559 antibody; or JQ1, eFT508, Osimetinib, PlatyrodinD, BMS-202, CA-170, TPP-1, DPPA-1, AUNP-12 small molecule inhibitors.

8. The pharmaceutical combination according to any one of claims 5 to 7, characterized in that: said¹⁸The F-labeled compound is selected from the group consisting of [ alpha ], [ beta ]¹⁸F]FDG、¹⁸F-Alfatide、[¹⁸F]FLT or Na¹⁸F is any one of the above.

9. The pharmaceutical combination according to any one of claims 5 to 7, characterized in that: said^99mTc-labelled compounds selected from Na^99mTcO4、^99mTc-MIBI、^99mTc-MAA or^99mTc-MDP.

10. The pharmaceutical combination according to any one of claims 5 to 7, characterized in that: said⁶⁴Cu-labelled compounds selected from⁶⁴CuCl₂、⁶⁴Cu-EB-RGD, or⁶⁴Any one of Cu-DOTATATE.

11. The pharmaceutical combination according to any one of claims 5 to 7, characterized in that: the radionuclide label is selected from Na¹⁸F、[¹⁸F]FDG、¹⁸F-Alfatide、^99mTc-MDP、^99mTc-MAA、^99mTc-RGD or⁶⁴Any one of Cu-RGD.

12. The pharmaceutical combination according to any one of claims 5 to 7, characterized in that: the radionuclide marker is selected from the group consisting of¹⁸F]FDG or^99mTc-RGD。

13. The pharmaceutical combination according to any one of claims 5 to 7, characterized in that: the immune checkpoint inhibitor is selected from any one of the following: the anti-mouse PD-L1 antibody BP0101, Atezolizumab (Atlizumab) or Avelumab.

14. The pharmaceutical combination according to any one of claims 5 to 7, characterized in that: the immune checkpoint inhibitor is an anti-mouse PD-L1 antibody BP 0101.

15. A kit for immunotherapy of tumors, characterized by: the kit comprises the pharmaceutical combination of any one of claims 5 to 7.

16. The kit of claim 15, wherein: said radionuclide or marker thereof and said immune checkpoint inhibitor are packaged separately.

17. The kit of claim 15, wherein: the dose of the radionuclide or the marker thereof is 1-10 times of the injection dose required by the imaging of the radionuclide or the marker thereof in the same injection object.

18. The kit of claim 15, wherein: the immune checkpoint inhibitor is further divided into two portions of equal dosage.