Anti-tumor effect of polyethylene glycol-modified interleukin-2 and immune mechanism thereof

Abstract

Disclosed is polyethylene glycol (PEG)-modified IL-2, wherein the molecular weight of PEG is approximately 40 kD. Also disclosed are a use thereof in treatment of tumors, and a method for using PEG-modified IL-2 for treatment of tumors.

Description

Antitumor effects and immune mechanisms of polyethylene glycol-modified interleukin-2 Technical Field

[0001] This invention relates to the field of medicine, and particularly to 40kd polyethylene glycol-modified interleukin-2, and its use in the treatment of tumors. Background Technology

[0002] Cancer is a global health problem that threatens human health. Cancer immunotherapy is a treatment method that activates immune cells in the body and enhances the body's anti-tumor immune response through active or passive means, specifically eliminating microresidual tumor lesions, inhibiting tumor growth, and breaking tumor immune tolerance. It has the advantages of few side effects and significant therapeutic effects. Interleukin-2 (IL-2 / IL2) was the first proven effective cancer immunotherapy (Rosenberg SA. IL-2: the first effective immunotherapy for human cancer. J Immunol. 2014; 192(12):5451-5458).

[0003] IL-2 is mainly secreted by activated T cells, but B cells, NK cells, and monocytes / macrophages can also secrete it. IL-2 has multiple functions, including stimulating the proliferation, development, homeostasis, activation, and function of various T cell subsets; stimulating NK cell proliferation and promoting NK cell killing activity and cytokine production, inducing lymphokine-activated killer cell production; and promoting B cell proliferation and antibody secretion (Abbas AK et al. Revisiting IL-2: Biology and therapeutic prospects. Sci Immunol. 2018; 3(25):eaat1482). However, IL-2 has a very short half-life in vivo, only a few minutes (<15 minutes) (Hernandez R et al. Engineering IL-2 for immunotherapy of autoimmunity and cancer. Nat Rev Immunol. 2022; 22(10):614-628), requiring multiple high-dose administrations within a short period. More importantly, IL-2 is a multifunctional cytokine that can promote the proliferation of effector cells and the anti-tumor effect mediated by T cells or NK cells. At the same time, it can promote the proliferation and development of regulatory T cells (Tregs) (Malek TR et al. CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice. Implications for the nonredundant function of IL-2. Immunity. 2002; 17(2):167-178; Fehérvari Z et al. The dichotomous role of IL-2:tolerance versus immunity. Trends Immunol. 2006; 27(3):109-111), maintain the homeostasis and immunosuppressive function of Treg cells, and to some extent assist tumor immune escape. The clinical use of high doses of IL-2 can lead to leakage of body fluids and proteins from capillaries, causing life-threatening vascular leakage syndrome and other toxic side effects (Siegel JP, Puri RK. Interleukin-2 toxicity. J Clin Oncol. 1991; 9(4): 694-704), which limits the application of IL-2.

[0004] Regulatory T cells constitutively express a high-affinity (Kd~10) protein composed of an α chain (CD25), a β chain (CD122), and a γ chain (CD132). -11 The heterotrimeric IL-2 receptor (M), and T cells and NK cells express medium affinity (Kd~10) molecules consisting of only two chains: the β chain (CD122) and the γ chain (CD132). -9 The heterodimeric IL-2 receptor (Hernandez R et al. as above; Suzuki H et al. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor beta. Science. 1995; 268(5216):1472-1476). The trimeric receptor has an affinity for IL-2 that is about 100 times higher than that of the dimeric receptor (Yu A et al. Selective IL-2 responsiveness of regulatory T cells through multiple intrinsic mechanisms supports the use of low-dose IL-2 therapy in type 1 diabetes. Diabetes. 2015; 64(6):2172-2183), which is why high doses of IL-2 must be used to activate the dimeric receptor on T cells when treating tumors. Modifying IL-2 using techniques such as structural biology and protein engineering to prevent it from binding to the α chain and instead preferentially bind to the dimer receptor to specifically activate effector T cells and limit off-target immune responses may help overcome the limitations of IL-2 in tumor treatment, making IL-2 therapy safer and more long-lasting.

[0005] PEGylation of IL-2 can alter its IL-2 bias, promoting the proliferation of effector cells such as T cells and NK cells. The change in the CD8 / Treg ratio suggests that the anti-tumor mechanism of PEG-modified IL-2 requires further investigation. Furthermore, one lesson learned from the failure of NKTR-214 (Nektar) is that overactivation of the immune system can induce increased PD-1 expression on the surface of T cells. Clinical trials of NKTR-214 have shown increased cytokine-related side effects, indicating that excessive activation of immune cells and the immune system should be avoided, as it can induce immune cell exhaustion. Therefore, the development and discovery of new strategies to increase the anti-tumor effect of IL-2 while minimizing the promotion of tumor immune escape are crucial.

[0006] Invention Summary

[0007] In a first aspect, IL-2 modified with polyethylene glycol (PEG) is provided, wherein the PEG has a molecular weight of about 40 kD.

[0008] In a second aspect, a pharmaceutical composition is provided comprising the PEG-modified IL-2 described in the first aspect, optionally comprising a pharmaceutically acceptable carrier, and particularly, the pharmaceutical composition is for treating tumors. Specifically, a pharmaceutical composition for treating lung cancer is provided, comprising the PEG-modified IL-2 described in the first aspect and an immune checkpoint inhibitor.

[0009] In a third aspect, a method for treating tumors is provided, comprising administering to a subject in need a therapeutically effective amount of the PEG-modified IL-2 of the first aspect or the pharmaceutical composition of the second aspect. In particular, a method for treating lung cancer is provided, comprising administering to a subject in need a therapeutically effective amount of the PEG-modified IL-2 of the first aspect and an immune checkpoint inhibitor.

[0010] In a fourth aspect, the use of the PEG-modified IL-2 described in the first aspect in the preparation of a medicament for treating tumors, or the use of the PEG-modified IL-2 described in the first aspect for treating tumors, is provided. In particular, the use of the PEG-modified IL-2 described in the first aspect and an immune checkpoint inhibitor in the preparation of a medicament for treating lung cancer, or the use for treating lung cancer, is provided.

[0011] In the fifth aspect, a method is provided for using the PEG-modified IL-2 described in the first aspect to promote spleen enlargement, reduce the proportion of regulatory T cells, increase the CD8 / Treg ratio, promote effector cell infiltration into the tumor interior, reduce the STAT5 phosphorylation level of regulatory T (Treg) cells, reduce the expression of transcription factors regulating Treg cells, reduce the expression of PD-1 on immune cells, reduce the expression of exhaustion-related transcription factors, increase the proportion of central memory cells, increase the proportion of CD8+ central memory T cells, and / or reduce tumor immune escape effects.

[0012] In a sixth aspect, the PEG-modified IL-2 described in the first aspect is provided for use in promoting spleen enlargement, reducing the proportion of regulatory T cells, increasing the CD8 / Treg ratio, promoting effector cell infiltration into tumors, reducing the phosphorylation level of STAT5 in regulatory T (Treg) cells, reducing the expression of transcription factors regulating Treg cells, reducing the expression of PD-1 on immune cells, reducing the expression of exhaustion-related transcription factors, increasing the proportion of central memory cells, increasing the proportion of CD8+ central memory T cells, and / or reducing tumor immune escape effects.

[0013] In a seventh aspect, the use of the PEG-modified IL-2 described in the first aspect in the preparation of agents for promoting spleen enlargement, reducing the proportion of regulatory T cells, increasing the CD8 / Treg ratio, promoting effector cell infiltration into tumors, reducing the phosphorylation level of STAT5 in regulatory T (Treg) cells, reducing the expression of transcription factors regulating Treg cells, reducing the expression of PD-1 on immune cells, reducing the expression of exhaustion-related transcription factors, increasing the proportion of central memory cells, increasing the proportion of CD8+ central memory T cells, and / or reducing tumor immune escape effects is provided. Attached Figure Description

[0014] Figure 1: Different concentrations of 40kd PEG-modified IL-2 altered the CD8 / Treg ratio in the spleen. (A) Spleen size. (B) CD8+ in spleen cells. + T cells and CD4 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are isolated from immune cells, specifically CD3. + Intragate analysis of T cells CD4 + T cells and CD8 + T cells in CD3 + The proportion of T cells. (C) CD25 in spleen cells. + FoxP3 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are used to isolate immune cells, using CD3 + CD4 in T cells + T cell separation CD4 + T cells. In CD4 + T cell gate analysis CD25 + FoxP3 + T cells in CD4 + The proportion of T cells. (D) CD8 in spleen cells. + T cells and CD4 + T cell ratio. 400,000U and 600,000U 40kb PEG-IL-2 can promote the body's immune function. (E) CD8 in spleen cells + T cells and CD4 + CD25 + FoxP3 + Treg cell ratio. 400,000U and 600,000U 40kd PEG-IL-2 can alter the body's immune homeostasis and promote positive immune function.

[0015] Figure 2: PEG-modified IL-2 of different molecular sizes can alter the CD8 / Treg ratio. (A) CD8 in spleen cells + T cells and CD4 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are isolated from immune cells, specifically CD3. + Intragate analysis of T cells CD4 + T cells and CD8 + T cells in CD3 + The proportion of T cells. (B) CD25 in spleen cells. + FoxP3 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are used to isolate immune cells, using CD3 + CD4 in T cells + T cell separation CD4 + T cells. In CD4 + T cell gate analysis CD25 + FoxP3 + T cells in CD4 + The proportion of T cells. (C) CD8 in spleen cells. + T cells and CD4 + T cell ratio: 400,000U 40kb PEG-IL-2 promotes immune function more effectively than 1,200,000U rhIL-2. (D) CD8+ in spleen cells. + T cells and CD4 + CD25 + FoxP3 + The Treg cell ratio is higher at 400,000U 40kb PEG-IL-2 than at 1,200,000U rhIL-2, which is more effective at altering the body's immune balance and promoting positive immune function.

[0016] Figure 3: 40kd PEG-modified IL-2 still promoted an increase in the CD8 / Treg ratio on day 7 after administration. (A) CD8 in spleen cells + T cells and CD4 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are isolated from immune cells, specifically CD3. + Intragate analysis of T cells CD4 + T cells and CD8 + T cells in CD3 + The proportion of T cells. (B) CD25 in spleen cells.+ FoxP3 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are used to isolate immune cells, using CD3 + CD4 in T cells + T cell separation CD4 + T cells. In CD4 + T cell gate analysis CD25 + FoxP3 + T cells in CD4 + The proportion of T cells. (C) CD8 in spleen cells. + T cells and CD4 + T cell ratio. (D) CD8 in spleen cells. + T cells and CD4 + CD25 + FoxP3 + Treg cell ratio.

[0017] Figure 4: Y-type 40kd PEG-modified IL-2 can alter immune homeostasis and increase the CD8 / Treg ratio. (A) Spleen size. (B) CD8 cells in spleen cells. + T cells and CD4 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are isolated from immune cells, specifically CD3. + Intragate analysis of T cells CD4 + T cells and CD8 + T cells in CD3 + The proportion of T cells. (C) CD25 in spleen cells. + FoxP3 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are used to isolate immune cells, using CD3 + CD4 in T cells + T cell separation CD4 + T cells. In CD4 + T cell gate analysis CD25 + FoxP3 + T cells in CD4 + The proportion of T cells. (D) CD8 in spleen cells. + T cells and CD4 + T cell ratio. (E) CD8 in spleen cells. + T cells and CD4 + CD25+ FoxP3 + Treg cell ratio. Y-type 40kd PEG-IL-2 can alter the body's immune balance and promote positive immune function.

[0018] Figure 5: The administration method does not affect the effect of 40kd PEG-modified IL-2 on immune homeostasis, but increases the CD8 / Treg ratio. (A) Spleen size. (B) CD8+ in spleen cells. + T cells and CD4 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are isolated from immune cells, specifically CD3. + Intragate analysis of T cells CD4 + T cells and CD8 + T cells in CD3 + The proportion of T cells. (C) CD25 in spleen cells. + FoxP3 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are used to isolate immune cells, using CD3 + CD4 in T cells + T cell separation CD4 + T cells. In CD4 + T cell gate analysis CD25 + FoxP3 + T cells in CD4 + The proportion of T cells. (D) CD8 in spleen cells. + T cells and CD4 + T cell ratio. (E) CD8 in spleen cells. + T cells and CD4 + CD25 + FoxP3 + Treg cell ratio.

[0019] Figure 6: 40kd PEG-modified IL-2 can delay melanoma growth. (A) Mice in each group of melanoma model mice after 7 days of administration. (B) Tumors, spleens, and lymph nodes in each group of melanoma model mice. (C) The proportion of melanoma model mice in each group whose tumor volume exceeds 4 times the initial tumor volume. (D) Daily trend of tumor volume change in each group of melanoma model mice.

[0020] Figure 7: 40kd PEG-modified IL-2 can delay lung cancer growth. (A) Mice in each lung cancer model group after 7 days of administration. (B) Tumors, spleens, and lymph nodes in each group of lung cancer model mice. (C) The proportion of lung cancer model mice in each group whose tumor volume exceeds 4 times the initial tumor volume. (D) Daily trend of tumor volume change in each group of lung cancer model mice.

[0021] Figure 8: 40kd PEG-modified IL-2 can delay the growth of B-cell lymphoma. (A) Mice in each group of B-cell lymphoma model mice after 7 days of administration. (B) Tumors, spleen, and lymph nodes in each group of B-cell lymphoma model mice. (C) The proportion of B-cell lymphoma model mice in each group whose tumor volume exceeds 4 times the initial tumor volume. (D) Daily trend of tumor volume change in each group of B-cell lymphoma model mice.

[0022] Figure 9: The effect of 40kd PEG-modified IL-2 in delaying melanoma growth is similar to that of high-dose rhIL-2. (A) Tumors, spleen, and lymph nodes in each group of melanoma model mice. (B) The proportion of melanoma model mice in each group whose tumor volume exceeds 4 times the initial tumor volume. (C) The daily trend of tumor volume change in each group of melanoma model mice.

[0023] Figure 10: 40kd PEG-modified IL-2 can alter the proportion of immune cells in lymph nodes, spleen, and tumor tissue. (A) In a melanoma model, CD3+ cells in lymph nodes, spleen, and tumor tissue... + The proportion of T cells in all cells. (B) In a melanoma model, CD3+ cells in lymph nodes, spleen, and tumor tissue. - NK + The proportion of T cells in all cells. (C) CD3 content in lymph node, spleen, and tumor tissue cells in a lung cancer model. + The proportion of T cells in all cells. (D) In ​​a lung cancer model, CD3+ cells in lymph nodes, spleen, and tumor tissue. - NK + The proportion of T cells in all cells. (E) CD3 content in lymph node, spleen, and tumor tissue cells in a B-cell lymphoma model. + The proportion of T cells in all cells. (F) CD3 content in lymph node, spleen, and tumor tissue cells in a B-cell lymphoma model. - NK + The proportion of T cells in all cells.

[0024] Figure 11: 40kd PEG-modified IL-2 can alter the proportion of melanoma immune cells. (A) CD8+ cells in lymph nodes, spleen, and tumor tissue. + T cells and CD4 +T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are isolated from immune cells, specifically CD3. + Intragate analysis of T cells CD4 + T cells and CD8 + T cells in CD3 + The proportion of T cells. (B) CD25 in lymph node, spleen, and tumor tissue cells. + FoxP3 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are used to isolate immune cells, using CD3 + CD4 in T cells + T cell separation CD4 + T cells. In CD4 + T cell gate analysis CD25 + FoxP3 + T cells in CD4 + The proportion of T cells. (C) CD4 in lymph nodes, spleen, and tumor tissue cells. + CD25 + FoxP3 + T cells in CD3 + The proportion of T cells. (D) CD8 in lymph nodes, spleen, and tumor tissue cells. + T cells and CD4 + CD25 + FoxP3 + Treg cell ratio.

[0025] Figure 12: 40kd PEG-modified IL-2 can alter the proportion of immune cells in lung cancer. (A) CD8+ cells in lymph nodes, spleen, and tumor tissue. + T cells and CD4 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are isolated from immune cells, specifically CD3. + Intragate analysis of T cells CD4 + T cells and CD8 + T cells in CD3 + The proportion of T cells. (B) CD25 in lymph node, spleen, and tumor tissue cells. + FoxP3 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are used to isolate immune cells, using CD3 + CD4 in T cells +T cell separation CD4 + T cells. In CD4 + T cell gate analysis CD25 + FoxP3 + T cells in CD4 + The proportion of T cells. (C) CD4 in lymph nodes, spleen, and tumor tissue cells. + CD25 + FoxP3 + T cells in CD3 + The proportion of T cells. (D) CD8 in lymph nodes, spleen, and tumor tissue cells. + T cells and CD4 + CD25 + FoxP3 + Treg cell ratio.

[0026] Figure 13: 40kd PEG-modified IL-2 can alter the proportion of immune cells in B-cell lymphoma. (A) CD8+ cells in lymph nodes, spleen, and tumor tissue. + T cells and CD4 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are isolated from immune cells, specifically CD3. + Intragate analysis of T cells CD4 + T cells and CD8 + T cells in CD3 + The proportion of T cells. (B) CD25 in lymph node, spleen, and tumor tissue cells. + FoxP3 + T cell flow cytometry results. Overlapping cells were removed using FSC-A and FSA-H, and CD3+ was used. + T cells are used to isolate immune cells, using CD3 + CD4 in T cells + T cell separation CD4 + T cells. In CD4 + T cell gate analysis CD25 + FoxP3 + T cells in CD4 + The proportion of T cells. (C) CD4 in lymph nodes, spleen, and tumor tissue cells. + CD25 + FoxP3 + T cells in CD3 + The proportion of T cells. (D) CD8 in lymph nodes, spleen, and tumor tissue cells. + T cells and CD4 + CD25 + FoxP3+ Treg cell ratio.

[0027] Figure 14: 40kd PEG-modified IL-2 can alter the infiltration and distribution of melanoma immune cells.

[0028] Figure 15: 40kd PEG-modified IL-2 can alter the infiltration and distribution of lung cancer immune cells.

[0029] Figure 16: 40kd PEG-modified IL-2 can alter the distribution of immune cell infiltration in B-cell lymphoma.

[0030] Figure 17: 40kd PEG-modified IL-2 after antagonizing CD8 cells did not delay melanoma growth. (A) Tumors, spleen, and lymph nodes in each group of melanoma model mice. (B) Tumor weight and tumor index (ratio of melanoma tumor tissue weight to mouse body weight) in each group of melanoma model mice. (C) Spleen mass and spleen index (ratio of spleen weight to mouse body weight) in each group of melanoma model mice.

[0031] Figure 18: PD-1 inhibitors and 40kd PEG-modified IL-2 did not have a synergistic effect in delaying melanoma growth. (A) Mice in each group of melanoma model mice 7 days after administration. (B) Tumors, spleens, and lymph nodes in each group of melanoma model mice. (C) Tumor weight and tumor index (ratio of melanoma tumor tissue to body weight) in each group of melanoma model mice. (D) Spleen weight and spleen index (ratio of spleen weight to mouse body weight) in each group of melanoma model mice.

[0032] Figure 19: PD-1 inhibitors and 40kd PEG-modified IL-2 have a synergistic effect in delaying lung cancer growth. (A) Mice in each lung cancer model group 7 days after administration. (B) Tumors, spleens, and lymph nodes in each group of lung cancer model mice. (C) Tumor weight and tumor index (ratio of lung cancer tumor tissue weight to mouse body weight) in each group of lung cancer model mice. (D) Spleen weight and spleen index (ratio of spleen weight to mouse body weight) in each group of lung cancer model mice.

[0033] Figure 20: 40kd PEG-modified IL-2 can reduce the expression level of CD8 immune checkpoints in tumor tissues of tumor-bearing models. (A) CD8 expression in tumor tissues of melanoma models. + (B) Expression levels of T cell surface immune checkpoints. + Expression levels of T cell surface immune checkpoints. (C) CD8 expression levels in tumor tissue of a B-cell lymphoma model. + Expression levels of immune checkpoints on the surface of T cells.

[0034] Figure 21: 40kd PEG-modified IL-2 can delay the depletion of CD8 cells in tumor tissues of a tumor-bearing model while preserving their IFN-γ secretion capacity. (A) CD8 cells in lymphoma, spleen, and tumor tissues of a melanoma model. + PD-1 + (B) T cell IFN-γ secretion levels. (B) CD8+ levels in lymphoma, spleen, and tumor tissue of a lung cancer model. + PD-1 + T cell IFN-γ secretion levels. (C) CD8 levels in B-cell lymphoma model, melanoma model, lymphoma, spleen, and tumor tissue. + PD-1 + T cell IFN-γ secretion levels.

[0035] Figure 22: 40kd PEG-modified IL-2 can delay the depletion of CD8 cells in tumor tissues of a tumor-bearing model, preserving their ability to secrete Granzyme B. (A) CD8 cells in lymphoma, spleen, and tumor tissues of a melanoma model. + PD-1 + T cell Granzyme B secretion levels. (B) CD8 levels in lymphoma, spleen, and tumor tissue in a lung cancer model. + PD-1 + T cell Granzyme B secretion levels. (C) CD8 levels in B-cell lymphoma model, melanoma model, lymphoma, spleen, and tumor tissue. + PD-1 + T cell Granzyme B secretion levels.

[0036] Figure 23: Preparation of PEG-modified IL2. (A) Recombinant human IL2 before (-) or after (+) PEG modification by SDS-PAGE followed by silver staining. M, marker. (B) Monitoring of purified PEG-modified IL-2. (C) Activity assays of recombinant human IL2 and PEG-modified recombinant human IL2 with different molecular weights and structures. (D) Activity assays of PEG-modified recombinant human IL2 (40kD-PEG-IL-2) and recombinant human IL2 (IL-2). (E) SDS-PAGE electrophoresis of Y-type 40kd-PEG-modified recombinant human IL2. (F) Activity assays of Y-type 40kd-PEG-modified recombinant human IL2 (Y-40kD-PEG-IL-2) and recombinant human IL2 (IL-2).

[0037] Figure 24: 40kd PEG-IL-2 decreased STAT5 phosphorylation in Treg cells, while rhIL-2 increased it. Mouse spleen mononuclear cells were treated with different concentrations of rhIL-2 and 40kd PEG-IL-2. Cells were collected after treatment. Flow cytometry was used to determine the level of phosphorylated STAT5 expression in the cells. (A) shows the percentage of pSTAT5+ cells in Treg cells after one day of treatment. (B) shows the percentage of p-STAT5+ cells in Treg cells after two days of treatment. (C) With increasing concentration, rhIL-2 promoted the increase of STAT5 phosphorylation in Treg cells, but 40kd PEG-IL-2 decreased it. A t-test was used to compare the two samples. Significant differences between the data were calculated, and P < 0.05 was considered statistically significant. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0038] Figure 25: The effects of 40kd PEG-IL-2 and rhIL-2 on STAT5 phosphorylation levels in effector cells were similar. Mouse spleen mononuclear cells were treated with different concentrations of rhIL-2 and 40kd PEG-IL-2. Cells were collected after treatment. Flow cytometry was used to determine the intracellular phosphorylated STAT5 expression levels. The percentage of p-STAT5+ cells in CD8+ T cells (A) and NK cells (B) is shown one day after treatment. The two samples were compared using a t-test. Significant differences between the data were calculated, and P < 0.05 was considered statistically significant. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0039] Figure 26: 40kd PEG-IL-2 reduces the mRNA levels of regulatory Treg transcription factors. Mouse spleen mononuclear cells were treated with different concentrations of rhIL-2 and 40kd PEG-IL-2. Cells were collected after treatment. The relative levels of Treg-related genes were measured 3 days after treatment. A t-test was used to compare the two samples. Significant differences between the data were calculated, and P < 0.05 was considered statistically significant. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0040] Figure 27: 40kd PEG-IL-2 reduces the expression of immune checkpoint molecules on the surface of immune cells. (A) Mouse bone marrow cells were treated with different concentrations of rhIL-2 and 40kd PEG-IL-2. Cells were collected after treatment. After three days of treatment, the expression of PD-1 on the surface of immune cells was detected by flow cytometry. The percentage of CD3+PD-1+ T cells is shown after three days of treatment. The dashed line represents the control group without rhIL-2 or 40kd PEG-IL-2 stimulation. (B) Mouse bone marrow cells and tumor cell lines were co-cultured and treated with different concentrations of rhIL-2 and 40kd PEG-IL-2. After three days of treatment, the expression of PD-1 on the surface of immune cells in the co-culture system was detected by flow cytometry. The percentage of CD3+PD-1+ T cells is shown. The two samples were compared using a t-test. Significant differences between the data were calculated, and P < 0.05 was considered statistically significant. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0041] Figure 28: 40kd PEG-IL-2 reduces mRNA levels of exhaustion-related transcription factors. Mouse spleen mononuclear cells were treated with different concentrations of rhIL-2 and 40kd PEG-IL-2. Cells were collected after treatment. The levels of genes associated with T cell exhaustion were detected 3 days after treatment. A t-test was used to compare the two samples. Significant differences between the data were calculated, and P < 0.05 was considered statistically significant. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0042] Figure 29: 40kD PEG-IL-2 stimulation in a melanoma model T cells, CD8+ activated T cells, and CD8+ T cells CM The percentage of cells increased, while rhIL-2 had no effect on these changes in cell percentage. A melanoma model was established. Once the model was successfully established, the rhIL-2 group received 400,000 U / day of rhIL-2 once daily for 5 days, and the 40kDa PEG-IL-2 group received 400,000 U / day of PEG-IL-2 once daily for 1 day. On day 7, tumor-bearing mice were sacrificed. Flow cytometry analysis was performed on lymph nodes, spleen, and tumor tissue. Flow cytometry analysis of gated CD3+ T cells was also performed. T cells are defined as CD3+CD8+CD44-CD62L+ cells. CD8+ activated T cells are defined as CD3+CD8+CD25+ cells. CD8+ central memory T cells (CD8+ T cells) are also defined as CD8+ T cells. CM CD8+ effector memory T cells are defined as CD3+CD8+CD4+CD62L+ cells.EM The cell type (CD3+CD8+CD44+CD62L–) is defined as a CD3+CD8+CD44+CD62L– cell. This is shown... T cells (A), CD8+ activated T cells (B), CD8+ T cells CM Cells (C) and CD8+ T EM Percentage of cells (D). Comparisons between multiple samples were performed using one-way ANOVA, and comparisons between groups were performed using Tukey's multiple comparison test. Significant differences between data were calculated; P < 0.05 was considered statistically significant. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0043] Figure 30: 40kD PEG-IL-2 stimulation in a lung cancer model T cells, CD8+ activated T cells, and CD8+ T cells CM The percentage of tumor-bearing mice increased, while rhIL-2 had no effect on these changes in cell percentage. A cancer model was constructed. After successful model establishment, mice were administered rhIL-2 (400,000 U / day) once daily for 5 days, and PEG-IL-2 (400,000 U / day) for 1 day. On day 7, tumor-bearing mice were sacrificed. Flow cytometry analysis was performed on lymph nodes, spleen, and tumor tissue. Flow cytometry analysis of gated CD3+ T cells was also performed. T cells are defined as CD3+CD8+CD44-CD62L+ cells. CD8+ activated T cells are defined as CD3+CD8+CD25+ cells. CD8+ central memory T cells (CD8+ T cells) are also defined as CD8+ T cells. CM CD8+ effector memory T cells are defined as CD3+CD8+CD4+CD62L+ cells. EM The cell type (CD3+CD8+CD44+CD62L–) is defined as a CD3+CD8+CD44+CD62L– cell. This is shown... T cells (A), CD8+ activated T cells (B), CD8+ T cells CM Cells (C) and CD8+ T EM Percentage of cells (D). Comparisons between multiple samples were performed using one-way ANOVA, and comparisons between groups were performed using Tukey's multiple comparison test. Significant differences between data were calculated, with P < 0.05 considered statistically significant. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0044] Figure 31: 40kD PEG-IL-2 stimulation in a B-cell lymphoma model T cells, CD8+ activated T cells, and CD8+ T cells CMThe percentage of cells increased, while rhIL-2 had no effect on these changes in cell percentage. A B-cell lymphoma model was established. After successful model establishment, the rhIL-2 group received 400,000 U / day of rhIL-2 once daily for 5 days; the 40kd PEG-IL-2 group received 400,000 U / day of PEG-IL-2 once daily for 1 day. On day 7, tumor-bearing mice were sacrificed. Flow cytometry analysis was performed on lymph nodes, spleen, and tumor tissue. Flow cytometry analysis of gated CD3+ T cells was also performed. T cells are defined as CD3+CD8+CD44-CD62L+ cells. CD8+ activated T cells are defined as CD3+CD8+CD25+ cells. CD8+ central memory T cells (CD8+ T cells) are also defined as CD8+ T cells. CM CD8+ effector memory T cells are defined as CD3+CD8+CD4+CD62L+ cells. EM The cell type (CD3+CD8+CD44+CD62L–) is defined as a CD3+CD8+CD44+CD62L– cell. This is shown... T cells (A), CD8+ activated T cells (B), CD8+ T cells CM Cells (C) and CD8+ T EM Percentage of cells (D). Comparisons between multiple samples were performed using one-way ANOVA, and comparisons between groups were performed using Tukey's multiple comparison test. Significant differences between data were calculated, with P < 0.05 considered statistically significant. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0045] Summary of the Invention

[0046] Unless otherwise stated, all technical and scientific terms have their common meanings to those skilled in the art. All patents, patent applications, publications, GenBank sequences, websites, and other public materials are included herein by reference unless otherwise stated. If multiple definitions exist for the terms used in this invention, the one used here shall prevail. When describing URLs or other identifiers or addresses, it should be understood that such identifiers are subject to change and that specific information on the Internet is constantly updated and can be found by searching the Internet. Their disclosure is intended to serve as a basis for public access.

[0047] As used herein, the terms “protein,” “peptide,” “polypeptide,” and “amino acid sequence” are used interchangeably to refer to polymers of any length, such as two or more amino acid residues. The term also includes amino acid polymers that are naturally or artificially modified; for example, through disulfide bonding, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation and modification, such as conjugation to tags or biologically active components. Conventional single-letter or three-letter amino acid residue encodings are used herein.

[0048] We investigated the effects of a single PEG-modified IL-2 on the body's immune system and its anti-tumor mechanism. We found that 40kd PEG-modified IL-2 (40kd PEG-IL-2) exerts its anti-tumor effect by altering immune homeostasis. A single low-dose dose of 40kd PEG-modified IL-2 promotes spleen enlargement, reduces the proportion of regulatory T cells, increases the CD8 / Treg ratio, and alters the bias of ordinary IL-2, promoting positive immunity. It is more effective than an equivalent dose of ordinary IL-2 in delaying the growth of melanoma, lung cancer, and B-cell lymphoma tumors. The anti-melanoma effect of low-dose 4kd PEG-modified IL-2 is more significant than that of high-dose IL-2. Furthermore, 40kd PEG-modified IL-2 does not promote tumor CD8+. + Even when T cells reach terminal exhaustion, they still retain the ability to secrete gamma interferon and granzyme B.

[0049] IL2 is composed of CD4 + T cells, CD8 + IL-2, secreted by cells such as T cells and NK cells, plays a crucial role in the immune system. Low-dose IL-2 preferentially targets the trimer IL-2 receptor, maintaining Treg cells and peripheral immune tolerance, and is used to treat autoimmune diseases. High-dose IL-2 preferentially targets the dimer IL-2 receptor, activating effector T cells and NK cells through the IL-2 receptor activation signaling pathway, and is used to treat solid tumors and hematologic malignancies. The short half-life of IL-2, the high dosage required, and serious side effects, such as vascular leakage syndrome, significantly limit its clinical application. To make IL-2 therapy safer and more long-lasting, various modifications to IL-2 have been proposed, including pegylation, Fc fusion, IL-2 mutant design, bispecific antibody design, and combination with immune checkpoint antibody drugs.

[0050] Nektar's Bempegaldesleukin (NKTR-214) is produced by coupling six polyethylene glycol (PEG) residues that can be gradually detached in vivo to the lysine residues on or near the IL-2 CD25 interface of the IL-2 molecule, forming 2-PEG and 1-PEG forms (Charych D et al. Modeling the receptor pharmacology, pharmacodynamics, and pharmacodynamics of NKTR-214, a kinetically-controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy. PLoS One. 2017; 12(7):e0179431), which prolongs the drug's half-life (approximately 12 times that of ordinary IL-2) (Charych DH et al. NKTR-214, an Engineered Cytokine with Biased IL2 Receptor Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse Tumor Models. Clin Cancer). Res. 2016; 22(3): 680-690).However, later clinical studies found that the primary endpoints of PFS and ORR were not met in the phase 3 clinical trials for the treatment of metastatic melanoma. In April 2022, all clinical studies of NKTR-214 in combination with nivolumab were terminated (Eggermont AM et al. PIVOT-12: a phase III study of adjuvant bempegaldesleukin plus nivolumab in resected stage III / IV melanoma at high risk for recurrence. Future Oncol. 2022; 18(8):903-913; Huddart RA et al. PIVOT-10: Phase II study of bempegaldesleukin plus nivolumab in cisplatin-ineligible advanced urothelial cancer. Future Oncol. 2021; 17(2):137-149; Khushalani NI et al. Bempegaldesleukin plus nivolumab in untreated, unresectable or metastatic). melanoma: Phase IIIPIVOT IO 001 study design. Future Oncol. 2020; 16(28):2165-2175).

[0051] This study primarily investigates the effects of PEG-modified IL2 on immune homeostasis and its anti-tumor mechanism. The coupling position of polyethylene glycol (PEG) affects IL2 and the α-chain of the IL2 receptor (CD25). Therefore, PEG-modified IL2 can preferentially bind to the β-chain of the IL2 receptor (CD122) on the surface of effector immune cells such as activated natural T cells and NK cells, promoting positive immunity and exerting an anti-tumor effect. We also found that in healthy physiological model mice, PEG-modified IL2 of different molecular sizes can affect immune homeostasis to varying degrees. A single small dose of 40 kDa PEG-modified IL2 promotes spleen enlargement, activates more immune cells, reduces the proportion of regulatory T cells, increases the CD8 / Treg ratio, and can alter the bias of ordinary IL2, promoting positive immunity. Ordinary IL2, on the other hand, exhibits a dual nature, activating CD8+ while also activating other immune cells. +T cells can promote Treg cell proliferation, but have little effect on the CD8 / Treg ratio (Malek TR. The biology of interleukin-2. Annu Rev Immunol. 2008; 26:453-479). The conjugation method and administration route of 40kd PEG do not change or reverse the positive immune-promoting effect of 40kd PEG-modified IL2.

[0052] 40kd PEG-modified IL-2 showed a greater ability to delay the growth of melanoma, lung cancer, and B-cell lymphoma tumors than the same dose of ordinary IL-2. The anti-melanoma effect of low-dose 40kd PEG-modified IL-2 was more significant than that of high-dose IL-2. On one hand, the PEG modification of 40kd PEG-modified IL-2 prolongs the half-life of IL-2, reducing the dosage and frequency of administration. This overcomes the need for high doses of ordinary IL-2, potentially increasing patient tolerance and reducing severe and potentially fatal toxic side effects such as vascular leakage syndrome caused by high doses. On the other hand, 40kd PEG-modified IL-2 can enhance the anti-tumor effect of IL-2 while altering its immune bias, minimizing tumor immune escape.

[0053] PEG-modified IL-2 reduces the proportion of regulatory T cells, increases the CD8 / Treg cell ratio, and alters the tumor immunophenotype. First, in tumor-bearing mice, a single low-dose dose of 40kD PEG-modified IL-2 promotes spleen enlargement, reduces the proportion of regulatory T cells in tumor tissue, and increases the CD8 / Treg cell ratio, thus altering the bias of ordinary IL-2 and promoting positive immunity. Ordinary IL-2, on the other hand, upregulates the proportion of Treg cells in tumor tissue, while decreasing or maintaining the CD8 / Treg cell ratio. Similar to Nektar's NKTR-214, 40kD PEG-modified IL-2 downregulates the Treg cell ratio and promotes an increase in the CD8 / Treg cell ratio, but the upregulation of the CD8 / Treg cell ratio by 40kD PEG-modified IL-2 is not as significant as that by NKTR-214. Ordinary IL-2, however, upregulates the proportion of regulatory T cells. Second, studies have shown that IL2 can alter the immunogenicity of "cold tumors" with poor immune infiltration (Raeber ME et al. Interleukin-2-based therapies in cancer. Sci Transl Med. 2022; 14(670): eabo5409), reversing tumor immune tolerance. Our immunohistochemistry also revealed that both ordinary IL2 and 40kd PEG-modified IL2 treatments resulted in greater immune cell infiltration in tumor tissues. However, the ordinary IL2 group was mostly distributed at the tumor periphery, exhibiting an "immune rejection" pattern, while the 40kd PEG-modified IL2 group promoted greater CD8+ infiltration.+ When T cells infiltrate tumor tissue, they often exhibit an "immune infiltration" pattern. 40kd PEG-modified IL2 can alter the tumor immune phenotype, promoting the infiltration of more effector cells into the tumor.

[0054] Antagonizing CD8 + Following T cell therapy, the antitumor effect of 40kD PEG-modified IL2 was inhibited, and CD8... +The number and function of T cells can affect the anti-tumor effect of 40kd PEG-modified IL2. Studies have found that IL2 can induce cell exhaustion (Arrieta O et al. Expression of PD-1 / PD-L1 and PD-L2 in peripheral T-cells from non-small cell lung cancer patients. Oncotarget. 2017; 8(60):101994-102005; Liu Y et al. IL-2 regulates tumor-reactive CD8(+)T cell exhaustion by activating the aryl hydrocarbon receptor. Nat Immunol.2021;22(3):358-369; Beltra JC et al.IL2Rβ-dependent signals drive terminal exhaustion and suppress memory development during chronic viral infection.Proc Natl Acad Sci US A.2016;113(37):E5444-5453;Hashimoto M et al.PD-1 combination therapy with IL-2 modifies CD8(+)T cell exhaustion. program.Nature.2022;610(7930):173-181;West EE et al.PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells.J Clin Invest.2013;123(6):2604-2615;Kwon B.The two faces of IL-2:a key driver of CD8(+)T-cell exhaustion.Cell Mol Immunol.2021;18(7):1641-1643), and studies have shown that IL2 can reverse cell exhaustion (Sun Q et al.BCL6 promotes a stem-like CD8(+)T cell program in cancer via antagonizing BLIMP1).Sci Immunol.2023;8(88):eadh1306;Liu X et al.IL-2 Restores T-Cell Dysfunction Induced by Persistent Mycobacterium tuberculosis Antigen Stimulation.Front Immunol.2019;10:2350;IL-2-STAT5 Activity Antagonizes TOX and Reverses CD8+T Cell Exhaustion.Cancer Discov.2023 Dec 21:OF1;Hu CY et al.Interleukin-2 reverses CD8(+)T cell exhaustion in clinical malignant pleural effusion of lung cancer.Clin Exp Immunol.2016;186(1):106-114)。.

[0055] Immunotherapy can enhance the host’s ability to eliminate tumor cells, but after long-term exposure to related antigens, T cells enter a specific differentiation state, namely T cell exhaustion (Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol. 2015; 15(8):486-499). T cell exhaustion is a state in which T cells gradually lose their effector function and memory T cell characteristics due to prolonged exposure to persistent antigens and continuous stimulation. Its characteristics include progressive loss of effector function, altered responsiveness to homeostatic cytokines, persistent expression of inhibitory receptors, changes in epigenetic and transcriptional profiles, and altered metabolic pathways (Wherry EJ, Kurachi M as above; Belk JA et al. Epigenetic regulation of T cell exhaustion. Nat Immunol. 2022; 23(6):848-860; Beltra JC et al. Developmental Relationships of Four Exhausted CD8(+) T Cell Subsets Reveals Underlying Transcriptional and Epigenetic Landscape Control Mechanisms. Immunity. 2020; 52(5):825-841.e828; Weisshaar N et al. Rgs16 promotes antitumor CD8(+) T cell exhaustion. Sci Immunol. 2022; 7(71):eabh1873). T cell exhaustion is one of the mechanisms that evolved to prevent the immune system from becoming overactive, and it is also a major obstacle to current anti-cancer immunotherapy.

[0056] We found that 40kd PEG-modified IL-2 could delay the depletion of CD8 cells in tumor tissues of melanoma and B-cell lymphoma models, and the terminally depleted state of cells (PD-1). + Tim-3 + LAG-3 +The proportion of CD8 cells is low, and they retain high levels of secreted cytokines IFN-γ and Granzyme B. PD-1 inhibitors and 40kD PEG-modified IL2 do not have a synergistic effect in anti-tumor efficacy; for example, 40kD PEG-modified IL2 and anti-PD-1 monoclonal antibodies do not have a synergistic effect in melanoma models. However, in lung cancer models, because 40kD PEG-modified IL2 does not affect PD-1 expression levels, CD8 cells... + PD-1 + TCF-1 + The proportion of T cells is reduced, but CD8 is promoted. + PD-1 + T cells retain higher granzyme B secretion levels while IFN-γ secretion levels are only slightly reduced, indicating a synergistic effect between PD-1 inhibitors and 40kd PEG-modified IL-2 in anti-lung cancer studies. Ordinary IL-2 cannot delay cell exhaustion; terminally exhausted cells (PD-1...) + Tim-3 + LAG-3 + With a high proportion of CD8 cells, the ability of CD8 cells to secrete cytokines IFN-γ and Granzyme B is lost.

[0057] In summary, 40kd PEG-modified IL2 has an anti-tumor effect and is of considerable value in reducing the impact of IL2 on Treg cells while minimizing the impact on CD8+ T cell exhaustion.

[0058] Tregs (Regulatory T cells) or suppressor T cells are a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent excessive immune responses and autoimmune diseases. Tregs typically suppress or downregulate the induction and proliferation of effector Th1 and Th2 cells. Tregs express CD4, CD25, and Foxp3 (CD4+). + CD25 hig FoxP3 + They also exhibit a characteristic phenotype of secreting the immunosuppressive cytokines TGFβ and IL-10. Tregs expand in subjects with cancer and contribute to immunosuppression, thereby helping cancer evade anti-cancer immune mechanisms.

[0059] In a first aspect, IL-2 modified with polyethylene glycol (PEG) is provided, wherein the PEG has a molecular weight of about 40 kD.

[0060] Polyethylene glycol (PEG) is a non-toxic, water-soluble, neutral polymer with good biocompatibility and blood compatibility, and is used for local, gastrointestinal, and intravenous administration in humans. PEG modification of proteins refers to the activation of one or two end groups of PEG, which acquire specific functional groups. These functional groups are active against at least one functional group in the protein to which they are to bind, allowing PEG to bind to the protein's terminal (N-terminus or C-terminus) or a specific amino acid through covalent bonding. PEG's binding sites are universal. PEG is a polymer of ethylene glycol and ethylene oxide, also known as carbon wax, with the structural formula CH2(OH)-(CH2CH2O)n-CH2OH. PEG used for drug modification can be branched (e.g., U / Y-shaped branched molecules) or unbranched (straight-chain) forms, as seen in EP0593868, EP0809996, and CN1243779C.

[0061] For example, branched PEG can include Y-type PEG, as shown in the molecular diagram below:

[0062] Among them, P a and P b They are the same or different polyethylene glycol molecules; j is an integer; R i H, substituted or unsubstituted alkyl, substituted aryl, aralkyl or heteroalkyl, etc.; X1 and X2 are each independently a linking group, for example selected from the following groups: (CH2)n, ... n OCO, (CH2) n NHCO, (CH2) n CO, where n is an integer from 1 to 10, and F is a terminal group, such as selected from the following: hydroxyl, carboxyl, ester, acyl chloride, acyl hydrazine, maleimide, pyridine disulfide, which can react with the amino, hydroxyl or thiol group on the therapeutic agent or matrix to form a covalent bond.

[0063] The molecular weight of PEG used in this article is determined by adding the atomic weights of the atoms that make up PEG.

[0064] Various methods for determining the molecular weight of a molecule are known in the art. For example, electrophoresis can be used to determine the molecular weight of a molecule by comparing it with a standard molecular weight marker. These methods are all within the capabilities of those skilled in the art.

[0065] The PEG used to modify IL-2 can be any suitable type of PEG, such as linear or non-linear PEG. In one embodiment, the PEG comprises branched or unbranched PEG.

[0066] As used herein, “IL-2” means IL-2 from any source, including mammalian sources such as humans, mice, rats, primates, and pigs, and can be natural or obtained through recombinant or synthetic techniques, including recombinant IL-2 peptides produced by a microbial host. IL-2 can be or contains a natural peptide sequence, or can be an active variant of a natural IL-2 peptide.

[0067] In one embodiment, the IL-2 is derived from human or non-human mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, horses, and cats.

[0068] In one embodiment, human IL-2 or its active variants are used herein, more preferably produced recombinantly. The nucleotide and amino acid sequences of human IL-2 are disclosed, for example, in Genbank ref3558 or P60568. Unless otherwise specified, the IL-2 used is in substantially pure form, for example, with a purity of 95% or higher, more preferably 96, 97, 98, or 99%. IL-2 can be used in monomeric or multisomatic protein form. Preferably, the IL-2 polypeptide or active variant is derived from a human source and includes recombinant human IL-2, particularly recombinant human IL-2 produced by a microbial host.

[0069] The IL-2 that can be used in this invention is any clinically available IL-2 for the treatment of tumors. Those skilled in the art can identify or determine which IL-2 or its derivatives or variants can be used in this invention. In one embodiment, the IL-2 of this invention is human IL-2, such as recombinant human IL-2. In one embodiment, the IL-2 used herein comprises the amino acid sequence shown in SEQ ID NO: 1.

[0070] As used herein, polyethylene glycol-modified IL-2 refers to IL-2 modified by PEG, such as unbranched PEG or branched PEG, through linkage with any suitable site on IL-2. The PEG modification described herein can be located at any suitable site in IL-2 for PEG modification, such as at lysine, serine, threonine residues, or the N-terminal α-amino group of IL-2. In one embodiment, the PEG modification is at an N-terminal amino acid residue of IL-2, such as an N-terminal lysine, serine, or threonine residue. In another embodiment, the PEG modification is at the N-terminal α-amino group of IL-2. The PEG modification can be, for example, a single-site modification or a multi-site modification, preferably a single-site modification.

[0071] As used herein, 40KD PEG-modified IL2 is IL-2 modified with PEG having a molecular weight of 40KD, wherein the PEG can be any suitable type of PEG, such as linear or nonlinear PEG. In a preferred embodiment, the 40KD PEG-modified IL2 is IL-2 with the N-terminus modified by PEG having a molecular weight of about 40KD (e.g., SEQ ID NO: 1). "About 40KD" means that the sum of the atomic weights of the atoms constituting the PEG is 40KD ± 10%, 40KD ± 5%, 40KD ± 4%, 40KD ± 3%, 40KD ± 2%, 40KD ± 1%, 40KD ± 0.5%, or even 40KD ± 0.1%.

[0072] In a second aspect, a pharmaceutical composition is provided comprising PEG-modified IL-2 as described in the first aspect, optionally comprising a pharmaceutically acceptable carrier, preferably, the pharmaceutical composition for treating tumors.

[0073] As used in this article, "treatment" refers to the partial or complete elimination or relief of symptoms, or their stabilization, following treatment. Therefore, treatment encompasses prevention, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing the worsening of symptoms or disease progression.

[0074] As used herein, "therapeutic effective dose" or "therapeutic dose" means a dose that is at least sufficient to produce a therapeutic effect in a subject. For the active ingredient described herein, a specific therapeutic effective dose can be initially estimated using a variety of techniques known in the art. A suitable dose range for human subjects can be determined, for example, using data from cell culture experiments and other animal studies. Dosage levels and regimens can be determined based on known doses and regimens, and if necessary, extrapolated based on known properties and / or empirically determined based on a variety of factors. These factors include, for example, the subject's weight, general health, age, activity of the specific compound used, sex, diet, time of administration, drug combination, disease severity and duration, as well as the patient's disease susceptibility and the physician's judgment. After the patient's condition improves, a maintenance dose of the compound or composition may be administered, and the dose, dosage form, and frequency of administration, or combinations thereof, may be varied if necessary. Precise dosage and regimens should be determined based on the physician's judgment and the specific patient's condition.

[0075] The therapeutically effective dose depends largely on the nature of the drug, the patient's (human) condition, and the nature and severity of the disease being treated. For example, when treating tumors, the therapeutically effective dose of 40kD polyethylene glycol (PEG) modified IL-2 can range from as low as 1 million units / kg body weight to as high as 10 million units / kg body weight, and more specifically, dose ranges of 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 100-150, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300. 00, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-900, 400-800, 400-700, 400-600, 400-500, 500-900, 500-800, 500-700, 500-600, 600-900, 600-800, 600-700, 700-900, 700-800 or 800-900 million U / kg body weight, preferably between 2 million units / kg body weight and 4 million units / kg body weight.

[0076] The term "drug-acceptable carrier" includes any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents. The use of such media and agents is well known in the art. The active ingredients used herein, such as glucocorticoids and polyethylene glycol-modified IL2, can be mixed with one or more pharmaceutically acceptable additives, diluents, or carriers. Examples of drug-acceptable carriers include, but are not limited to, lactose, sucrose, dextran, mannitol or glucose starch, talc, magnesium stearate, magnesium oxide, crystalline cellulose, methylcellulose, carboxymethyl cellulose, gelatin, glycerin, sodium alginate, saline, and water, and may also contain additives such as fillers, binders, humectants, flow aids, stabilizers, preservatives, emulsifiers, and additional solvents or solubilizers or substances that achieve a storage effect.

[0077] The pharmaceutical composition can be formulated in any suitable form (e.g., for parenteral delivery), such as solution, suspension, tablet, dispersible tablet, pill, capsule, powder, sustained-release formulation or elixir, for oral administration, and transdermal patch formulation and dry powder inhaler. Typically, compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, 4th Edition, 1985, 126). The pharmaceutical composition can be formulated as a solid, liquid, gel, or other form. The pharmaceutical composition can be formulated for subcutaneous or intravenous delivery.

[0078] The pharmaceutical compositions are available in bulk and unit dose forms, as well as in forms known in the art such as openable or perforated implants, capsules, blister packs, or cartridges. Kits are also provided, which include a delivery device, an individual container containing the pharmaceutical compositions described herein, and optional other suitable additives, such as other therapeutic compounds, excipients, surfactants (used as therapeutic agents and formulation ingredients), antioxidants, flavoring and coloring agents, fillers, volatile oils, buffers, dispersants, surfactants, antioxidants, flavoring agents, swelling agents, propellants, and preservatives, along with instructions for use of the kit components.

[0079] The pharmaceutical composition can be formulated for administration via any route known to those skilled in the art, including but not limited to intramuscular, intravenous, intradermal, intralesional, intraperitoneal, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, ear, inhalation, buccal (e.g., sublingual), and transdermal administration. Administration can be local, topical, or systemic.

[0080] As used in this article, a tumor (also called a neoplasm) is an abnormal mass of tissue that results from cells proliferating at an abnormally high rate. Tumors can be benign (non-cancerous) or malignant (cancerous). As used in this article, tumors encompass both hematologic malignancies and solid tumors.

[0081] In this document, the tumor can be any tumor known in the art or proven in the future to be treatable with IL-2. The tumor can be a solid tumor or a metastatic tumor, such as glioma, sarcoma, adenocarcinoma, adenosarcoma, or adenoma. In some embodiments, the tumor includes, but is not limited to, tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, cervix, or liver. In one implementation, the tumors described herein are solid tumors, such as lung and bronchial tumors, breast tumors, colon and rectal tumors, kidney tumors, stomach tumors, esophageal tumors, liver and intrahepatic bile duct tumors, bladder tumors, brain and other nervous system tumors, head and neck tumors, oral and pharyngeal tumors, cervical tumors, uterine body tumors, thyroid tumors, ovarian tumors, testicular tumors, prostate tumors, malignant melanoma, cholangiocarcinoma, thymoma, non-melanoma skin cancer, and hematologic malignancies and / or malignancies, such as childhood leukemia and lymphoma, multiple myeloma, Hodgkin's disease, lymphocytic and cutaneous lymphomas, acute and chronic leukemias such as acute lymphoblastic leukemia, acute myeloid or chronic myeloid leukemia, plasma cell vegetations, lymphoid vegetations, and AIDS-related cancers. Typically, combination therapies are used to treat solid tumors such as stromal cancers. Exemplary tumors include, for example, pancreatic tumors, ovarian tumors, lung tumors, colon tumors, prostate tumors, cervical tumors, and breast tumors. In one embodiment, the tumor is selected from melanoma, lung cancer, lymphomas such as B-cell lymphoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, cervical cancer, head and neck cancer, and breast cancer. Preferably, the tumor is selected from melanoma, lung cancer, and lymphomas such as B-cell lymphoma.

[0082] Optionally, the pharmaceutical composition may also contain other agents for treating tumors. These agents may be agents that assist the therapeutic effect of IL-2, agents that alone have a tumor therapeutic effect, or agents that have other effects (e.g., reducing the side effects of IL-2). In one embodiment, the agent comprises, but is not limited to, anticancer agents, including but not limited to chemotherapeutic agents, radiotherapy agents, cytokines, anti-angiogenic agents, apoptosis inducers, or anticancer immunotoxins or coagulation ligands, such as paclitaxel, bevacizumab, vincristine, vinblastine, angiostatin, nucleoside analogs or other antimetabolites, glucocorticoids such as dexamethasone, budesonide, cortisone, prednisone, triamcinolone acetonide and its derivatives, arubicin, bleomycin, actinomycin C, carboplatin, cyclophosphamide, cytarabine, interferons such as interferon α-2a, α-2b, β, γ; and anticancer antibodies, including but not limited to anti-CEA antibodies, anti-EGFR antibodies, anti-EpCAM antibodies, anti-Her2 antibodies, anti-PSA antibodies, anti-TGF-β antibodies, anti-TRAIL-R1 and TRAIL- R2 antibodies; immune checkpoint inhibitors, including, for example, inhibitors of immunosuppressive molecules such as anti-cytotoxic T-lymphocyte antigen 4 (CTLA4 or CD152), programmed cell death protein 1 (PD-1), or programmed cell death protein 1 ligand 1 (PD-L1), such as antibodies or aptamers against CTLA4, PD-1, or PD-L1; immunomodulators that can increase or decrease the production of one or more cytokines, upregulate or downregulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation of one or more types of immune cells, such as including but not limited to nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, steroids (e.g., glucocorticoids), cytokines such as TGF-β, cytokines, chemokines, or receptor antagonists, heterologous anti-lymphocyte globulins, etc.

[0083] In particular, inhibitors of the immunosuppressive molecules CTLA4, PD-1, and PD-L1 are intended for use in the combinations and methods provided by this invention. In particular instances, the immune checkpoint inhibitors are antibodies or aptamers. Examples of CTLA4, PD-1, and PD-L1 inhibitors include anti-CTLA4, anti-PD-1, and anti-PD-L1 antibodies and aptamers.

[0084] Programmed cell death protein 1 (PD-1): The PD-1 molecule is a member of the immunoglobulin gene superfamily. Human PD-1 has an extracellular region containing an immunoglobulin superfamily domain, a transmembrane domain, and an intracellular region including an immunoreceptor tyrosine repressor motif (ITIM) (Ishida et al., EMBO J.11:3887, 1992; Shinohara et al., Genomics 23:704, 1994; US Patent No. 5,698,520). PD-1 is expressed on T cells, B cells, and macrophages. The ligands for PD-1 are B7 family members PD-ligand 1 (PD-L1, also known as B7-H1) and PD-L2 (also known as B7-DC). Exemplary amino acid sequences of human PD-1 are shown in Ishida et al., EMBO J.11:3887, 1992; Shinohara et al. Genomics 23:704, 1994; and U.S. Patent No. 5,698,520. PD-1 inhibitors include substances that reduce the expression or activity of PD ligand 1 (PD-L1) or PD ligand 2 (PD-L2) or reduce the interaction between PD-1 and PD-L1 or PD-L2. Exemplary compounds include antibodies (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies), RNAi molecules (e.g., anti-PD-1 RNAi molecules, anti-PD-L1 RNAi, and anti-PD-L2 RNAi), antisense molecules (e.g., anti-PD-1 antisense RNA, anti-PD-L1 antisense RNA, and anti-PD-L2 antisense RNA), and dominant-negative proteins (e.g., dominant-negative PD-1 protein, dominant-negative PD-L1 protein, and dominant-negative PD-L2 protein), see, for example, PCT Publication 2008 / 083174, which is incorporated herein by reference. Anti-PD-1 antibodies include, but are not limited to, any of those described in U.S. Patent Nos. 7,943,743, 8,008,449, 8,779,105, 8,735,553, 8,217,149, 8,679,767, and 8,779,108, and International Patent Publications WO 2008 / 156712, WO 2010 / 077634, and WO 2013 / 019906. These anti-PD-1 antibodies have been involved in clinical trials for the treatment of various cancers, such as melanoma, non-small cell lung cancer, renal cell carcinoma, hematologic malignancies, lymphoma, leukemia, pancreatic cancer, prostate cancer, lung cancer, and multiple myeloma.

[0085] In one particular embodiment, a pharmaceutical composition for treating lung cancer is provided, comprising the PEG-modified IL-2 of any one of claims 1-3 and an immune checkpoint inhibitor. In a preferred embodiment, the immune checkpoint inhibitor is a CTLA4, PD-1, or PD-L1 inhibitor such as an anti-CTLA4, anti-PD-1, or anti-PD-L1 antibody or aptamer. In a further preferred embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor, such as an anti-PD-1 antibody or aptamer. In a particularly preferred embodiment, the PD-1 antibody is anti-mouse PD-1 (clone number 29F.1A12, biolegend, catalog number 135220).

[0086] In a third aspect, a method of treating a tumor includes administering a therapeutically effective amount of the PEG-modified IL-2 of the first aspect or the pharmaceutical composition of the second aspect to a subject in need.

[0087] The tumor may be a tumor known in the art or proven in the future to be treatable with IL-2, such as, but not limited to, solid tumors or metastatic tumors, such as gliomas, sarcomas, adenocarcinomas, adenosarcomas, or adenomas. In some embodiments, the tumor includes, but is not limited to, tumors of the breast, heart, lungs, small intestine, colon, spleen, kidneys, bladder, head and neck, ovaries, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, cervix, or liver. In one implementation, the tumors described herein are solid tumors, such as lung and bronchial tumors, breast tumors, colon and rectal tumors, kidney tumors, stomach tumors, esophageal tumors, liver and intrahepatic bile duct tumors, bladder tumors, brain and other nervous system tumors, head and neck tumors, oral and pharyngeal tumors, cervical tumors, uterine body tumors, thyroid tumors, ovarian tumors, testicular tumors, prostate tumors, malignant melanoma, cholangiocarcinoma, thymoma, non-melanoma skin cancer, and hematologic malignancies and / or malignancies, such as childhood leukemia and lymphoma, multiple myeloma, Hodgkin's disease, lymphocytic and cutaneous lymphomas, acute and chronic leukemias such as acute lymphoblastic leukemia, acute myeloid or chronic myeloid leukemia, plasma cell vegetations, lymphoid vegetations, and AIDS-related cancers. Typically, combination therapies are used to treat solid tumors such as stromal cancers. Exemplary tumors include, for example, pancreatic tumors, ovarian tumors, lung tumors, colon tumors, prostate tumors, cervical tumors, and breast tumors. In one embodiment, the tumor is selected from melanoma, lung cancer, lymphomas such as B-cell lymphoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, cervical cancer, head and neck cancer, and breast cancer. Preferably, the tumor is selected from melanoma, lung cancer, and lymphomas such as B-cell lymphoma. In one embodiment, the drug described herein is used to treat tumors selected from melanoma, lung cancer, and B-cell lymphoma.

[0088] In one embodiment, the method may further include administering other agents for treating tumors. These agents may be agents that assist the therapeutic effect of IL-2, agents that alone have a tumor therapeutic effect, or agents that have other effects (e.g., reducing the side effects of IL-2). In one embodiment, the agent comprises, but is not limited to, anticancer agents, including but not limited to chemotherapeutic agents, radiotherapy agents, cytokines, anti-angiogenic agents, apoptosis inducers, or anticancer immunotoxins or coagulation ligands, such as paclitaxel, bevacizumab, vincristine, vinblastine, angiostatin, nucleoside analogs or other antimetabolites, glucocorticoids such as dexamethasone, budesonide, cortisone, prednisone, triamcinolone acetonide and its derivatives, arubicin, bleomycin, actinomycin C, carboplatin, cyclophosphamide, cytarabine, interferons such as interferon α-2a, α-2b, β, γ; and anticancer antibodies, including but not limited to anti-CEA antibodies, anti-EGFR antibodies, anti-EpCAM antibodies, anti-Her2 antibodies, anti-PSA antibodies, and anti-TGF-β antibodies. Antibodies against TRAIL-R1 and TRAIL-R2; immune checkpoint inhibitors, such as antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA4 or CD152), programmed cell death protein 1 (PD-1), or programmed cell death protein 1 ligand 1 (PD-L1); immunomodulators that can increase or decrease the production of one or more cytokines, upregulate or downregulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation of one or more types of immune cells, including but not limited to nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, steroids (e.g., glucocorticoids), cytokines such as TGF-β, cytokines, chemokines, or receptor antagonists, heterologous anti-lymphocyte globulins, etc.

[0089] In one embodiment, the PEG-modified IL-2 or pharmaceutical composition is administered orally, intravenously (IV), subcutaneously, intramuscularly, intratumorally, intradermally, topically, percutaneously, transrectally, intrathecally, or subepidermally, or formulated for such administration. In a particular embodiment, the PEG-modified IL-2 or pharmaceutical composition is administered intravenously or subcutaneously. The most suitable route depends on a variety of factors, such as the nature of the disease, the course of the disease, the severity of the disease, and the specific composition used, which can be determined by those skilled in the art.

[0090] In one embodiment, the PEG-modified IL-2 or pharmaceutical composition is administered twice weekly, once weekly, once every 14 days, once every 21 days, or once monthly. In other examples, the PEG-modified IL-2 or pharmaceutical composition is administered twice weekly, once weekly, once every 14 days, once every 21 days, or once monthly.

[0091] In one embodiment, the PEG-modified IL-2 or pharmaceutical composition is administered or formulated for administration in dose ranges between or approximately between the following: 100,000 units / kg body weight to 10,000,000 units / kg body weight (subject), for example 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 50 -1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 150-1000, 150-100, 150-900, 150-800 150-700, 150-600, 150-500, 150-400, 150-300, 150-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400 -1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700, 700-1000, 700-900, 700-800, 800-1000 or 800-900 million units / kg body weight (subject).

[0092] The PEG-modified IL-2 described in the first aspect or the pharmaceutical composition described in the second aspect may be administered in any proportion and / or sequentially or simultaneously with other pharmaceutical agents, as determined by those skilled in the art based on the patient's condition.

[0093] The object is a human or non-human object suffering from a tumor, and non-limiting examples include humans, other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, horses, cats, and other non-mammals. In some embodiments, the object is a human.

[0094] In one particular embodiment, a method of treating lung cancer is provided, comprising administering to a subject of need a therapeutically effective amount of the PEG-modified IL-2 and immune checkpoint inhibitor described in the first aspect. In a preferred embodiment, the immune checkpoint inhibitor is a CTLA4, PD-1, or PD-L1 inhibitor such as an anti-CTLA4, anti-PD-1, or anti-PD-L1 antibody or aptamer. In a further preferred embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor, such as an anti-PD-1 antibody or aptamer. In a particularly preferred embodiment, the PD-1 antibody is anti-mouse PD-1 (clone number 29F.1A12, biolegend, catalog number 135220).

[0095] In a fourth aspect, the use of the PEG-modified IL-2 described in the first aspect in the preparation of a medicament for treating tumors is provided, or the use of the PEG-modified IL-2 described in the first aspect for treating tumors is provided.

[0096] The tumor may be a tumor known in the art or proven in the future to be treatable with IL-2, such as, but not limited to, solid tumors or metastatic tumors, such as gliomas, sarcomas, adenocarcinomas, adenosarcomas, or adenomas. In some embodiments, the tumor includes, but is not limited to, tumors of the breast, heart, lungs, small intestine, colon, spleen, kidneys, bladder, head and neck, ovaries, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, cervix, or liver. In one implementation, the tumors described herein are solid tumors, such as lung and bronchial tumors, breast tumors, colon and rectal tumors, kidney tumors, stomach tumors, esophageal tumors, liver and intrahepatic bile duct tumors, bladder tumors, brain and other nervous system tumors, head and neck tumors, oral and pharyngeal tumors, cervical tumors, uterine body tumors, thyroid tumors, ovarian tumors, testicular tumors, prostate tumors, malignant melanoma, cholangiocarcinoma, thymoma, non-melanoma skin cancer, and hematologic malignancies and / or malignancies, such as childhood leukemia and lymphoma, multiple myeloma, Hodgkin's lymphoma, lymphocytic and cutaneous lymphomas, acute and chronic leukemias such as acute lymphoblastic leukemia, acute myeloid or chronic myeloid leukemia, plasma cell vegetations, lymphoid vegetations, and AIDS-related cancers. Typically, combination therapies are used to treat solid tumors such as stromal cancers of solid tumors. Exemplary tumors include, for example, pancreatic tumors, ovarian tumors, lung tumors, colon tumors, prostate tumors, cervical tumors, and breast tumors. In one embodiment, the tumor is selected from melanoma, lung cancer, lymphomas such as B-cell lymphoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, cervical cancer, head and neck cancer, and breast cancer. Preferably, the tumor is selected from melanoma, lung cancer, and lymphomas such as B-cell lymphoma. In one embodiment, the drug described herein is used to treat tumors selected from melanoma, lung cancer, and B-cell lymphoma.

[0097] In a particular embodiment, the use of the PEG-modified IL-2 and immune checkpoint inhibitors described in the first aspect is provided in the preparation of a medicament for treating lung cancer, or for the treatment of lung cancer. In a preferred embodiment, the immune checkpoint inhibitor is a CTLA4, PD-1, or PD-L1 inhibitor such as an anti-CTLA4, anti-PD-1, or anti-PD-L1 antibody or aptamer. In a further preferred embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor, such as an anti-PD-1 antibody or aptamer. In a particularly preferred embodiment, the PD-1 antibody is anti-mouse PD-1 (Selleck China, catalog number A2122), which is an in vivo antibody against mouse programmed death-1 (PD-1) antibody that reacts with mouse PD-1, also known as CD279, to inhibit PD-1 function. Studies in mice have demonstrated its significant anti-tumor effect (https: / / www.selleck.cn / products / anti-mouse-pd-1-cd279--invivo.html).

[0098] In the fifth aspect, PEG-modified IL-2 as described in the first aspect is provided for promoting spleen enlargement, reducing the proportion of regulatory T cells, increasing the CD8 / Treg ratio, and promoting immune cells such as T cells like CD8. + Methods for T cell infiltration into tumors, reducing STAT5 phosphorylation levels in regulatory T (Treg) cells, reducing the expression of transcription factors regulating Treg cells, reducing PD-1 expression on immune cells, reducing the expression of exhaustion-related transcription factors, increasing the proportion of central memory cells, increasing the proportion of CD8+ central memory T cells, and / or reducing tumor immune escape effects. These methods can be in vitro (e.g., in vitro cell experiments) or in vivo.

[0099] In a sixth aspect, PEG-modified IL-2 as described in the first aspect is provided for promoting spleen enlargement, reducing the proportion of regulatory T cells, increasing the CD8 / Treg ratio, and promoting the growth of immune cells such as T cells like CD8. + Uses include T cell infiltration into tumors, reducing STAT5 phosphorylation levels in regulatory T (Treg) cells, reducing the expression of transcription factors that regulate Treg cells, reducing PD-1 expression in immune cells, reducing the expression of exhaustion-related transcription factors, increasing the proportion of central memory cells, increasing the proportion of CD8+ central memory T cells, and / or reducing tumor immune escape effects.

[0100] In the seventh aspect, the PEG-modified IL-2 described in the first aspect is provided for the preparation of IL-2 for promoting spleen enlargement, reducing the proportion of regulatory T cells, increasing the CD8 / Treg ratio, and promoting immune cells such as T cells such as CD8+. + Use in agents or kits for T cell infiltration into tumors, reducing STAT5 phosphorylation levels in regulatory T (Treg) cells, reducing the expression of transcription factors that regulate Treg cells, reducing PD-1 expression in immune cells, reducing the expression of exhaustion-related transcription factors, increasing the proportion of central memory cells, increasing the proportion of CD8+ central memory T cells, and / or reducing tumor immune escape effects.

[0101] The kit may also include other reagents (e.g., solvents, buffers, etc.) required to formulate the PEG-modified IL-2 into a suitable pharmaceutical preparation, and optionally, instructions.

[0102] Unless the context otherwise indicates, the word “or” as used in this article is intended to include “and”.

[0103] As used herein, “optional” or “optionally” means that the event or condition described below occurs or does not occur, including both the occurrence and non-occurrence of said event or condition. For example, an optionally included step means that the step is present or absent; an optionally included pharmaceutically acceptable carrier means that said pharmaceutically acceptable carrier is included or not included.

[0104] As used herein, the term "about" refers to a range of values ​​that includes a specific value and that a person skilled in the art would reasonably consider similar to that specific value. In some embodiments, the term "about" refers to within the standard error of a measurement commonly accepted in the art. For example, in some embodiments, "about" refers to ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, or even ±0.5% of a specific value.

[0105] As used in this article, when a specification lists specific values ​​or proportions for a feature, it also covers any range of any two of those values ​​or proportions. For example, listing the values ​​1, 2, 3, and 4 also covers 1-2, 1-3, 1-4, 2-3, 2-4, and 3-4. Example

[0106] The present invention is further illustrated by the following embodiments, but any embodiment or combination thereof should not be construed as limiting the scope or implementation of the invention. The scope of the invention is defined by the appended claims, and those skilled in the art can clearly understand the scope defined by the claims in conjunction with this specification and common knowledge in the art. Without departing from the spirit and scope of the invention, those skilled in the art can make any modifications or changes to the technical solutions of the invention, and such modifications and changes are also included within the scope of the invention. Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0107] Materials and Methods

[0108] 1. Cells

[0109] Mouse melanoma B16F10 cells, mouse Lewis's lung adenocarcinoma LLC cells, and mouse B-cell lymphoma A20 cells were purchased from the Slack Laboratory Animal Center of the Chinese Academy of Sciences.

[0110] B16F10 and LLC cells were cultured in DMEM + 10% FBS + 1× penicillin antibody, with the medium changed every 2-3 days. A20 cells were cultured in RPMI 1640 + 10% FBS + 0.05mM 2-β-mercaptoethanol + 1× penicillin antibody, with the medium changed every 2-3 days.

[0111] 2. Constructing a subcutaneous tumor-bearing mouse model

[0112] Six-week-old SPF-grade female C57BL / 6j and BALB / c mice were purchased from the Slack Animal Experiment Center, Chinese Academy of Sciences. They were housed in the SPF-grade laboratory of the Fudan University Animal Experiment Center. Newly purchased mice were kept in a quiet environment in an SPF-grade animal facility for at least 7 days to ensure acclimatization. Hair was removed from the left axilla area of ​​the mice before cell injection to fully expose the skin and ensure the subcutaneous injection area and surrounding tumor were hairless. The subcutaneous injection area was disinfected with 75% alcohol. Using a 1ml syringe, 200μL of cell suspension was subcutaneously injected into the left axilla of each 5-6 week old female C57BL / 6j mouse, i.e., 1×10⁻⁶ cells per mouse. 6 A subcutaneous melanoma model was constructed using 1×10 B16F10 cells. 6 Lung cancer model mice were constructed using LLC cells, 1×10 6 A20 cells were used to construct a mouse model of B-cell lymphoma. After 6-8 days, the subcutaneous tumor grew to the size of a mung bean (2.5-4 mm long and 2.5-3 mm wide), indicating successful model establishment.

[0113] 3. Drug intervention

[0114] Both recombinant human IL-2 (rhIL-2) and polyethylene glycol modified IL-2 (PEG IL-2) were provided by Xiamen Tebo Co., Ltd. The preparation method is described in "8. Preparation of polyethylene glycol (PEG) modified IL2 (see Figure 23)" below.

[0115] PEG-IL-2 modified with polyethylene glycol (PEG) at different molecular weights includes 10kDa PEG-IL-2, 20kDa PEG-IL-2, and 40kDa PEG-IL-2. 40kDa PEG-IL-2 with different structural modifications includes Y-type (branched) 40kDa PEG-IL-2 and M-type (unbranched) PEG-IL-2, 20kDa PEG-IL-2, and 40kDa PEG-IL-2. The following "10kDa PEG-IL-2, 20kDa PEG-IL-2, 40kDa PEG-IL-2" all refer to the M-type (unbranched). All of the above drugs are stored at -20 degrees Celsius.

[0116] Subcutaneous tumor-bearing mice were randomly divided into three groups of six mice each: a PBS group, an rhIL-2 group, and a 40kDa PEG-IL-2 group. Administered medications via intraperitoneal injection. The PBS group received sterile, calcium-free injections. 2+ Mg 2+ The PBS buffer solution was injected once daily for five consecutive days, with one week constituting one cycle. The rhIL-2 group was injected with recombinant human IL-2 (rhIL-2), 400,000 U / animal (approximately 2.5 mg / kg), once daily for five consecutive days, with one week constituting one cycle. The 40kd PEG-IL-2 group was injected with 40kd PEG-modified IL-2 (40kd PEG-IL-2), 400,000 U / animal (approximately 2.5 mg / kg), once weekly, with one week constituting one cycle.

[0117] Anti-mouse PD-1-inVivo was purchased from Selleck China (product number A2122). Administer 100 μg of anti-mouse PD-1-inVivo per device via intraperitoneal injection once every two days, for a total of three doses.

[0118] 4. Tumor proliferation marker detection

[0119] Monitor tumor size in mice every 1-2 days. After 1 week, or when the tumor size becomes too large (exceeding 1,500 mm²), monitor the tumor size again. 3 Mice were euthanized when the tumor size exceeded 10% of the animal's body weight or when the tumor surface ulcerated. Subcutaneous tumors, spleen, and lymph nodes were obtained through dissection, and the corresponding mouse body weight, tumor tissue weight, and spleen weight were weighed and recorded. The dissected tissues were fixed in formalin solution (a 35% or higher concentration of formaldehyde aqueous solution).

[0120] One week later, or when the tumor becomes too large (tumor volume exceeds 1,500 mm). 3 Mice were euthanized when the tumor size exceeded 10% of the animal's body weight or when the tumor surface ulcerated. Subcutaneous tumors, spleens, and lymph nodes were obtained through dissection, and the corresponding mouse body weight, tumor tissue weight, and spleen weight were weighed and recorded. The spleen index was calculated as follows: Spleen index = Spleen weight (g) / Mouse body weight (g) × 100%. The dissected tissues were fixed in formalin solution (a 35% or higher concentration of formaldehyde aqueous solution). Relative tumor volume (RTV) = Tumor volume in the treatment group / Tumor volume in the control group. The tumor growth inhibition value (TGI) = (1 - Average tumor weight in the treatment group / Average tumor weight in the control group) × 100%.

[0121] 5. Flow cytometry analysis

[0122] Mice were dissected, and spleens were harvested. The spleen was ground to prepare a single-cell suspension. Red blood cells from the single-cell suspension were lysed and incubated with anti-mouse CD16 / 32 (TruStain FcX) for 15 minutes, followed by washing three times with stain buffer. Then, the cells were incubated with flow cytometry antibodies. Depending on the specific flow cytometry antibody staining requirements, the antibodies were either incubated on ice in the dark for 30 minutes or, after membrane perforation and fixation, incubated on ice in the dark for 30 minutes, followed by washing three times with stain buffer. Flow cytometry analysis was then performed. All processed samples were analyzed within 2 hours. Overlapping cells were removed using FSC-A and FSC-H. After removing overlapping cells, CD3 was analyzed. - NK1.1 + The proportion of T cells. Using CD3 + T cells are isolated from immune cells, specifically CD3. + Intra-gate analysis of T cells and the presence of various immune cells in CD3 + The proportion of T cells.

[0123] Flow cytometry antibodies include: FITC-labeled anti-mouse CD3ε (clone 145-2C11, biolegend, catalog number 100340), APC-labeled anti-mouse CD4 (clone RM4-5, biolegend, catalog number 100516), PE / Cyanine7-labeled anti-mouse CD25 (clone PC61, biolegend, catalog number 102016), PE-labeled anti-mouse FoxP3 (clone 150D, biolegend, catalog number 320008), Pacific Blue-labeled anti-mouse CD8a (clone 53-6.7, biolegend, catalog number 100725), PE / Cyanine7-labeled anti-mouse CTLA-4 (clone UC10-4B9, biolegend, catalog number 106314), and PE-labeled anti-mouse... Tim-3 (clone RMT3-23, biolegend, catalog number 119704), Brilliant Violet 605 labeled anti-mouse PD-1 (clone 29F.1A12, biolegend, catalog number 135220), Brilliant Violet 650 labeled anti-mouse LAG-3 (clone C9B7W, biolegend, catalog number 125227), PE / Cyanine labeled anti-mouse CTLA-4 (clone UC10-4B9, biolegend, catalog number 106314), Alexa 700-marked anti-mouse NK-1.1 (clone number UC10-4B9, biolegend, catalog number 106314).

[0124] 6. Pathological examination of tumor tissue sections

[0125] At the end of the experiment, tumor tissues from all tumor-bearing model mice that were euthanized were fixed in 10% formalin, paraffin-embedded, dewaxed and hydrated, and then immunohistochemically stained. The tissues were incubated with diluted mouse anti-CD3, CD4, and CD8 antibodies. The chromogenic agent of the labeled antibodies was developed through chemical reaction to determine the intracellular antigens. The localization, qualitative and quantitative studies of indicators such as CD3, CD4, and CD8 were conducted.

[0126] 7. Data Analysis

[0127] Experimental data were statistically analyzed using Graphpad Prism 9 software. Statistical differences between data points were calculated, with P < 0.05 considered statistically significant. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0128] 8. Preparation of polyethylene glycol (PEG) modified IL2 (see Figure 23)

[0129] PEG with molecular weights of 10kDa, 20kDa, and 40kDa has two structures: branched and unbranched.

[0130] The IL-2 (SEQ ID NO: 1) replacement buffer is sodium acetate acetate buffer (pH value). ), and M-ALD-40kD and Y-ALD-40KD (Beijing Jiankai Technology Co., Ltd.) linear and branched PEG were mixed in the following proportions (mass ratio: The PEG-2 crude product was obtained by reduction with sodium cyanoborohydride at 2-10℃ for 3-18 hours. The crude PEG-2 was initially separated by cation exchange chromatography in a sodium acetate-acetate buffer system. The target protein peak was collected and subjected to reverse-phase chromatography with gradient elution in an ethyl acetate-trifluoroacetic acid system. The target protein peak was collected and concentrated by cation exchange chromatography in a sodium acetate-acetate buffer system. Finally, the target protein peak was replaced with sodium acetate-acetate buffer by ultrafiltration. 10kDa and 20kDa unbranched (linear) PEG-modified IL-2 were also prepared using the same process as for the 40kDa unbranched (linear) PEG-modified IL-2 (PEG). The different molecular weights and structures of PEG-modified IL2 obtained were represented as follows: unbranched IL2 included 10kD PEG-IL-2, 20kD PEG-IL-2, and 40kD PEG-IL-2; 40kD (branched)-IL-2, 20kD (branched)-IL-2, 10kD PEG (branched)-IL-2 and other types of electrophoresis diagrams were similar but not shown.

[0131] The selected PEG type primarily modifies the N-terminus of the peptide chain. After modification with 20kD unbranched PEG, the molecular weight of recombinant human IL-2 (ordinary) increased from 15kD to 35kD, with high purity (Figure 1a, Figure 1b). Activity assays using the CTLL-2 cell method confirmed that the PEG-modified IL-2 had comparable activity to the IL-2 working standard (Figure 1c, Figure 1d).

[0132] The sequence of the human IL-2 used is shown in SEO ID NO: 1 (5'-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT-3').

[0133] Example 1: 400,000U 40kd PEG-modified IL-2 increases the CD8 / Treg ratio

[0134] To preliminarily verify whether 40kD PEG-modified IL-2 has a regulatory effect on the immune system, this study first observed the spleen tissue and detected the proportion of immune cells in each group of mice. Different doses of 40kD PEG-modified IL-2 (400,000U 40kD PEG-IL-2) were administered intraperitoneally at 50,000U / mouse, 100,000U / mouse, 200,000U / mouse, 400,000U / mouse, and 600,000U / mouse, respectively. An intraperitoneal injection of the same dose of PBS served as a control group. Mice were sacrificed on day 7, and their spleens were dissected. The size and weight of the spleen were observed, revealing that 40kD PEG-IL-2 promoted spleen enlargement and spleen cell proliferation (see Figure 1A). Flow cytometry analysis of spleen cells showed that 400,000U and 600,000U 40kD PEG-IL-2 promoted CD8+. + T cells and CD4 + The T cell ratio (CD8 / CD4) (see Figure 1B) promotes the body's immune function; CD8 from spleen cells... + T cells and CD4 + CD25 + FoxP3 + In terms of the CD8 / Treg ratio, 400,000 U and 600,000 U 40 kDa PEG-IL-2 can alter immune homeostasis (see Figure 1C) and promote positive immune function. The minimum concentration required to alter the CD8 / Treg ratio in mouse spleen is 400,000 U (see Figure 1).

[0135] Example 2: 40kd PEG-modified IL-2 can alter immune homeostasis and increase the CD8 / Treg ratio.

[0136] To further investigate the effects of polyethylene glycol (PEG)-modified IL-2 of different molecular sizes on the immune system, the effects of 10kd PEG, 20kd PEG, and 40kd PEG-modified IL-2 on the immune system were compared. Mice were intraperitoneally injected with 400,000 U / mouse of PEG-modified IL-2 of different molecular sizes: 10kd PEG-IL-2 (10kd PEG-IL-2), 20kd PEG-modified IL-2 (20kd PEG-IL-2), and 40kd PEG-modified IL-2 (40kd PEG-IL-2). An intraperitoneal injection of an equal dose of PBS served as a blank control group, while intraperitoneal injection of 1,200,000 U of recombinant human IL-2 (rhIL-2, IL-2) for 5 consecutive days served as the experimental control group. Mice were sacrificed on day 7. Spleens were harvested from the mice, and the proportion of immune cells in the spleen cells was detected by flow cytometry. We found that both 20kd PEG-IL-2 and 40kd PEG-IL-2 upregulated CD8+ in mouse spleen cells. + T cells and CD4 + The T cell ratio (CD8 / CD4) (see Figures 2A and 2C) showed that a single dose of 40 kDa PEG-IL-2 upregulated the CD8 / CD4 ratio more significantly than continuous 5-day administration of a high dose (1,200,000 U) of conventional IL-2 (see Figure 2C). CD8 from spleen cells... + T cells and CD4 + CD25 + FoxP3 + Regarding the Treg cell ratio (CD8 / Treg), both 20kd PEG-IL-2 and 40kd PEG-IL-2 upregulated the CD8 / Treg ratio in mouse spleen cells (see Figure 2B). However, 40kd PEG-IL-2 upregulated the CD8 / Treg ratio in mouse spleen cells more significantly than high-dose (1,200,000U) IL-2. Furthermore, a single administration of 400,000U 40kd PEG-IL-2 significantly increased the CD8 / Treg ratio compared to continuous high-dose IL-2 for 5 days (see Figure 2D). IL-2 modified with polyethylene glycol of different molecular sizes can affect the proportion of immune cells in mouse spleen to varying degrees. Low-dose 40kd PEG-IL-2 significantly promoted the increase in both the CD8 / CD4 cell ratio and the CD8 / Treg cell ratio, with effects more pronounced than high-dose ordinary IL-2.

[0137] Example 3: 40kd PEG-modified IL-2 still promoted an increase in the CD8 / Treg ratio on day 7 after administration.

[0138] To investigate the duration of the immune homeostasis effect of 40kd PEG-modified IL-2, this study observed the proportion of immune cells in the spleen tissue of healthy mice at different time points. Mice were intraperitoneally injected with 400,000 U / mouse of 40kd PEG-modified IL-2, while a control group received an equivalent dose of PBS intraperitoneally. Mice were sacrificed on days 3, 5, and 7, and spleen cells were analyzed by flow cytometry (see Figures 3A and 3B). We found that 40kd PEG-IL-2 significantly increased the CD8 / CD4 cell ratio (see Figure 3C) and the CD8 / Treg cell ratio (see Figure 3D) on day 5, and this effect persisted until day 7 (see Figure 3).

[0139] Example 4: 40kd PEG-modified IL2 with different structures can all promote an increase in the CD8 / Treg ratio.

[0140] Examples 1-3 demonstrated that M-type (unbranched) PEG-modified IL2 promoted an increase in the CD8 / Treg ratio. To further investigate the effects of different 40kd PEG-modified IL2 structures on mouse immune homeostasis, the effects of Y-type (branched) 40kd PEG-modified IL2 (Y-type 40kd PEG-IL-2) on the immune system were compared. After intraperitoneal injection of 400,000 U / mouse of Y-type 40kd PEG-modified IL2, and spleen harvesting, we found that Y-type 40kd PEG-modified IL2 promoted spleen enlargement and increased spleen mass (see Figure 4A). Flow cytometry was used to detect the proportion of immune cells in spleen cells. We found that Y-type 40kd PEG-modified IL2 upregulated CD8+. + T cells and CD8 + T cells and CD4 + T cell ratio (CD8 / CD4) (see Figures 4B and 4D), from spleen cells CD8 + T cells and CD4 + CD25 + FoxP3 + Based on the Treg cell ratio (CD8 / Treg), Y-type 40kD PEG-repaired IL2 did not affect the Treg cell ratio but upregulated spleen CD8. + T cells and CD4 + CD25 + FoxP3 + Treg cell ratio (CD8 / Treg) (see Figures 4C and 4E). Different structures of 40kd PEG-modified IL2 can affect the proportion of immune cells in mouse spleen. Y-type 40kd PEG-IL-2 can significantly promote the increase of CD8 / CD4 cell ratio and CD8 / Treg cell ratio, and its effect is more significant than that of 40kd PEG-IL-2.

[0141] Example 5: Effects of different administration methods

[0142] To investigate the effects of different administration routes of 40kd PEG-IL-2 on immune homeostasis in mice, this study compared subcutaneous and intraperitoneal administration. Mice were injected intraperitoneally and subcutaneously with 400,000 U / mouse of 40kd PEG-modified IL-2 (40kd PEG-IL-2), with an equivalent dose of PBS serving as a blank control. Mice were sacrificed on day 7, and spleen size was observed. We found that in the control group, subcutaneous and intraperitoneal administration did not affect spleen size. However, in the 40kd PEG-IL-2 group, the spleen size and mass of mice administered subcutaneously were larger than those administered intraperitoneally (Figure 5A). Flow cytometry analysis of the proportion of immune cells in spleen cells revealed that both administration routes promoted the upregulation of CD8+ in mouse spleen cells. + T cells and CD4 + The T cell ratio (CD8 / CD4) (see Figures 5B and 5D) and the CD8 / Treg ratio (see Figures 5C and 5E) showed similar changes in immune balance between the two administration methods, with no statistically significant differences (see Figures 5D and 5E).

[0143] Example 6: 40kd PEG-modified IL-2 can delay tumor growth

[0144] 40kd PEG-modified IL-2 can alter immune homeostasis in mice, increasing the proportion of immune cells with cytotoxic effects and promoting positive immune homeostasis. To investigate the antitumor effect of 40kd PEG-modified IL-2, we constructed three different subcutaneous tumor-bearing mouse models: subcutaneous melanoma, subcutaneous lung adenocarcinoma, and subcutaneous B-cell lymphoma. In each model mouse, the control group received intraperitoneal injection of 400,000 U / mouse of 40kd PEG-modified IL-2, the control group received intraperitoneal injection of an equal dose of PBS, and the experimental control group received intraperitoneal injection of 400,000 U / mouse of recombinant human IL-2 (rhIL-2, IL-2) for 5 consecutive days. To obtain quantifiable data, we weighed the tumors in the mice. In a mouse model of melanoma, the mean tumor weights on day 7 were 2.20±0.74 g, 2.18±0.99 g, and 0.89±0.65 g in the PBS group, rhIL-2 group, and 40 kDa PEG-IL-2 group, respectively (P = 0.0189); the mean tumor growth inhibition rates on day 7 were 1.24% and 59.55% in the rhIL-2 group and 40 kDa PEG-IL-2 group, respectively (P = 0.0179). In the B-cell lymphoma model, on day 7, the mean tumor weights in the PBS group, rhIL-2 group, and 40kd PEG-IL-2 group were 1.40±0.286g, 1.02±0.467g, and 0.21±0.103g, respectively (P=0.0007); the mean tumor growth inhibition rates in the rhIL-2 group and the 40kd PEG-IL-2 group on day 7 were 22.48% and 82.32%, respectively (P=0.0260). In the lung cancer model, on day 7 after drug administration, the lung cancer tumors in two mice in the 40kd PEG-IL-2 group completely disappeared, and no tumor tissue could be obtained by dissection (the mean tumor weights of the three groups were 1.29±0.326g, 1.04±0.188g, and 0.21±0.144g, respectively (P=0.0481). On day 9, the mean tumor growth inhibition rates of the rhIL-2 group and the 40kd PEG-IL-2 group were 19.66% and 83.75%, respectively (P=0.0120).

[0145] We found that 40kd PEG-IL-2 delayed melanoma growth more effectively than regular IL-2 (see Figure 6). On day 4, the relative tumor volume (relative to the PBS group) in the rhIL-2 group and the 40kd PEG-IL-2 group were 0.969 and 0.341, respectively (P = 0.0268), as shown in Table 1-1. On days 4, 5, 6, and 7, the relative tumor volume in the 40kd PEG-IL-2 group was 0.35, 0.65, 0.44, and 0.48 times that of the regular IL-2 group, respectively (P < 0.05), as detailed in Table 1-1. We also found that 40kd PEG-IL-2 delayed B-cell lymphoma growth more effectively than regular IL-2 (see Figure 8). To further compare the differences between 40kd PEG-IL-2 and regular IL-2 in inhibiting B-cell lymphoma growth, we analyzed the relative tumor volume of the two groups. On day 5, the relative tumor volumes in the rhIL-2 group and the 40kd PEG-IL-2 group were 0.72 and 0.11, respectively (P = 0.0125). On days 3, 4, 5, 6, and 7, the relative tumor volumes in the 40kd PEG-IL-2 group were 0.37, 0.14, 0.15, 0.10, and 0.05 times that of the ordinary IL-2 group, respectively. Relative tumor volumes (relative to the PBS group) are shown in Tables 1-3. In both melanoma and B-cell lymphoma models, 40kd PEG-IL-2 promoted spleen volume and mass increase, accompanied by reactive lymph node enlargement. 40kd PEG-IL-2 also delayed lung cancer growth. To further compare the differences in the inhibitory effects of 40kd PEG-IL-2 and IL-2 on lung cancer growth, we compared the relative tumor volumes of the two groups. On day 3, the relative tumor volumes in the rhIL-2 group and the 40kd PEG-IL-2 group were 3.60 and 0.58, respectively (P = 0.0116). On days 3, 4, 5, 6, and 9, the relative tumor volumes in the 40kd PEG-IL-2 group were 0.16, 0.24, 0.11, 0.23, and 0.27 times that of the ordinary IL-2 group, respectively (P < 0.05). Relative tumor volumes (relative to the PBS group) are shown in Tables 1-2. Based on the daily changes in tumor volume and relative tumor volume, 40kd PEG-IL-2 had an effect of delaying lung cancer growth, while ordinary IL-2 did not affect the growth rate of lung cancer. In a mouse model of lung cancer, 40kd PEG-IL-2 did not affect spleen volume and weight, but it could promote lymph node enlargement (see Figure 7).

[0146] Table 1-1: Relative tumor volume (RTV) in melanoma models. *The table shows the average value ± SD.

[0147] Table 1-2: Relative tumor volume (RTV) in lung cancer models. *The table shows the mean ± SD. In the #40kd PEG-IL-2 group, only 3 mice were observed on day 5, two of which had unmeasurable tumor volume.

[0148] Table 1-3: Relative tumor volume (RTV) in B-cell lymphoma models. *The table shows the average value ± SD.

[0149] Because different cell surfaces express IL2 receptors with varying affinities, low-dose IL2 can promote the proliferation of regulatory T cells, while promoting effector cell proliferation requires high-dose IL2. To further investigate and compare the antitumor effects of 40kD PEG-IL-2 with high-dose IL2, we administered a single intraperitoneal injection of 400,000 U / mouse of 40kD PEG-modified IL-2 to classic immunotherapy-bearing tumor-bearing mice (melanoma mouse model). A control group received an equivalent dose of PBS intraperitoneally, while an experimental control group received 1,200,000 U / mouse of recombinant human IL-2 (rhIL-2, IL-2) intraperitoneally for 5 consecutive days. We found that both low-dose 40kD PEG-IL-2 and high-dose IL2 could delay melanoma development (see Figure 9), promote spleen enlargement and weight increase, and simultaneously induce reactive lymph node enlargement. However, in promoting spleen enlargement and lymph node hyperplasia, low-dose 40kD PEG-IL-2 was more effective than high-dose conventional rhIL-2.

[0150] Example 7: 40kd PEG-modified IL2 promotes greater infiltration of immune cells into tumors.

[0151] To further verify the effect of 40kd PEG-modified IL2 on the proportion of immune cells within tumors, flow cytometry was used to detect the proportion of immune cells in lymph node tissues, spleen tissues, and tumor tissues of three tumor-bearing mouse models. The results showed that 40kd PEG-modified IL2 could affect the proportion of tissue immune cells to varying degrees in different lymphoid organs (Figures 10, 11, 12, and 13). Both ordinary IL2 and 40kd PEG-modified IL2 promoted the entry of more T cells and NK cells into tumor tissues (see Figure 10), with 40kd PEG-modified IL2 significantly promoting the entry of T cells and CD8+ cells into tumor tissues. + T cell proliferation (Figs. 11, 12, and 13). Most notably, ordinary IL-2 upregulated the proportion of regulatory T cells (Tregs), but 40kD PEG-modified IL-2 did not promote an increase in the proportion of regulatory T cells. CD8 +The CD8 / Treg ratio can reflect the body's immune homeostasis to some extent. We found that 40kd PEG-modified IL-2 significantly promoted the CD8 / Treg ratio in tumor tissues of three tumor-bearing mouse models of melanoma, lung cancer, and B-cell lymphoma, promoting a positive immune response (see Tables 2-1, 2-2, and 2-3). In melanoma tumor tissues, the CD8 / Treg ratio in the 40kd PEG-IL-2 group was 1.7 times that in the PBS group (6.9 in the PBS group, P = 0.0019), while the ratio in the rhIL-2 group was similar to that in the PBS group, with no statistically significant difference (P = 0.9814). The CD8 / Treg ratios in the 40kd PEG-IL-2 group and the rhIL-2 group were 11.7 and 6.6, respectively, with the CD8 / Treg ratio in the 40kd PEG-IL-2 group being 1.8 times that in the rhIL-2 group (P = 0.0109, see Table 2-1). In lung cancer tumor tissues, the CD8 / Treg ratio in the 40kd PEG-IL-2 group was similar to that in the PBS group, with no statistically significant difference (P = 0.4916), while the ratio in the rhIL-2 group was 0.5 times that of the PBS group (PBS group: 3.4, P = 0.0263). The CD8 / Treg ratios in the 40kd PEG-IL-2 group and the rhIL-2 group were 2.9 and 1.8, respectively, with the CD8 / Treg ratio in the 40kd PEG-IL-2 group being 1.6 times that of the rhIL-2 group (P = 0.0481, see Table 2-2). In lymphoma tumor tissues, the CD8 / Treg ratio in the 40kd PEG-IL-2 group was similar to that in the PBS group, with no statistically significant difference (P = 0.0509), while the ratio in the rhIL-2 group was 0.4 times that of the PBS group (PBS group: 1.7, P = 0.0160). The CD8 / Treg ratios in the 40kd PEG-IL-2 group and the rhIL-2 group were 1.1 and 0.7, respectively, with the CD8 / Treg ratio in the 40kd PEG-IL-2 group being 1.6 times that of the rhIL-2 group (P = 0.0468, see Tables 2-3). In contrast, ordinary IL-2 promoted a decrease in the CD8 / Treg ratio in tumor tissue, promoting a negative immune response (Figures 11, 12, and 13).

[0152] Table 2-1: CD8 in melanoma models + The ratio of T cells to Tregs. Sample size: *, Lymph nodes: PBS, n=9; rhIL-2, n=6; 40kd PEG-IL-2, n=9. #, Spleen: PBS, n=9; rhIL-2, n=6; 40kd PEG-IL-2, n=9. &, Tumor: PBS, n=5; rhIL-2, n=6; 40kd PEG-IL-2, n=7. The table shows the mean ± SD.

[0153] Table 2-2: CD8 in lung cancer models + The ratio of T cells to Tregs. Note: Sample size: n = 6. The table shows the mean ± SD.

[0154] Table 2-3: CD8 in B-cell lymphoma models + The ratio of T cells to Tregs. Note: Sample size: n = 6. The table shows the mean ± SD.

[0155] Currently, most scientists agree that tumor immunophenotypes are divided into three main categories (Desbois M et al. Integrated digital pathology and transcriptome analysis identifies molecular mediators of T-cell exclusion in ovarian cancer. Nat Commun. 2020; 11(1):5583): First, the immune-infiltrating type, CD8 + T cells can infiltrate into the tumor; secondly, there is the immune rejection type, although there are more CD8 cells. + The degree of T cell infiltration is high, but it is concentrated around the tumor periphery; third, it is an immune desert type, with very few CD8 cells within the tumor. + T cell infiltration. To further investigate the anti-tumor immune-related mechanism of 40kd PEG-modified IL2, we performed immunohistochemical fluorescence staining on tumor tissues from three mouse models. We found that in melanoma mouse tumor tissues, both ordinary IL2 and 40kd PEG-modified IL2 significantly promoted the entry of more immune cells into the tumor tissue (see Figure 14). 40kd PEG-modified IL2 promoted the entry of more immune cells into the tumor tissue, including more CD8+ cells. + When T cells enter the tumor tissue, the tumor tissue immunophenotyping shifts towards an "immune infiltration" pattern. However, ordinary IL2 promotes the distribution of immune cells at the tumor tissue periphery, shifting the tumor tissue immunophenotyping towards an "immune rejection" pattern (see Figure 14). Similar phenomena were observed in lung cancer model mice (see Figure 15) and B-cell lymphoma model mice (see Figure 16). 40kd PEG-modified IL2 promoted the entry of more T cells into the tumor tissue, including more CD8+ cells. + T cells are distributed within tumor tissue, promoting the transformation of tumor tissue into a "hot tumor." Regular IL-2 promotes the growth of more T cells and CD8+. + T cells are distributed to the periphery of the tumor tissue.

[0156] Immunohistochemical and flow cytometry results of tumor tissue both indicated that 40kD PEG-modified IL2 could affect CD8. + The distribution and proportion of T cells in tumor tissue, in order to further verify CD8 + To investigate whether T cells play a key role in the antitumor effect of 40kD PEG-modified IL2, we first used the monoclonal antibody anti-mouse CD8a-InVivo (100ug / mouse, twice a day for a total of three doses) to antagonize CD8a in mice. + T cells, while intraperitoneal injection of CD8 + On the first day after T-cell antagonism, a melanoma model was treated with 40kDa PEG-modified IL-2. When CD8+ in vivo... + After T cells were antagonized by monoclonal antibodies, 40kd PEG-modified IL2 did not delay the growth of melanoma (see Figure 17).

[0157] Example 8: 40kd PEG-modified IL2 delays CD8 + T cells enter a state of terminal exhaustion.

[0158] 40kd PEG-modified IL2 can affect CD8 + T cells exert anti-tumor effects when they antagonize CD8 + The anti-tumor effect of T cells is suppressed. CD8 + The number and function of T cells affect the antitumor effect of 40kD PEG-modified IL-2. 40kD PEG-modified IL-2 does not induce Treg cell proliferation, but it does affect CD8 cells. + The proportion of T cells was not significantly affected. To further investigate the effect of 40kD PEG-modified IL2 on CD8... + The effect on T cell function was detected by flow cytometry in lymph nodes, spleen, and tumor cells of melanoma, lung cancer, and B-cell lymphoma-bearing mouse models. + The expression of T cell surface immune checkpoints and cytokine secretion function were observed. We found that treatment with 40kd PEG-modified IL2 increased the expression of CD8+ of immune checkpoint molecules (PD-1, Tim-3, LAG-3, and CTLA-4) in tumor cells of three tumor-bearing mouse models. + The proportion of T cells was reduced compared to that after ordinary IL2 treatment, and in some cases, the expression rate was even lower than that in the control group. Although 40kd PEG-modified IL2 treatment did not reduce the expression of the immune checkpoint CD8 in B-cell lymphoma model mice, this was not observed. + The proportion of T cells was reduced, but the expression of the immune checkpoint CD8 was decreased in B-cell lymphoma model mice. +The absolute number of T cells. In melanoma-bearing mice treated with 40kd PEG-modified IL2, the proportion of CD8+ T cells expressing PD-1, Tim-3, and CTLA-4 was lower than in the control group. In a lung cancer model, 40kd PEG-modified IL2 did not interfere with the expression level of PD-1 on the surface of CD8+ T cells. Using anti-PD-1 antagonists in both lung cancer and melanoma models, we found that 40kd PEG-modified IL2 combined with a PD-1 inhibitor could delay lung cancer progression (Figure 18) but had little effect on melanoma tumor growth (Figure 19).

[0159] In a mouse model of melanoma, to compare the synergistic effect of Anti-mouse PD-1-In Vivo and 40kd PEG-IL-2 in anti-tumor efficacy, tumor weight was analyzed and compared among the three groups. The mean tumor weights of the PBS group, anti-PD-1 group, 40kd PEG-IL-2 group, and anti-PD-1+40kd PEG-IL-2 group were 3.9±0.87g, 1.8±0.92g, 1.1±0.48g, and 1.62±0.13g, respectively. The differences in mean tumor weight among the four groups were statistically significant (P<0.0001). The mean tumor weights in the anti-PD-1 group, the 40kd PEG-IL-2 group, and the anti-PD-1+40kd PEG-IL-2 group were all smaller than those in the PBS group, and the differences were statistically significant (anti-PD-1 group vs PBS group: P = 0.0017; 40kd PEG-IL-2 group vs PBS group: P < 0.0001; anti-PD-1+40kd PEG-IL-2 group vs PBS group: P = 0.0010). When comparing the anti-PD-1 group, the 40kd PEG-IL-2 group, and the anti-PD-1+40kd PEG-IL-2 group pairwise, there was no statistically significant difference in mean tumor weight (anti-PD-1 group vs. 40kd PEG-IL-2 group: P = 0.3651; anti-PD-1 group vs. anti-PD-1+40kd PEG-IL-2 group: P = 0.9691; 40kd PEG-IL-2 group vs. anti-PD-1+40kd PEG-IL-2 group: P = 0.6671). The mean tumor growth inhibition rates of the anti-PD-1 group, the 40kd PEG-IL-2 group, and the anti-PD-1+40kd PEG-IL-2 group were 53.0%, 73.2%, and 58.7%, respectively, with no statistically significant difference (P = 0.1955).

[0160] In a lung cancer model, to investigate whether 40kd PEG-IL-2 and PD-1 inhibitors have a synergistic effect in antitumor efficacy, we administered Anti-mouse PD-1-In Vivo, 40kd PEG-IL-2, and a combination of these drugs, respectively, and observed tumor growth. We found that Anti-mouse PD-1-In Vivo, 40kd PEG-IL-2, and the combination therapy all exhibited antitumor effects.

[0161] To compare whether Anti-mouse PD-1-In Vivo and 40kd PEG-IL-2 have a synergistic effect in anti-tumor efficacy, tumor weight was analyzed and compared among the three groups. The mean tumor weights of the PBS group, anti-PD-1 group, 40kd PEG-IL-2 group, and anti-PD-1+40kd PEG-IL-2 group were 2.0±0.73g, 1.34±0.64g, 0.5±0.19g, and 0.1±0.07g, respectively, with statistically significant differences (P<0.0001). The mean tumor weight of both the 40kd PEG-IL-2 group and the anti-PD-1+40kd PEG-IL-2 group was smaller than that of the PBS group (40kd PEG-IL-2 group vs. PBS group: P = 0.0011; anti-PD-1+40kd PEG-IL-2 group vs. PBS group: P = 0.0001). However, the mean tumor weight of the anti-PD-1 group was similar to that of the PBS group, and the difference was not statistically significant (anti-PD-1 group vs. PBS group: P = 0.2000).

[0162] The mean tumor growth inhibition rates in the anti-PD-1 group, the 40kd PEG-IL-2 group, and the anti-PD-1 + 40kd PEG-IL-2 group were 33.2%, 73.7%, and 92.7%, respectively, with statistically significant differences (P = 0.0015). Pairwise comparisons revealed that the 40kd PEG-IL-2 group had a higher mean tumor growth inhibition rate than the PD-1 inhibitor monotherapy group (P = 0.0166), and the combination therapy group had a higher mean tumor growth inhibition rate than the PD-1 inhibitor monotherapy group (P = 0.0014). However, the mean tumor inhibition rates in the combination therapy group and the 40kd PEG-IL-2 monotherapy group were similar, with no statistically significant difference (P = 0.3501).

[0163] To further investigate whether 40kD PEG-modified IL2 affects CD8 + T cell terminal exhaustion was observed, and the 40kd PEG-modified IL2 treatment group simultaneously expressed CD8 markers of PD-1, Tim-3, and LAG-3. +The proportion of T cells was lower in the group with normal IL2, and it did not promote CD8 in tumor tissue. + T cells enter a state of terminal exhaustion (see Figure 20).

[0164] In a melanoma model, 40kd PEG-modified IL2 promoted the expression of TCF-1 in CD8+ cells. + PD-1 + Increased proportion of T cells (CD8) + PD-1 + TCF-1 + T cell percentage: PBS vs rhIL-2 vs 40kb PEG-IL-2: 3.44% vs 7.40% vs 6.69%, P<0.05) and CD8 + PD-1 + Increased expression levels of TCF-1 (CD8) on T cells + PD-1 + Mean fluorescence intensity of TCF-1 on T cell surface: PBS vs rhIL-2 vs 40kd PEG-IL-2: 730.0 vs 821.3 vs 822.3 (P<0.05). In a lung cancer model, 40kd PEG-modified IL2 reduced CD8 expression of TCF-1. + PD-1 + T cell ratio (CD8) + PD-1 + TCF-1 + T cell percentage: PBS vs rhIL-2 vs 40kb PEG-IL-2: 1.95% vs 1.80% vs 0.56%, P<0.05) and CD8 + PD-1 + Reduced TCF-1 expression levels on T cells (CD8) + PD-1 + Mean fluorescence intensity of TCF-1 on T cell surface: PBS vs rhIL-2 vs 40kd PEG-IL-2: 1100.3 vs 947.7 vs 738.0, P<0.05).

[0165] CD8 + T cells primarily exert their cytotoxic effects by secreting granzyme B (Gzmb) and interferon-gamma (IFN-γ). Detection of CD8... + T cells and CD8 + PD-1 + We found that the ability of T cells to secrete Gzmb and IFN-γ increased CD8 levels in tumor tissues of the 40kd PEG-modified IL2 group. + T cells and CD8+ PD-1 + T cells still retain the ability to secrete IFN-γ (see Figure 21) and Gzmb (see Figure 22), but CD8 in tumor tissues of the ordinary IL2 group... + T cells and CD8 + PD-1 + The ability of T cells to secrete Gzmb and IFN-γ is reduced (see Figures 21 and 22). In the melanoma model, CD8 + PD-1 + T cells retain the ability to secrete Gzmb and IFN-γ (see Figures 21 and 22).

[0166] However, in the lung cancer model, CD8+PD-1+ T cells retained the ability to secrete Gzmb, but lost their ability to secrete IFN-γ (see Figures 21 and 22). From the perspective of cytokine synthesis and secretion, CD8+ T cells mainly exert their cytotoxic effects by secreting granzyme B (GzmB) and interferon-gamma (IFN-γ). By examining the ability of CD8+ T cells and CD8+PD-1+ T cells to secrete GzmB and IFN-γ, we found that CD8+ T cells and CD8+PD-1+ T cells in the 40kd PEG-modified IL2 group and rhIL-2 group tumor tissues still retained the ability to secrete GzmB (see Figures 21 and 22), but showed a trend of losing their IFN-γ secretion ability. From the perspective of cytokine secretion levels, 40kd PEG-IL-2 promoted lung cancer CD8+ T cells to enter the intermediate stage of exhaustion, but these CD8+ T cells still retained cytotoxic activity, explaining why 40kd PEG-IL-2 combined with a PD-1 inhibitor can achieve better results.

[0167] In a mouse model of B-cell lymphoma, regarding the expression levels of immune checkpoint molecules, the proportions of CD8+PD-1+ T cells, CD8+Tim-3+ T cells, and CD8+LAG-3+ T cells were higher in the 40kd PEG-IL-2 group than in the PBS group (see Figure 20). However, in terms of absolute counts, 40kd PEG-IL-2 reduced the absolute numbers of CD8+PD-1+ T cells, CD8+Tim-3+ T cells, CD8+LAG-3+ T cells, and CD8+CTLA-4+ T cells in the tumor tissue. rhIL-2, on the other hand, promoted the expression of immune checkpoint molecules on CD8+ T cells, resulting in an increase in the proportions of CD8+PD-1+ T cells, CD8+Tim-3+ T cells, CD8+LAG-3+ T cells, and CD8+CTLA-4+ T cells. In terms of absolute cell count, rhIL-2 can increase the absolute counts of CD8+PD-1+ T cells and CD8+CTLA-4+ T cells. Regarding cytokine synthesis and secretion, CD8+ T cells primarily exert their cytotoxic effects by secreting granzyme B (GzmB) and interferon-gamma (IFN-γ). Detecting the ability of CD8+ T cells and CD8+PD-1+ T cells to secrete GzmB and IFN-γ, we found that CD8+ T cells and CD8+PD-1+ T cells in the 40kd PEG-modified IL2 group and the rhIL-2 group still retained the ability to secrete IFN-γ (Figure 21) and GzmB (Figure 22). Regarding cytokine secretion, 40kd PEG-IL-2 does not promote cells to enter the intermediate or late exhaustion state because both CD8+ T cells and CD8+PD-1+ T cells still retain the ability to secrete IFN-γ and GzmB.

[0168] Example 9: 40kd PEG-modified IL2 reduces STAT5 phosphorylation levels in regulatory T cells.

[0169] To investigate the differences between 40kd PEG-IL-2 and rhIL-2 in influencing the proportion of Treg cells, we measured the level of p-STAT5 (phosphorylation site at Tyr694) in Treg cells using the p-STAT5 detection method reported by Michael Bitar et al. (BITAR M et al. Evaluating STAT5 Phosphorylation as a Mean to Assess T Cell Proliferation[J]. Front Immunol, 2019, 10:722). When analyzing the p-STAT5 level in Treg cells, we found that at concentrations of 40 kDa PEG-IL-2 (10,000 U / mL and 100,000 U / mL), the level of p-STAT5 phosphorylation (Tyr694) in Treg cells treated with 40 kDa PEG-IL-2 was lower than that treated with an equal dose of rhIL-2 (see Figures 24A and 24B). Furthermore, with increasing concentration, 40 kDa PEG-IL-2 significantly reduced the level of STAT5 phosphorylation (Tyr694) in Treg cells compared to rhIL-2 (see Figure 24C).

[0170] In addition, we analyzed p-STAT5 levels in effector cells. Analysis of STAT5 phosphorylation levels in effector cells revealed that 10,000 U / mL and 100,000 U / mL concentrations of 40 kDa PEG-IL-2 and equivalent concentrations of rhIL-2 similarly promoted STAT5 phosphorylation (Tyr694) levels in CD8+ T cells (see Figure 25A) and NK cells (see Figure 25B). This is similar to the previous results regarding the biological activity of rhIL-2 and 40 kDa PEG-IL-2. This also explains why 40 kDa PEG-IL-2 (400,000 U / cell) and rhIL-2 did not show an advantage in promoting NK cell and CD8+ T cell proportions.

[0171] In summary, 40kd PEG-IL-2 reduces the proportion of Treg cells and inhibits their effector function by decreasing STAT5 phosphorylation in Treg cells; however, 40kd PEG-IL-2 does not affect the phosphorylation level of STAT5 in NK cells and CD8+ T cells. Therefore, compared with rhIL-2, 40kd PEG-IL-2 does not show any advantage in promoting effector cells.

[0172] Example 10: 40kd PEG-modified IL2 reduces the expression of Treg transcription factors.

[0173] 40kd PEG-IL-2 significantly reduced the transcription of Foxp3 (P<0.0001, see Figure 26) and CTLA4 (P<0.0001, see Figure 26) compared to rhIL-2, indicating that at the transcriptional level, 40kd PEG-IL-2 inhibited the inhibitory function of Treg cells, while rhIL-2 promoted the inhibitory function of Treg cells.

[0174] Studies have shown that IL-2 / IL-2R binding activates the JAK / STAT signaling pathway, leading to phosphorylation of downstream STAT5, which mediates Prdm1 gene transcription and BLIMP-1 protein expression. BLIMP-1 is the molecular basis for IL-10 secretion by Treg cells. Furthermore, IRF4 gene expression also depends on IL-2-inducible T cell kinase (ITK) and mammalian target of rapamycin (mTOR) signaling. Simultaneously, IRF4 transcription is regulated by Foxp3 expression; when Foxp3 is absent, IRF4 mRNA is significantly reduced. This study found that 40kd PEG-IL-2 significantly reduced the transcriptional levels of Blimp-1 (P = 0.0020, see Figure 26), IRF4 (P < 0.0001, see Figure 26), Nfatc1 (P = 0.0007, see Figure 26), Nfatc2 (P = 0.0001, see Figure 26), and Stat5 (P = 0.0175, see Figure 26) compared to rhIL-2. Therefore, combined with previous findings, 40kd PEG-IL-2 showed a weaker ability to activate the IL-2 / IL-2R-STAT5 signaling pathway in Treg cells than rhIL-2. 40kd PEG-IL-2 reduced the transcription of Blimp-1 and IRF4 genes by decreasing Foxp3 transcription and STAT5 phosphorylation, thereby reducing the proportion of Foxp3+ T cells and inhibiting the effector function of Treg cells.

[0175] Example 11: 40kd PEG-modified IL2 reduces PD-1 expression in immune cells

[0176] To investigate whether 40kD polyethylene glycol-modified interleukin-2 induces immune cells into a state of exhaustion, we examined the expression of PD-1 on the surface of immune cells. In the absence of antigen stimulation, neither 40kD PEG-IL-2 nor rhIL-2 affected the proportion of CD3+PD-1+ T cells (see Figure 27A), and there was no significant difference between 40kD PEG-IL-2 and rhIL-2 in influencing the expression of immune checkpoint molecules on the surface of immune cells (see Figure 27A).

[0177] To investigate whether 40kd PEG-IL-2 and rhIL-2 differ in their effects on cell exhaustion under antigen stimulation, we co-cultured different types of tumor cells and immune cells, and examined the expression of PD-1 on the surface of immune cells. Under tumor cell stimulation, both 40kd PEG-IL-2 and rhIL-2 affected the expression levels of immune checkpoints on the surface of immune cells to varying degrees. When B-cell lymphoma A20 cells were co-cultured with immune cells, 40kd PEG-IL-2 significantly reduced the proportion of CD3+PD-1+ T cells compared to rhIL-2 (see Figure 27B), with a statistically significant difference. In summary, 40kd PEG-IL-2 reduced the expression levels of immune checkpoint molecules on the surface of immune cells in the A20 and immune cell co-culture system compared to rhIL-2.

[0178] In summary, 40kd PEG-IL-2 can reduce the expression level of PD-1 on the surface of immune cells in the presence of antigen stimulation, while rhIL-2 promotes the expression of PD-1 by immune cells.

[0179] Example 12: 40kd PEG-modified IL2 reduces the expression of exhaustion-related transcription factors

[0180] Studies have shown that transcription factors such as T-bet, Eomes, Blimp-1, and NFAT are involved in affecting T cell exhaustion [WEULERSSE M et al. Eomes-Dependent Loss of the Co-activating Receptor CD226 Restrains CD8(+)T Cell Anti-tumor Functions and Limits the Efficacy of Cancer Immunotherapy[J].Immunity,2020,53(4):824-39.e10.]. This study found that 40kd PEG-IL-2 significantly reduced the transcriptional levels of Pdcd1 (P<0.0001, see Figure 28), CTLA-4 (P<0.0001, see Figure 28), Tox (P=0.0007, see Figure 28), Blimp-1 (P=0.0033, see Figure 28), Eomes (P=0.0002, see Figure 28), T-bet (P<0.0001, see Figure 28), Nfatc1 (P=0.0046), and Nfatc2 (P=0.0013, see Figure 28) compared to rhIL-2.

[0181] Example 13: In a mouse model of melanoma, 40kd PEG-modified IL2 increased the proportion of central memory cells, superior to IL-2; however, it did not increase effector memory CD8+ T cells.

[0182] To investigate the effect of 40kD PEG-IL-2 on the balance of CD8+ effector T cells and CD8+ memory T cells, flow cytometry was used to determine the proportions of CD8+ activated T cells and CD8+ memory T cells in lymph nodes, spleen, and tumor tissues. We defined cells as CD8+CD44-CD62L+. T cells are defined as CD8+CD25+ cells as CD8+ activated T cells, and CD8+CD44+CD62L+ cells as CD8+ central memory T cells (T cells). CM CD8+CD44+CD62L- cells are defined as CD8+ effector memory T cells (T cells). EM (cells). In a melanoma model, 40kd PEG-IL-2 promoted the growth of tumor cells. T cells, CD8+ activated T cells and CD8+ T cells CM The proportion of cells increased, while rhIL-2 did not affect the proportion of cells in tumor tissue. T cells, CD8+ activated T cells, CD8+ T cells CM Cells and CD8+ T EM Cell proportions (see Figure 29). Regarding naïve CD8+ T cells, in melanoma tumor tissue, 40kd PEG-IL-2... The proportion of T cells was 1.75 times that of the PBS group (40kD PEG-IL-2 vs PBS: 10.16 vs 5.805, P = 0.0125), while the proportion of rhIL-2 was... The proportion of T cells was similar in the PBS group (rhIL-2 vs PBS: 7.360 vs 5.805, P = 0.0697), as detailed in Figure 29A. 40kD PEG-IL-2 The proportion of T cells was higher in the 40kd PEG-IL-2 group than in the rhIL-2 group, a statistically significant difference (P = 0.0354). Regarding activated CD8+ T cells, in melanoma tumor tissue, the proportion of CD8+ activated T cells in the 40kd PEG-IL-2 group was 1.74 times that in the PBS group (40kd PEG-IL-2 vs PBS: 18.27 vs 10.48, P = 0.0019), while the proportion of CD8+ activated T cells in the rhIL-2 group was similar to that in the PBS group (rhIL-2 vs PBS: 13.80 vs 10.48, P = 0.0822), as detailed in Figure 29B. The proportion of CD8+ activated T cells in the 40kd PEG-IL-2 group was 1.32 times higher than that in the rhIL-2 group, a statistically significant difference (P = 0.0266).

[0183] Regarding central memory CD8+ T cells, in melanoma tumor tissue, CD8+ T cells with 40kd PEG-IL-2... CM The ratio was 2.58 times that of the PBS group (40kd PEG-IL-2 vs PBS: 21.0 vs 8.148, P = 0.0133), while rhIL-2's CD8+ T... CM The cell proportions were similar to those in the PBS group (rhIL-2 vs PBS: 11.47 vs 8.148, P = 0.0829), as detailed in Figure 29C. 40kd PEG-IL-2 CD8+ T cells CM The proportion of cells was higher than that of rhIL-2, and was 1.83 times higher than that of the rhIL-2 group, with a statistically significant difference (P = 0.0281).

[0184] Regarding effector memory CD8+ T cells, in melanoma tumor tissue, CD8+ T cells with 40kd PEG-IL-2... EM The ratios were similar to those in the PBS group (40kd PEG-IL-2 vs PBS: 16.99 vs 17.01, P = 0.9999), while rhIL-2's CD8+ T... EM The cell proportions were similar to those in the PBS group (rhIL-2 vs PBS: 17.81 vs 17.01, P = 0.7670), as detailed in Figure 29D. 40kd PEG-IL-2 CD8+ T cells CM The proportion of cells was similar to that of rhIL-2, and the difference was not statistically significant (P = 0.7579).

[0185] In summary, compared with rhIL-2, 40kD PEG-IL-2 can promote the growth of melanoma tumor tissue. T cells, CD8+ activated T cells and CD8+ T cells CM The proportion of these cells increases, while rhIL-2 cannot alter the proportion of these cells in tumor tissue. 40kD PEG-IL-2 promotes the production of memory cells, maintaining long-term protection of the body by the immune system and preventing tumor recurrence. Compared to rhIL-2, 40kD PEGIL-2 has an advantage in promoting anti-tumor memory immune responses.

[0186] Example 14: In a mouse model of lung adenocarcinoma, 40kd PEG-modified IL-2 promoted an increase in the proportion of CD8+ central memory T cells, which was superior to that of ordinary IL-2; it also increased effector memory CD8+ T cells, but not as much as ordinary IL-2.

[0187] To investigate the effect of 40kD PEG-IL-2 on the balance of CD8+ effector T cells and CD8+ memory T cells, flow cytometry was used to determine the proportions of CD8+ activated T cells and CD8+ memory T cells in lymph nodes, spleen, and tumor tissues. We defined cells as CD8+CD44-CD62L+. T cells are defined as CD8+CD25+ cells as CD8+ activated T cells, and CD8+CD44+CD62L+ cells as CD8+ central memory T cells (T cells). CM CD8+CD44+CD62L- cells are defined as CD8+ effector memory T cells (T cells). EM (Cells). In a lung adenocarcinoma model, neither 40kd PEG-IL-2 nor rhIL-2 affected the levels of PEG-IL-2 in lung adenocarcinoma tumor tissue. The proportion of T cells. 40kD PEG-IL-2 promotes the growth of CD8+ T cells in tumor tissue. EM The proportion of cells increases, but it does not affect CD8+ activated T cells and CD8+ T cells. CM The proportion of CD8+ activated T cells and CD8+ T cells was increased by rhIL-2. EM Cell proportion, but does not affect CD8+ T cells. CM Cell proportions (see Figure 30). Regarding naïve CD8+ T cells, in lung adenocarcinoma tumor tissue, 40kd PEG-IL-2... The proportion of T cells was similar in the PBS group (40kD PEG-IL-2 vs PBS: 1.622 vs 1.436, P = 0.9369), while the proportion of rhIL-2 was similar in the PBS group. The T cell ratio was also similar to that of the PBS group (rhIL-2 vs PBS: 0.946 vs 1.436, P = 0.6536), see Figure 30A for details. 40kd PEG-IL-2 The proportion of T cells was similar to that of rhIL-2, with no statistically significant difference (P = 0.4664). Regarding activated CD8+ T cells, in lung adenocarcinoma tumor tissue, the proportion of CD8+ activated T cells with 40kd PEG-IL-2 was similar to that with the PBS group (40kd PEG-IL-2 vs PBS: 35.27 vs 32.23, P = 0.4182), while the proportion of activated CD8+ T cells with rhIL-2 was 1.38 times that of the PBS group (rhIL-2 vs PBS: 44.33 vs 32.23, P = 0.0040), as detailed in Figure 30B. The proportion of CD8+ activated T cells with 40kd PEG-IL-2 was lower than that with rhIL-2, being 0.80 times lower, a statistically significant difference (P = 0.0157).

[0188] Regarding central memory CD8+ T cells, in lung adenocarcinoma tumor tissue, CD8+ T cells with 40kd PEG-IL-2... CM The ratio was 1.11 times that of the PBS group (40kd PEG-IL-2 vs PBS: 12.78 vs 11.49, P = 0.2606), while rhIL-2's CD8+ T... CM The cell proportion was 0.84 times that of the PBS group (rhIL-2 vs PBS: 9.673 vs 11.49, P = 0.1022), see Figure 30C for details. 40kd PEG-IL-2 CD8+ T cells CM The proportion of cells was higher than that of rhIL-2, and was 1.32 times higher than that of the rhIL-2 group, with a statistically significant difference (P = 0.0126).

[0189] Regarding effector memory CD8+ T cells, in lung adenocarcinoma tumor tissue, CD8+ T cells with 40kd PEG-IL-2... EM The ratio was 1.40 times that of the PBS group (40kd PEG-IL-2 vs PBS: 34.22 vs 24.37, P = 0.0106), while rhIL-2's CD8+ T... EM The cell proportion was 1.24 times that of the PBS group (rhIL-2 vs PBS: 30.15 vs 24.37, P = 0.0377), see Figure 30D for details. 40kd PEG-IL-2 CD8+ T cells EM The cell proportions were similar to those of rhIL-2, and the difference was not statistically significant (P = 0.0593).

[0190] In summary, compared with rhIL-2, 40kD PEG-IL-2 promoted the growth of CD8+ T cells in lung adenocarcinoma tumor tissue. CMThe number of cells increased, but the number of CD8+ activated T cells decreased, and it did not affect... T cells and CD8+ T cells CM Cell percentage. In summary, 40kD PEG-IL-2 promotes the growth of CD8+ T cells in tumor tissue. CM The proportion of cells increased compared to the rhIL-2 treated group (CD8+ T cells). CM The higher proportion of cells indicates that tumor cells were cleared after treatment with 40kd PEG-IL-2. The antigen stimulation was less than that of the rhIL-2 group, which promoted the production of CD8+ memory T cells and the anti-tumor memory immune response. In terms of preventing tumor recurrence, 40kd PEG-IL-2 has an advantage over rhIL-2.

[0191] Example 15: In a mouse model of B-cell lymphoma, 40kd PEG-modified IL2 promoted an increase in the proportion of CD8+ central memory T cells, superior to IL-2; however, it did not promote the growth of effector memory CD8+ T cells as effectively as ordinary interleukin-2.

[0192] To investigate the effect of 40kD PEG-IL-2 on the balance of CD8+ effector T cells and CD8+ memory T cells, flow cytometry was used to determine the proportions of CD8+ activated T cells and CD8+ memory T cells in lymph nodes, spleen, and tumor tissues. We defined cells as CD8+CD44-CD62L+. T cells are defined as CD8+CD25+ cells as CD8+ activated T cells, and CD8+CD44+CD62L+ cells as CD8+ central memory T cells (T cells). CM CD8+CD44+CD62L- cells are defined as CD8+ effector memory T cells (T cells). EM (cells). In a B-cell lymphoma model, 40kd PEG-IL-2 promoted the growth of tumor cells in tumor tissue. T cells, CD8+ T cells CM Cells and CD8+ T EM The proportion of cells increased, but the proportion of CD8+ activated T cells decreased, while rhIL-2 reduced the proportion of cells in tumor tissue. T cells increased CD8+ T cells EM The cell ratio is affected, but not the CD8+ activated T cells and CD8+ T cells. CM Cell proportions (see Figure 31).

[0193] Regarding naïve CD8+ T cells, in B-cell lymphoma tumor tissue, 40kd PEG-IL-2 The proportion of T cells was similar in the PBS group (40kD PEG-IL-2 vs PBS: 3.160 vs 2.386, P = 0.214), while the proportion of rhIL-2 was higher. The proportion of T cells was 0.49 times that of the PBS group (rhIL-2 vs PBS: 1.176 vs 2.386, P = 0.0406), see Figure 31A for details. 40kd PEG-IL-2 The proportion of T cells was higher in the group with rhIL-2, which was 2.69 times higher than that in the rhIL-2 group, and the difference was statistically significant (P = 0.0090).

[0194] Regarding activated CD8+ T cells, in B-cell lymphoma tumor tissue, the proportion of CD8+ activated T cells with 40kd PEG-IL-2 was 0.88 times that of the PBS group (40kd PEG-IL-2 vs PBS: 10.34 vs 11.76, P = 0.0454), while the proportion of activated CD8+ T cells with rhIL-2 was similar to that of the PBS group (rhIL-2 vs PBS: 12.68 vs 11.76, P = 0.7077), as detailed in Figure 31B. Proportion of CD8+ activated T cells with 40kd PEG-IL-2.

[0195] Regarding central memory CD8+ T cells, in B-cell lymphoma tumor tissue, CD8+ T cells with 40kd PEG-IL-2... CM The ratio was 2.34 times that of the PBS group (40kd PEG-IL-2 vs PBS: 3.152 vs 1.349, P = 0.0068), while rhIL-2's CD8+ T... CM The cell proportions were similar to those in the PBS group (rhIL-2 vs PBS: 1.872 vs 1.349, P = 0.6291), as detailed in Figure 31C. 40kd PEG-IL-2 CD8+ T cells CM The proportion of cells was higher than that of rhIL-2, and was 1.68 times higher than that of the rhIL-2 group, with a statistically significant difference (P = 0.0182).

[0196] Regarding effector memory CD8+ T cells, 40kd PEG-IL-2 in CD8+ T cells... EM The ratio was 1.28 times that of the PBS group (40kd PEG-IL-2 vs PBS: 22.79 vs 17.83, P = 0.0306), while rhIL-2's CD8+ T... EMThe cell proportion was 1.59 times that of the PBS group (rhIL-2 vs PBS: 28.38 vs 17.83, P = 0.0247), see Figure 31D for details. 40kd PEG-IL-2 CD8+ T cells EM The proportion of cells was less than that of rhIL-2, which was 0.80 times that of the rhIL-2 group, and the difference was statistically significant (P = 0.0241).

[0197] In summary, compared with rhIL-2, 40kD PEG-IL-2 promoted the growth of B-cell lymphoma tumor tissue. T cells and CD8+ T cells CM Cell proliferation increased, but CD8+ activated T cells decreased. 40kD PEG-IL-2 promoted CD8+ T cells more effectively than rhIL-2. CM Increased cell proportion. 40kD PEG-IL-2 promotes CD8+ T cells in tumor tissue. CM The proportion of cells increased compared to the rhIL-2 treated group (CD8+ T cells). CM The higher proportion of cells indicates that tumor cells were cleared after treatment with 40kd PEG-IL-2. The antigen stimulation was less than that of the rhIL-2 group, which promoted the production of CD8+ memory T cells and the anti-tumor memory immune response. In terms of preventing tumor recurrence, 40kd PEG-IL-2 has an advantage over rhIL-2.

Claims

1. A polyethylene glycol (PEG)-modified interleukin-2 (IL-2), wherein the PEG has a molecular weight of about 40 kD, preferably, the PEG is attached to the N-terminus of IL-2, more preferably, the IL-2 is unitarily modified at its N-terminus with the PEG.

2. The PEG-modified IL-2 of claim 1, wherein the IL-2 is human IL-2, preferably human recombinant IL-2, particularly preferably comprising the amino acid sequence set forth in SEQ ID NO:

1.

3. The PEG-modified IL-2 of claim 1 or 2, wherein the PEG is branched, e.g., Y-shaped, or linear, preferably linear.

4. A pharmaceutical composition for treating a tumor, comprising the PEG-modified IL-2 of any one of claims 1-3, optionally comprising a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of claim 4, wherein the tumor is a solid tumor, preferably a tumor selected from the group consisting of a breast, cardiac, lung, small intestine, colon, rectum, spleen, kidney, stomach, esophagus, oral cavity, pharynx, urinary bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, cervix, or liver, more preferably a tumor selected from the group consisting of a glioma, sarcoma, melanoma, lung cancer, lymphoma, e.g., B-cell lymphoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, cervical cancer, head and neck cancer, and breast cancer, more preferably a tumor selected from the group consisting of melanoma, lung cancer, and lymphoma, e.g., B-cell lymphoma.

6. The pharmaceutical composition of claim 4 or 5, further comprising another tumor therapeutic agent, e.g., a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, an anti-angiogenic agent, an apoptosis-inducing agent, an anti-cancer immunotoxin, an anti-cancer antibody, an immune checkpoint inhibitor, or an immunomodulatory agent, preferably selected from the group consisting of paclitaxel, bevacizumab, vincristine, vinblastine, vasostatin, a nucleoside analogue or other anti-metabolite, a glucocorticoid, e.g., dexamethasone, budesonide, cortisone, prednisone, triamcinolone acetonide and derivatives thereof, aclarubicin, bleomycin, actinomycin D, carboplatin, cyclophosphamide, cytarabine, an interferon, e.g., interferon alpha-2a, alpha-2b, beta, gamma, an anti-CEA antibody, an anti-EGFR antibody, an anti-EpCAM antibody, an anti-Her2 antibody, an anti-PSA antibody, an anti-TGF-beta antibody, an anti-TRAIL-R1, TRAIL-R2 antibody, an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an NSAID such as aspirin, a cytokine such as TGFb, a cytokine, a chemokine, or a receptor antagonist.

7. A pharmaceutical composition for treating lung cancer, comprising the PEG-modified IL-2 of any one of claims 1-3 and an immune checkpoint inhibitor, preferably the immune checkpoint inhibitor is a CTLA4, PD-1, or PD-L1 inhibitor, e.g., an anti-CTLA4, anti-PD-1, or anti-PD-L1 antibody or aptamer, more preferably a PD-1 inhibitor, e.g., an anti-PD-1 antibody or aptamer, more preferably an antibody.

8. A method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of the PEG-modified IL-2 of any one of claims 1-3 or the pharmaceutical composition of any one of claims 4-6, preferably, the tumor is a solid tumor, preferably a tumor selected from the group consisting of a breast, cardiac, lung, small intestine, colon, rectum, spleen, kidney, stomach, esophagus, oral cavity, pharynx, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicle, cervix, or liver, more preferably a tumor selected from the group consisting of a glioma, sarcoma, melanoma, lung cancer, lymphoma such as B-cell lymphoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, cervical cancer, head and neck cancer, and breast cancer, more preferably a tumor selected from the group consisting of a melanoma, lung cancer, and lymphoma such as B-cell lymphoma.

9. The method of claim 8, wherein the subject is a human subject or a non-human subject, preferably a human or other mammal such as a cow, rat, mouse, dog, monkey, goat, sheep, cow, deer, horse, cat, and other non-mammal, more preferably a human.

10. The method of claim 8 or 9, comprising administering the PEG-modified IL-2 of any one of claims 1-3 and another tumor therapeutic agent simultaneously or sequentially, the other tumor therapeutic agent comprising a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, an anti-angiogenic agent, an apoptosis-inducing agent, an anti-cancer immunotoxin, an anti-cancer antibody, an immune checkpoint inhibitor, or an immunomodulatory agent, preferably selected from the group consisting of paclitaxel, bevacizumab, vincristine, vinblastine, vasostatin, a nucleoside analogue or other anti-metabolite, a glucocorticoid such as dexamethasone, budesonide, cortisone, prednisone, triamcinolone acetonide and derivatives thereof, aclarubicin, bleomycin, actinomycin D, carboplatin, cyclophosphamide, cytarabine, an interferon such as interferon alpha-2a, alpha-2b, beta, gamma, an anti-CEA antibody, an anti-EGFR antibody, an anti-EpCAM antibody, an anti-Her2 antibody, an anti-PSA antibody, an anti-TGF-beta antibody, an anti-TRAIL-R1, TRAIL-R2 antibody, an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an NSAID such as aspirin, a cytokine such as TGFb, a cytokine, a chemokine, or a receptor antagonist.

11. A method of treating lung cancer comprising administering to a subject in need thereof a therapeutically effective amount of the PEG-modified IL-2 of any one of claims 1-3 and an immune checkpoint inhibitor, preferably the immune checkpoint inhibitor is a CTLA4, PD-1, or PD-L1 inhibitor such as an anti-CTLA4, anti-PD-1, or anti-PD-L1 antibody or aptamer, more preferably a PD-1 inhibitor such as an anti-PD-1 antibody or aptamer, more preferably an antibody.

12. Use of the PEG-modified IL-2 of any one of claims 1-3 for the manufacture of a medicament for the treatment of a tumor, preferably a solid tumor, preferably a tumor selected from the group consisting of a tumor of the breast, heart, lung, small intestine, colon, rectum, spleen, kidney, stomach, esophagus, oral cavity, pharynx, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicle, cervix, or liver, more preferably a tumor selected from the group consisting of a glioma, sarcoma, melanoma, lung cancer, lymphoma such as B-cell lymphoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, cervical cancer, head and neck cancer, and breast cancer, more preferably a tumor selected from the group consisting of a melanoma, lung cancer, and lymphoma such as B-cell lymphoma.

13. Use of the PEG-modified IL-2 of any one of claims 1-3 and an immune checkpoint inhibitor for the manufacture of a medicament for the treatment of lung cancer, preferably the immune checkpoint inhibitor is a CTLA4, PD-1 or PD-L1 inhibitor such as an anti-CTLA4, anti-PD-1 or anti-PD-L1 antibody or aptamer, more preferably a PD-1 inhibitor such as an anti-PD-1 antibody or aptamer, more preferably an antibody.

14. The PEG-modified IL-2 of any one of claims 1-3 for use in a method of promoting splenomegaly, reducing the proportion of regulatory T cells, increasing the CD8 / Treg ratio, promoting immune cell, e.g., T cell, e.g., CD8 + T cell, infiltration into tumors, reducing the level of STAT5 phosphorylation in regulatory T (Treg) cells, reducing the expression of transcription factors that regulate Treg cells, reducing the expression of PD-1 by immune cells, reducing the expression of exhaustion-associated transcription factors, increasing the proportion of central memory cells, increasing the proportion of CD8+ central memory T cells, increasing the proportion of CD8+ central memory T cells, and / or reducing the effects of tumor immune escape.

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