收稿日期: 2024-10-21
网络出版日期: 2025-04-12
版权
Biological activity and antitumor effect of long-acting recombinant human interleukin-2 drug
Received date: 2024-10-21
Online published: 2025-04-12
Copyright
目的: 考察运用非天然氨基酸定点偶联聚乙二醇(polyethylene glycol，PEG)获得的PEG化重组人白介素2(pegylated recombinant human interleukin 2，PEG-rhIL-2)的生物学活性和抑瘤作用，并探索其抑瘤机制。方法: 用表面等离子共振(surface plasmon resonance，SPR)技术检测T41、Y45和V91三个不同位点PEG-rhIL-2与人IL-2受体α(interleukin 2 receptor α，IL-2Rα)和IL-2受体β(interleukin 2 receptor β，IL-2Rβ)的平衡解离常数(equilibrium dissociation constant，KD)。采用Western blot检测不同剂量rhIL-2和PEG-rhIL-2激活CTTL-2和YT细胞中Janus激酶-信号转导和转录激活因子5(Janus kinase-signal transducer and activator of transcription 5，JAK-STAT5)信号通路的水平。将小鼠单次给药后采血，检测不同时间点药物的浓度，评估Y45-PEG-rhIL-2的药代动力学参数。选择小鼠肝癌细胞系Hepa1-6、胰腺癌细胞系Pan-02和结肠癌细胞系MC-38，用C57BL/6品系小鼠构建肿瘤模型，分别注射不同剂量的Y45-PEG-rhIL-2及赋形剂对照，评估药物的抑瘤作用，并在MC-38模型中评估Y45-PEG-rhIL-2与抗小鼠程序性死亡受体-1(programmed death-1，PD-1)单抗联用的抑瘤作用。构建Hepa1-6小鼠肿瘤模型，分别注射rhIL-2、Y45-rhIL-2和Y45-PEG-rhIL-2，用流式细胞仪分析肿瘤浸润淋巴细胞的比例。结果: SPR检测结果显示PEG-rhIL-2与IL-2Rα/IL-2Rβ亲和力均降低，Y45-PEG-rhIL-2与IL-2Rα亲和力降低至约1/250，与IL-2Rβ亲和力降低至1/3。Western blot结果显示Y45-PEG-rhIL-2激活表达IL-2受体异源三聚体αβγ(heterotrimeric IL-2 receptor complex αβγ，IL-2Rαβγ)的CTLL-2细胞JAK-STAT5信号的活性降低至约1/300，激活表达IL-2受体异源二聚体βγ(heterodimeric IL-2 receptor complex βγ，IL-2Rβγ)的YT细胞JAK-STAT5信号的活性降低至1/3。小鼠单次给药后药代动力学评估结果显示Y45-PEG-rhIL-2消除半衰期为17.7 h，具有优于rhIL-2的药代动力学特征。在小鼠肿瘤模型中，Y45-PEG-rhIL-2显示出剂量依赖的抑瘤作用，且与抗PD-1抗体联用抑瘤作用优于Y45-PEG-rhIL-2单用和抗PD-1抗体单用。流式结果表明Y45-PEG-rhIL-2给药72 h后，肿瘤浸润CD8+T细胞比例增加86.84%，给药120 h后CD8+T细胞与调节T细胞(regulatory T cell，Treg)比值提高75.10%。结论: 采用非天然氨基酸定点偶联得到的Y45-PEG-rhIL-2通过改变受体亲和力，调节CD8+T细胞杀伤肿瘤，在小鼠多个移植瘤模型上具有剂量依赖的抑瘤作用。
梁学军 , 张凤霞 , 靳婷 , 祝静静 . 长效重组人白介素2药物的生物学活性和抑瘤作用[J]. 北京大学学报（医学版）, 2025 , 57(2) : 253 -261 . DOI: 10.19723/j.issn.1671-167X.2025.02.005
Objective: To investigate the biological activity and antitumor effect of pegylated recombinant human interleukin 2 (PEG-rhIL-2) obtained by site-specific conjugation of polyethylene glycol (PEG) with non-natural amino acids, and to explore its antitumor mechanism. Methods: The binding activities of PEG-rhIL-2 at three different sites (T41, Y45, and V91) to human interleukin 2 receptors α (IL-2Rα) and β (IL-2Rβ) and were detected by surface plasmon resonance (SPR) technology. Western blot was used to detect the levels of the Janus kinase-signal transducer and activator of transcription 5 (JAK-STAT5) signaling pathway activated by different doses of rhIL-2 and PEG-rhIL-2 in CTTL-2 and YT cells. Blood was collected after a single administration in mice to detect the drug concentration at different time points and evaluate the pharmacokinetic parameters of Y45-PEG-rhIL-2. Mouse hepatoma cell line Hepa1-6, pancreatic cancer cell line Pan-02, and colon cancer cell line MC-38 were selected. Tumor models were constructed in C57BL/6 mice. Different doses of Y45-PEG-rhIL-2 and excipient control were administrated respectively to evaluate the tumor suppression effect of the drug. In the MC-38 colon cancer model, the tumor suppression effect of Y45-PEG-rhIL-2 combined with anti-programmed death-1 (PD-1) monoclonal antibody was evaluated. Hepa1-6 mouse tumor models were constructed and rhIL-2, Y45-rhIL-2 and Y45-PEG-rhIL-2 were administrated respectively. The proportion of tumor-infiltrating lymphocytes was analyzed by flow cytometry. Results: The SPR detection results showed that the binding activities of PEG-rhIL-2 to IL-2Rα/IL-2Rβ were both reduced. The affinity of Y45-PEG-rhIL-2 to IL-2Rα was reduced to approximately 1/250, and its affinity to IL-2Rβ was reduced to 1/3. Western blot results showed that the activity of Y45-PEG-rhIL-2 in stimulating JAK-STAT5 signaling in CTLL-2 cells expressing heterotrimeric IL-2 receptor complex IL-2Rαβγwas reduced to approximately 1/300, while its activity in YT cells expressing heterodimeric IL-2 receptor complex IL-2Rβγwas reduced to approximately 1/3. The pharmacokinetic evaluation after a single dose in the mice showed that the elimination half-life of Y45-PEG-rhIL-2 was 17.7 h. Y45-PEG-rhIL-2 has pharmacokinetic characteristics superior to those of rhIL-2. Y45-PEG-rhIL-2 showed dose-dependent tumor suppression activity, and the combination of Y45-PEG-rhIL-2 and anti-PD-1 antibody had a better tumor-inhibiting effect than the single use of Y45-PEG-rhIL-2 or anti-PD-1 antibody. Flow cytometry analysis demonstrated that 72 h after the administration of Y45-PEG-rhIL-2, the proportion of tumor-infiltrating cytotoxic T lymphocytes (CD8+T cells) increased by 86.84%. At 120 h after administration, the ratio of CD8+T cells to regulatory T cells (Treg) increased by 75.10%. Conclusion: Y45-PEG-rhIL-2 obtained by site-specific conjugation via non-natural amino acids changed its receptor binding activity and inhibited tumor growth in dose-dependent manner in multiple tumor models by regulating CD8+T cells.
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