PD-L1 targeting compound HF12 as well as preparation method and application thereof

Abstract

The invention provides a PD-L1 targeting compound HF12 as well as a preparation method and application thereof, and relates to the technical field of medicine synthesis. The radioactive probe [68Ga] Ga-HF12 provided by the invention has the biological characteristic of specifically targeting PD-L1, the uptake level of PD-L1 positive cells is high, the affinity is good, the SUV value of tumors of PD-L1 positive cell tumor-bearing mice in PET (Polyethylene Terephthalate) imaging is high, and the radioactive probe [68Ga] Ga-HF12 can be used for early diagnosis and pathological research of PD-L1 high-expression tumors and can be used for clinical application. Disease course development and curative effect evaluation are evaluated.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug synthesis, and specifically relates to a compound HF12 targeting PD-L1 and a preparation method and application thereof. Background Art

[0002] Tumor immune escape is an important way for tumors to occur and develop. Immunotherapy targeting tumor escape activates the body's ability to recognize and eliminate tumor cells by inhibiting negative immune regulatory signaling pathways. PD-1 / PD-L1 has become a key target for immunotherapy, but not all patients can benefit from it. With further understanding of the tumor microenvironment, researchers have found that the expression level of PD-L1 in tumor lesions and tumor microenvironment is related to whether patients can benefit from immunotherapy. Therefore, accurately evaluating the PD-L1 expression level of tumors is of great clinical significance for screening patients.

[0003] PD-1 is a type I transmembrane protein of the CD28 family, expressed in human T cells, B cells, and macrophages. PD-L1 is the main ligand of PD-1 and is overexpressed in a variety of malignant tumors, including non-small cell lung carcinoma (NSCLC), breast cancer, and melanoma. Activation of the PD-1 / PD-L1 pathway inhibits CD4 + T cells and CD8 + The proliferation of T cells reduces the function of T cells to kill tumor cells, thus leading to tumor immune escape and making the immune system unable to attack and kill tumor cells. At present, the expression of PD-L1 in tumors is mainly evaluated by immunohistochemistry (IHC). However, IHC has great shortcomings in detecting the expression level of PD-L1 in patients, mainly due to tumor heterogeneity, different quantitative standards, and different detection antibodies. In addition, repeated biopsies are impractical to monitor treatment response. Molecular imaging techniques such as PET and SPECT have the advantages of non-invasive, whole-body quantitative positioning, real-time, repeatable, and simultaneous display of primary and metastatic lesions. Therefore, radiolabeled macromolecules (such as antibodies, antibody fragments, and adhesion molecules) and small molecules (such as peptides, peptide mimetics, and non-peptide) are very attractive as radiotracers for imaging up- and down-regulation of PD-L1 in cancer patients to guide clinical treatment decisions.

[0004] Previous studies have shown that radiolabeled PD-L1 antibodies can be used for imaging in tumor-bearing models with different PD-L1 expressions, such as 89Zr-atezolizumab, this type of tracer has disadvantages such as long blood circulation time, slow clearance from the body, and weak tumor penetration, which means that it takes several days after injection to obtain optimized images and tumor-background contrast. Single-domain antibodies, nanobodies, and small molecule proteins are also used for imaging PD-L1 expression in tumors due to their small size and good pharmacokinetics. For example, 18 F-labeled PD-L1 adhesin ( 18 F-BMS-986192) has been studied for imaging and quantification of PD-L1 expression in patients with non-small cell lung cancer and metastatic melanoma. Peptides have lower molecular weight, can be cleared more quickly from the blood pool and non-target organs, have higher affinity and specificity, and are easily chemically modified. Recently, peptide-based PD-L1 tracers such as 68 Ga-NOTA-WL12 and 18 F-DK222 has also been developed. These tracers can quantify the expression of tumors with different PD-L1 levels and can be used to guide the dynamic changes of PD-L1 in tumors after PD-L1 monoclonal antibody treatment. However, no tracer has been approved for clinical use so far, so there is a need to explore tracers with better biological properties. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention combines cyclic peptide with DOTA to improve the structure of PD-L1 drugs so that they can be 68 Ga labeling as a diagnostic drug. The technical solution is as follows:

[0006] A compound HF12 targeting PD-L1 or a pharmaceutically acceptable salt thereof, which has the structural formula shown in Formula I:

[0007]

[0008] The method for preparing the compound HF12 or a pharmaceutically acceptable salt thereof comprises:

[0009] 1) Solid phase synthesis of peptides on Rink Amide MBHAResin using the Fmoc-tBu strategy with an amide at the C-terminus. Each coupling step was activated with DIC and HOBT in DMF, and Fmoc deprotection was performed with 20% piperidine / DMF;

[0010] 2) Starting from Gly, Fmoc-protected amino acids are coupled sequentially. After synthesis, chloroacetic anhydride and DIPEA are reacted, and TFA is used to cleave the peptide segment;

[0011] 3) The crude peptide obtained in step 2) was precipitated with ice-cold ether, dissolved in DMF and then cyclized with DIEA. After concentration, it was coupled with DOTA-NHS-ester and deprotected with TFA; and

[0012] 4) The crude peptide obtained in step 3) is precipitated with ether and collected by filtration.

[0013] Use of the compound HF12 or a pharmaceutically acceptable salt thereof in the preparation of a radioactive probe targeting PD-L1.

[0014] A radioactive probe targeting PD-L1, comprising the compound HF12 or a pharmaceutically acceptable salt thereof labeled with a radionuclide; the radionuclide is selected from 68 Ga.

[0015] The method for preparing the PD-L1 targeted radioactive probe comprises:

[0016] 1) dissolving the compound HF12 according to (1) in dimethyl sulfoxide to obtain a HF12 solution;

[0017] 2) adding sodium acetate solution to the HF12 solution to obtain a labeling precursor sodium acetate solution; and

[0018] 3) 68 Ga]GaCl3 hydrochloric acid solution is added to the labeling precursor sodium acetate solution, and after reaction, it is cooled to room temperature to obtain [ 68 Ga]Ga-HF12.

[0019] Optionally, the reaction temperature in step 3) is 80-100°C, preferably 90°C.

[0020] Optionally, the method further comprises:

[0021] 4) After getting [ 68 Ga]Ga-HF12, and then the labeling rate was determined in a high performance liquid chromatography (radio-HPLC) with a radioactivity detector;

[0022] Preferably, the high performance liquid chromatography (radio-HPLC) with a radioactive detector includes: the first mobile phase is an aqueous solution of 0.1% trifluoroacetic acid, the second mobile phase is an acetonitrile solution, and the gradient elution conditions are: 0 min, 100% of the first mobile phase; 0-10 min, 100%-0% of the first mobile phase; 10-15 min, 100% of the first mobile phase; the flow rate of the mobile phase is 1 mL / min.

[0023] A tumor imaging agent targeting PD-L1, comprising the compound HF12 or a pharmaceutically acceptable salt thereof, or the radioactive probe targeting PD-L1.

[0024] Use of the compound HF12 or a pharmaceutically acceptable salt thereof, or the radioactive probe targeting PD-L1, or the tumor imaging agent in the preparation of a kit for tumor diagnosis.

[0025] A tumor diagnosis kit, comprising: the compound HF12 or a pharmaceutically acceptable salt thereof, or the radioactive probe targeting PD-L1, or the tumor imaging agent.

[0026] The beneficial effects brought about by the technical solution provided by the embodiment of the present invention include at least:

[0027] The present invention provides 68 The Ga]Ga-HF12 radioactive probe has the biological characteristics of specifically targeting PD-L1, with high uptake levels and good affinity in PD-L1-positive cells. The SUV value of tumors in PD-L1-positive cell-bearing mice in PET imaging is high, and it can be used for early diagnosis, pathological research, disease course development, and efficacy evaluation of tumors with high PD-L1 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 In Example 3 of the present invention, 68 Ga]Ga-HF12 cell uptake and inhibition experimental results;

[0030] Figure 2 HF12 IC in Example 4 of the present invention 50 Value determination result diagram;

[0031] Figure 3 In Example 5 of the present invention, 68 Ga]Ga-HF12 outflow experimental results;

[0032] Figure 4 In Example 6 of the present invention, 68 Ga]Ga-HF12 internalization experimental results;

[0033] Figure 5 For the biodistribution experiment in Example 7 of the present invention [ 68 Distribution of Ga]Ga-HF12 in tumor-bearing mice;

[0034] Figure 6 In Example 8 of the present invention, 68 MicroPET / CT images of Ga]Ga-HF12 in CHO-PD-L1 tumor-bearing mice and blocking experiments;

[0035] Figure 7 is the molecular formula of the compound HF12 targeting PD-L1 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0037] The present invention specifically relates to a novel radionuclide labeled ligand and a preparation method thereof, and more particularly to a 68 A Ga-labeled inhibitor targeting programmed cell death ligand 1 (PD-L1) HF12 radionuclide-labeled ligand, and a preparation method and application thereof.

[0038] Terms and Definitions

[0039] PD-L1, Programmed Cell Death Ligand 1, programmed cell death ligand 1.

[0040] PD-1, Programmed Death Receptor 1, programmed cell death receptor 1.

[0041] NSCLC, Non-small cell lung carcinoma, non-small cell lung cancer.

[0042] IHC, Immunohistochemistry, immunohistochemistry.

[0043] PET,Positron Emission Computed Tomography, positron emission computed tomography.

[0044] SPECT, Single-Photon Emission Computed Tomography, single photon emission computed tomography.

[0045] DMSO, Dimethylsulfoxide.

[0046] PBS, phosphate buffer saline, phosphate buffered saline.

[0047] DMF, N,N-Dimethylformamide, N,N-dimethylformamide.

[0048] DIC, N,N-Diisopropylcarbodiimide, N,N-Diisopropylcarbodiimide.

[0049] HOBT, 1-Hydroxybenzotriazole, 1-hydroxybenzotriazole.

[0050] DIPEA, N,N-Diisopropylethylamine, N,N-Diisopropylethylamine.

[0051] Example 1: Synthesis of radiolabeled ligand HF12

[0052]

[0053] Synthesis method: The peptides were synthesized by standard solid phase synthesis method, and the peptides were constructed in Rink Amide MBHAResin with acid-labile side chain protecting groups, in which the C-terminal amide was formed by Fmoc-tBu orthogonal protection strategy. In brief, each synthesis step was activated and coupled with DIC and HOBT in DMF for 1 h. After each coupling step, Fmoc deprotection was performed using piperidine / DMF (20%, v / v) solution. Gly was selected as the C-terminal residue, and Fmoc-Cys(Trt)-OH, Fmoc-Orn(Boc)-OH, Fmoc-NMe-Nle-OH, Fmoc-NMe-Nle-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Sar-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-NMeAla-OH, and Fmoc-Tyr(tBu)-OH were coupled in sequence. After the peptide sequence was assembled, chloroacetic anhydride solution (dissolved in DMF) was added to the reaction vessel, and then DIPEA was added for reaction. After the reaction was completed, the peptide was cleaved from the resin using TFA for more than 4 hours. After collecting the filtrate, drain it, add ice-cold ether to precipitate the crude peptide, and then collect the solid by filtration. The overall deprotected crude peptide solid was dissolved in DMF in a 50mL centrifuge tube at room temperature, and DIEA was added to the solution for cyclization. The reaction solution was concentrated to dryness on a high-speed vacuum or Geneva EZ-2, and then the crude residue was dissolved in DMF, DOTA-NHS-ester and DIEA were added for coupling, and after the reaction was completed, TFA was used to remove the protecting group. Then drain it, add ice-cold ether to precipitate the crude peptide, and then collect the solid by filtration. The crude peptide was purified by preparative HPLC equipped with an analytical C18 column. The peptide was further characterized by analytical HPLC and online ESI mass spectrometry detection (LC-MS).

[0054] Example 2 Radiolabeled ligand HF12 68 Ga radiolabeling

[0055] (1) Dissolve 1 mg of the compound in DMSO to prepare a HF12 solution with a concentration of 1 μg / μL;

[0056] (2) Elute the germanium-gallium generator with 4 mL of high-purity 0.05 N hydrochloric acid solution, and discard the first 1.5 mL and the last 2 mL of the eluent;

[0057] (3) 0.5 mL of [ 6845 μL of 0.5N sodium acetate solution was added to the hydrochloric acid solution of Ga]GaCl3, at which the pH of the solution was about 4.0, and 10 μL of HF12 solution prepared in step (1) was added thereto, mixed evenly, reacted at 90°C for 10 min, cooled to room temperature, and the labeling rate was determined by high performance liquid chromatography (radio-HPLC) with a radioactive detector to obtain [ 68 Ga]Ga-HF12;

[0058] In the high performance liquid chromatography (radio-HPLC) with a radioactivity detector in step (3), the first mobile phase is an aqueous solution of 0.1% trifluoroacetic acid, the second mobile phase is an acetonitrile solution, and the gradient elution conditions are: 0 min, 100% of the first mobile phase; 0-10 min, 100%-0% of the first mobile phase; 10-15 min, 100% of the first mobile phase; the flow rate of the mobile phase is 1 mL / min.

[0059] Example 3 Cellular uptake and inhibition experiments

[0060] (1) CHO-PD-L1 (PD-L1 positive) cells (purchased from Puzhou Gene Biotechnology Co., Ltd.) (~1×10 5 / well) were inoculated in a 24-well plate and cultured in an incubator for 48 hours until the cell coverage was 90-100%. The culture medium was aspirated, the cells were washed twice with phosphate buffered saline, and 0.5 ml of 2 μCi / well of [ 68 Ga]Ga-HF12 was incubated at 37°C for 10, 30, 60 and 120 min, and 30 μM / L PD-L1 inhibitor BMS1166 was used to block cellular uptake. After the incubation, the solution was aspirated and the cells were quickly washed three times with cold phosphate-buffered saline to terminate cellular uptake; all cells were lysed with sodium hydroxide, and the lysate was collected for radioactivity counting.

[0061] (2) Figure 1 As shown, the results of in vitro cell uptake experiments show that: 68 The uptake of Ga]Ga-HF12 in CHO-PD-L1 (PD-L1 positive) cells in vitro was very high, much higher than that in CHO (PD-L1 negative) cells; after adding the PD-L1 inhibitor BMS1166, [ 68 Ga]Ga-HF12 uptake was blocked, indicating that [ 68 Ga]Ga-HF12 specifically binds to programmed cell death ligand 1.

[0062] Example 4IC 50 Value determination

[0063] CHO-PD-L1 (PD-L1 positive) cells (~1×105 / well) were inoculated in a 24-well plate and cultured in an incubator for 48 hours until the cell coverage was 90-100%. The culture medium was aspirated, the cells were washed twice with phosphate buffered saline, and 0.5 ml of 2 μCi / well of [ 68 Ga]Ga-HF12 and cold compound HF12, so that the final concentration of HF12 was 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 nM, respectively. After incubation at 37°C for 60 minutes, the solution was aspirated, and the cells were quickly washed three times with phosphate buffered saline to terminate cell uptake. All cells were lysed with sodium hydroxide, and the lysate was collected for radioactivity counting;

[0064] By IC 50 The experimental results of value determination show that: Figure 2 As shown, HF12 has a high binding affinity to PD-L1, with an IC 50 The value is 12.47±2.89nM.

[0065] Example 5 Cell efflux experiment

[0066] CHO-PD-L1 (PD-L1 positive) cells (~1×10 5 / well) were inoculated in a 24-well plate and cultured in an incubator for 48 hours until the cell coverage was 90-100%. The culture medium was aspirated, the cells were washed twice with phosphate buffered saline, and 0.5 ml of 2 μCi / well of [ 68 Ga]Ga-HF12, incubate at 37°C for 30 minutes, aspirate the solution, quickly wash the cells twice with phosphate-buffered saline, add radioactive culture medium and incubate for 0, 10, 30, 60, and 120 minutes, remove the culture medium, quickly wash the cells three times with phosphate-buffered saline to terminate cell uptake; lyse all cells with sodium hydroxide, and collect the lysate for radioactivity counting.

[0067] Results: Figure 3 As shown, 68 The efflux rate of Ga]Ga-HF12 increased with time, and the efflux rate within 2 h was 78.94±3.26%ID / well.

[0068] Example 6 Cell internalization experiment

[0069] CHO-PD-L1 positive cells (~1×10 5 / well) were inoculated in a 24-well plate and cultured in an incubator for 48 hours until the cell coverage was 90-100%. The culture medium was aspirated, the cells were washed twice with phosphate buffered saline, and 0.5 ml of 2 μCi / well of [ 68Ga]Ga-HF12, incubate at 37°C for 30 minutes, then aspirate the solution and quickly wash the cells twice with phosphate buffered saline. Add 0.5 mL of glycine-HCl solution to each well, collect the glycine-HCl solution into a tube after 5 minutes, wash the cells with PBS, and collect the PBS into the same tube. Repeat glycine-HCl and PBS once, and collect the solution. Lyse all cells with sodium hydroxide, and collect the lysate for radioactivity counting.

[0070] Results: Figure 4 As shown, 68 The internalization rate of Ga]Ga-HF12 was 31.14±1.84%ID / well within 2 h.

[0071] Example 7 Biodistribution in tumor-bearing mice

[0072] 1. Establishment of CHO-PD-L1 and CHO xenograft tumor nude mouse models: After 16-22 g NOD / SCID mice aged 5-8 weeks (purchased from the Experimental Animal Center of Sun Yat-sen University) were adaptively raised for one week, CHO-PD-L1 (high expression of PD-L1) and CHO (low expression of PD-L1) cells in the logarithmic phase were digested and centrifuged, and resuspended in PBS to make 5×10 7 The CHO-PD-L1 cell suspension was inoculated into the right axilla of the mouse, and the CHO cell suspension was inoculated into the left axilla of the mouse, with 100 μL of cell suspension inoculated on each side of each mouse.

[0073] 2. Randomly divide 9 model mice into 3 groups, 3 mice in each group. 68 After the Ga]Ga-HF12 solution was diluted with normal saline, each model mouse was injected with about 25 μCi of the drug solution through the tail vein. The mice were killed by cervical dislocation 60 and 120 minutes later, and the tissues of interest were taken to measure the radioactivity count.

[0074] Blocking experiment: excessive HF12 (50 μg / mouse) and [ 68 After mixing with Ga]Ga-HF12 solution (about 25 μCi), the mixture was injected through the tail vein. After 60 minutes, the mice were killed by cervical dislocation and the tissues of interest were obtained to measure the radioactivity count.

[0075] 3. From the results of the biodistribution experiment, we can see that Figure 5 As shown, 68 Ga]Ga-HF12 had a higher uptake in mouse CHO-PD-L1 tumors, but no significant uptake was observed in CHO tumors. 68 The uptake of Ga]Ga-HF12 reached its highest level at 60 min; the tumor uptake of CHO-PD-L1 in the blocking group was significantly reduced.

[0076] Example 8 Imaging of Tumor-Bearing Mice

[0077] Establishment of nude mouse models of CHO-PD-L1 and CHO xenograft tumors: After 16-22 g NOD / SCID mice aged 5-8 weeks were adaptively fed for one week, CHO-PD-L1 and CHO cells in logarithmic phase were digested and centrifuged, and resuspended in PBS to make 5×10 7 The CHO-PDL1 cell suspension was inoculated into the right armpit of the mouse, and the CHO cell suspension was inoculated into the left armpit of the mouse, with 100 μL of cell suspension inoculated into each side of each mouse. After 10 to 15 days of feeding and observation, the tumor diameter was about 5 mm to 10 mm.

[0078] [ 68 [Ga]Ga-HF12 static imaging: After the labeled solution is diluted with physiological saline, 0.1 mL (about 100 μCi) of the drug solution is taken with an insulin needle and injected into the tail vein of the model mouse. MicroPET / CT imaging is performed 1 hour later.

[0079] Blocking imaging experiment: excessive HF12 (50 μg / mouse) and [ 68 The mixture was mixed with Ga]Ga-HF12 solution (about 100 μCi) and injected into the tail vein, followed by microPET / CT imaging 1 hour later.

[0080] From the results of microPET / CT imaging experiments, we can see that Figure 6 As shown, 68 Ga]Ga-HF12 was taken up in mouse CHO-PD-L1 tumors, and the uptake in CHO-PD-L1 tumors was significantly higher than that in CHO tumors. Blocking imaging experiments showed that after blocking, the uptake of CHO-PD-L1 tumors was significantly inhibited.

[0081] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A compound HF12 targeting PD-L1 or a pharmaceutically acceptable salt thereof, characterized in that: It has the structural formula shown in Formula I:

2. A method for preparing the compound HF12 or a pharmaceutically acceptable salt thereof according to claim 1, comprising: 1) Solid phase synthesis of peptides on Rink Amide MBHA Resin using the Fmoc-tBu strategy, with an amide at the C-terminus, activation with DIC and HOBT in DMF for each coupling step, and Fmoc deprotection with 20% piperidine / DMF; 2) Starting from Gly, Fmoc-protected amino acids are coupled sequentially. After synthesis, chloroacetic anhydride and DIPEA are reacted, and TFA is used to cleave the peptide segment; 3) The crude peptide obtained in step 2) was precipitated with ice-cold ether, dissolved in DMF, cyclized with DIEA, concentrated, coupled with DOTA-NHS-ester, and deprotected with TFA; and 4) The crude peptide obtained in step 3) is precipitated with ether and collected by filtration.

3. Use of the compound HF12 or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a radioactive probe targeting PD-L1.

4. A radioactive probe targeting PD-L1, characterized in that: Comprising the compound HF12 or a pharmaceutically acceptable salt thereof according to claim 1 labeled with a radionuclide; the radionuclide is selected from 68 Ga.

5. The method for preparing a PD-L1 targeted radioactive probe according to claim 4, comprising: 1) dissolving the compound HF12 according to claim 1 in dimethyl sulfoxide to obtain a HF12 solution; 2) adding sodium acetate solution to the HF12 solution to obtain a labeling precursor sodium acetate solution; as well as 3) 68 Ga]GaCl3 hydrochloric acid solution is added to the labeling precursor sodium acetate solution, and after reaction, it is cooled to room temperature to obtain [ 68 Ga]Ga-HF12.

6. The preparation method according to claim 5, characterized in that: The reaction temperature in step 3) is 80-100°C, preferably 90°C.

7. The preparation method according to claim 5, characterized in that: The method further comprises: 4) After getting [ 68 Ga]Ga-HF12, and then the labeling rate was determined in a high performance liquid chromatography with a radioactivity detector; Preferably, the high performance liquid chromatography with a radioactive detector includes: the first mobile phase is an aqueous solution of 0.1% trifluoroacetic acid, the second mobile phase is an acetonitrile solution, and the gradient elution conditions are: 0 min, 100% of the first mobile phase; 0-10 min, 100%-0% of the first mobile phase; 10-15 min, 100% of the first mobile phase; the flow rate of the mobile phase is 1 mL / min.

8. A tumor imaging agent targeting PD-L1, comprising the compound HF12 or a pharmaceutically acceptable salt thereof according to claim 1, or the radioactive probe targeting PD-L1 according to claim 4.

9. Use of the compound HF12 or a pharmaceutically acceptable salt thereof according to claim 1, or the radioactive probe targeting PD-L1 according to claim 4, or the tumor imaging agent according to claim 8 in the preparation of a kit for tumor diagnosis.

10. A tumor diagnosis kit, comprising: The compound HF12 or a pharmaceutically acceptable salt thereof according to claim 1, or the radioactive probe targeting PD-L1 according to claim 4, or the tumor imaging agent according to claim 8.

Images