Biological activity and antitumor effect of long-acting recombinant human interleukin-2 drug.
- Author:
Xuejun LIANG
1
;
Fengxia ZHANG
1
;
Ting JIN
1
;
Jingjing ZHU
1
Author Information
1. Novocodex Biopharmaceuticals Company Limited, Shaoxing 312000, Zhejiang, China.
- Publication Type:Journal Article
- Keywords:
Immunotherapy;
Non-natural amino acids;
Recombinant interleukin 2
- MeSH:
Interleukin-2/pharmacokinetics*;
Animals;
Mice;
Humans;
Recombinant Proteins/pharmacology*;
Polyethylene Glycols/chemistry*;
Cell Line, Tumor;
Antineoplastic Agents/pharmacokinetics*;
Signal Transduction/drug effects*;
STAT5 Transcription Factor/metabolism*;
Interleukin-2 Receptor alpha Subunit/metabolism*;
Interleukin-2 Receptor beta Subunit/metabolism*
- From:
Journal of Peking University(Health Sciences)
2025;57(2):253-261
- CountryChina
- Language:Chinese
-
Abstract:
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.
