NUCLIDE-LABELED INHIBITORY PEPTIDE, AND PREPARATION METHOD THEREFOR AND USE THEREOF

Abstract

A nuclide-labeled inhibitory peptide, and a preparation method therefor and use thereof are provided. The nuclide-labeled inhibitory peptide is prepared by labeling ASF1a peptide with .sup.68Ga/.sup.177Lu by DOTA, and an amino acid sequence of the ASF1a peptide is YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA (as shown in SEQ ID NO: 1). The .sup.68Ga labeled ASF1a inhibitory peptide of the present invention displays the expression level of ASF1a of a tumor through PET/CT imaging, has good imaging sensitivity, can specifically screen high-expression and low-expression individuals, and achieves noninvasive prediction of the efficacy of tumor immunotherapy. The .sup.177Lu-labeled ASF1a inhibitory peptide provides a novel and effective therapeutic strategy for a tumor that highly expresses ASF1a and is not effective for immunotherapy.

Claims

1. A nuclide-labeled inhibitory peptide, prepared by labeling an ASF1a peptide with .sup.68Ga or .sup.177Lu by 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), wherein an amino acid sequence of the ASF1a peptide is YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA, as shown in SEQ ID NO: 1.

2. A preparation method for the nuclide-labeled inhibitory peptide according to claim 1, comprising the following steps: (1) mixing a DOTA-coupled ASF1a peptide with a nuclide solution to obtain a mixed solution, mixing the mixed solution with sodium acetate to obtain a resulting solution, adjusting a pH of the resulting solution, and placing the resulting solution in a water bath to obtain a reaction solution; (2) enabling the reaction solution obtained in step (1) to pass through a chromatography column, and collecting a product.

3. The preparation method for the nuclide-labeled inhibitory peptide according to claim 2, wherein the nuclide solution in step (1) is a .sup.68GaCl.sub.3 solution or a .sup.177LuCl.sub.3/HCl solution, and a radiation amount of the .sup.68GaCl.sub.3 solution or the .sup.177LuCl.sub.3/HCl solution is independently 111-185 MBq.

4. The preparation method for the nuclide-labeled inhibitory peptide according to claim 3, wherein a preparation method for the .sup.68GaCl.sub.3 solution comprises: rinsing a .sup.68Ge.sup.68Ga generator by hydrochloric acid, and collecting an intermediate product .sup.68GaCl.sub.3; wherein an amount of the hydrochloric acid is 4 mL, and the intermediate product is .sup.68GaCl.sub.3, and the .sup.68GaCl.sub.3 is 2.sup.nd to 3.sup.rd mL flowing out of the .sup.68Ge.sup.68Ga generator.

5. The preparation method for the nuclide-labeled inhibitory peptide according to claim 4, wherein the hydrochloric acid has a concentration of 0.04-0.06 M, and the .sup.68Ge.sup.68Ga generator has a flow rate of 0.8-1.2 mL/min.

6. The preparation method for the nuclide-labeled inhibitory peptide according to claim 2, wherein the DOTA-coupled ASF1a peptide in step (1) has a concentration of 0.8-1.2 mg/mL, and a volume ratio of the DOTA-coupled ASF1a peptide to the nuclide solution is (0.01-0.03):(1.00-3.00).

7. The preparation method for the nuclide-labeled inhibitory peptide according to claim 2, wherein a volume ratio of the DOTA-coupled ASF1a peptide to the sodium acetate in step (1) is (15-25):(250-350), the sodium acetate has a concentration of 0.23-0.27 M, and the pH is adjusted to 3.8-4.2.

8. The preparation method for the nuclide-labeled inhibitory peptide according to claim 2, wherein the water bath in step (1) has a temperature of 93-97? C., and the chromatography column in step (2) is a C18 small column.

9. The preparation method for the nuclide-labeled inhibitory peptide according to claim 3, wherein when the nuclide solution is the .sup.68GaCl.sub.3 solution, the water bath is performed for 8-12 min, and when the nuclide solution is the .sup.177LuCl.sub.3/HCl solution, the water bath is performed for 25-35 min.

10. A method for preparing an anti-tumor drug, comprising: using the nuclide-labeled inhibitory peptide according to claim 1, wherein the nuclide-labeled inhibitory peptide is a nuclide .sup.177Lu-labeled inhibitory peptide, and a tumor is one of melanoma, lung cancer, lung metastatic cancer, and breast cancer.

11. A method for preparing a PET/CT imaging agent, comprising: using the nuclide-labeled inhibitory peptide according to claim 1, wherein the nuclide-labeled inhibitory peptide is a nuclide .sup.68Ga-labeled inhibitory peptide.

12. A method for preparing an anti-tumor drug, comprising: using a nuclide-labeled inhibitory peptide prepared by the preparation method for the nuclide-labeled inhibitory peptide according to claim 2, wherein the nuclide-labeled inhibitory peptide is a nuclide .sup.177Lu-labeled inhibitory peptide, and a tumor is one of melanoma, lung cancer, lung metastatic cancer, and breast cancer.

13. The method for preparing the anti-tumor drug according to claim 12, wherein in the preparation method for the nuclide-labeled inhibitory peptide, the nuclide solution in step (1) is a .sup.68GaCl.sub.3 solution or a .sup.177LuCl.sub.3/HCl solution, and a radiation amount of the .sup.68GaCl.sub.3 solution or the .sup.177LuCl.sub.3/HCl solution is independently 111-185 MBq.

14. The method for preparing the anti-tumor drug according to claim 13, wherein a preparation method for the .sup.68GaCl.sub.3 solution comprises: rinsing a .sup.68Ge.sup.68Ga generator by hydrochloric acid, and collecting an intermediate product .sup.68GaCl.sub.3; wherein an amount of the hydrochloric acid is 4 mL, and the intermediate product is .sup.68GaCl.sub.3, and the .sup.68GaCl.sub.3 is 2.sup.nd to 3.sup.rd mL flowing out of the .sup.68Ge.sup.68Ga generator.

15. The method for preparing the anti-tumor drug according to claim 14, wherein the hydrochloric acid has a concentration of 0.04-0.06 M, and the .sup.68Ge.sup.68Ga generator has a flow rate of 0.8-1.2 mL/min.

16. The method for preparing the anti-tumor drug according to claim 12, wherein in the preparation method for the nuclide-labeled inhibitory peptide, the DOTA-coupled ASF1a peptide in step (1) has a concentration of 0.8-1.2 mg/mL, and a volume ratio of the DOTA-coupled ASF1a peptide to the nuclide solution is (0.01-0.03):(1.00-3.00).

17. The method for preparing the anti-tumor drug according to claim 12, wherein in the preparation method for the nuclide-labeled inhibitory peptide, a volume ratio of the DOTA-coupled ASF1a peptide to the sodium acetate in step (1) is (15-25):(250-350), the sodium acetate has a concentration of 0.23-0.27 M, and the pH is adjusted to 3.8-4.2.

18. The method for preparing the anti-tumor drug according to claim 12, wherein in the preparation method for the nuclide-labeled inhibitory peptide, the water bath in step (1) has a temperature of 93-97? C., and the chromatography column in step (2) is a C18 small column.

19. The method for preparing the anti-tumor drug according to claim 13, wherein when the nuclide solution is the .sup.68GaCl.sub.3 solution, the water bath is performed for 8-12 min, and when the nuclide solution is the .sup.177LuCl.sub.3/HCl solution, the water bath is performed for 25-35 min.

20. A method for preparing a PET/CT imaging agent, comprising: using a nuclide-labeled inhibitory peptide prepared by the preparation method for the nuclide-labeled inhibitory peptide according to claim 2, wherein the nuclide-labeled inhibitory peptide is a nuclide .sup.68Ga-labeled inhibitory peptide.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the drawings required to be used in the description of the embodiments or the prior art are briefly introduced below. It is obvious that the drawings in the description below are merely embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings according to the drawings provided without creative efforts.

[0033] FIG. 1 is a schematic diagram of a .sup.68Ga/.sup.177Lu-labeled AP1 polypeptide;

[0034] FIG. 2 is a diagram of identification and purification of a .sup.68Ga/.sup.177Lu-labeled AP1 product;

[0035] FIGS. 3A-3B show biological safety of AP1 at 24 hours and 48 hours after incubation in B16F10 cells for 24 hours, where FIG. 3A shows the inhibition of cell growth at 24 hours after incubation of AP1 in B16F10 cells for 24 hours, and FIG. 3B shows the inhibition rate of cell growth at 48 hours after incubation of AP1 in B16F10 cells for 24 hours;

[0036] FIG. 4 shows biological safety of .sup.68Ga-AP1 at different doses;

[0037] FIGS. 5A-5B show a competitive binding and inhibition experiment between .sup.68GaAP1 and AP1;

[0038] FIG. 6 shows a specific uptake and inhibition of .sup.177Lu-AP1 in B16F10 cells;

[0039] FIGS. 7A-7B show inhibitory effect of .sup.177Lu-AP1 on B16F10 cell growth, where FIG. 7A shows cell growth inhibition rate at 24 hours after incubation for 24 hours, and FIG. 7B shows cell growth inhibition rate at 48 hours after incubation for 24 hours;

[0040] FIGS. 8A-8C show in vivo targeting and specificity of a .sup.68Ga-AP1 imaging probe, where FIG. 8A shows PET/CT imaging of an individual that is sensitive to immunotherapy; FIG. 8B shows PET/CT imaging of an individual that is not insensitive to immunotherapy, and FIG. 8C shows PET/CT imaging of a high-uptake tumor that is insensitive to immunotherapy after addition of AP1 inhibition;

[0041] FIGS. 9A-9C show in vivo biodistribution and hemodynamic characteristics of FIG. 9A .sup.68Ga-AP1 imaging probe, where a shows biodistribution of .sup.68Ga-AP1 in major organs in a body, FIG. 9B shows distributions of .sup.68Ga-AP1 in ASF1a high-expression and low-expression tumors, respectively, and FIG. 9C shows pharmacokinetics of .sup.68Ga-AP1 in vivo;

[0042] FIGS. 10A-10B show the correlation between different expressions of ASF1a and the efficacy of immunotherapy in a melanoma B16F10 model, where FIG. 10A shows a tumor volume growth curve of mice after treatment with the immunosuppressant BMS-1, purple represents an effective immunosuppressive treatment group, and black represents an ineffective immunosuppressive treatment group; FIG. 10B shows ratios of the tumors to contralateral muscle uptake in different response groups of immunotherapy;

[0043] FIG. 11 shows tumor volume growth curves of immunotherapy-insensitive individuals treated with BMS-1 alone and BMS-1 combined with .sup.177Lu-AP1.

DETAILED DESCRIPTION OF EMBODIMENTS

[0044] The present invention provides a nuclide-labeled inhibitory peptide prepared by labeling ASF1a peptide with .sup.68Ga or .sup.177Lu by DOTA, wherein the ASF1a peptide has an amino acid sequence of YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA (as shown in SEQ ID NO: 1) (ASF1a Peptide, AP1) and a molecular weight (MW) of 4952.62. The nuclide-labeled position is a position represented by in the schematic diagram 1 on the tetraazacyclo ring of DOTA.

[0045] The present invention further provides a preparation method for the nuclide-labeled inhibitory peptide, comprising the following steps: [0046] (1) mixing the DOTA-coupled ASF1a peptide with a nuclide solution to obtain a mixed solution, mixing the mixed solution with sodium acetate, adjusting pH, and placing in a water bath to obtain a reaction solution; [0047] (2) enabling the reaction solution obtained in the step (1) to pass through a chromatography column, and collecting a product.

[0048] In the present invention, the nuclide solution in the step (1) is a .sup.68GaCl.sub.3 solution or a .sup.177LuCl.sub.3/HCl solution, preferably a .sup.68GaCl.sub.3 solution.

[0049] In the present invention, a radiation amount of the .sup.68GaCl.sub.3 solution or the .sup.177LuCl.sub.3/HCl solution in the step (1) is independently 111-185 MBq, preferably 121-175 MBq, further preferably 131-165 MBq, and more preferably 145 MBq.

[0050] In the present invention, a preparation method for the .sup.68GaCl.sub.3 solution comprises: rinsing a .sup.68Ge.sup.68Ga generator by hydrochloric acid, and collecting an intermediate product .sup.68GaCl.sub.3; wherein an amount of the hydrochloric acid is 4 mL, and the intermediate product is .sup.68GaCl.sub.3 that is 2.sup.nd to 3.sup.rd mL flowing out of the .sup.68Ge.sup.68Ga generator.

[0051] In the present invention, the hydrochloric acid has a concentration of 0.04-0.06 M, preferably 0.05 M.

[0052] In the present invention, the flow rate of the .sup.68Ge.sup.68Ga generator is 0.8-1.2 mL/min, preferably 0.9-1.1 mL/min, and further preferably 1 mL/min.

[0053] In the present invention, the DOTA-coupled ASF1a peptide in the step (1) has a concentration of 0.8-1.2 mg/mL, preferably 0.9-1.1 mg/mL, and further preferably 1 mg/mL.

[0054] In the present invention, a volume ratio of the DOTA-coupled ASF1a peptide to the nuclide solution in the step (1) is (0.01-0.03):(1.00-3.00), preferably 0.02:(1.0-2.5), and further preferably 0.02:2.05.

[0055] In the present invention, a volume ratio of the DOTA-coupled ASF1a peptide to the sodium acetate in the step (1) is (15-25):(250-350), preferably (17-23):(270-330), further preferably (19-21):(290-310), and more preferably 20:300.

[0056] In the present invention, the sodium acetate in the step (1) has a concentration of 0.23-0.27 M, preferably 0.24-0.26 M, and further preferably 0.25 M.

[0057] In the present invention, the pH in the step (1) is adjusted to 3.8-4.2, preferably 3.9-4.1, and further preferably 4.0.

[0058] In the present invention, the water bath in step (1) has a temperature of 93-97? C., preferably 94-96? C., and further preferably 95? C.

[0059] In the present invention, the chromatography column in the step (1) is a C18 small column.

[0060] In the present invention, when the nuclide solution is a .sup.68GaCl.sub.3 solution, the water bath is performed for 8-12 min, preferably 9-11 min, and further preferably 10 min.

[0061] In the present invention, when the nuclide solution is a .sup.177LuCl.sub.3/HCl solution, the water bath is performed for 25-35 min, preferably 27-33 min, further preferably 29-31 min, and more preferably 30 min.

[0062] The present invention further provides use of the nuclide-labeled inhibitory peptide or a nuclide-labeled inhibitory peptide prepared by the preparation method for a nuclide-labeled inhibitory peptide in preparing a PET/CT imaging agent, wherein the tumor is one of melanoma, lung cancer, lung metastatic cancer, and breast cancer, preferably melanoma.

[0063] The technical solutions provided by the present invention will be described in detail below with reference to the examples, which, however, should not be construed as limiting the scope of the present invention.

Main Instruments or Equipment

[0064]

TABLE-US-00001 PET/CT SIEMENS, Germany .sup.68Ge.sup.68Ga generator ITG, Germany Multifunctional microplate reader Bio-Tek, USA 2480 WIZARD2 Automatic Gamma Counter PerkinElmer, USA High performance liquid chromatograph Shimadzu, Japan LC-20AT

Reagents and Consumables

[0065]

TABLE-US-00002 BMS-1 MCE, China CCK8 kit Beyotime, China C18 column Sep-Pak, Ireland 0.05M HCl, 528 ?L of 30% HCl was diluted to a volume of 100 mL by adding ultrapure water 0.25M sodium acetate (NaOAc), 2.05 g of anhydrous sodium acetate powder was weighed and dissolved in 100 mL of ultrapure water, and dissolved by ultrasonic

EXAMPLE 1

Synthesis of ASF1a Peptide, AP1

[0066] (1) Estimating a feeding amount of each amino acid according to the weight and molecular weight of the target polypeptide, wherein each amino acid and DOTA use protected raw materials. [0067] (2) Placing 2-Cl(Trt)-Cl resin into a 150 mL reactor, and adding 80 mL of DCM for soaking for 2 h. [0068] (3) Washing the resin with DMF and then draining, and repeating 4 times to drain the resin. [0069] (4) Weighing Fmoc-Ala-OH (the first amino acid at the C terminal, CAS No. 154445-77-9)+80 mL of DCM and DIEA and adding into a reactor, and then placing the reactor into a shaker at 30? C. for reaction for 2 h. [0070] (5) Adding 0.5 mL of DIEA and 0.5 mL of methanol through a pipette for reaction for 20 min, and blocking unreacted groups on the resin. [0071] (6) Adding an appropriate amount of piperidine solution (piperidine/DMF=1:4) in a volume ratio of 20% which is 3 times of the volume of the resin into a reactor, reacting for 20 min, removing an Fmoc protecting group, washing with DMF 4 times after the protection is removed, and then draining; [0072] (7) Taking 10-20 resins in the reactor by using a long-neck pipette, detecting by using a ninhydrin method, wherein if the resins are colored, indicating that deprotection is successful; if no color is developed, repeating the deprotection-washing-detection operation. [0073] (8) Weighing Fmoc-Cys(trt)-OH (the second amino acid at the C-terminal, with a molar amount that is 3 times that of the first amino acid) and an appropriate amount of HOBT and DIC and adding to a reactor, and then placing the reactor into a shaker at 30? C. for reaction for 1 h. [0074] (9) Washing the resin with DMF and then draining, and repeating 4 times to drain the resin. [0075] (10) Taking 10-20 resins in the reactor by using a long-neck pipette, detecting by using a ninhydrin method, wherein if the resins are colored, indicating that condensation is incomplete, continuing to react; if the resins are colorless, indicating that the reaction is complete. [0076] (11) Adding a piperidine solution (piperidine/DMF=1:4) in a volume ratio of 20% which is 3 times of the volume of the resin into a reactor, reacting for 20 min, removing an Fmoc protecting group, washing with DMF 4 times after the protection is removed, and then draining; taking 10-20 resins in the reactor by using a long-neck pipette, detecting by using a ninhydrin method, wherein if the resins are colored, indicating that deprotection is successful; if no color is developed, repeating the deprotection-washing-detection operation. [0077] (12) Linking the remaining amino acids and DOTA in sequence according to the steps 8-11. [0078] (13) Cutting off all the polypeptide protecting groups by using a cutting reagent, cutting off the polypeptide protecting groups from the resin, adding a cutting fluid containing the polypeptide into the glacial ethyl ether, and normally settling the polypeptide in the glacial ethyl ether in a precipitation state; after the polypeptide is settled, centrifuging the system in a low-temperature centrifuge to remove a supernatant; resuspending and washing the precipitate with glacial ethyl ether, centrifuging again to remove the supernatant, and washing away residual impurities; repeating the operations 4 times to obtain a crude product of the target polypeptide. [0079] (14) Separating the target peptide fragment from impurities by a high performance liquid chromatograph (HPLC), freeze-drying the inoculated target peptide fragment solution into powder to obtain the DOTA-coupled ASF1a peptide (ASF1a Peptide, AP1) with a molecular weight (MW) of 4952.62, and sending the peptide to QC quality inspection.

EXAMPLE 2

[0080] A nuclide-labeled inhibitory peptide is prepared by labeling ASF1a peptide with .sup.68Ga by DOTA, wherein an amino acid sequence of the ASF1a peptide is YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA (as shown in SEQ ID NO: 1).

[0081] A preparation method for the nuclide-labeled inhibitory peptide comprises the following steps: [0082] (1) rinsing a .sup.68Ge.sup.68Ga generator with 4 mL of hydrochloric acid at a concentration of 0.05 M (with a flow rate of 0.8 mL/min), and collecting an intermediate product .sup.68GaCl.sub.3 of the 2.sup.nd to 3.sup.rd mL; [0083] (2) mixing 15 ?L of DOTA-coupled ASF1a peptide (with a concentration of 1 mg/mL) with 1 mL of a .sup.68GaCl.sub.3 solution (with a radiation amount of 111 MBq) to obtain a mixed solution, mixing the mixed solution with 250 ?L of sodium acetate (with a concentration of 0.23 M), adjusting the pH to 3.8, and placing in a metal bath at 93? C. for 8 min to obtain a reaction solution; [0084] (3) dropwise activating the C18 small column by using 5 mL of 70% ethanol, washing by using 5 mL of normal saline, then pushing 10 mL of air, adding the reaction solution obtained in the step (2), washing by using 1 mL of normal saline to remove residual water (collected as free radionuclide), washing the C18 small column by using 0.3 mL of 60% ethanol, and collecting a product, namely the purified labeled product.

EXAMPLE 3

[0085] A nuclide-labeled inhibitory peptide is prepared by labeling ASF1a peptide with .sup.177Lu by DOTA, wherein an amino acid sequence of the ASF1a peptide is YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA (as shown in SEQ ID NO: 1).

[0086] A preparation method for the nuclide-labeled inhibitory peptide comprises the following steps: [0087] (1) mixing 25 ?L of DOTA-coupled ASF1a peptide (with a concentration of 1.2 mg/mL) with 1.1 mL of a .sup.177LuCl.sub.3/HCl solution (with a radiation amount of 185 MBq) to obtain a mixed solution, mixing the mixed solution with 350 ?L of sodium acetate (with a concentration of 0.27 M), adjusting the pH to 4.2, and placing in a metal bath at 97? C. for 35 min to obtain a reaction solution; [0088] (2) dropwise activating the C18 small column by using 6 mL of 70% ethanol, washing by using 6 mL of normal saline, adding the reaction solution obtained in the step (1), washing by using 3 mL of normal saline to remove residual water (collected as free radionuclide), washing the C18 small column by using 0.4 mL of 60% ethanol, and collecting a product, namely the purified labeled product.

EXAMPLE 4

[0089] A nuclide-labeled inhibitory peptide is prepared by labeling ASF1a peptide with .sup.68Ga by DOTA, wherein an amino acid sequence of the ASF1a peptide is YGRKKRRQRRRCASTEEKWARLARRIAGAGGVTLDGFGGCA (as shown in SEQ ID NO: 1).

[0090] A preparation method for the nuclide-labeled inhibitory peptide comprises the following steps: [0091] (1) rinsing a .sup.68Ge.sup.68Ga generator with 4 mL of hydrochloric acid at a concentration of 0.05 M (with a flow rate of 1 mL/min), and collecting an intermediate product .sup.68GaCl.sub.3 of the 2.sup.nd to 3.sup.rd mL; [0092] (2) mixing 20 ?L of DOTA-coupled ASF1a peptide (with a concentration of 1 mg/mL) with 2.05 mL of a .sup.68GaCl.sub.3 solution (with a radiation amount of 145 MBq) to obtain a mixed solution, mixing the mixed solution with 500 ?L of sodium acetate (with a concentration of 0.25 M), adjusting the pH to 4, placing in a metal bath at 95? C. for 10 min to obtain a reaction solution, and cooling to room temperature; [0093] (3) dropwise activating the C18 small column by using 5 mL of 70% ethanol, washing by using 5 mL of normal saline, then pushing 13 mL of air, adding the reaction solution obtained in the step (2), washing by using 2 mL of normal saline to remove residual water (collected as free radionuclide), washing the C18 small column by using 0.35 mL of 60% ethanol, and collecting a product, namely the purified labeled product.

EXPERIMENTAL EXAMPLE 1

Identification and Stability Analysis of .SUP.68.Ga/.SUP.177.Lu Labeled Product

[0094] (1) The HPLC mobile phases were water and acetonitrile (each containing 0.1% TFA), and the gradient was set from 20% to 50% acetonitrile concentration over a period of 30 minutes. [0095] (2) The injection analysis of .sup.68Ga/.sup.177Lu-AP1 in PBS at different time points (0, 1.5, 3.5, and 24 h) under the same condition is shown in FIG. 2 (identification and purification of .sup.68Ga/.sup.177Lu-labeled AP1 product) (The radiation peak of the product is mainly the peak of the polypeptide. Since the half-life of .sup.68Ga is only 68 minutes, and the amount of radiation injected is very small, the stability data is determined by .sup.177Lu (6.71 days) with a longer half-life).

AP1 Safety Analysis

[0096] (1) The skin melanoma B16F10 cells of mice in the logarithmic growth phase were taken and digested by using 0.25% pancreatin, and the cell concentration was adjusted to 6?10.sup.4/mL; the cells were plated in a 96-well plate at a volume of 100 ?L per well, the cell concentration was 6?10.sup.3 cells per well, and the cells were incubated overnight in an incubator at 37? C./5% CO.sub.2; [0097] (2) the medium was replaced for each group of cells, and 100 ?L of a culture solution with a specified concentration was added, wherein the concentration of each dose was 0, 0.5, 1, 5, 10, 20, 50 and 100 ?g/mL; after 24 h, a DMEM medium (containing 10% of North American fetal bovine serum and 1% of double antibody) was used, and the culture was continued; [0098] (3) 10 ?L of CCK8 solution (10%) was added at 0 h and 24 h after the normal culture medium was used, and the culture was continued in the incubator for 1 h; [0099] (4) after 1 h, the absorbance at 450 nm of each well was measured using a microplate reader. The relative survival rate of the cells was calculated according to the measured OD value. Cell viability=(material added group?blank group)/(control group?blank group)?100%. FIGS. 3A-3B show biological safety of AP1 at 24 h and 48 h after incubation in B16F10 cells for 24 h. FIG. 3A shows the inhibition of cell growth at 24 h after incubation of AP1 in B16F10 cells for 24 h, and FIG. 3B shows the inhibition rate of cell growth at 48 h after incubation of AP1 in B16F10 cells for 24 h.

[0100] Safety of radiolabeled .sup.68Ga-AP1 imaging probe [0101] (1) The B16F10 cells of mice in the logarithmic growth phase were taken and digested by using 0.25% pancreatin, and the cell concentration was adjusted to 6?10.sup.4 cells/mL; the cells were plated in a 96-well plate at a volume of 100 ?L per well, the cell concentration was 6?10.sup.3 cells per well, and the cells were incubated overnight in an incubator at 37? C./5% CO.sub.2; [0102] (2) the medium was replaced for each group of cells, 100 ?L of a culture solution with a specified concentration was added, and the radioactive doses were taken as 0, 1, 2.5, 10, 25, 50, 100, and 200 ?Ci/mL; after 12 h, the normal culture medium was used and 10 ?L of CCK8 solution (10%) was added, and the culture was continued in an incubator for 1 h; [0103] (3) after 1 h, the absorbance at 450 nm of each well was measured using a microplate reader. The relative survival rate of the cells was calculated according to the measured OD value. Cell viability=(material added group?blank group)/(control group?blank group)?100%. FIG. 4 shows biological safety of .sup.68Ga-AP1 at different doses.

EXPERIMENTAL EXAMPLE 2

Cell Binding and Inhibition Assay

Binding and Inhibition Assay of B16F10 Cells to .SUP.68.Ga-AP1

[0104] (1) When the cells grew to more than 90%, the cells were digested with 0.25% pancreatin, the cell concentration was adjusted to 1?10.sup.5 cells/mL, the cells were plated in a 24-well plate, and 0.5 mL of culture medium was added to each well. The cells were incubated overnight in an incubator at 37? C./5% CO.sub.2. [0105] (2) On the next day, after the cells were completely adhered to the wall, the culture medium added with 0.11 MBq/mL .sup.68Ga-AP1 and different concentrations of AP1 (0, 0.36, 3.6, 36, 180, and 360 ?g/mL) was used, and 3 replicate wells were set up in each group. [0106] (3) The culture medium added with the radiolabeled drug was incubated with B16F10 cells for 1 h. [0107] (4) After the supernatant was removed and the cells were washed twice with PBS, the cells at the bottom of the well were lysed with 0.5 mL of 0.2 M NaOH and washed with PBS, all cells were collected in a radioimmunotube. 0.5 mL of 0.11 MBq/mL .sup.68Ga-AP1 was the source count T. [0108] (5) The radioactivity of the liquid (.sup.68Ga-AP1) detected and collected by the radioimmunotube of each concentration of AP1 was denoted as count B. [0109] (6) The cellular uptake was B/T?100%, and the half inhibitory concentrations were fitted based on different concentrations of AP1. FIGS. 5A-5B show a competitive binding and inhibition experiment between .sup.68GaAP1 and AP1.

In Vitro Targeting Verification of .SUP.177.Lu-AP1

[0110] (1) When the B16F10 cells grew to more than 90%, the cells were digested with 0.25% pancreatin, the cell concentration was adjusted to 1?10.sup.5 cells/mL, the cells were plated in a 24-well plate, and 0.5 mL of culture medium was added to each well. The cells were incubated overnight in an incubator at 37? C./5% CO.sub.2. [0111] (2) On the next day, after the cells were completely adhered to the wall, the culture medium added with 0.11 MBq/mL .sup.177Lu-AP1 and excessive AP1 (100 ?g/mL) was used, and 2 replicate wells were set up in each group. [0112] (3) The medium added with the radiolabeled drug was allowed to act on B16F10 cells for different periods of time, and an inhibition group was set for each time point. [0113] (4) At different time points, the supernatants were removed, the cells were washed twice with PBS, the cells at the bottom of the wells were lysed with 0.5 mL of 0.2 M NaOH, collected and washed with PBS, and all cells were collected in a radioimmunotube. 0.5 mL of 0.11 MBq/mL of .sup.177Lu-AP1 was the source count T. [0114] (4) The total radioactive binding of the liquid (count B) detected and collected by the radioimmunotube of each concentration of AP1 was denoted as TB, the non-specific binding of the uptake rate inhibited by the addition of excess AP1 was denoted as NSB, and the specific binding was denoted as SB=TB?NSB. FIG. 6 shows a specific uptake and inhibition of .sup.177Lu-AP1 in B16F10 cells.

EXPERIMENTAL EXAMPLE 3

Experiment of Growth Inhibitory Effect of .SUP.177.Lu-AP1 on B16F10 Cells

[0115] (1) The B16F10 cells of mice in the logarithmic growth phase were taken and digested by using pancreatin, and the cell concentration was adjusted to 4?10.sup.4 cells/mL; the cells were plated in a 96-well plate at a volume of 100 ?L per well, the cell concentration was 4?10.sup.3 cells per well, and the cells were incubated overnight in an incubator at 37? C./5% CO.sub.2; [0116] (2) the medium was replaced for each group of cells, 100 ?L of culture solution with a specified dosage was added, and .sup.177LuCl.sub.3 and .sup.177Lu-AP1 with the same dosage concentration were added into two 96-well plates, wherein dosage concentrations were 0, 10, 100, 200, 300, 400, 500 and 600 ?Ci/mL; after 24 h, the normal culture medium was used to continue culturing; [0117] (3) a culture medium with 10 ?L of CCK8 solution (10%) was added at 24 h and 48 h after the normal culture medium was used, and the culture was continued in the incubator for 1 h; [0118] (4) after 1 h, the absorbance at 450 nm of each well was measured using a microplate reader. The relative survival rate of the cells was calculated according to the measured OD value. Cell viability=(material added group?blank group)/(control group?blank group)?100%. FIGS. 7A-7B show inhibitory effect of .sup.177Lu-AP1 on B16F10 cell growth, where FIG. 7A shows cell growth inhibition rate at 24 h after incubation for 24 h, and FIG. 7B shows cell growth inhibition rate at 48 h after incubation for 24 h.

EXPERIMENTAL EXAMPLE 4

Verification of In Vivo Targeting and Specificity of .SUP.68.Ga-AP1 Imaging Probe

[0119] (1) The B16F10 cells in the logarithmic growth phase were trypsinized and resuspended to a concentration of 1?10.sup.6 cells/mL, and the amount of cells injected per mouse was 1?10.sup.5 in 6-week-old C57BL/6 tumor-bearing left forelimbs. [0120] (2) On the day 10 after tumor bearing, 30-100 ?Ci of .sup.68Ga-AP1 was injected into tail veins of different mice, and Micro-PET/CT static development was performed for 10 min 1.5, 3.5 and 5.5 h after the administration (after the isoflurane-oxygen mixed gas with a volume fraction of 3% was used for preanesthesization, the mice were placed in a PET/CT scanning bed, and then the isoflurane-oxygen mixed gas with a volume fraction of 1.5% was used for maintaining the anesthesia). [0121] (3) Highly expressed tumors were imaged and injected with the same dose of .sup.68Ga-AP1 and 100 ?g AP1 the next day for in vivo uptake inhibition experiments, and Micro-PET/CT static imaging was performed for 10 min at 1, 3.5, and 5.5 h after intravenous injection, where the white arrows were tumors. FIGS. 8A-8C show in vivo targeting and specificity of a .sup.68Ga-AP1 imaging probe, where FIG. 8A shows PET/CT imaging of an individual that is sensitive to immunotherapy; FIG. 8B shows PET/CT imaging of an individual that is not insensitive to immunotherapy, and FIG. 8C shows PET/CT imaging of a high-uptake tumor that is insensitive to immunotherapy after addition of AP1 inhibition.

EXPERIMENTAL EXAMPLE 5

In Vivo Biodistribution and Hemodynamic Characteristics of .SUP.68.Ga-AP1 Imaging Probe

[0122] (1) The .sup.68Ga-AP1 imaging probe was used for screening a high expression group and a low expression group of tumor ASF1a, and 20 ?Ci of .sup.68Ga-AP1 was injected into the tail vein of each mouse. After 0.5, 1.5, 3.5 and 5.5 h after injection, each important organ was dissected and separated. Each organ weight was measured and calculated as % ID/g=organ radioactivity count/(organ weight?source count)?100% for 3 mice per time point. [0123] (2) Mice were sacrificed at 10 min, 20 min, 30 min, 1.5 h, 3.5 h, 5.5 h respectively after tail vein injection of .sup.68Ga-AP1, the radioactivity of the organs in the blood of the mice was measured, n=3 was measured at each time point, and the mean value of each time point was taken, and an intravascular two-compartment model was selected to fit hemodynamic parameters using PKSover software. FIGS. 9A-9C show in vivo biodistribution and hemodynamic characteristics of a .sup.68Ga-AP1 imaging probe, where FIG. 9A shows biodistribution of 68Ga-AP1 in major organs in a body, FIG. 9B shows distributions of .sup.68Ga-AP1 in ASF1a high-expression and low-expression tumors, respectively, and FIG. 9C shows pharmacokinetics of .sup.68Ga-AP1 in vivo.

EXPERIMENTAL EXAMPLE 6

Evaluation of Correlation Between Different Expressions of ASF1a and the Efficacy of Immunotherapy in a Melanoma B16F10 Model

[0124] (1) The B16F10 tumor-bearing mouse model was established, and each mouse was born with 1?10.sup.5 cells in the left forelimb. From day 4, 0.1 mg of BMS-1 was injected intraperitoneally every 3 days, and the tumor volume was measured; from day 7, PET imaging was performed every 3 days, and the ratio of tumor uptake/contralateral muscle uptake was recorded. [0125] (2) When the tumor grew to day 16, the tumor volume was less than 1000 mm.sup.3, which was regarded as the immunotherapy-effective group (immunotherapy-sensitive group), and the correlation of .sup.68Ga-AP1 PET imaging uptake in the immunotherapy-sensitive group and the immunotherapy-insensitive group was compared. FIGS. 10A-10B show the correlation between different expressions of ASF1a and the efficacy of immunotherapy in a melanoma B16F10 model, where FIG. 10A shows a tumor volume growth curve of mice after treatment with the immunosuppressant BMS-1, purple represents an effective immunosuppressive treatment group, and black represents an ineffective immunosuppressive treatment group; FIG. 10B shows ratios of the tumors to contralateral muscle uptake in different response groups of immunotherapy.

EXPERIMENTAL EXAMPLE 7

[0126] Individuals with high expression of ASF1a predicted to be insensitive to immunotherapy were screened by early .sup.68Ga-AP1 PET imaging, and .sup.177Lu-AP1 targeted radionuclide therapy was performed and the efficacy was evaluated [0127] (1) A B16F10 tumor mouse model was established, the 68Ga-AP1 PET imaging was performed on days 7-8 after treatment, individuals with high expression of ASF1a were screened, one group was administrated with PDL1 inhibitor BMS-1, and the other group was administrated with BMS-1 combined with .sup.177Lu-AP1. The tumor growth was measured and recorded every 2-3 days. FIG. 11 shows tumor volume growth curves of immunotherapy-insensitive individuals treated with BMS-1 alone and BMS-1 combined with .sup.177Lu-AP1.

[0128] The present invention establishes a method for labeling ASF1a inhibitory peptide (AP1) with .sup.68Ga/.sup.177Lu by using .sup.68GaCl.sub.3 produced by a .sup.68Ge.sup.68Ga generator or purchased .sup.177LuCl.sub.3 and designed ASF1a inhibitory peptide, evaluates the pharmacological characteristics and the biological characteristics of the ASF1a inhibitory peptide in a B16F10 tumor model mouse, is further used in the ASF1a targeted imaging study, and analyzes the correlation of the imaging result with immunotherapy. The efficacy of radionuclide-targeted therapy on screened ASF1a individuals is evaluated. The preclinical study shows that the labeling rate of .sup.68Ga-AP1 is 81.98?7.55%, the labeling rate of .sup.177Lu-AP1 is 78.34?13.59%, the radiochemical purity of the product measured by an HPLC method is greater than 95%, and the product has good stability within 24 h.

[0129] The above descriptions are only preferred embodiments of the present invention. It should be noted that those of ordinary skill in the art can also make several improvements and modifications without departing from the principle of the present invention, and such improvements and modifications shall fall within the protection scope of the present invention.