Preparation method and application of an interleukin-2 mutant radiopharmaceutical

Abstract

This invention relates to a method for preparing and applying a radiopharmaceutical containing an interleukin-2 mutant, specifically belonging to the field of molecular biology pharmaceuticals. The radiopharmaceutical comprises the interleukin-2 mutant (sIL-2), a radionuclide, and a chelating agent. The radionuclide is labeled with GGGGK-HYNIC and linked to the LPETG tag of sIL-2 under the action of Sortase A enzyme. This invention is based on a radiopharmaceutical prepared from an interleukin-2 mutant. Through protein sequence mutation, the resulting mutant sIL-2 exhibits lower toxicity than interleukin-2 (IL-2) and higher receptor affinity, which is more conducive to T cell imaging in vivo. Site-specific labeling of the radionuclide using Sortase A enzyme and residual cysteine ​​residues at the C-terminus of the protein structure ensures the homogeneity of the prepared radiopharmaceutical. Furthermore, HYNIC is used as a bifunctional chelating agent, and TPPTS and tricine are used as co-ligands to achieve… 99m The Tc-HYNIC core exhibits better in vivo and in vitro stability.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying an interleukin-2 mutant radiopharmaceutical, specifically belonging to the field of molecular biology drugs. Background Technology

[0002] Over the past decade, tumor immunotherapy has rapidly developed with the clinical approval of various immune checkpoint-targeting antibodies and T-cell-mediated cell immunotherapies. Tumor immunotherapy has shown promising application prospects in clinical practice, even revolutionizing the clinical treatment of certain cancers, such as melanoma, non-small cell lung cancer, advanced lymphoma, and liquid B-cell tumors. Despite this rapid development, a large proportion of patients do not respond to immunotherapy (e.g., immune checkpoint-targeting antibody therapy is effective in 20-30% of patients), and many patients experience side effects. For example, 60% of patients receiving the CTLA-4 antibody Yervoy experience immune-related adverse events, of which 10-30% are serious (grade 3-4). These side effects are mainly caused by excessive T-cell activation. Extensive infiltration of T lymphocytes within the tumor is a sign of good prognosis, while the accumulation of T lymphocytes in normal, non-immune organs or tissues can lead to adverse reactions due to excessive immunity. Therefore, it is particularly important to monitor the dynamic distribution of T cells in the tumor microenvironment and normal organs, visualize the immune response, identify patients earlier during treatment, and stop ineffective immunotherapy in a timely manner, so as to terminate the serious side effects and meaningless economic consumption caused by it as soon as possible.

[0003] Currently, monitoring tumor immune responses typically involves measuring the levels of circulating lymphocytes, cytokines, and immunoglobulins in blood samples, or assessing them through biopsies of tumor tissue, spleen, and lymph nodes. However, these methods are invasive and do not provide information on the tumor microenvironment and tumor metastasis, nor do they offer dynamic and spatial information on T cells in heterogeneous tumors. This results in unreliable data that fails to accurately reflect the outcomes of tumor immunotherapy. Furthermore, morphological assessment methods used in solid tumor treatment often rely solely on imaging findings of tumor volume, leading to unreliable results when evaluating early responses to tumor immunotherapy, as tumor size may remain unchanged or even increase in patients sensitive to early immunotherapy. Therefore, developing imaging probes targeting T cells to non-invasively monitor changes in systemic and tumor microenvironmental immune responses during treatment is of significant clinical importance. It can guide immunotherapy, improve treatment efficiency, and optimize treatment strategies.

[0004] Interleukin 2 (IL-2) is a functional protein that plays a stimulatory and regulatory role in the immune system and is crucial for immune homeostasis. IL-2 mediates its action by binding to the interleukin 2 receptor (IL-2R). IL-2R exists in a trimeric form composed of IL-2Rα, IL-2Rβ, and IL-2Rγ, and a dimer form composed of IL-2Rβ and IL-2Rγ. Both receptor forms can transduce IL-2 signals. However, the affinity of IL-2 for the trimeric αβγ IL-2R is approximately 10-100 times greater than that for the dimer βγ IL-2R, indicating that IL-2Rα only enhances the binding of the trimeric αβγ IL-2R to IL-2, but is not important for signal transduction. The distribution of these two receptors also differs across different T cell subsets; for example, the trimeric receptor is highly expressed on Treg cells, specifically on Memory CD8 cells. + T cells and NK cells highly express dimeric receptors. In addition, activated CD4+... + and CD8 + T lymphocytes also highly express IL-2R on their surface. Therefore, constructing molecular probes using radiolabeled IL-2 to image IL-2R on the surface of T cells allows for non-invasive monitoring of T cell changes throughout the body and within the tumor microenvironment during treatment, thereby guiding tumor immunotherapy. The radionuclide fluorine-19 (… 18 Interleukin-2 (F) labeled 18 F-FB-IL-2 was designed as a PET imaging probe for T cell imaging. 18 F-FB-IL-2 can accurately quantify the degree of lymphocyte infiltration. In mouse models of lung cancer following radiotherapy or radiotherapy combined with immunotherapy... 18 The uptake of F-FB-IL-2 was 10 times and 27 times higher than that of untreated lung cancer mice, respectively, indicating that the probe can be used as an imaging agent to monitor active T lymphocytes in the tumor microenvironment after immunotherapy. 18 F-FB-IL-2 is currently undergoing Phase I clinical trials.

[0005] IL-2 is also used clinically to treat melanoma and metastatic renal cell carcinoma, but it often causes serious side effects, mainly pulmonary edema caused by vascular leakage syndrome, due to the expression of a certain level of trimer αβγ IL-2R by pulmonary endothelial cells. Therefore, one of the current focuses of IL-2 research is to reduce its toxicity. IL-2v is one such mutant. Through mutation, IL-2v no longer binds to IL-2Rα, thus reducing toxicity and prolonging its blood half-life compared to IL-2. Therefore, IL-2v may be more suitable as an imaging probe ligand for T cells. However, because the mutation reduces the affinity of IL-2v for the trimer receptor αβγ IL-2R, it also weakens its binding to activated T cells (which mainly express trimer αβγ IL-2R), which may be detrimental to imaging. Super IL-2 is an IL-2 mutant with high receptor affinity selected by phage cloning; through mutation, it enhances the binding affinity to IL-2Rβ. Introducing the F42A mutation into super IL-2 (super IL-2-F42A, sIL-2) can further reduce its interaction with IL-2Rα, thereby reducing toxicity. However, sIL-2 has a higher affinity for both the trimer and dimer receptors than IL-2v. Therefore, compared to IL-2v, sIL-2 may be more suitable as an imaging ligand for T cells to develop molecular probes. Molecular imaging probes constructed using radiolabeled sIL-2 will have a higher target / background ratio, lower toxicity, and are more suitable for in vivo T cell imaging. Summary of the Invention

[0006] The purpose of this invention is to provide a radiopharmaceutical containing an interleukin-2 mutant. In 1998, the U.S. Food and Drug Administration (FDA) approved IL-2, a typical cytokine that specifically binds to and proliferates T cells, for the treatment of stage IV (metastatic) melanoma patients. IL-2 receptors (IL-2Rs) have three subunits: IL-2Rα (CD25), IL-2β (CD122), and IL-2γ (CD132), which are highly expressed on the surface of T cells. IL-2Rs exist in two forms: IL-2Rαβγ (trimer) and IL-2Rβγ (dimer). Regulatory T cells (Tregs) highly express the trimer receptor IL-2Rαβγ, while native CD8+ T cells, CD4+ / CD8+ T cells, and NK cells highly express the dimer receptor IL-2Rβγ. Wild-type IL-2 has a higher affinity for binding to IL-2Rαβγ than IL-2Rβγ (Kd value 10). -11 vs 10 -9 Therefore, IL-2 preferentially binds to immunosuppressive Treg cells rather than tumor-killing CD8 cells. +T cells. Sum IL-2 is an artificial mutant of IL-2 selected through phage screening (abbreviated as sIL-2), with six mutations. Among them, the L80F, R81D, L85V, I86V, and I92F mutations enhance the binding affinity with IL-2Rβ, while the F42A mutation reduces the interaction with IL-2Rα. Therefore, unlike wild-type IL-2, which preferentially binds to immunosuppressive cells, sIL-2 preferentially binds to tumor-infiltrating effector T cells that play a major killing role. sIL-2-based imaging is also more reflective of tumor immune activity, guiding tumor immunotherapy. Reports indicate that... 18 F-labeled wild-type IL-2 was used for non-invasive PET imaging of activated T cells (CD25+ IL-2Rα) in patients receiving immune checkpoint inhibitory therapy, but its uptake in tumors was relatively low due to the low expression of CD25 detected in tumor tissue. Furthermore, it cannot reliably reflect treatment-related immune responses because the upregulation of IL-2Rα on T cells after immune checkpoint inhibitory therapy is transient, and IL-2Rα expression in tumors is primarily limited to immunosuppressive Treg cells rather than activated CD8 T cells. Radionuclide-labeled sIL-2, through its targeting action, preferentially binds to effector CD8 cells infiltrating the tumor microenvironment. + T cells, using nuclear medicine techniques such as single-photon emission computed tomography (SPECT) or positron emission tomography (PET), are used to image and evaluate tumor-infiltrating cytotoxic effector T cells. This imaging is more representative of tumor immune activity than wild-type IL2 imaging, and the uptake in tumors is relatively higher. This allows for more effective screening of patients suitable for immunotherapy and more reliable assessment of efficacy after immune checkpoint inhibitory therapy. Furthermore, previous literature... 18 F or 99m Tc-labeled wild-type IL-2 was prepared using a non-specific site labeling method, making it difficult to control the uniformity and reproducibility of the labeled samples. Furthermore, non-specific site labeling may affect the binding of IL-2 to T cells. We designed a flexible GGGGS chain to link an LPETG motif at the C-terminus of sIL-2. This motif can bind to the radiolabeled GGGGK-HYNIC-α motif at a location far from the sum IL-2 and IL-2R binding site via Sortase A. 99m Tc is coupled at a fixed point to avoid affecting 99m The binding affinity of Tc-sIL-2 to IL-2R. The site-specific labeling method of this invention can obtain uniform and reproducible labeled samples, and the labeling site is far from the active region at the C-terminus, thus not affecting the activity of the label. Furthermore, compared to non-specifically labeled IL-2 reported in the literature, site-specific labeling... 99mTc-sIL-2 exhibits a lower liver background ratio, resulting in better imaging contrast. Furthermore, different labeling methods and different radionuclides can also affect the in vivo behavior of the markers. Regarding SPECT radionuclides, 99m The half-life of Tc is 6 hours, which is suitable for our sIL-2 imaging evaluation. We have also developed... 99m The Tc-GH labeling method for sIL-2 is a site-directed labeling method for directly expressing the GGGC sequence at the C-terminus of sIL-2. Regarding PET nuclides, it is similar to previously reported methods... 18 F is different; we mainly use 68 Ga marker, 68 Ga is easier to obtain through a generator, and the marking process is simpler.

[0007] The objective of this invention is achieved through the following technical solution: a radiopharmaceutical containing an interleukin-2 mutant. It comprises an interleukin-2 mutant sIL-2 and a radionuclide, wherein sIL-2 is an interleukin-2 mutant; the radionuclide is labeled using a bifunctional chelating agent to label the interleukin-2 mutant; the radionuclide is... 99m Tc, 68 Ga 64 Cu, 111 In, 90 Y and 177 The bifunctional chelating agent is any one of HYNIC, GGGC, NOTA, NODAGA, DOTA, and DTPA.

[0008] Furthermore, the radioactive nuclide is 99m When Tc, (1) by 99m Tc-tagged GGGGK-HYNIC- 99m Tc is linked to the LPETG tag of sIL-2 by the action of Sortase A enzyme; (2) by 99m Tc-labeled sodium gluconate 99m Tc-GH is coupled to the GGGC tag of sIL-2 to form the radiopharmaceutical based on the interleukin-2 mutant.

[0009] Furthermore, the radioactive nuclide is 68 Ga 64 Cu, 111 In, 90 Y and 177 Lu Shi, 68 Ga 64 Cu, 111 In, 90 Y and 177Lu is formed by labeling sIL-2 with bifunctional chelators such as NOTA, NODAGA, DOTA, or DTPA to create the interleukin-2-based radiopharmaceutical.

[0010] A method for preparing and applying an interleukin-2 mutant radiopharmaceutical, and a radionuclide. 99m Tc will... 99m Tc-tagged GGGGK-HYNIC- 99m Tc is linked to the LPETG tag of sIL-2 by Sortase A enzyme to form the radiopharmaceutical based on the interleukin-2 mutant, the method comprising the following steps:

[0011] a. Preparation of LPETG-tagged sIL-2

[0012] The sIL-2 gene was constructed into a vector, and a signal peptide was added to the N-terminus of the protein to promote its secretion extracellularly. An LPETG motif was fused to the C-terminus for radionuclide labeling, and a 10-histanidine tag (His tag) was fused for purification. After expressing the sIL-2 protein using the expression system, the supernatant was purified using a nickel column and molecular sieve to obtain the target protein. SDS-PAGE results showed that the purified protein had a purity greater than 95%, ready for further biological analysis and in vivo applications.

[0013] b、GGGGK-HYNIC- 99m Preparation of Tc

[0014] Prepare 1 mL of a mixture containing sodium triphenylphosphine trisulfonate (TPPTS), trimethylolglycine, disodium succinate, succinic acid, and GGGGK-HYNIC, with a mass ratio of 4:6:6:7:38:39:12:13:0.01. Freeze-dry the mixture for later use. Add 1 mL of Na... 99m TcO4 solution, heated in a 100 °C water bath for 20-25 minutes, then cooled to room temperature after the reaction is complete to prepare GGGGK-HYNIC- 99m Tc. Analyzed by HPLC for later use;

[0015] c, sIL-2-HYNIC- 99m Preparation of Tc

[0016] GGGGK-HYNIC- was dissolved in 2M NaOH solution. 99m Adjust the pH of the Tc solution to 7.4-7.8. Add sIL-2 and GGGGK-HYNIC- labeled with LPETG. 99mTc, Sortase A enzyme, and 1M CaCl2 solution were mixed, and the pH was readjusted to 7.4-7.8 with 2M NaOH solution. The reaction was carried out at room temperature for 30 minutes, and the labeling efficiency was analyzed by radioactive thin-layer chromatography (ITLC). Superose was used... TM Purification was performed using a 75-cell exclusion column, and the fraction of the target compound was collected to prepare the interleukin-2 mutant. 99m Tc-labeled radiopharmaceutical sIL-2-HYNIC 99m Tc. Further radioactive ITLC analysis confirmed the presence of sIL-2-HYNIC- 99m After determining the radiochemical purity of Tc, it will be ready for use.

[0017] A method for preparing and applying a radiopharmaceutical containing an interleukin-2 mutant, and a radionuclide. 99m Tc will... 99m Tc-labeled sodium gluconate 99m Tc-GH is coupled to the GGGC tag of sIL-2 to form the interleukin-2 mutant-based radiopharmaceutical, the method comprising the following steps:

[0018] a. Preparation of GGGC-tagged sIL-2

[0019] The sIL-2 gene was constructed into a vector, and a signal peptide was added to the N-terminus of the protein to promote its secretion extracellularly. A GGGC motif was fused to the C-terminus for coupling and radionuclide labeling. After expressing the sIL-2 protein using the expression system, the supernatant was purified by molecular sieve to obtain the target protein. SDS-PAGE results showed that the purified protein had a purity greater than 95%, ready for further biological analysis and in vivo applications.

[0020] b、 99m Preparation of Tc-GH

[0021] Add 1 mL of Na to the sodium gluconate (GH) box. 99m TcO4 solution, let stand at room temperature for 10 minutes to prepare 99m Tc-labeled sodium gluconate 99m Tc-GH. Prepared for radioactive ITLC analysis.

[0022] c, sIL-2-GGGC- 99m Preparation of Tc

[0023] sIL-2-GGGC, 99mTc-GH and disodium ethylenediaminetetraacetate (EDTA-2Na) solution were mixed, and the pH of the mixture was adjusted to 4.5-5.0 using 0.5 M succinate buffer. The mixture was reacted in a 37 °C water bath for 1.0 hour to prepare the interleukin-2 mutant. 99m Tc-labeled radiopharmaceutical sIL-2-GGGC- 99m Tc. Prepared for use after radioactive ITLC analysis.

[0024] A method for preparing and applying a radiopharmaceutical containing an interleukin-2 mutant, and a radionuclide. 68 Ga is labeled onto sIL-2 by the bifunctional chelator NOTA or NODAGA to form the interleukin-2 mutant-based radiopharmaceutical, the method comprising the following steps:

[0025] a. Preparation of Nota-sIL-2

[0026] Mix sIL-2 and Mal-NOTA labeled with GGGC, and adjust the pH of the mixture to 7.4-7.8 with a solution. React overnight at 4°C. Utilize Superose TM Purification was performed using a 75 μm size exclusion column, and the fraction containing the target compound was collected. The collected solutions were combined to obtain the target product sIL-2-NOTA. The preparation method for sIL-2 conjugated with NODAGA, DOTA, and DTPA is the same as above.

[0027] b, sIL-2-NOTA- 68 Preparation of Ga

[0028] Fresh rinse from germanium-gallium generator 68 GaCl3 was added, and the pH was adjusted to 3.5-4.0 using 2.5M NH4OAc. sIL-2-NOTA was added, and the reaction was carried out at room temperature for 20 minutes. This prepared the interleukin-2 mutant. 68 Ga-labeled radiopharmaceutical sIL-2-NOTA- 68 Ga. Prepared for use after radioactive ITLC analysis. sIL-2-NODAGA- 68 The Ga preparation process is the same as described above. 64 Cu, 111 In, 90 Y and 177 The Lu tagging method is the same as... 68 Ga.

[0029] The HPLC method described is using an Agilent 1260 HPLC system equipped with a YMC-Pack ODS-A C 18The analytical column (250 × 4.6 mm, ID S-5 μm, 12 nm) was used for gradient elution for 20 min. The mobile phase A consisted of deionized water (containing 0.05% TFA), and the mobile phase B consisted of acetonitrile (containing 0.05% TFA). The flow rate was 1 mL / min, with the elution gradient as follows: initially 90% A and 10% B, at 17.5 min 60% A and 40% B, and at 20 min 90% A and 10% B.

[0030] The size exclusion column purification method was performed using an Agilent 1260 HPLC system equipped with Superose. TM 75-row barrier column, elution for 50 minutes, flow rate 0.8 mL / min, mobile phase 0.1% PBST.

[0031] The radioactive ITLC method uses transient thin-layer chromatography strips or polyamide strips, with physiological saline or 1% EDTA-2Na aqueous solution as the developing solvent.

[0032] The interleukin-2 mutant radiopharmaceutical was used for T cell imaging.

[0033] The beneficial effects of this invention are:

[0034] 1. This invention is a radiopharmaceutical prepared based on the interleukin-2 mutant. Through protein sequence mutation, the mutant sIL-2 is formed with lower toxicity than interleukin-2 (IL-2) and higher receptor affinity than IL-2, which is more conducive to T cell in vivo imaging.

[0035] 2. This invention utilizes Sortase A enzyme and radionuclide site-directed labeling mediated by residual cysteine ​​residues at the C-terminus of the protein structure, resulting in radiopharmaceuticals with uniform properties. Furthermore, HYNIC is used as a bifunctional chelating agent, while TPPTS and tricine are employed as synergistic ligands to achieve… 99m The Tc-HYNIC core exhibits better in vitro and in vivo stability.

[0036] 3. The interleukin-2 mutant radiopharmaceutical of the present invention is a novel molecular imaging probe targeting the interleukin-2 receptor. It can be applied to nuclear medicine molecular imaging of T cells to non-invasively monitor changes in T cells infiltrating the whole body and tumor microenvironment during tumor immunotherapy, thereby guiding tumor immunotherapy and being used for prognostic evaluation. Attached Figure Description

[0037] Figure 1 (A) sIL-2-LPETG-His tag, (C) sIL-2-GGGC, (D) sIL-2-NOTA, (E) sIL-2-NODAGA structural diagrams.

[0038] Figure 2 A comparison of the binding strength of IL-2 and sIL-2 to different T cells.

[0039] Figure 3 Polyacrylamide gel electrophoresis was used to verify the purity of the sIL-2-LPETG-His tag.

[0040] Figure 4 (A) sIL-2-HYNIC- 99m Tc, (B)sIL-2-GGGC- 99m Tc,(C)sIL-2-NOTA- 68 Ga,(D)sIL-2-NODAGA- 68 Schematic diagram of the structure of Ga radiopharmaceutical.

[0041] Figure 5 (A) sIL-2-HYNIC- 99m (A) Tc labeling rate; (B) After purification, sIL-2-HYNIC- 99m Radiochemical purity of Tc.

[0042] Figure 6 Injecting sIL-2-HYNIC into the MC38 tumor model 99m (A) SPECT / CT image after 0.5 h; (B) SPECT / CT image of the unlabeled protein blocking group after 0.5 h.

[0043] Figure 7 Injection of sIL-2-HYNIC 99m Comparison of in vivo biodistribution results in the MC38 tumor model after Tc 0.5 h with those in the non-blocking and blocking experiments.

[0044] Figure 8 (A) Injection of sIL-2-HYNIC into the MC38 tumor model 99m Tc and IL-2-HYNIC 99m SPECT / CT images after 0.5 h Tc; (B) Injection of sIL-2-HYNIC- into the MC38 tumor model. 99m Tc and IL-2-HYNIC 99m Comparison of in vivo biodistribution results after Tc 0.5 h.

[0045] Figure 9 (A) In the MC38 tumor model, injection of sIL-2-HYNIC before and after anti-PD-L1 antibody treatment. 99mSPECT / CT images after 0.5 h of Tc; (B) In the MC38 tumor model, the anti-PD-L1 antibody treatment group and the untreated group were injected with sIL-2-HYNIC- 99m Comparison of in vivo biodistribution results after Tc 0.5 h.

[0046] Figure 10 Before and after adoptive T-cell therapy (ACT) in MC38-Ova and MC38 tumor models, (A) injection of sIL-2-HYNIC- 99m SPECT / CT image after Tc 0.5 h; (B) Injection of sIL-2-HYNIC- 99m Comparison of in vivo biodistribution results after Tc 0.5 h.

[0047] Figure 11 (A) 99m Tc-GH labeling rate; (B) sIL-2-GGGC- 99m Tc labeling rate.

[0048] Figure 12 After purification, (A) sIL-2-NOTA- 68 Ga and (B) sIL-2-NODAGA- 68 Radiochemical purity of Ga. Detailed Implementation

[0049] Materials used in the embodiments of this invention:

[0050] Succinic acid, disodium succinate, sodium triphenylphosphine trisulfonate (TPPTS), and trimethylolamine (tricine) were all purchased from Sigma-Aldrich, USA. HYNIC-KGGGG was purchased from Shanghai Jier Biochemical Co., Ltd. Mal-NOTA, Mal-NODAGA, Mal-DOTA, and Mal-DTPA were purchased from Xi'an Ruixi Biotechnology Co., Ltd. Sortase A enzyme was purchased from Nanjing Detai Biotechnology Co., Ltd. Na 99m The TcO4 eluent and germanium-gallium generator were purchased from Beijing Atomic High-Tech Co., Ltd. and China Isotope & Radiation Corporation.

[0051] Example 1:

[0052] This example compares the binding strength of interleukin-2 (IL-2) and its mutant form (sIL-2) to different T cells.

[0053] The binding strength of IL-2 and sIL-2 to different T cells was detected by flow cytometry. Figure 2As shown, sIL-2 and Memory CD8 + T cells bind more readily to IL-2; conversely, IL-2 binds more readily to Treg cells than to sIL-2. This is due to Memory CD8. + T cells are useful for tumor immunotherapy, while Treg cells are detrimental to immunotherapy. Therefore, in vivo imaging of radiopharmaceuticals constructed using the interleukin-2 mutant sIL-2 is more conducive to predicting the effectiveness of tumor immunotherapy.

[0054] Example 2:

[0055] This embodiment uses sIL-2-HYNIC- 99m Taking the preparation method and application of Tc radiopharmaceuticals as an example.

[0056] sIL-2-HYNIC- 99m In Tc, sIL-2 is an interleukin-2 mutant, a radioactive isotope. 99m Tc is by... 99m Tc-tagged GGGGK-HYNIC- 99m Tc is linked to the LPETG tag of sIL-2 by Sortase A enzyme. The interleukin-2 mutant radiopharmaceutical is sIL-2-HYNIC- 99m Tc, the interleukin-2 mutant radiopharmaceutical sIL-2-HYNIC- 99m Tc is a colorless, transparent liquid injection.

[0057] sIL-2-HYNIC- 99m The preparation method of Tc is as follows:

[0058] Preparation of sIL-2: The sIL-2 gene was constructed into a vector. A signal peptide was constructed at the N-terminus of the protein to promote protein secretion outside the cell. The LPETG motif was fused to the C-terminus of the protein for radionuclide labeling, and a 10-histag was fused for purification. Figure 1 A). After expressing the sIL-2 protein using the expression system, the supernatant was purified using a nickel column and molecular sieve to obtain the target protein. Polyacrylamide gel electrophoresis (SDS-PAGE) showed that the purified protein purity was greater than 95%. Figure 3 ).

[0059] GGGGK-HYNIC- 99mPreparation of Tc: Prepare 1 mL of a mixture containing 5.0 mg TPPTS, 6.5 mg tricine, 38.5 mg disodium succinate, 12.7 mg succinic acid, and 10 μg GGGGK-HYNIC in a 10 mL vial, and freeze-dry the mixture. Add 1 mL of Na to the freeze-dried powder. 99m TcO4 solution (10-35 mCi), heat the vial in a 100 °C water bath for 20-25 minutes, and after the reaction is complete, cool to room temperature to prepare GGGGK-HYNIC- 99m Tc markers.

[0060] sIL-2-HYNIC- 99m Tc (or IL-2-HYNIC-) 99m Preparation of Tc: Adjust GGGGK-HYNIC- with 2 M NaOH solution (approximately 50 μL). 99m Adjust the pH of the Tc solution to 7.4-7.8 and divide it into three portions. Take one portion of GGGGK-HYNIC- 99m Add 100 μg of LPETG-tagged interleukin-2 mutant sIL-2, 100 μg of Sortase A enzyme, and 10 μL of 1 M CaCl2 to the Tc solution. Adjust the pH of the mixture to 7.4–7.8 with approximately 5 μL of 2 M NaOH solution. Mix thoroughly and incubate at room temperature for 30 minutes. Develop using transient thin-layer chromatography strips with physiological saline as the developing solvent. Analyze the labeling efficiency using a radiometric thin-layer chromatography scanner. Utilize Superose... TM Purification was performed using a 75-cell exclusion column, and the target fraction was collected to prepare the interleukin-2 mutant. 99m Tc-labeled radiopharmaceutical sIL-2-HYNIC 99m Tc ( Figure 4 A). Using transient thin-layer chromatography strips again, with physiological saline as the developing solvent, sIL-2-HYNIC- was analyzed using a radiometric thin-layer chromatography scanner. 99m The radiochemical purity of Tc. For example... Figure 5 As shown, sIL-2-HYNIC- obtained after the reaction mediated by Sortase A enzyme. 99m The Tc product labeling rate is approximately 40-50% ( Figure 5 A). After purification, sIL-2-HYNIC- 99m The radiochemical purity of Tc is greater than 95% ( Figure 5 B). Radionuclides 99m Tc-labeled interleukin-2 (IL-2-HYNIC-) 99m Tc) is made using the same method.

[0061] sIL-2-HYNIC- 99m SPECT / CT imaging of Tc in tumor-bearing mice: sIL-2-HYNIC- in the MC38 colorectal cancer tumor model. 99m Tc can provide clear SPECT imaging of tumor-infiltrating T cells 0.5 hours after administration. Figure 6 A). Except for high uptake in the kidneys, background uptake in other organs was low. The probe remained at the tumor site for a longer period. In the blockade group, tumor uptake was significantly reduced ( Figure 6 B), indicating sIL-2-HYNIC- 99m Tc binds specifically to tumor-infiltrating T cells.

[0062] sIL-2-HYNIC- 99m Biodistribution of Tc in tumor-bearing mice: MC38 colorectal cancer tumor models were divided into two groups of four mice each. One group of mice was injected via tail vein with 100 μL (~74 kBq) of sIL-2-HYNIC- 99m Tc mice were sacrificed 0.5 hours after injection; another group of mice were injected via tail vein with a total of 100 μL (~74 kBq) sIL-2-HYNIC- 99m Tc and 100 μg sIL-2 were injected, and the patients were sacrificed 0.5 h after injection. Blood and major organs were collected, weighed, and radioactivity counts were measured. After decay correction, the percentage injection dose rate per gram of tissue (%ID / g) was calculated. The experimental results verified the imaging results and showed the distribution of the probe in various tissues and organs. Figure 7 ). sIL-2-HYNIC- 99m Tc exhibits good uptake in MC38 tumors. In the blockade assay, excess unlabeled sIL-2 significantly reduced sIL-2-HYNIC- 99m Tc uptake in spleen, lymph nodes and MC38 tumors ( P < 0.05), which indicates that sIL-2-HYNIC- 99m The binding of Tc to T cells is specific.

[0063] sIL-2-HYNIC- 99m Tc and IL-2-HYNIC 99m Comparison of SPECT / CT imaging and biodistribution of Tc in the MC38 colorectal cancer tumor model: SPECT / CT imaging results showed that 0.5 h after drug administration, sIL-2-HYNIC- 99m Tc and IL-2-HYNIC 99m Tc is taken up by tumors ( Figure 8A). Aside from high uptake in the kidneys, both probes showed low background uptake in other organs. Biodistribution results indicated that sIL-2-HYNIC- 99m Tc uptake was significantly higher than IL-2-HYNIC in MC38 tumors, spleen, and lymph nodes. 99m Tc ( P < 0.01)( Figure 8 (B) This is because sIL-2 has a higher affinity for binding to T cells than IL-2.

[0064] sIL-2-HYNIC- 99m Comparison of SPECT / CT imaging and biodistribution of Tc before and after anti-PD-L1 antibody treatment in the MC38 colorectal cancer model: SPECT / CT imaging and biodistribution results showed that after anti-PD-L1 antibody treatment, sIL-2-HYNIC- 99m Tc uptake in MC38 tumors was significantly increased ( P < 0.01)( Figure 9 This is because after anti-PD-L1 antibody treatment, the number of T cells infiltrating MC38 tumors increased significantly, leading to increased probe uptake.

[0065] sIL-2-HYNIC- 99m SPECT / CT imaging and biodistribution of Tc in an adoptive T-cell therapy (ACT) model: SPECT / CT imaging and biodistribution results show ( Figure 10 ), prior to ACT, sIL-2-HYNIC- 99m Tc uptake is similar in MC38-Ova and MC38 tumors. In Ova-specific CD8... + Following T-cell ACT, sIL-2-HYNIC- 99m Tc uptake in MC38-Ova tumors was significantly higher than before ACT ( P < 0.01), and the probe uptake in MC38-Ova tumors was also significantly higher than in MC38 tumors ( P < 0.05). Furthermore, sIL-2-HYNIC- 99m Tc uptake in MC38-Ova tumor-draining lymph nodes (TdLNs) was significantly higher than before ACT ( P < 0.001). This indicates that sIL-2-HYNIC- 99m Tc can be used for in vivo SPECT / CT imaging tracing of adoptive T cells.

[0066] Example 3:

[0067] This embodiment uses sIL-2-GGGC- 99mThe preparation method of Tc radiopharmaceuticals is taken as an example.

[0068] Preparation of sIL-2-GGGC: The sIL-2 gene was constructed into a vector, a signal peptide was constructed at the N-terminus of the protein to promote protein secretion outside the cell, and a GGGC motif was fused to the C-terminus of the protein for coupling and radionuclide labeling. Figure 1 B). After expressing the sIL-2 protein using the expression system, the supernatant was purified by molecular sieve to obtain the target protein. Polyacrylamide gel electrophoresis (SDS-PAGE) showed that the purified protein had a purity greater than 95%.

[0069] 99m Preparation of Tc-GH: Add 1 mL of Na to the sodium gluconate (GH) kit. 99m TcO4 solution (10⁻³⁵ mCi), incubated at room temperature for 10 minutes, to prepare 99m Tc-labeled sodium gluconate 99m Tc-GH. Developed using transient thin-layer paper chromatography strips with physiological saline as the developing solvent, and analyzed using a radiometric thin-layer chromatography scanner. (Example: ...) Figure 11 As shown in A, 99m The labeling rate of Tc-GH is greater than 95%.

[0070] sIL-2-GGGC- 99m Preparation of Tc: 99m Divide the Tc-GH solution into three portions. Take one portion. 99m Tc-GH solution was mixed with 100 μg sIL-2-GGGC and 10 μL disodium ethylenediaminetetraacetate solution (1 mg / mL). The pH of the mixture was adjusted to 4.5-5.0 using 0.5 M succinate buffer. The mixture was reacted in a 37 °C water bath for 1.0 h to prepare the interleukin-2 mutant. 99m Tc-labeled radiopharmaceutical sIL-2-GGGC- 99m Tc ( Figure 4 B). Using transient thin-layer paper chromatography strips and physiological saline as the developing solvent, the mixture was developed and analyzed using a radiometric thin-layer chromatography scanner. (e.g.,...) Figure 11 As shown in B, sIL-2-GGGC- 99m The labeling rate of Tc is greater than 95%.

[0071] Example 4:

[0072] This embodiment uses sIL-2-NOTA- 68 Ga or sIL-2-NODAGA- 68 The preparation method of Ga radiopharmaceuticals is taken as an example.

[0073] Preparation of sIL-2-NOTA: 1 mg of the GGGC-tagged interleukin-2 mutant sIL-2-GGGC and 100 μg of Mal-NOTA were dissolved in PB buffer (pH = 7.4). The mixture was thoroughly mixed and reacted overnight at 4 °C. Superose was then used to prepare the solution. TM Purification was performed using a 75 μm size exclusion column, and the fraction containing the target analyte was collected. The collected solutions were combined to obtain the target product sIL-2-NOTA ( Figure 1 C).

[0074] Preparation of sIL-2-NODAGA: 1 mg of the GGGC-tagged interleukin-2 mutant sIL-2-GGGC and 100 μg of Mal-NODAGA were dissolved in PB buffer (pH = 7.4). The mixture was thoroughly mixed and reacted overnight at 4 °C. Superose was then used to prepare the solution. TM Purification was performed using a 75 μm size exclusion column, and the fraction containing the target analyte was collected. The collected fractions were combined to obtain the target product sIL-2-NODAGA ( Figure 1 D).

[0075] sIL-2-NOTA- 68 Ga or sIL-2-NODAGA- 68 Ga preparation: Fresh rinsing from germanium-gallium generator 68 Adjust the pH of GaCl3 to 3.5-4.0 using 2.5M NH4OAc. Take 1-2 mL of the solution. 68 GaCl3 solution (10-20 mCi) was mixed with 100 μg sIL-2-NOTA or sIL-2-NODAGA, and the mixture was reacted at room temperature for 20 minutes to prepare the interleukin-2 mutant. 68 Ga-labeled radiopharmaceutical sIL-2-NOTA- 68 Ga ( Figure 4 C) or sIL-2-NODAGA- 68 Ga ( Figure 4 D). Analysis was performed using transient thin-layer chromatography strips with a 1% EDTA-2Na aqueous solution as the developing solvent, and analyzed using a radiometric thin-layer chromatography scanner. Figure 12 As shown, sIL-2-NOTA- 68 Ga or sIL-2-NODAGA- 68 The radiochemical purity of Ga is greater than 95%.

Claims

1. A method for preparing an interleukin-2 mutant radiopharmaceutical, characterized in that: The method includes the following steps: S1. Preparation of SUM IL-2 with GGGC tag The sum IL-2 gene was constructed into a vector, and a signal peptide was built at the N-terminus of the protein to promote its secretion into the extracellular space. A GGGC motif was fused to the C-terminus of the protein for coupling and radionuclide labeling. After expressing the sum IL-2 protein in a supernatant, the protein was purified using molecular sieves to obtain the target protein sum IL-2-GGGC. Polyacrylamide gel electrophoresis showed that the purified protein had a purity greater than 95%, ready for further biological analysis and in vivo applications. Preparation of S2 and 99mTc-GH Add Na99mTcO4 solution to the sodium gluconate kit and let it stand at room temperature for 10 minutes to prepare 99mTc-labeled sodium gluconate 99mTc-GH; analyze it by radioactive ITLC for later use. Preparation of S3, sum IL-2-GGGC-99mTc The solutions of sum IL-2-GGGC, 99mTc-GH, and disodium EDTA were mixed, and the pH of the mixture was adjusted to 4.5-5.0 using 0.5M succinate buffer. The mixture was reacted in a water bath at 37°C for 1.5 hours to prepare the radiopharmaceutical sum IL-2-GGGC-99mTc labeled with the interleukin-2 mutant 99mTc. The radiopharmaceutical was then prepared for use after radioactive ITLC analysis.

2. The method for preparing the interleukin-2 mutant radiopharmaceutical according to claim 1, characterized in that: The radioactive ITLC method uses transient thin-layer chromatography strips or polyamide strips, with physiological saline or 1% EDTA-2Na aqueous solution as the developing solvent.

3. An interleukin-2 mutant radiopharmaceutical obtained by the preparation method as described in claim 1 or 2.

4. The application of a radiopharmaceutical containing an interleukin-2 mutant as described in claim 3, characterized in that: The interleukin-2 mutant radiopharmaceutical is used to prepare radiopharmaceuticals for in vivo T-cell nuclear medicine imaging.

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