WO2023226530A1 - 一种核素标记的抑制肽及其制备方法和应用 - Google Patents

一种核素标记的抑制肽及其制备方法和应用 Download PDF

Info

Publication number
WO2023226530A1
WO2023226530A1 PCT/CN2023/081067 CN2023081067W WO2023226530A1 WO 2023226530 A1 WO2023226530 A1 WO 2023226530A1 CN 2023081067 W CN2023081067 W CN 2023081067W WO 2023226530 A1 WO2023226530 A1 WO 2023226530A1
Authority
WO
WIPO (PCT)
Prior art keywords
nuclide
inhibitory peptide
asf1a
labeled
solution
Prior art date
Application number
PCT/CN2023/081067
Other languages
English (en)
French (fr)
Inventor
杨凯
施秀敏
Original Assignee
苏州大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州大学 filed Critical 苏州大学
Publication of WO2023226530A1 publication Critical patent/WO2023226530A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/008—Peptides; Proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the technical field of tumor prognosis, and in particular to a radionuclide-labeled inhibitory peptide and its preparation method and application.
  • ASF1a promotes the suppression of tumor immunity.
  • ASF1a is overexpressed in a variety of primary human tumors, including melanoma and LUAD. Studies have shown that high expression of ASF1a is associated with significantly worse prognosis in patients with hepatocellular carcinoma. ASF1a is a potential therapeutic target.
  • ASF1 is a histone H3-H4 chaperone conserved from yeast to human cells.
  • ASF1a and ASF1b are mammalian isoforms that participate in DNA replication coupling and DNA replication-non-replication nucleosome assembly pathways.
  • ASF1 also plays a role in the regulation of gene transcription. For example, ASF1a resolves bivalent chromatin domains to induce lineage-specific genes during embryonic stem cell differentiation. Functional and mechanistic studies indicate that ASF1a deficiency sensitizes LUAD tumors to anti-PD-1 therapy by promoting M1-like macrophage polarization and enhancing T cell activation.
  • ASF1a is a negative regulator of immunotherapy.
  • the designed PET probe can be used to visualize and dynamically monitor tumor ASF1a expression levels, and formulate treatment strategies for cancer patients based on ASF1a expression levels.
  • the purpose of the present invention is to design and provide a gallium-labeled polypeptide targeting ASF1a for use as a PET/CT imaging agent for predicting tumor immunotherapy resistance. Passed 1.11 ⁇ After administration of 3.7 MBq, it can be clearly visualized and predict the efficacy of immunotherapy. The imaging can be repeated in a short period of time to dynamically monitor immunotherapy. At the same time, it can conduct isotope-labeled peptide targeted therapy for selected immune-resistant individuals with high ASF1a expression, providing a This is an effective treatment strategy.
  • the method of the present invention is not limited to skin melanoma, but is more suitable for tumors with high expression of ASF1a, such as lung cancer, lung metastasis cancer, breast cancer, etc.
  • the invention provides a radionuclide-labeled inhibitory peptide, which labels ASF1a peptide with 68 Ga/ 177 Lu through DOTA; the amino acid sequence of the ASF1a peptide is YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA (as shown in SEQ NO: 1 ) (ASF1a Peptide, AP1), molecular weight (MW) 4952.62.
  • the position of the nuclide label is shown in Figure 1 The position represented is on the four-aza-heterocyclic ring of DOTA.
  • the invention also provides a method for preparing the nuclide-labeled inhibitory peptide, which includes the following steps:
  • step (2) Pass the reaction solution obtained in step (1) through the chromatographic column and collect the product.
  • the nuclide solution in step (1) is a 68 GaCl 3 solution or a 177 LuCl 3 /HCl solution; the radiation dose of the 68 GaCl 3 solution or 177 LuCl 3 /HCl solution is independently 111 to 185 MBq.
  • the preparation method of the 68 GaCl 3 solution is: rinse the 68 Ge- 68 Ga generator with hydrochloric acid, and collect the intermediate product 68 GaCl 3 ;
  • the amount of hydrochloric acid used is 4 mL, and the intermediate product is the 2nd to 3 mL flowing out of the 68 Ge- 68 Ga generator.
  • the concentration of hydrochloric acid is 0.04-0.06M; the flow rate of the 68 Ge- 68 Ga generator is 0.8-1.2 mL/min.
  • the concentration of the DOTA-coupled ASF1a peptide in step (1) is 0.8-1.2 mg/mL; the volume ratio of the DOTA-coupled ASF1a peptide to the nuclide solution is 0.01-0.03:1.00-3.00.
  • the volume ratio of the DOTA-coupled ASF1a peptide and sodium acetate in step (1) is 15-25:250-350; the concentration of sodium acetate is 0.23-0.27M; the adjusted pH is 3.8-4.2 .
  • the temperature of the water bath in step (1) is 93-97°C; the chromatographic column in step (2) is a C18 column.
  • the water bath time is 8 to 12 minutes; when the nuclide solution is a 177 LuCl 3 /HCl solution, the water bath time is 25 to 35 minutes.
  • the present invention further provides the nuclide-labeled inhibitory peptide and the preparation method of the nuclide-labeled inhibitory peptide.
  • the nuclide-labeled inhibitory peptide prepared by the method can be used in the preparation of anti-tumor drugs or PET/CT imaging.
  • the tumor is one of melanoma, lung cancer, lung metastasis cancer and breast cancer.
  • the present invention has the following beneficial effects:
  • 68 Ga is produced by 68 Ge- 68 Ga generator. It can be produced repeatedly every 4 hours and can be used for more than 1 year. The nuclide cost is low, and the half-life of 68 Ga is only 68 minutes. The radiation dose is low, which meets the short-term repetitive dynamics. Monitoring and can be used as an imaging agent for dynamic monitoring of tumor immunotherapy.
  • the present invention uses 68 GaCl 3 produced by a 68 Ge- 68 Ga generator or purchased 177 LuCl 3 and the designed ASF1a inhibitory peptide to establish a method for labeling 68 Ga/ 177 Lu ASF1a inhibitory peptide (AP1) and evaluate its pharmacology Characteristics and biological properties in B16F10 tumor model mice, and further used for ASF1a targeted imaging research, while analyzing the correlation of imaging results with immunotherapy.
  • the efficacy of radionuclide targeted therapy was evaluated in selected ASF1a individuals. Preclinical studies have shown that the 68 Ga-AP1 labeling rate is 81.98 ⁇ 7.55% and the 177 Lu-AP1 labeling rate is 78.34 ⁇ 13.59%.
  • the radiochemical purity of the product measured by HPLC is >95%, and it has good stability within 24 hours. .
  • the designed and synthesized ASF1a peptide has good biocompatibility.
  • the cell survival rate is 94.73 ⁇ 10.96% at 24 hours and 102.73 ⁇ 5.76% at 48 hours. There is no significant difference between each concentration. difference.
  • the synthesized 68 Ga-AP1 was used as a PET/CT imaging probe.
  • the maximum radioactive activity was 200 ⁇ Ci/mL
  • the cell survival rate was 95.31 ⁇ 9.05%, with no significant difference among each dose group. All the above indicate that the peptide and PET/CT imaging probe have good biocompatibility.
  • the synthesized 177 Lu-AP1 was studied to have a better killing effect in B16F10 cells than the free 177 LuCl 3. The difference was statistically significant, and the effect on B16F10 cells was continuously observed in the next 24 hours and 48 hours. Inhibitory effect on tumor cell proliferation.
  • two groups of drugs were added to B16F10 cells and incubated for 24 hours, and then the normal medium was replaced. When the dose was 100 ⁇ Ci/mL, 177 Lu-AP1 was significantly better than 177 LuCl 3. As the dose increased, the inhibitory effect was more Significantly.
  • the cell survival rate of the 177 Lu-AP1 group after 24 hours was 65.31 ⁇ 13.64%, 177 LuCl 3 group was 82.19 ⁇ 16.69%; after 48 hours, the cell survival rate of the 177 Lu-AP1 group was 64.59 ⁇ 8.28%, and that of the 177 LuCl 3 group was 86.98.19 ⁇ 3.22%.
  • the T/N ratio of this group was 1.11 ⁇ 0.2362, and the T/N ratio of the immunotherapy-ineffective group was 2.32 ⁇ 0.5997. The difference between the two groups was statistically significant. This study shows that in 68 Ga-AP1 imaging, the higher the uptake, the more likely the immunotherapy will be ineffective. It is expected to be used to non-invasively predict individuals who are ineffective for immunotherapy, or to dynamically monitor during the immunotherapy process to guide treatment.
  • the 68 Ga-labeled ASF1a inhibitory peptide of the present invention can display the expression level of tumor ASF1a through PET/CT imaging, has good imaging sensitivity, can specifically screen high-expressing and low-expressing individuals, and achieve non-invasive prediction of tumor immunity. Treatment efficacy. 177 Lu-labeled ASF1a inhibitory peptide provides a new and effective treatment strategy for tumors that highly express ASF1a but are ineffective in immunotherapy.
  • Figure 1 is a schematic diagram of 68 Ga/ 177 Lu labeled AP1 polypeptide
  • Figure 2 shows the identification and purification diagram of 68 Ga/ 177 Lu labeled AP1 product
  • Figure 3 shows the biosafety of AP1 in B16F10 cells after 24 hours of incubation at 24 hours and 48 hours.
  • the left picture shows the cell growth inhibition rate of AP1 in B16F10 cells after 24 hours of incubation.
  • the right picture shows the cell growth inhibition rate of AP1 in B16F10 cells. Cell growth inhibition rate 48 hours after incubation for 24 hours;
  • Figure 4 shows the biological safety of different doses of 68 Ga-AP1
  • Figure 5 shows the competitive binding and inhibition experiments between 68 GaAP1 and AP1;
  • Figure 6 shows the specific uptake and inhibition of 177 Lu-AP1 in B16F10 cells
  • Figure 7 shows the inhibitory effect of 177 Lu-AP1 on B16F10 cell growth. a: 24-hour cell growth inhibition rate after 24 hours of incubation; b: 48-hour cell growth inhibition rate after 24 hours of incubation;
  • Figure 8 shows the targeting and specificity of 68 Ga-AP1 imaging probe in vivo.
  • a PET/CT imaging of individuals sensitive to immunotherapy
  • b PET/CT imaging of individuals insensitive to immunotherapy
  • c Immunotherapy PET/CT imaging after adding AP1 inhibition to treatment-insensitive high-uptake tumors;
  • Figure 9 shows the biodistribution and hemodynamic characteristics of 68 Ga-AP1 imaging probe in vivo.
  • a Biodistribution of 68 Ga-AP1 in major organs in vivo
  • b 68 Ga-AP1 in tumors with high and low expression of ASF1a respectively.
  • Distribution c: Pharmacokinetic characteristics of 68 Ga-AP1 in vivo;
  • Figure 10 shows the correlation of different expressions of ASF1a to the efficacy of immunotherapy in the melanoma B16F10 model.
  • a The tumor volume growth curve of mice after treatment with the immunosuppressant BMS-1. Purple represents the effective group of immunosuppressive treatment, and black represents the ineffective immunosuppressive treatment.
  • b Ratio of tumor to contralateral muscle uptake in different response groups to immunotherapy;
  • Figure 11 shows the growth curve of tumor volume in immunotherapy-insensitive individuals treated with BMS-1 alone and BMS-1 combined with 177 Lu-AP1.
  • the invention provides a radionuclide-labeled inhibitory peptide, which labels 68 Ga/ 177 Lu ASF1a peptide through DOTA; the amino acid sequence of the ASF1a peptide is YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA (as shown in SEQ NO: 1) (ASF1a Peptide, AP1), molecular weight (MW) 4952.62.
  • the position of the nuclide label is shown in Figure 1 The position represented is on the four-aza-heterocyclic ring of DOTA.
  • the invention also provides a method for preparing the nuclide-labeled inhibitory peptide, which includes the following steps:
  • step (2) Pass the reaction solution obtained in step (1) through the chromatographic column and collect the product.
  • the nuclide solution in step (1) is a 68 GaCl 3 solution or a 177 LuCl 3 /HCl solution; preferably, it is a 68 GaCl 3 solution.
  • the radiation dose of the 68 GaCl 3 solution or the 177 LuCl 3 /HCl solution in step (1) is independently 111 to 185 MBq; preferably 121 to 175 MBq; further preferably 131 to 165 MBq; more preferably 145 MBq.
  • the preparation method of the 68 GaCl 3 solution is: rinse the 68 Ge- 68 Ga generator with hydrochloric acid, and collect the intermediate product 68 GaCl 3 ;
  • the amount of hydrochloric acid used is 4 mL, and the intermediate product is the 2nd to 3 mL flowing out of the 68 Ge- 68 Ga generator.
  • the concentration of hydrochloric acid is 0.04-0.06M; preferably 0.05M.
  • the flow rate of the 68 Ge- 68 Ga generator is 0.8 to 1.2 mL/min; preferably 0.9 to 1.1 mL/min, and more preferably 1 mL/min.
  • the concentration of DOTA-coupled ASF1a peptide in step (1) is 0.8-1.2 mg/mL; preferably 0.9-1.1 mg/mL; further preferably 1 mg/mL.
  • the volume ratio of the DOTA-coupled ASF1a peptide and the nuclide solution in step (1) is 0.01-0.03:1.00-3.00; preferably 0.02:1.0-2.5; further preferably 0.02:2.05.
  • the volume ratio of the DOTA-coupled ASF1a peptide and sodium acetate in step (1) is 15-25:250-350; preferably 17-23:270-330; further preferably 19-21:290 ⁇ 310; more preferably 20:300.
  • the concentration of sodium acetate in step (1) is 0.23-0.27M; preferably 0.24-0.26M; further preferably 0.25M.
  • the pH adjusted in step (1) is 3.8-4.2; preferably 3.9-4.1; further One step is preferably 4.0.
  • the temperature of the water bath in step (1) is 93-97°C; preferably 94-96°C; further preferably 95°C.
  • the chromatographic column in step (1) is a C18 column.
  • the water bath time is 8 to 12 minutes; preferably 9 to 11 minutes; further preferably 10 minutes.
  • the water bath time is 25 to 35 minutes; preferably 27 to 33 minutes; further preferably 29 to 31 minutes; more preferably 30 minutes.
  • the present invention further provides the nuclide-labeled inhibitory peptide and the nuclide-labeled inhibitory peptide prepared by the method for preparing anti-tumor drugs or PET/CT imaging.
  • the tumor is one of melanoma, lung cancer, lung metastasis cancer and breast cancer; preferably, it is melanoma.
  • a radionuclide-labeled inhibitory peptide which labels ASF1a peptide with 68 Ga through DOTA; the amino acid sequence of the ASF1a peptide is YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA;
  • step (3) Use 5 mL of 70% ethanol to activate the C18 cartridge drop by drop, then rinse with 5 mL of normal saline and push 10 mL of air, then add the reaction solution obtained in step (2), rinse with 1 mL of normal saline and remove the residual water (collected as free radionuclides), and then rinse the C18 column with 0.3 mL of 60% ethanol, and the collected product is the purified labeled product.
  • a radionuclide-labeled inhibitory peptide which 177 Lu labels ASF1a peptide through DOTA;
  • the amino acid sequence of the ASF1a peptide is YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA;
  • a radionuclide-labeled inhibitory peptide which labels ASF1a peptide with 68 Ga through DOTA; the amino acid sequence of the ASF1a peptide is YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA;
  • step (3) Activate the C18 cartridge drop by drop with 5 mL of 70% ethanol, rinse with 5 mL of normal saline, push 13 mL of air, then add the reaction solution obtained in step (2), rinse with 2 mL of normal saline and remove the residual water (collect as free radionuclide), and then rinse the C18 column with 0.35 mL of 60% ethanol, and the collected product is the purified labeled product.
  • HPLC mobile phases are water and acetonitrile (each containing 0.1% TFA), and the gradient is set to an acetonitrile concentration from 20% to 50% for 30 minutes.
  • B16F10 cells grow to more than 90%, digest the cells with 0.25% trypsin, adjust the cell concentration to 1 ⁇ 10 5 /mL, spread on a 24-well plate, and add 0.5 mL of culture medium to each well. Incubate overnight in a 37 °C, 5% CO2 incubator.
  • B16F10 cells in the logarithmic growth phase were trypsinized and resuspended to a cell concentration of 1 ⁇ 10 6 /mL.
  • the left forelimb tumor-bearing cells of 6-week-old C57BL/6 mice were injected with a cell volume of 1 ⁇ 10 5 .
  • the present invention uses 68 GaCl 3 produced by a 68 Ge -68 Ga generator or purchased 177 LuCl 3 and a designed ASF1a inhibitory peptide to establish a method for labeling 68 Ga/ 177 Lu ASF1a inhibitory peptide (AP1), and evaluate its pharmacological characteristics and Biological properties in B16F10 tumor model mice, and further used for ASF1a targeted imaging research, while analyzing the correlation of imaging results with immunotherapy.
  • the efficacy of radionuclide targeted therapy was evaluated in selected ASF1a individuals. Preclinical studies have shown that the 68 Ga-AP1 labeling rate is 81.98 ⁇ 7.55% and the 177 Lu-AP1 labeling rate is 78.34 ⁇ 13.59%.
  • the radiochemical purity of the product measured by HPLC is >95%, and it has good stability within 24 hours. .

Abstract

一种核素标记的抑制肽及其制备方法和应用。通过DOTA将68Ga/177Lu标记ASF1a肽；所述ASF1a肽的氨基酸序列为YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA。所述68Ga标记的ASF1a抑制肽，通过PET/CT显像显示肿瘤ASF1a表达水平，显像灵敏度好，能够特异性的筛选高表达及低表达个体，实现无创预测肿瘤免疫治疗疗效。177Lu标记的ASF1a抑制肽对高表达ASF1a而免疫治疗无效的肿瘤提供一种新的有效的治疗策略。

Description

一种核素标记的抑制肽及其制备方法和应用 技术领域
本发明涉及肿瘤预后技术领域，尤其涉及一种核素标记的抑制肽及其制备方法和应用。
背景技术
近年来，免疫疗法通过利用免疫系统的力量来抗击癌症，从而彻底改变了癌症研究领域。免疫治疗已成为当今癌症治疗的重要手段，但事实上只有少数(不到20％)的患者可以从抗PD-1/PD-L1免疫治疗中受益，而且这些患者中有20～40％发生了严重的不良事件。识别哪些患者更有可能从免疫检查点阻断(ICB)中受益，并将疗效最大化和毒性降至最低，具有重要意义。
ASF1a促进了肿瘤免疫的抑制。ASF1a在包括黑色素瘤、LUAD在内的多种原发人类肿瘤中过度表达。已有研究表明在肝细胞癌患者中，ASF1a的高表达与显著较差的预后相关。ASF1a是一个潜在的治疗靶点。
ASF1是一种组蛋白H3-H4伴侣，从酵母到人类细胞都是保守的。ASF1a和ASF1b是哺乳动物的异构体，参与DNA复制偶联和DNA复制非复制的核小体组装途径。ASF1还在基因转录调控中发挥作用。例如，ASF1a在胚胎干细胞分化过程中解析二价染色质结构域以诱导谱系特异性基因。功能和机制研究表明，ASF1a缺陷通过促进M1样巨噬细胞极化和增强T细胞活化，使LUAD肿瘤对抗PD-1治疗增敏。ASF1a是免疫治疗的负性调节因子，通过设计的PET探针可视化动态监测肿瘤ASF1a表达水平，并根据ASF1a表达水平来制定癌症患者的治疗策略。
因此，如何针对ASF1a靶点，设计并提供一种靶向ASF1a的核素标记的多肽，并将其应用在肿瘤免疫治疗的预后当中，是本领域技术人员亟需解决的问题。
发明内容
本发明的目的在于针对ASF1a靶点，设计并提供一种靶向ASF1a的镓标记的多肽在预测肿瘤免疫治疗耐药的PET/CT显像剂的应用。通过1.11～ 3.7MBq给药即可清晰显影，预测免疫治疗疗效，可在短期内重复显像，动态监测免疫治疗，同时针对筛选出的ASF1a高表达的免疫耐药个体进行同位素标记多肽靶向治疗，提供一种有效的治疗策略，本发明方法不仅限于皮肤黑色素瘤，更适用于ASF1a高表达的肿瘤，如肺癌、肺转移癌、乳腺癌等。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种核素标记的抑制肽，所述核素标记的抑制肽通过DOTA将68Ga/177Lu标记ASF1a肽；所述ASF1a肽的氨基酸序列为YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA(如SEQ NO:1所示)(ASF1a Peptide，AP1)，分子量(MW)4952.62。核素标记位置是示意图1中代表的位置，DOTA的四氮杂环上。
本发明还提供了所述核素标记的抑制肽的制备方法，包括如下步骤：
(1)将DOTA偶联的ASF1a肽与核素溶液混合，得混合液，将混合液与醋酸钠混合，调整pH，水浴，得反应液；
(2)将步骤(1)得到的反应液过色谱柱，收集产物。
优选的，步骤(1)所述核素溶液为68GaCl3溶液或177LuCl3/HCl溶液；所述68GaCl3溶液或177LuCl3/HCl溶液的放射量独立为111～185MBq。
优选的，所述68GaCl3溶液的制备方法为：用盐酸淋洗68Ge-68Ga发生器，收集中间产物68GaCl3；
盐酸用量为4mL，所述中间产物为68Ge-68Ga发生器流出的第2～3mL。
优选的，所述盐酸的浓度为0.04～0.06M；所述68Ge-68Ga发生器的流速为0.8～1.2mL/min。
优选的，步骤(1)所述DOTA偶联的ASF1a肽的浓度为0.8～1.2mg/mL；所述DOTA偶联的ASF1a肽与核素溶液的体积比为0.01～0.03：1.00～3.00。
优选的，步骤(1)所述DOTA偶联的ASF1a肽与醋酸钠的体积比为15～25:250～350；所述醋酸钠的浓度为0.23～0.27M；所述调整pH为3.8～4.2。
优选的，步骤(1)所述水浴的温度为93～97℃；步骤(2)所述色谱柱为C18小柱。
优选的，当核素溶液为68GaCl3溶液时，所述水浴的时间为8～12min；当核素溶液为177LuCl3/HCl溶液时，所述水浴的时间为25～35min。
本发明还进一步的提供了所述的核素标记的抑制肽、所述的一种核素标记的抑制肽的制备方法制备得到的核素标记的抑制肽在制备抗肿瘤药物或PET/CT显像剂中的应用，所述肿瘤为黑色素瘤、肺癌、肺转移癌和乳腺癌中的一种。
与现有技术相比，本发明具有如下的有益效果：
1、68Ga为68Ge-68Ga发生器生产，可以每4小时重复生产，可使用1年以上，核素成本低，而且68Ga的半衰期仅为68分钟，辐射剂量低，满足短期重复动态监测，可作为肿瘤免疫治疗动态监测显像剂。
2、本发明以68Ge-68Ga发生器生产的68GaCl3或购买的177LuCl3以及设计的ASF1a抑制肽，建立68Ga/177Lu标记ASF1a抑制肽(AP1)的方法，评价其药理学特点以及在B16F10肿瘤模型鼠中的生物学特性，并进一步用于ASF1a靶向成像研究，同时分析成像结果于免疫治疗的相关性。对筛选的ASF1a个体进行放射性核素靶向治疗评估其疗效。临床前研究表明，68Ga-AP1标记率为81.98±7.55％，177Lu-AP1标记率为78.34±13.59％，HPLC法测定产品放化纯＞95％，而且在24小时内有良好的稳定性。
3、设计合成的ASF1a肽有良好的生物相容性，多肽最大浓度为100μg/mL时，24小时细胞的存活率为94.73±10.96％，48小时为102.73±5.76％，各浓度间均无明显差异。合成的68Ga-AP1作为PET/CT显像探针，在放射性活度最大为200μCi/mL时，细胞存活率为95.31±9.05％，各剂量组无明显差异。以上均说明多肽及PET/CT显像探针具有良好的生物相容性。
4、研究合成的68Ga-AP1及177Lu-AP1在B16F10细胞中摄取，均能被AP1所抑制，差异有统计学意义，说明68Ga-AP1及177Lu-AP1在细胞水平的特异性摄取。
5、研究合成的177Lu-AP1在B16F10细胞中相较游离的177LuCl3有更优异的杀伤效应，差异有统计学意义，而且在接下来的24小时及48小时均能持续的观察到对肿瘤细胞增殖的抑制作用。实验中在B16F10细胞中加入两组药物孵育24小时后，更换正常培养基，当剂量为100μCi/mL时，177Lu-AP1明显优于177LuCl3，随着剂量的增大，抑制作用更为显著。当放射性剂量为600μCi/mL时，24小时后177Lu-AP1组细胞存活率为65.31±13.64％，177LuCl3 组为82.19±16.69％；48小时后177Lu-AP1组细胞存活率为64.59±8.28％，177LuCl3组为86.98.19±3.22％。
6、在B16F10荷瘤鼠模型中，68Ga-AP1显像显示不同个体摄取高低不同，最佳显像时间为注射显像剂后3.5-5.5小时，在体内的生物分布数据也显示，该显像剂主要通过肝、肾代谢，在肿瘤高摄取组，5.5小时肿瘤％ID/g为18.95±0.2479％，低摄取组肿瘤％ID/g为5.243±1.734％，差异有统计学意义。在血液动力学分析中，68Ga-AP1在血液内半排时间为2.1933小时，体内平均驻留时间(MRT)为3.1643小时。
7、分析B16F10肿瘤模型在早期显像中，肿瘤摄取最大值与对侧肌肉摄取最大值比值与小鼠免疫抑制剂BMS-1治疗疗效的相关性。治疗组所有小鼠于荷瘤后第4天开始腹腔注射BMS-1，每隔3天注射0.05mg/每只小鼠，并记录肿瘤体积，观察至第16天结束，在治疗过程中分别于第7天或第10天进行68Ga-AP1显像，并记录小鼠肿瘤摄取/对侧肌肉摄取最大值的比值T/N。以第16天肿瘤体积小于1000mm3认为免疫治疗有效，该组T/N比值为1.11±0.2362，免疫治疗无效组T/N为2.32±0.5997，两组差异有统计学意义。该研究表明68Ga-AP1显像中，摄取越高，免疫治疗越可能无效，有望用于无创预测免疫治疗无效个体，或者免疫治疗过程中动态监测，指导治疗。
8、筛选68Ga-AP1显像T/N比值大于2.32的个体进行单独BMS-1及联合177Lu-AP1治疗，177Lu-AP1对肿瘤的增殖有明显抑制作用，抑瘤率为63.94％，差异有统计学意义。
综上，本发明所述的68Ga标记的ASF1a抑制肽，通过PET/CT显像显示肿瘤ASF1a表达水平，显像灵敏度好，能够特异性的筛选高表达及低表达个体，实现无创预测肿瘤免疫治疗疗效。177Lu标记的ASF1a抑制肽对高表达ASF1a而免疫治疗无效的肿瘤提供一种新的有效的治疗策略。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面 描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为68Ga/177Lu标记AP1多肽的示意图；
图2为68Ga/177Lu标记AP1产物的鉴定和纯化图；
图3为AP1在B16F10细胞中孵育24小时后24小时及48小时的生物安全性，其中左图为AP1在B16F10细胞中孵育24小时后24小时细胞生长抑制率；右图为AP1在B16F10细胞中孵育24小时后48小时细胞生长抑制率；
图4为不同剂量的68Ga-AP1的生物安全性；
图5为68GaAP1与AP1竞争性结合与抑制实验；
图6为177Lu-AP1在B16F10细胞的特异性摄取与抑制；
图7为177Lu-AP1对B16F10细胞生长抑制效应.a：孵育24小时后的24小时细胞生长抑制率；b：孵育24小时后的48小时细胞生长抑制率；
图8为68Ga-AP1显像探针体内靶向性及特异性.a：对免疫治疗敏感个体PET/CT显像；b：对免疫治疗不敏感个体PET/CT显像；c：对免疫治疗不敏感的高摄取肿瘤加入AP1抑制后PET/CT显像；
图9为68Ga-AP1显像探针体内生物分布及血液动力学特征.a：68Ga-AP1在体内主要器官的生物分布；b：68Ga-AP1分别在ASF1a高表达及低表达肿瘤中的分布；c：68Ga-AP1在体内药代动力学特征；
图10为黑色素瘤B16F10模型中ASF1a不同表达对免疫治疗的疗效相关性.a：免疫抑制剂BMS-1治疗后小鼠肿瘤体积生长曲线，紫色代表免疫抑制治疗有效组，黑色代表免疫抑制治疗无效组；b：免疫治疗不同反应组肿瘤与对侧肌肉摄取的比值；
图11为免疫治疗不敏感个体单独BMS-1治疗以及BMS-1联合177Lu-AP1治疗肿瘤体积生长曲线。
具体实施方式
本发明提供了一种核素标记的抑制肽，所述核素标记的抑制肽通过DOTA将68Ga/177Lu标记ASF1a肽；所述ASF1a肽的氨基酸序列为 YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA(如SEQ NO:1所示)(ASF1a Peptide，AP1)，分子量(MW)4952.62。核素标记位置是示意图1中代表的位置，DOTA的四氮杂环上。
本发明还提供了所述核素标记的抑制肽的制备方法，包括如下步骤：
(1)将DOTA偶联的ASF1a肽与核素溶液混合，得混合液，将混合液与醋酸钠混合，调整pH，水浴，得反应液；
(2)将步骤(1)得到的反应液过色谱柱，收集产物。
在本发明中，步骤(1)所述核素溶液为68GaCl3溶液或177LuCl3/HCl溶液；优选为68GaCl3溶液。
在本发明中，步骤(1)所述68GaCl3溶液或177LuCl3/HCl溶液的放射量独立为111～185MBq；优选为121～175MBq；进一步优选为131～165MBq；更优选为145MBq。
在本发明中，所述68GaCl3溶液的制备方法为：用盐酸淋洗68Ge-68Ga发生器，收集中间产物68GaCl3；
盐酸用量为4mL，所述中间产物为68Ge-68Ga发生器流出的第2～3mL。
在本发明中，所述盐酸的浓度为0.04～0.06M；优选为0.05M。
在本发明中，所述68Ge-68Ga发生器的流速为0.8～1.2mL/min；优选为0.9～1.1mL/min，进一步优选为1mL/min。
在本发明中，步骤(1)所述DOTA偶联的ASF1a肽的浓度为0.8～1.2mg/mL；优选为0.9～1.1mg/mL；进一步优选为1mg/mL。
在本发明中，步骤(1)所述DOTA偶联的ASF1a肽与核素溶液的体积比为0.01～0.03：1.00～3.00；优选为0.02：1.0～2.5；进一步优选为0.02：2.05。
在本发明中，步骤(1)所述DOTA偶联的ASF1a肽与醋酸钠的体积比为15～25:250～350；优选为17～23:270～330；进一步优选为19～21:290～310；更优选为20:300。
在本发明中，步骤(1)所述醋酸钠的浓度为0.23～0.27M；优选为0.24～0.26M；进一步优选为0.25M。
在本发明中，步骤(1)所述调整pH为3.8～4.2；优选为3.9～4.1；进 一步优选为4.0。
在本发明中，步骤(1)所述水浴的温度为93～97℃；优选为94～96℃；进一步优选为95℃。
在本发明中，步骤(1)所述色谱柱为C18小柱。
在本发明中，当核素溶液为68GaCl3溶液时，所述水浴的时间为8～12min；优选为9～11min；进一步优选为10min。
在本发明中，当核素溶液为177LuCl3/HCl溶液时，所述水浴的时间为25～35min；优选为27～33min；进一步优选为29～31min；更优选为30min。
本发明还进一步提供了所述的核素标记的抑制肽、所述的一种核素标记的抑制肽的制备方法制备得到的核素标记的抑制肽在制备抗肿瘤药物或PET/CT显像剂中的应用，所述肿瘤为黑色素瘤、肺癌、肺转移癌和乳腺癌中的一种；优选为黑色素瘤。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
主要仪器或设备
试剂与耗材
实施例1
ASF1a Peptide，AP1的合成
(1)根据目标多肽的重量及分子量预估每个氨基酸的投料量，每个氨基酸及DOTA均使用带保护原料。
(2)将2-Cl(Trt)-Cl resin放入150mL反应器中，并加入80mL DCM浸泡2小时；
(3)用DMF洗涤树脂，然后抽干，如此重复4次，将树脂抽干。
(4)称取Fmoc-Ala-OH(C端第一个氨基酸，CAS No.154445-77-9)+80mL DCM和DIEA加入到反应器中，然后将反应器置于30℃的摇床中反应2小时；
(5)用吸管补加0.5mL DIEA，0.5mL甲醇，反应20min，封闭树脂上未反应的基团。
(6)向反应器中加入适量体积比20％的哌啶溶液(哌啶/DMF＝1:4)，为树脂体积的3倍，反应20min，脱去Fmoc保护基团，脱完保护后用DMF洗涤4次，然后抽干；
(7)用长颈吸管取反应器中树脂10～20颗，用茚三酮法检测，树脂有颜色，说明脱保护成功；若不显色，重复脱保护-洗涤-检测操作。
(8)称取Fmoc-Cys(trt)-OH(C端第二个氨基酸，摩尔量为第一个氨基酸的3倍)+适量的HOBT和DIC加入到反应器中，然后将反应器置于30℃的摇床中反应1小时。
(9)用DMF洗涤树脂，然后抽干，如此重复4次，将树脂抽干。
(10)用长颈吸管取反应器中树脂10～20颗，用茚三酮法检测，若树脂有颜色，说明缩合不完全，继续反应；若树脂为无色，说明反应完全。
(11)向反应器中加入体积比20％的哌啶溶液(哌啶/DMF＝1:4)，为树脂体积的3倍，反应20min，脱去Fmoc保护基团，脱完保护后用DMF洗涤4次，然后抽干；用长颈吸管取反应器中树脂10～20颗，用茚三酮法检测，树脂有颜色，说明脱保护成功；若不显色，重复脱保护-洗涤-检测操作。
(12)按照步骤8-11依次连接剩余氨基酸以及DOTA。
(13)用切割试剂将多肽保护基团全部切除，并从树脂上切割下来，含有多肽的剪切液加入到冰乙醚中，正常情况下，多肽将以沉淀状态在冰乙醚 中沉降出来；多肽沉降后，体系在低温离心机中，离心去除上清；将沉淀以冰乙醚重悬洗涤，再次离心除去上清，洗去残留的杂质；重复操作4次后，得到目标多肽的粗产品。
(14)通过高效液相色谱仪器(HPLC)将目标肽段与杂质分离，将接取目标肽段溶液冻干成粉末，得到DOTA偶联的ASF1a肽(ASF1a Peptide，AP1)，分子量(MW)4952.62，并送QC质检。
实施例2
一种核素标记的抑制肽，所述核素标记的抑制肽通过DOTA将68Ga标记ASF1a肽；所述ASF1a肽的氨基酸序列为YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA；
所述核素标记的抑制肽的制备方法，步骤如下：
(1)用4mL浓度为0.05M盐酸淋洗68Ge-68Ga发生器(流速为0.8mL/min)，收集第2～3mL中间产物68GaCl3；
(2)将15μLDOTA偶联的ASF1a肽(浓度为1mg/mL)与1mL68GaCl3溶液(放射量为111MBq)混合，得混合液，将混合液与250μL醋酸钠(浓度为0.23M)混合，调整pH为3.8，93℃金属浴8min，得反应液；
(3)用5mL的70％乙醇逐滴活化C18小柱，再用5mL生理盐水冲洗后推10mL空气，再加入步骤(2)得到的反应液，用1mL生理盐水冲洗后去除残留水分(收集为游离放射性核素)，再用0.3mL60％乙醇冲洗C18小柱，收集产物即为纯化后标记产物。
实施例3
一种核素标记的抑制肽，所述核素标记的抑制肽通过DOTA将177Lu标记ASF1a肽；所述ASF1a肽的氨基酸序列为YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA；
所述核素标记的抑制肽的制备方法，步骤如下：
(1)将25μLDOTA偶联的ASF1a肽(浓度为1.2mg/mL)与1.1mL177LuCl3/HCl溶液(放射量为185MBq)混合，得混合液，将混合液与350μL醋酸钠(浓度为0.27M)混合，调整pH为4.2，97℃金属浴35min，得反应液；
(2)用6mL的70％乙醇逐滴活化C18小柱，再用6mL生理盐水冲洗后加入步骤(1)得到的反应液，用3mL生理盐水冲洗后去除残留水分(收集为游离放射性核素)，再用0.4mL60％乙醇冲洗C18小柱，收集产物即为纯化后标记产物。
实施例4
一种核素标记的抑制肽，所述核素标记的抑制肽通过DOTA将68Ga标记ASF1a肽；所述ASF1a肽的氨基酸序列为YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA；
所述核素标记的抑制肽的制备方法，步骤如下：
(1)用4mL浓度为0.05M盐酸淋洗68Ge-68Ga发生器(流速为1mL/min)，收集第2～3mL中间产物68GaCl3；
(2)将20μLDOTA偶联的ASF1a肽(浓度为1mg/mL)与2.05mL68GaCl3溶液(放射量为145MBq)混合，得混合液，将混合液与500μL醋酸钠(浓度为0.25M)混合，调整pH为4，95℃金属浴10min，得反应液，冷却至室温；
(3)用5mL的70％乙醇逐滴活化C18小柱，再用5mL生理盐水冲洗后推13mL空气再加入步骤(2)得到的反应液，用2mL生理盐水冲洗后去除残留水分(收集为游离放射性核素)，再用0.35mL60％乙醇冲洗C18小柱，收集产物即为纯化后标记产物。
实验例1
68Ga/177Lu标记产物鉴定及稳定性分析
(1)HPLC流动相分别是水和乙腈(各含0.1％TFA)，梯度设置为乙腈浓度从20％到50％，时长30分钟。
(2)分别在不同时间点(0、1.5、3.5、24h)取PBS中的68Ga/177Lu-AP1的相同条件进样分析如图2(68Ga/177Lu标记AP1产物的鉴定和纯化)所示(产物的放射峰主要是多肽的峰，由于68Ga半衰期只有68分钟，进样的放射量很微量，所以稳定性的数据通过半衰期更长的177Lu(6.71天)来测定)。
AP1安全性分析
(1)取对数生长期小鼠皮肤黑色素瘤B16F10细胞，用0.25％胰酶消化 调整成6×104/mL，铺96孔板，每孔100μL，细胞浓度为6×103个每孔，在37℃，5％CO2培养箱中过夜；
(2)给每组细胞换液，加入规定浓度培养液100μL，各剂量浓度为0、0.5、1、5、10、20、50，100μg/mL；24小时后更换DMEM培养基(含10％北美胎牛血清+1％双抗)继续培养；
(3)分别在更换正常培养基后的0小时，24小时加10μL CCK8溶液(10％)，继续培养箱中培养1小时；
(4)1小时后用酶标仪测量各孔450nm处吸光值。再根据测得的OD值计算细胞相对存活率。细胞活力＝(加材料组-空白)/(对照组-空白)×100％。图3.AP1在B16F10细胞中孵育24小时后24小时及48小时的生物安全性。(左图)AP1在B16F10细胞中孵育24小时后24小时细胞生长抑制率；(右图)AP1在B16F10细胞中孵育24小时后48小时细胞生长抑制率
放射性标记的68Ga-AP1显像探针的安全性
(1)取对数生长期B16F10细胞，用0.25％胰酶消化调整成6×104/mL，铺96孔板，每孔100μL，细胞浓度为6×103个每孔，在37℃，5％CO2培养箱中过夜；
(2)给每组细胞换液，加入规定浓度培养液100μL，取放射性剂量为0，1，2.5，10，25，50，100，200μCi/mL；12小时后更换正常培养基并加10μL CCK8溶液(10％)，继续培养箱中培养1小时；
(3)1小时后用酶标仪测量各孔450nm处吸光值。再根据测得的OD值计算细胞相对存活率。细胞活力＝(加材料组-空白)/(对照组-空白)×100％。图4：不同剂量的68Ga-AP1的生物安全性。
实验例2
细胞结合与抑制实验
B16F10细胞对68Ga-AP1的结合抑制实验
(1)细胞长至90％以上用0.25％胰酶消化细胞，调整细胞浓度为1×105/mL，铺24孔板，每孔加入培养基0.5mL。在37℃，5％CO2培养箱中过夜。
(2)次日细胞完全贴壁后更换加0.11MBq/mL 68Ga-AP1和不同浓度 AP1的培养基(0，0.36，3.6，36，180，360μg/mL)，每组设3个复孔。
(3)将加入放射性标记药物的培养基分别与B16F10细胞孵育1小时
(4)去除上清液用PBS清洗两次后用0.5mL 0.2M NaOH裂解孔底细胞，并用PBS清洗全部收集于放免管中。0.5mL的0.11MBq/mL 68Ga-AP1为源计数T。
(5)将每个浓度AP1的放免管测收集液体(68Ga-AP1)放射性计数B。
(6)细胞摄取率为B/T×100％，根据不同浓度AP1拟合半数抑制浓度。图5：68GaAP1与AP1竞争性结合与抑制实验。
177Lu-AP1的体外靶向性验证
(1)B16F10细胞长至90％以上用0.25％胰酶消化细胞，调整细胞浓度为1×105/mL，铺24孔板，每孔加入培养基0.5mL。在37℃，5％CO2培养箱中过夜。
(2)次日细胞完全贴壁后更换加0.11MBq/mL 177Lu-AP1和过量AP1的培养基(100μg/mL)，每组设2个复孔。
(3)将加入放射性标记药物的培养基分别与B16F10细胞作用不同的时间，每个时间点均设置抑制组。
(4)在不同的时间点分别去除上清液用PBS清洗两次后用0.5mL 0.2M NaOH裂解孔底细胞，收集后再用PBS清洗全部收集于放免管中。0.5mL的0.11MBq/mL 0.11MBq/mL 177Lu-AP1为源计数T。
(4)将每个浓度AP1的放免管测收集液体计数B总的放射性结合记为TB，加入过量AP1抑制后的摄取率非特异性结合记为NSB，特异性结合记为SB＝TB-NSB。图6：177Lu-AP1在B16F10细胞的特异性摄取与抑制。
实验例3
177Lu-AP1对B16F10细胞生长抑制效应实验
(1)取对数生长期B16F10细胞，胰酶消化调整成4×104/mL，铺96孔板，每孔100μL，细胞浓度为4×103个每孔，在37℃，5％CO2培养箱中过夜；
(2)给每组细胞换液，加入规定剂量培养液100μL，两块96孔板分别加入同等剂量浓度的177LuCl3及177Lu-AP1，各剂量浓度为0，10，100，200， 300，400，500，600μCi/mL；24小时后更换正常培养基继续培养；
(3)分别在更换正常培养基后的24小时，48小时加入含有10μL CCK8溶液(10％)的培养基，继续培养箱中培养1小时；
(4)1小时后用酶标仪测量各孔450nm处吸光值。再根据测得的OD值计算细胞相对存活率。细胞活力＝(加材料组-空白)/(对照组-空白)×100％。图7：177Lu-AP1对B16F10细胞生长抑制效应.a：孵育24小时后的24小时细胞生长抑制率；b：孵育24小时后的48小时细胞生长抑制率。
实验例4
68Ga-AP1显像探针体内靶向性及特异性的验证。
(1)取对数生长期的B16F10细胞胰酶消化重悬为1×106/mL细胞浓度，在6周龄的C57BL/6的左前肢荷瘤，每只小鼠注射细胞量为1×105。
(2)在荷瘤后第10天，给不同小鼠尾静脉注射30-100μCi 68Ga-AP1，并在给药后1.5、3.5、5.5h分别进行Micro-PET/CT静态显影10分钟(采用体积分数为3％的异氟烷-氧气混合气体预麻醉后，置于PET/CT扫描床，再采用体积分数为1.5％的异氟烷-氧气混合气体维持麻醉)。
(3)显像高表达肿瘤于次日注射相同剂量68Ga-AP1及100μgAP1进行体内摄取抑制实验，并同样在静脉注射后1、3.5、5.5h进行Micro-PET/CT静态显影10分钟，白色箭头处为肿瘤。图868Ga-AP1显像探针体内靶向性及特异性.a：对免疫治疗敏感个体PET/CT显像；b：对免疫治疗不敏感个体PET/CT显像；c：对免疫治疗不敏感的高摄取肿瘤加入AP1抑制后PET/CT显像。
实验例5
68Ga-AP1显像探针体内生物分布及血液动力学特征
(1)68Ga-AP1显像探针筛选出肿瘤ASF1a高表达组及低表达组，分别给每只小鼠尾静脉注射20μCi68Ga-AP1。注射后0.5、1.5、3.5、5.5h后解剖分离每个重要脏器。测量每个脏器重量，计算每个脏器％ID/g＝器官放射性计数/(器官重量×源计数)×100％，每个时间点3只小鼠；
(2)在尾静脉注射68Ga-AP1后分别于10分钟、20分钟、30分钟、1.5小时、3.5小时、5.5小时处死小鼠，测量小鼠血液进行器官的放射性计数， 每个时间点n＝3，取各个时间点的平均值用PKSover软件选择脉管内二室模型拟合血液动力学参数。图9：68Ga-AP1显像探针体内生物分布及血液动力学特征.a：68Ga-AP1在体内主要器官的生物分布；b：68Ga-AP1分别在ASF1a高表达及低表达肿瘤中的分布；c：68Ga-AP1在体内药代动力学特征。
实验例6
评估黑色素瘤B16F10模型中ASF1a不同表达对免疫治疗的疗效相关性
(1)建立B16F10荷瘤鼠模型，每只小鼠在左前肢荷1×105细胞数，从第4天开始，每隔3天腹腔注射BMS-1 0.1mg并测量肿瘤体积，第7天开始每隔3天做PET显像，记录肿瘤摄取/对侧肌肉摄取比值。
(2)肿瘤生长至第16天时肿瘤体积小于1000mm3视为免疫治疗有效组(免疫治疗敏感组)，比较免疫治疗敏感组与不敏感组中68Ga-AP1 PET显像摄取的相关性。图10黑色素瘤B16F10模型中ASF1a不同表达对免疫治疗的疗效相关性.a：免疫抑制剂BMS-1治疗后小鼠肿瘤体积生长曲线，紫色代表免疫抑制治疗有效组，黑色代表免疫抑制治疗无效组；b：免疫治疗不同反应组肿瘤与对侧肌肉摄取的比值
实验例7
通过早期68Ga-AP1 PET显影筛选预测免疫治疗不敏感的ASF1a高表达个体，进行177Lu-AP1靶向放射性核素治疗并评估疗效
(1)建立B16F10肿瘤鼠模型，于治疗后第7-8天进行68Ga-AP1 PET显像，筛选出ASF1a高表达个体，一组予PDL1抑制剂BMS-1，一组BMS-1联合177Lu-AP1治疗，每2-3天测量并记录肿瘤生长情况。图11：免疫治疗不敏感个体单独BMS-1治疗以及BMS-1联合177Lu-AP1治疗肿瘤体积生长曲线。
本发明以68Ge-68Ga发生器生产的68GaCl3或购买的177LuCl3以及设计的ASF1a抑制肽，建立68Ga/177Lu标记ASF1a抑制肽(AP1)的方法，评价其药理学特点以及在B16F10肿瘤模型鼠中的生物学特性，并进一步用于ASF1a靶向成像研究，同时分析成像结果于免疫治疗的相关性。对筛选的ASF1a个体进行放射性核素靶向治疗评估其疗效。临床前研究表明，68Ga-AP1标记率为81.98±7.55％，177Lu-AP1标记率为78.34±13.59％，HPLC法测定产品放化纯＞95％，而且在24小时内有良好的稳定性。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (11)

  1. 一种核素标记的抑制肽，其特征在于，所述核素标记的抑制肽通过DOTA将68Ga或177Lu标记ASF1a肽；所述ASF1a肽的氨基酸序列为YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA。
  2. 权利要求1所述核素标记的抑制肽的制备方法，其特征在于，包括如下步骤：
    (1)将DOTA偶联的ASF1a肽与核素溶液混合，得混合液，将混合液与醋酸钠混合，调整pH，水浴，得反应液；
    (2)将步骤(1)得到的反应液过色谱柱，收集产物。
  3. 根据权利要求2所述的一种核素标记的抑制肽的制备方法，其特征在于，步骤(1)所述核素溶液为68GaCl3溶液或177LuCl3/HCl溶液；所述68GaCl3溶液或177LuCl3/HCl溶液的放射量独立为111～185MBq。
  4. 根据权利要求3所述的一种核素标记的抑制肽的制备方法，其特征在于，所述68GaCl3溶液的制备方法为：用盐酸淋洗68Ge-68Ga发生器，收集中间产物68GaCl3；
    盐酸用量为4mL，所述中间产物为68Ge-68Ga发生器流出的第2～3mL。
  5. 根据权利要求4所述的一种核素标记的抑制肽的制备方法，其特征在于，所述盐酸的浓度为0.04～0.06M；所述68Ge-68Ga发生器的流速为0.8～1.2mL/min。
  6. 根据权利要求2所述的一种核素标记的抑制肽的制备方法，其特征在于，步骤(1)所述DOTA偶联的ASF1a肽的浓度为0.8～1.2mg/mL；所述DOTA偶联的ASF1a肽与核素溶液的体积比为0.01～0.03：1.00～3.00。
  7. 根据权利要求2所述的一种核素标记的抑制肽的制备方法，其特征在于，步骤(1)所述DOTA偶联的ASF1a肽与醋酸钠的体积比为15～25:250～350；所述醋酸钠的浓度为0.23～0.27M；所述调整pH为3.8～4.2。
  8. 根据权利要求2所述的一种核素标记的抑制肽的制备方法，其特征在于，步骤(1)所述水浴的温度为93～97℃；步骤(2)所述色谱柱为C18小柱。
  9. 根据权利要求3所述的一种核素标记的抑制肽的制备方法，其特征在于，当核素溶液为68GaCl3溶液时，所述水浴的时间为8～12min；当核素 溶液为177LuCl3/HCl溶液时，所述水浴的时间为25～35min。
  10. 权利要求1所述的核素标记的抑制肽、权利要求2～9任一项所述的一种核素标记的抑制肽的制备方法制备得到的核素标记的抑制肽在制备抗肿瘤药物中的应用，其特征在于，所述核素标记的抑制肽为核素177Lu标记的抑制肽；
    所述肿瘤为黑色素瘤、肺癌、肺转移癌和乳腺癌中的一种。
  11. 权利要求1所述的核素标记的抑制肽、权利要求2～9任一项所述的一种核素标记的抑制肽的制备方法制备得到的核素标记的抑制肽在制备PET/CT显像剂中的应用，其特征在于，所述核素标记的抑制肽为核素68Ga标记的抑制肽。
PCT/CN2023/081067 2022-05-26 2023-03-13 一种核素标记的抑制肽及其制备方法和应用 WO2023226530A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210578560.XA CN114874308B (zh) 2022-05-26 2022-05-26 一种核素标记的抑制肽及其制备方法和应用
CN202210578560.X 2022-05-26

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/510,683 Continuation US20240131207A1 (en) 2022-05-26 2023-11-16 Nuclide-labeled inhibitory peptide, and preparation method therefor and use thereof

Publications (1)

Publication Number Publication Date
WO2023226530A1 true WO2023226530A1 (zh) 2023-11-30

Family

ID=82677304

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/081067 WO2023226530A1 (zh) 2022-05-26 2023-03-13 一种核素标记的抑制肽及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN114874308B (zh)
WO (1) WO2023226530A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114874308B (zh) * 2022-05-26 2023-02-17 苏州大学 一种核素标记的抑制肽及其制备方法和应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150025016A1 (en) * 2011-11-18 2015-01-22 Commissariat A L'energie Atomique Et Aux Energies Alternatives Peptides that inhibit the interaction between asf1 and histones, and use thereof
CN104491890A (zh) * 2014-11-21 2015-04-08 北京肿瘤医院 放射性核素标记的新型生长抑素类似物分子探针及其应用
US20150132219A1 (en) * 2012-05-08 2015-05-14 Rigshospitalet 177-Lu LABELED PEPTIDE FOR SITE-SPECIFIC uPAR-TARGETING
CN107021998A (zh) * 2017-04-24 2017-08-08 北京大学第医院 一种用于肿瘤显像的正电子核素标记多肽
WO2021238842A1 (zh) * 2020-05-27 2021-12-02 北京市肿瘤防治研究所 Her2 亲和体和诊疗核素标记物及其制备方法与应用
WO2022021528A1 (zh) * 2020-07-28 2022-02-03 北京肿瘤医院(北京大学肿瘤医院) 一种ace2受体靶向核素多肽探针及其制备方法和应用
CN114874308A (zh) * 2022-05-26 2022-08-09 苏州大学 一种核素标记的抑制肽及其制备方法和应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4022094A4 (en) * 2019-08-26 2022-11-30 Liquid Lung DX BIOMARKERS FOR DIAGNOSIS OF LUNG CANCER
KR20230044463A (ko) * 2020-07-31 2023-04-04 폴 슈레 앙스띠뛰 갈륨-표지된 가스트린 유사체 및 cckb 수용체 양성 종양 또는 암의 영상화 방법에서의 용도
CN112079900B (zh) * 2020-09-21 2023-04-07 中国工程物理研究院核物理与化学研究所 一种环状ngr多肽、放射性核素标记分子探针及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150025016A1 (en) * 2011-11-18 2015-01-22 Commissariat A L'energie Atomique Et Aux Energies Alternatives Peptides that inhibit the interaction between asf1 and histones, and use thereof
US20150132219A1 (en) * 2012-05-08 2015-05-14 Rigshospitalet 177-Lu LABELED PEPTIDE FOR SITE-SPECIFIC uPAR-TARGETING
CN104491890A (zh) * 2014-11-21 2015-04-08 北京肿瘤医院 放射性核素标记的新型生长抑素类似物分子探针及其应用
CN107021998A (zh) * 2017-04-24 2017-08-08 北京大学第医院 一种用于肿瘤显像的正电子核素标记多肽
WO2021238842A1 (zh) * 2020-05-27 2021-12-02 北京市肿瘤防治研究所 Her2 亲和体和诊疗核素标记物及其制备方法与应用
WO2022021528A1 (zh) * 2020-07-28 2022-02-03 北京肿瘤医院(北京大学肿瘤医院) 一种ace2受体靶向核素多肽探针及其制备方法和应用
CN114874308A (zh) * 2022-05-26 2022-08-09 苏州大学 一种核素标记的抑制肽及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JIANG LI-SHA; WANG XIN-LU; YIN JI-LIN; WANG CHENG;: "68ga Labeled Somatostatin Analogues for Tumor Imaging", JOURNAL OF CHINA CLINIC MEDICAL IMAGING, vol. 26, no. 05, 20 May 2015 (2015-05-20), pages 365 - 368, XP009550619, ISSN: 1008-1062 *

Also Published As

Publication number Publication date
CN114874308B (zh) 2023-02-17
CN114874308A (zh) 2022-08-09

Similar Documents

Publication Publication Date Title
Mansur et al. Cell cycle regulation of the glyceraldehyde3phosphate dehydrogenaseluracil DNA glycosylase gene in normal human cells
Lu et al. Mutated PPP1R3B is recognized by T cells used to treat a melanoma patient who experienced a durable complete tumor regression
Banerjee et al. A modular strategy to prepare multivalent inhibitors of prostate-specific membrane antigen (PSMA)
WO2023226530A1 (zh) 一种核素标记的抑制肽及其制备方法和应用
Li et al. Effect and mechanism of LRP6 on cardiac myocyte ferroptosis in myocardial infarction
Bonnet‐Duquennoy et al. Targeted radionuclide therapy of melanoma: anti‐tumoural efficacy studies of a new 131I labelled potential agent
Tian et al. Noninvasive Molecular Imaging of MYC mRNA Expression in Human Breast Cancer Xenografts with a [99mTc] Peptide− Peptide Nucleic Acid− Peptide Chimera
Ren et al. Radiosynthesis of a novel antisense imaging probe targeting LncRNA HOTAIR in malignant glioma
La Lee et al. Combined radionuclide–chemotherapy and in vivo imaging of hepatocellular carcinoma cells after transfection of a triple-gene construct, NIS, HSV1-sr39tk, and EGFP
CN111320675A (zh) 放射性锝标记的pd-l1靶向多肽及其制备方法与应用
JP5796008B2 (ja) 壊死マーカーＥＲｐ２９に対するモノクローナル抗体及びその用途
Price et al. Metastatic potential of cloned murine melanoma cells transfected with activated c-Ha-ras
CN108472394A (zh) 用于检测、诊断和治疗胰腺癌的受体结合结构域配体
US20240131207A1 (en) Nuclide-labeled inhibitory peptide, and preparation method therefor and use thereof
Li et al. Development of a novel radiofluorinated riboflavin probe for riboflavin receptor-targeting PET imaging
Yan et al. Identification of a glypican-3 binding peptide from a phage-displayed peptide library for PET imaging of hepatocellular carcinoma
US20050123981A1 (en) Method of diagnosing, monitoring, staging, imaging and treating gastrointestinal cancer
Guan et al. Genetic and functional analyses of the novel KLF11 Pro193Thr variant in a three-generation family with MODY7
Weng et al. Peptide-based PET imaging agent of tumor TIGIT expression
CN109862903A (zh) 治疗性sall4肽
Yamasaki et al. Imaging of peripheral-type benzodiazepine receptor in tumor: in vitro binding and in vivo biodistribution of N-benzyl-N-[11C] methyl-2-(7-methyl-8-oxo-2-phenyl-7, 8-dihydro-9H-purin-9-yl) acetamide
Hu et al. Radiosynthesis, optimization and pharmacokinetic study of the 99mTc-labeled human epidermal growth factor receptor 2 affibody molecule probe 99mTc-(HE) 3ZHER2: V2
Prante et al. Characterization of uptake of 3-[131I] iodo-α-methyl-L-tyrosine in human monocyte-macrophages
Sharma et al. Design, synthesis and evaluation of 177Lu-labeled inverso and retro-inverso A9 peptide variants targeting HER2-overexpression
Shikano et al. Radiolabeled Molecular Imaging Agents for Amino Acid Transport in Tumors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23810605

Country of ref document: EP

Kind code of ref document: A1