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Journal of Peking University (Health Sciences) ›› 2025, Vol. 57 ›› Issue (2): 253-261. doi: 10.19723/j.issn.1671-167X.2025.02.005

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Biological activity and antitumor effect of long-acting recombinant human interleukin-2 drug

Xuejun LIANG, Fengxia ZHANG, Ting JIN, Jingjing ZHU*()   

  1. Novocodex Biopharmaceuticals Company Limited, Shaoxing 312000, Zhejiang, China
  • Received:2024-10-21 Online:2025-04-18 Published:2025-04-12
  • Contact: Jingjing ZHU E-mail:zhujingjing@novocodex.cn

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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.

Key words: Recombinant interleukin 2, Non-natural amino acids, Immunotherapy

CLC Number: 

  • R966

Table 1

Binding affinity between various PEG-rhIL-2 and human IL-2 receptors"

Items Equilibrium dissociation constant /(nmol/L)
IL-2Rα Test article/rhIL-2 IL-2Rβ Test article/rhIL-2
rhIL-2 23.0 1.0 355.0 1.0
T41-PEG-rhIL-2 1 130.0 49.1 994.0 2.8
Y45-PEG-rhIL-2 5 650.0 245.7 1 080.0 3.0
V91-PEG-rhIL-2 779.0 33.9 11 400.0 32.1

Figure 1

Activation of CTLL-2 and YT cells by PEG-rhIL-2 at different sites A, CTLL-2 cells; B, YT cells. rhIL-2, recombinant human interleukin 2; PEG-rhIL-2, pegylated recombinant human interleukin 2; EC50, median effect concentration; pSTAT5, phosphorylated signal transducer and activator of transcription 5."

Table 2

Activity parameters of pSTAT5 activation by various PEG-rhIL-2"

Items CTLL-2 cells YT cells
EC50/(μg/L) Test article/rhIL-2 EC50/(μg/L) Test article/rhIL-2
rhIL-2 0.1 1.0 47.6 1.0
T41-PEG-rhIL-2 1.1 8.3 305.1 6.4
Y45-PEG-rhIL-2 44.0 338.0 161.2 3.4
V91-PEG-rhIL-2 0.5 4.1 5 542.0 117.0

Table 3

Pharmacokinetics properties of Y45-PEG-rhIL-2"

Test article AUC0-last/(h × μg/L) Cmax /(μg/L) Tmax/h T1/2/h CL/ [L/(h·kg)]
rhIL-2 2 503.99 6 580.94 0.08 0.80 0.41
Y45-PEG-rhIL-2 161 868.20 26 651.07 0.42 17.70 0.01

Figure 2

Anti-tumor activity of Y45-PEG-rhIL-2 in mouse models A and D, Hepa1-6; B and E, Pan-02; C and F, MC-38. rhIL-2, recombinant human interleukin 2; PEG-rhIL-2, pegylated recombinant human interleukin 2; PD-1, programmed death-1."

Table 4

Tumor growth inhibition of test articles in mouse models"

Dose of drug of model/(mg/kg) TGI/%
Hepa1-6
  Control
    0 -
  Y45-PEG-rhIL-2
    0.1 67.17
    0.3 80.28***
    1.0 105.17***
Pan-02
  Control
    0 -
  Y45-PEG-rhIL-2
    1.0 88.17***
    5.0 106.68***
MC-38
  Control
    0 -
  Y45-PEG-rhIL-2
    1.0 88.11***
    5.0 98.42***
  Anti-PD-1 antibody
    10.0 77.75***
  Y45-PEG-rhIL-2+ anti-PD-1 antibody
    1.0+5.0 98.24***###△△△
    5.0+10.0 98.77***△△△

Figure 3

Body weight changes of mouse models after administration of drugs A, Hepa1-6; B, Pan-02; C, MC-38. PEG-rhIL-2, pegylated recombinant human interleukin 2."

Figure 4

Percentage of tumor infiltrated lymphocytes 72 h and 120 h after Y45-PEG-rhIL-2 administration respectively A, CD45+ in live cells; B, CD4+ in CD45+ cells; C, CD8+ in CD45+ cells; D, Treg in CD45+ cells; E, CD8+/Treg ratio. * P < 0.05; * * P < 0.01; * * * P < 0.001. G1, vehicle control; G2, Y45-PEG-rhIL-2; G3, Y45-rhIL-2; G4, rhIL-2. rhIL-2, recombinant human interleukin 2; PEG-rhIL-2, pegylated recombinant human interleukin 2."

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