Immunoregulatory factor composition and application thereof

Abstract

The invention discloses an immunoregulatory factor composition, which comprises a first component and a second component, the first component is a type I cytokine or a long-acting form thereof, and the second component is a type II cytokine or a long-acting form thereof; the long-acting form is formed by fusing a cell factor with a fusion partner through a connecting peptide or performing PEGylation modification on the cell factor, and the fusion partner is an Fc fragment of IgG or human serum albumin or an engineered mutant of one of the Fc fragment and the human serum albumin. The immunomodulatory factor composition synergistically activates precursor depleted CD8 + T cells and terminal depleted CD8 + T cells through a combination therapy of type I and type II cytokines. The invention also discloses nucleic acid, a carrier and a cell related to the immunomodulatory factor composition. The invention also discloses an application of the immunoregulatory factor composition in preparation of drugs for treating cancers. The immunoregulatory factor composition can relieve the depletion state of depleted T cells and improve the effector function and proliferation capacity of the depleted T cells so as to control tumor progression.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an immunomodulatory factor composition and applications thereof. Background Art

[0002] Immunotherapies, represented by immune checkpoint inhibitors (ICIs) and adoptive cell therapy (ACT), have brought significant clinical progress to cancer treatment. However, the average response rate of ICIs in various tumors is only 20%-30%, and most patients have difficulty maintaining a sustained response, accompanied by adverse reactions such as tissue-specific toxicity. The efficacy of CAR-T therapy in solid tumors is also not ideal. These adverse outcomes are partly due to the exhaustion of tumor-infiltrating lymphocytes (TILs). During chronic infections and cancers where antigen stimulation persists, memory T cells fail to develop effectively and become exhausted, manifesting as a loss of effector function, persistently elevated expression of inhibitory receptors, and altered epigenetic and transcriptional profiles and metabolic patterns.

[0003] Research aimed at revitalizing exhausted cells has been a hot topic in recent years, especially in the complex tumor microenvironment of solid tumors. Based on the expression of T cell surface molecules and transcription factor TCF-1, exhausted CD8 + TILs are divided into precursor-depleted CD8 + T cells (TCF-1 + PD-1 inter TIM-3 - ) and terminally exhausted CD8 + T cells (TCF-1 - PD-1 high TIM-3 + ).

[0004] In recent years, a variety of drugs have been shown to target certain exhausted T cell subsets, effectively expand and activate them to achieve enhanced cytotoxic function and anti-tumor cytokine secretion. For example, PD-1 inhibitors have been shown to act only on precursor exhausted CD8 T cells in chronic virus and tumor models. + T cell subsets, while terminally exhausted CD8 + T cells have no effect. Reference 1 (Yugang Guo, Li Tang. Metabolic reprogramming of terminally exhausted CD8 +T cells by IL-10 enhances anti-tumor immunity. Nat Immunol. 22, 746-756, 2021, doi: 10.1038 / s41590-021-00940-2) discloses a recombinant half-life extended fusion protein of human IL-10 and IgG1 Fc (IL-10–Fc). The IL-10–Fc fusion protein effectively expands terminally exhausted CD8 T cells by promoting oxidative phosphorylation. + T cells and reinvigorate their cytotoxic function, but deplete CD8 + T cells have no effect.

[0005] IL-2, a multipotent cytokine, has been shown to significantly expand antigen-activated CD8 + T cells, induce differentiation into effector cells and upregulate inhibitory receptors such as PD-1 and TIM-3. Under continuous antigen stimulation, IL-2 induces CD8 + T cells differentiate into terminally exhausted T cells. Reference 2 (Rafi Ahmed, Masao Hashimoto. PD-1 combination therapy with IL-2 modifies CD8 + T cell exhaustion program. Nature. 610, 173-181, 2022, doi: 10.1038 / s41586-022-05257-0) discloses that the combined treatment of PD-1 and IL-2 in chronic lymphocytic choriomeningitis virus (LCMV)-infected mice alters CD8 + T cell exhaustion program, demonstrated in the LCMV mouse model, that IL-2 only expands precursor-exhausted CD8 + Terminally exhausted CD8 + T cells are ineffective.

[0006] Research on the functional revitalization and quantitative expansion of exhausted T cells is ongoing, but is limited to the exploration of a single subset and cannot achieve better tumor clearance. In current preclinical or clinical studies, there are few strategies that synergistically activate different exhausted cell subsets. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides an immunomodulatory factor composition, which synergistically activates precursor-depleted CD8 through a combination therapy of type I and type II cytokines. + T cells and terminally exhausted CD8 + T cells.

[0008] An immunomodulatory factor composition comprises a first component and a second component, wherein the first component is a type I cytokine or a long-acting form thereof, and the second component is a type II cytokine or a long-acting form thereof; the long-acting form is a cytokine fused to a fusion partner via a connecting peptide or modified by PEGylation, and the fusion partner is the Fc fragment of IgG or human serum albumin, or an engineered mutant of one of them.

[0009] In the present invention, the engineered mutant may be a protein mutant achieved by mutation of amino acid sites.

[0010] Type I and II cytokines have been shown to act on precursor-depleted CD8 + T cells and terminally exhausted CD8 + In the present invention, an immunomodulatory factor composition is formed by arbitrarily combining type I and type II cytokines or their respective long-acting forms, which can synergistically activate precursor-exhausted CD8 + T cells and terminally exhausted CD8 + T cells can solve the problem of T cell exhaustion induced by continuous antigen stimulation in the complex tumor immune microenvironment of solid tumors, and better achieve the purpose of tumor treatment.

[0011] Preferably, the immunomodulatory factor composition is a fusion protein of type I cytokines and a fusion protein of type II cytokines, wherein the fusion protein is formed by fusion of the cytokine with a fusion partner via a connecting peptide, and the fusion partner is the Fc fragment of human IgG or human serum albumin, or an engineered mutant of one of them.

[0012] Preferably, type I or type II cytokines are fused to a fusion partner via a linker peptide to form a fusion protein, which can increase the molecular volume and make it less susceptible to glomerular filtration, thereby effectively extending the half-life of the cytokine and overcoming the short half-life of the cytokine.

[0013] Preferably, the type I cytokine is at least one of IL-2, IL-12, IL-15, IL-17, IL-21, H9T, IL-15m3, or an engineered mutant of one of them.

[0014] Preferably, the type II cytokine is at least one of IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-33, or an engineered mutant of one of them.

[0015] In the present invention, the amino acid sequences of type I cytokines and type II cytokines and their reference sequence numbers in NCBI are shown in Table 1.

[0016] Table 1: Amino acid sequences of type I and type II cytokines.

[0017]

[0018]

[0019] Preferably, the IgG is at least one of human IgG1, IgG2, IgG3, and IgG4.

[0020] In the present invention, the amino acid sequence of the human IgG1 Fc fragment is shown as SEQ ID NO.4; the amino acid sequence of the human IgG4 Fc fragment is shown as SEQ ID NO.5; and the GenBank number of human serum albumin in NCBI is AAA98797.1.

[0021] Preferably, the amino acid sequence of the connecting peptide is shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9.

[0022] In the present invention, when the fusion protein is formed by linking the carboxyl terminus of a cytokine to the amino terminus of an immunoglobulin IgG Fc fragment, the two are connected via the hinge region of the IgG Fc fragment (amino acid sequence as shown in SEQ ID NO. 6 or SEQ ID NO. 7). When the fusion protein is formed by linking the amino terminus of a cytokine to the carboxyl terminus of an immunoglobulin IgG Fc fragment, the two are connected via a connecting peptide with an amino acid sequence as shown in SEQ ID NO. 8 or SEQ ID NO. 9.

[0023] Preferably, the amino acid sequence of the type I cytokine fusion protein is shown as SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 or SEQ ID NO.19, SEQ ID NO.28, and the amino acid sequence of the type II cytokine fusion protein is shown as SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26 or SEQ ID NO.27.

[0024] Preferably, in the immunomodulatory factor composition, the weight ratio of the first component to the second component is 1-20:1-20.

[0025] The present invention also provides a nucleic acid encoding the cytokine or a long-acting form thereof in the above-mentioned immunomodulatory factor composition.

[0026] The present invention also provides a vector carrying the above nucleic acid.

[0027] The present invention also provides a cell, wherein the cell contains the above nucleic acid or vector.

[0028] The present invention also provides the use of the above-mentioned immunomodulatory factor composition in the preparation of a drug for treating cancer.

[0029] Functional exhaustion of tumor antigen-specific T cells in the tumor suppressive microenvironment is one of the key reasons why the body cannot effectively eliminate tumor cells. Compared with memory T cells induced by acute infection or vaccine immunity, exhausted T cells gradually lose their effector function, proliferation ability, and memory potential, and continue to express immune checkpoint molecules at high levels.

[0030] In the present invention, the immunomodulatory factor composition synergistically activates precursor-depleted CD8 + T cells and terminally exhausted CD8 + T cells can, to a certain extent, "reverse" the exhausted state of exhausted T cells, improve their effector function and proliferation ability to control tumor progression.

[0031] In the present invention, the expression "cancer" includes lung cancer (small cell lung cancer and non-small cell lung cancer), breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer and skin cancer.

[0032] Preferably, the cancer is at least one of colon cancer, melanoma, and head and neck squamous cell carcinoma.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The immunomodulatory factor composition of the present invention provides a combination therapy of type I and type II cytokines, which can synergistically activate precursor-depleted CD8 + T cells and terminally exhausted CD8 + T cells to address T cell exhaustion induced by continuous antigen stimulation in the complex tumor immune microenvironment of solid tumors.

[0035] (2) The fusion protein of the present invention effectively prolongs the half-life of endogenous drugs by introducing a fusion partner. At the same time, the selective amplification of exhausted T cells and antigen-specific T cells by the fusion protein reduces the damage of the combined therapy to normal tissues and organs throughout the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic diagram of an immunomodulatory factor composition, wherein:

[0037] A is a schematic diagram of the immunomodulatory factor composition obtained in Examples 1-4;

[0038] B is a schematic diagram of an immunomodulatory factor composition formed by a fusion protein formed by fusing the amino terminus of a cytokine with the carboxyl terminus of the Fc fragment of human IgG;

[0039] C is a schematic diagram of the immunomodulatory factor composition formed by the fusion protein of cytokine and human serum albumin.

[0040] Figure 2 This is a diagram showing the expression and biological activity of IL-2–Fc fusion protein, where:

[0041] A is a schematic diagram of the structure of IL-2–Fc fusion protein;

[0042] B is the result of size exclusion chromatography of IL-2–Fc fusion protein; C is the SDS-PAGE molecular band identification of IL-2–Fc fusion protein under reducing and non-reducing conditions;

[0043] D is the CD8 after treatment with IL-2–Fc fusion protein at different concentration gradients + Fold change plot of T cell counts;

[0044] E is a graph showing changes in STAT5 phosphorylation levels in YT-1 cells after stimulation with IL-2–Fc fusion protein at different concentration gradients for 30 minutes;

[0045] F is a graph showing the T cell proliferation ability test after 3 days of culture with 10 ng / mL IL-2–Fc fusion protein.

[0046] Figure 3 This is a graph showing the efficacy evaluation results of the immunomodulatory factor composition obtained in Example 1 after co-incubation of CD8+ T cells from OT-1 mice with B16F10-OVA tumor cells, wherein:

[0047] A is a schematic diagram of the in vitro OT-1 extraction, treatment and co-incubation experimental scheme;

[0048] B, C, and D are CD8 + T cell count statistics, tumor cell count statistics and CD8 + Statistical graph of T cell secretion of GrzmB, IFN-γ, and TNF-α.

[0049] Figure 4 This is a graph showing the anti-tumor activity of the immunomodulatory factor composition obtained in Example 1 in the MC38 mouse colon cancer tumor cell model, the CT26 mouse colon cancer model, and the highly invasive B16F10-OVA mouse melanoma model, wherein:

[0050] AC, DF, and GI are respectively the tumor growth curves, mouse survival curves, and mouse survival curves after re-inoculation in the MC38 mouse colon cancer tumor cell model, CT26 mouse colon cancer model, and B16F10-OVA mouse melanoma model after treatment with the immunomodulatory factor composition obtained in Example 1.

[0051] Figure 5 The anti-tumor activity of the immunomodulatory factor composition obtained in Example 1 in the refractory B16F10 mouse melanoma model and the 4MOSC-1 mouse head and neck squamous cell carcinoma model is shown in FIG.

[0052] AB and CD are the tumor growth curves and mouse survival curves in the B16F10 mouse melanoma model and the 4MOSC-1 mouse head and neck squamous cell carcinoma model respectively after being treated with the immunomodulatory factor composition obtained in Example 1.

[0053] Figure 6 This is a graph showing the anti-tumor activity of the immunomodulatory factor composition obtained in Example 2 in the MC38 mouse colon cancer tumor cell model and the B16F10 mouse melanoma model, wherein:

[0054] A and C are respectively the tumor growth curves in the MC38 mouse colon cancer tumor cell model and the B16F10 mouse melanoma model after being treated with the immunomodulatory factor composition obtained in Example 2;

[0055] B is the mouse survival curve in the MC38 mouse colon cancer tumor cell model after being treated with the immunomodulatory factor composition obtained in Example 2;

[0056] D and E are statistical diagrams of the number of immune cells and CD8 in the B16F10 mouse melanoma model after treatment with the immunomodulatory factor composition obtained in Example 2. + T cell count statistics.

[0057] Figure 7 This is a graph showing the anti-tumor activity of the immunomodulatory factor composition obtained in Example 3 in the B16F10 mouse melanoma model, wherein A and B are the tumor growth curve and mouse survival curve after treatment with the immunomodulatory factor composition obtained in Example 3, respectively.

[0058] Figure 8This is a graph showing the anti-tumor activity of the immunomodulatory factor composition obtained in Example 4 in the B16F10-OVA mouse melanoma model, wherein A and B are the tumor growth curve and mouse survival curve after treatment with the immunomodulatory factor composition obtained in Example 4, respectively.

[0059] Figure 9 Graph showing the safety evaluation results of the immunomodulatory factor composition obtained in Example 1 and Example 2 in C57BL / 6 mice, wherein A is a schematic diagram of the safety evaluation experimental scheme;

[0060] B is the weight change curve of mice after being treated with the immunomodulatory factor composition obtained in Example 1;

[0061] C and D are CD8 in the liver after treatment with the immunomodulatory factor composition obtained in Example 1 and Example 2, respectively. + Statistics of the number of T cells and NK cells and the changes in ALT and AST in mouse serum.

[0062] Figure 10 This is a diagram showing the anti-tumor immune results of the immunomodulatory factor composition obtained in Example 1 in the B16F10 melanoma model, wherein A is a schematic diagram of the tumor infiltration experimental scheme;

[0063] B and C are respectively a tumor growth curve and a tumor weight statistical graph after treatment with the immunomodulatory factor composition obtained in Example 1;

[0064] DF are statistical diagrams of the number of immune cells after treatment with the immunomodulatory factor composition obtained in Example 1;

[0065] GI are different CD8 + Statistics of the number of T cell exhaustion subpopulations;

[0066] J is the CD8 + Statistics of GrzmB secreted by T cells. DETAILED DESCRIPTION

[0067] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited to the following examples.

[0068] The raw materials used in the present invention are all commercially available.

[0069] The preparation method of the fusion protein is as follows:

[0070] The corresponding sequence was digested with HindIII and NotI restriction endonucleases and then ligated with the corresponding pcDNA3.1+ plasmid. After transformation into Top10 competent cells, single clones were picked for sequencing. The correctly sequenced single clone strain was expanded and cultured for plasmid extraction and transfected into HEK293F human embryonic kidney cells. After 3-4 days of culture, the supernatant was filtered through a 0.22μm membrane and collected. After purification through a Protein A affinity chromatography column, the protein was further ultrafiltered using a 30kDa ultrafiltration tube. The final purification of the protein was completed using the molecular exclusion method to obtain the fusion protein. The schematic diagram of the fusion protein is shown below. Figure 1 shown.

[0071] Figure 1 is a schematic diagram of an immunomodulatory factor composition, wherein: Figure 1 A is a schematic diagram of the immunomodulatory factor composition obtained in Examples 1-4, wherein when the fusion protein is a cytokine carboxyl terminus linked to the amino terminus of the Fc fragment of human IgG, the two are connected via the hinge region of the Fc fragment of IgG; Figure 1 B is a schematic diagram of an immunomodulatory factor composition formed by a fusion protein formed by fusing the amino terminus of a cytokine with the carboxyl terminus of the Fc fragment of human IgG; Figure 1 C is a schematic diagram of the immunomodulatory factor composition formed by the fusion protein of cytokine and human serum albumin.

[0072] Example 1

[0073] According to the above-mentioned preparation method of fusion protein, IL-2-Fc fusion protein and IL-10-Fc fusion protein were obtained. The two fusion proteins were mixed at a weight ratio of 1:20 to obtain an immunomodulatory factor composition. The amino acid sequence is shown in Table 2, and the structural diagram is shown in Figure 1 shown.

[0074] Example 2

[0075] According to the above-mentioned preparation method of fusion protein, IL-15SA fusion protein and IL-10–Fc fusion protein were obtained. The two fusion proteins were mixed at a weight ratio of 1:10 to obtain an immunomodulatory factor composition, the amino acid sequence of which is shown in Table 2, and the structural diagram is shown in Figure 1 shown.

[0076] Example 3

[0077] According to the above-mentioned preparation method of fusion protein, IL-2-Fc fusion protein and IL-4-Fc fusion protein were obtained. The two fusion proteins were mixed at a weight ratio of 1:10 to obtain an immunomodulatory factor composition, the amino acid sequence of which is shown in Table 2, and the structural diagram is shown in Figure 1 shown.

[0078] Example 4

[0079] According to the above-mentioned preparation method of fusion protein, IL-2-Fc fusion protein and IL-5-Fc fusion protein were obtained. The two fusion proteins were prepared at a weight ratio of 1:10 to obtain an immunomodulatory factor composition, the amino acid sequence of which is shown in Table 2, and the structural diagram is shown in Figure 1 shown.

[0080] Table 2: Amino acid sequences of the fusion proteins of Examples 1-4.

[0081]

[0082] Activity test:

[0083] 1. Expression of IL-2–Fc fusion protein and identification of its biological activity.

[0084] (1) Schematic diagram of the structure of IL-2–Fc fusion protein:

[0085] Figure 2 A in FIG is a schematic diagram of the structure of the IL-2–Fc fusion protein, wherein the IL-2–Fc fusion protein is obtained by connecting the carboxyl terminus of human IL-2 to the amino terminus of human IgG1 via a flexible linker in the hinge region.

[0086] (2) Size and purity of IL-2–Fc fusion protein:

[0087] Figure 2 Figure B is the result of size exclusion chromatography of IL-2–Fc fusion protein. As shown in the figure, peak 2 was collected and analyzed, and the purity of the fusion protein was detected by SDS-PAGE results.

[0088] Figure 2 Figure C is the SDS-PAGE molecular band identification diagram of IL-2–Fc fusion protein under reducing and non-reducing conditions. The protein size under reducing conditions is about 43 kDa, and the dimer protein size under non-reducing conditions is about 86 kDa.

[0089] (3) Evaluation of the biological activity of IL-2–Fc fusion protein:

[0090] Figure 2 D in the figure is CD8 IL-2-Fc fusion protein treated with different concentration gradients. + Fold change of T cell counts. Splenocytes from C57 / BL6 mice were preactivated with 1 μg / ml anti-CD3 antibody and cultured for 3 days in the presence of different IL-2–Fc concentrations. CD8 T cells were detected by flow cytometry. + The expansion capacity of T cells was found to be induced by low concentrations of IL-2–Fc treatment.+ Proliferation of T cells.

[0091] Figure 2 E in the figure is the STAT5 phosphorylation level of YT-1 cells after 30 minutes of stimulation with IL-2–Fc fusion protein at different concentration gradients. After YT-1 cells were treated with IL-2–Fc fusion protein at different concentration gradients, the STAT5 phosphorylation function of the downstream signaling pathway was detected. EC 50 It is 16.19pM.

[0092] Figure 2 F in the figure is a test chart of T cell proliferation ability after 3 days of culture with 10 ng / mL IL-2–Fc fusion protein. Among them, IL-2–Fc fusion protein is compared with wild-type IL-2, and the two have similar proliferation-promoting abilities.

[0093] 2. In vitro efficacy evaluation of the immunomodulatory factor combination.

[0094] CD8 + T cells were co-incubated with B16F10-OVA tumor cells for 2 days and treated with different groups of drugs (PBS, IL-2–Fc 10 ng / ml, IL-10–Fc 200 ng / ml, and a combination of IL-2–Fc and IL-10–Fc (i.e., Example 1)). The number of tumor cells and lymphocytes and cytokine secretion function were analyzed.

[0095] Figure 3 This is a graph showing the in vitro efficacy evaluation results of the immunomodulatory factor composition obtained in Example 1, wherein: Figure 3 A is a schematic diagram of the in vitro OT-1 extraction and co-incubation experimental scheme. + T cells were co-cultured with B16F10-OVA tumor cells, OT-1CD8 + T cells can specifically recognize OVA antigen and kill B16F10 melanoma cells expressing OVA antigen under the activation of different drug groups. The results showed that the Example 1 group fully exerted the CD8 + The proliferation of T cells is promoted and the killing ability of tumor cells is significantly enhanced. The combined stimulation of IL-2 and IL-10 on CD8 + T cell exhaustion decreased, and the number of two exhausted cell subsets increased and exhibited better effector function ( Figure 3 BD).

[0096] 3. In vivo efficacy evaluation of the immunomodulatory factor composition.

[0097] (1) Evaluation of the efficacy of the immunomodulatory factor composition obtained in Example 1 in the MC38 mouse colon cancer tumor cell model, the CT26 mouse colon cancer model, and the highly invasive B16F10-OVA mouse melanoma model:

[0098] CD45.2 + C57BL / 6 mice were subcutaneously inoculated with MC38 mouse colon cancer cells (1×10 6 ), B16F10-OVA melanoma cells (5×10 5 ) or CD45.2 + BALB / C mice were subcutaneously inoculated with CT26 colon cancer cells (8×10 5 ), and started to administer the drug in different tumor size ranges (MC38 50-60mm 2 ;B16F10-OVA 20-30mm 2 CT26 30-40mm 2 ), and then different drugs (PBS, IL-2–Fc 2 μg, IL-10–Fc 20 μg, and IL-2–Fc combined with IL-10–Fc (ie, Example 1)) were injected intratumorally every three days for a total of six doses. The tumor growth of the mice was monitored and the survival time was recorded.

[0099] Figure 4 The anti-tumor activity of the immunomodulatory factor composition obtained in Example 1 in the MC38 mouse colon cancer cell model, the CT26 mouse colon cancer model, and the highly invasive B16F10-OVA mouse melanoma model. In the MC38 mouse colon cancer cell model, treatment was performed every three days within a reasonable dosing window ( Figure 4 A). The experimental results showed that compared with the single-drug group, the tumor growth was only slightly controlled, and most mice died due to increased tumor burden. After 5-6 treatments, the Example 1 group achieved complete tumor cure and significantly prolonged the survival of the mice ( Figure 4 D). We subsequently validated this finding in a cold tumor CT26 mouse colon cancer model and a highly invasive B16F10-OVA mouse melanoma model ( Figure 4 BC), it is worth noting that the Example 1 group showed significant tumor control effect and achieved tumor clearance ( Figure 4 EF). In addition, in the tumor recurrence experiment conducted three months after the last administration, the surviving mice in the Example 1 group that achieved long-term cure resisted the secondary attack of tumor cells ( Figure 4 GI), demonstrating that it achieves long-lasting immune memory.

[0100] (2) Evaluation of the efficacy of the immunomodulatory factor composition obtained in Example 1 in the refractory B16F10 mouse melanoma model and the 4MOSC-1 mouse head and neck squamous cell carcinoma model:

[0101] CD45.2 + C57BL / 6 mice were subcutaneously inoculated with B16F10 melanoma cells (5×10 5 ) or inoculated 4MOSC-1 head and neck squamous cell carcinoma cells (4×10 5 ), and started to administer the drug in different tumor size ranges (B16F10 15-25mm 2 , n = 10; 4MOSC-1 15-40mm 2 , n = 5), and then different drugs (PBS, IL-2–Fc 2 μg, IL-10–Fc 20 μg, and IL-2–Fc combined with IL-10–Fc (ie, Example 1)) were injected intratumorally every three days for a total of six doses. The tumor growth of the mice was monitored and the survival time was recorded.

[0102] Figure 5 The following is a graph showing the anti-tumor activity of the immunomodulatory factor composition obtained in Example 1 in the refractory B16F10 mouse melanoma model and the 4MOSC-1 mouse head and neck squamous cell carcinoma model. The results showed that the group in Example 1 had a superior therapeutic effect and significantly improved the survival of the tumor-implanted mice ( Figure 5 ).

[0103] (3) The efficacy evaluation of the immunomodulatory factor composition obtained in Examples 2-4 in the MC38 mouse colon cancer tumor cell model, the B16F10-OVA mouse melanoma model or the B16F10 mouse melanoma model was similar to the methods (1) and (2) in Experiment 3.

[0104] Figure 6 The anti-tumor activity of the immunomodulatory factor composition obtained in Example 2 in the MC38 mouse colon cancer tumor cell model and the B16F10 mouse melanoma model is shown. The Example 2 group has a more superior therapeutic effect, significantly improves the survival of tumor-implanted mice, and can significantly expand tumor-infiltrating lymphocytes ( Figure 6 ).

[0105] Figure 7 The anti-tumor activity of the immunomodulatory factor composition obtained in Example 3 in the B16F10 mouse melanoma model is shown in FIG. Among them, the Example 3 group has a more superior therapeutic effect and significantly improves the survival time of the tumor-implanted mice ( Figure 7 ).

[0106] Figure 8The anti-tumor activity of the immunomodulatory factor composition obtained in Example 4 in the B16F10-OVA mouse melanoma model is shown in FIG. Among them, the Example 4 group has a more superior therapeutic effect and significantly improves the survival time of the tumor-implanted mice ( Figure 8 ).

[0107] 4. Safety assessment of immunomodulatory factor compositions.

[0108] C57BL / 6 mice were subcutaneously inoculated with B16F10 melanoma cells and then subcutaneously injected with different drug combinations (IL-2–Fc combined with IL-10–Fc (i.e., Example 1), and IL-15SA combined with IL-10–Fc (i.e., Example 2)) every three days. The spleen, liver, and blood of the mice were analyzed on the seventh day after two doses.

[0109] Figure 9 The safety evaluation results of the immunomodulatory factor composition obtained in Example 1 and Example 2 in C57BL / 6 mice are shown in Figure A, wherein Figure A is a schematic diagram of the safety evaluation experimental scheme. Figure 9 As shown in Figure B, the weight of mice was monitored during the treatment period. No weight loss was found in the Example 1 and Example 2 groups, and they maintained normal growth. Furthermore, the liver tissue and serum of the mice were analyzed. The results showed that the immunomodulatory factor composition of the Example 2 group induced the liver CD8 + T cell and NK cell expansion ( Figure 9 C), and the ALT and AST in the serum of Example 1 and Example 2 groups were similar to those of the PBS group, with no significant difference ( Figure 9 D).

[0110] 5. Evaluation of the anti-tumor immune ability of the immunomodulatory factor combination:

[0111] C57BL / 6 mice were subcutaneously inoculated with 0.5×10 6 B16F10 melanoma cells, when the tumor grows to an area of ​​25-45mm 2 The first administration was carried out within the range of 1:1, and different drugs (PBS, IL-2–Fc 2 μg, IL-10–Fc 20 μg, and IL-2–Fc combined with IL-10–Fc (ie, Example 1)) were injected once every 3 days for a total of four times. On the 21st day, the spleen and tumor tissues of the mice were removed for immune cell flow cytometric analysis.

[0112] Figure 10 The results of the anti-tumor immune effect of the immunomodulatory factor composition obtained in Example 1 in the B16F10 melanoma model are shown in Figure A, where Figure A is a schematic diagram of the tumor infiltration experimental scheme. The results show that the immunomodulatory factor composition of Example 1 exhibits a significant tumor control effect ( Figure 10BC). The number of infiltrating immune cells in the combined administration group with the same tumor mass was greatly increased, including CD8 + T cells, CD4 + T cells and NK cells showed significant differences ( Figure 10 DF), which is thought to be CD8 + As the main subpopulation that responds to IL-2 and IL-10, T cells play a major role in killing tumor cells and effectively improve the tumor microenvironment, thereby increasing the infiltration of other immune cells in the tumor and releasing therapeutic potential. + T cells were further analyzed for the expansion of their exhausted subsets under different drug administration groups. It was consistent with the prediction that compared with the single drug group, the precursor exhausted CD8 + T cells and terminally exhausted CD8 + T cells were significantly expanded, and the proportions of different subpopulations also showed an increasing trend ( Figure 10 GI). Subsequently, analysis of cytokines that play an important role in tumor immunity showed that the CD8 + T cells still have a high cytokine secretion capacity and show a stronger GrzmB secretion capacity ( Figure 10 J), which proves that under the activation of Example 1 group, CD8 + T cells still maintain good effector function, effectively slowing down and curbing the exhaustion process, and significantly enhancing the exhaustion of CD8 + T cell proliferation ability and anti-tumor immune response.

[0113] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An immunomodulatory factor composition, comprising a first component and a second component, characterized in that: The first component is a type I cytokine or a long-acting form thereof, and the second component is a type II cytokine or a long-acting form thereof; the long-acting form is a cytokine fused to a fusion partner via a connecting peptide or modified by PEGylation, and the fusion partner is an IgG Fc fragment or human serum albumin, or an engineered mutant of one of them.

2. The immunomodulatory factor composition according to claim 1, characterized in that The type I cytokine is at least one of IL-2, IL-7, IL-12, IL-15, IL-17, IL-21, H9T, and IL-15m3, or an engineered mutant of one of them; the type II cytokine is at least one of IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, and IL-33, or an engineered mutant of one of them.

3. The immunomodulatory factor composition according to claim 1, characterized in that The IgG is at least one of human IgG1, IgG2, IgG3, and IgG4.

4. The immunomodulatory factor composition according to claim 1, characterized in that The amino acid sequence of the connecting peptide is shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.

9.

5. The immunomodulatory factor composition according to claim 1, characterized in that: The amino acid sequence of the type I cytokine fusion protein is shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.28, and the amino acid sequence of the type II cytokine fusion protein is shown in SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26 or SEQ ID NO.

27.

6. The immunomodulatory factor composition according to claim 1, characterized in that: In the immunomodulatory factor composition, the weight ratio of the first component to the second component is 1-20:1-20.

7. A nucleic acid, characterized in that The nucleic acid encodes the cytokine or its long-acting form in the immunomodulatory composition according to any one of claims 1 to 6.

8. A carrier, characterized in that The vector carries the nucleic acid of claim 7.

9. A cell, characterized in that The cell contains the nucleic acid of claim 7 or the vector of claim 8.

10. Use of the immunomodulatory factor composition according to any one of claims 1 to 6 in the preparation of drugs for treating cancer.

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