Bispecific cytokine fusion protein and application thereof

Abstract

The invention discloses a bispecific cytokine fusion protein, which comprises an amino acid sequence of a type I cytokine and an amino acid sequence of a type II cytokine, and the amino acid sequence of the type I cytokine and the amino acid sequence of the type II cytokine are directly connected through a connecting peptide or are connected in series or in parallel through a fusion partner and the connecting peptide. Amino acid sequences of type I and type II cytokines are connected in series or in parallel to form the bispecific cytokine fusion protein, and the fusion protein can synergistically activate precursor depleted CD8 + T cells and terminal depleted CD8 + T cells, improve immunosuppression of a solid tumor complex microenvironment and release the potential of tumor immunotherapy. The invention further discloses nucleic acid, a carrier, a cell, a medicine or a medicine composition related to the bispecific cytokine fusion protein. The invention also discloses application of the bispecific cytokine fusion protein in preparation of drugs for treating cancers.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a bispecific cytokine fusion protein and applications thereof. Background Art

[0002] Tumor immunotherapy has made great progress in the past few decades, including cytokines, immune checkpoint inhibitors, oncolytic viruses, tumor vaccines and adoptive cell therapy. For example, immune checkpoint inhibitors (ICB) such as PD-1 / PD-L1 have achieved some results in restoring the immunosuppressive tumor microenvironment. However, the average response rate of ICB in various tumors is only 20% to 30%, and most patients have difficulty maintaining a lasting response and are accompanied by adverse reactions such as tissue-specific toxicity. The efficacy of chimeric antigen receptor T cell (CAR-T) therapy in solid tumors is also not ideal. In addition, more than a dozen cytokines have been used in clinical studies of tumor immunotherapy, but the efficacy is still unclear. One of the main reasons is the exhaustion of T cells in the tumor microenvironment, which is manifested by changes in transcriptional profiles, metabolic patterns, and continued increase in inhibitory receptor expression and loss of effector function.

[0003] Research aimed at rejuvenating exhausted T cells has been a hot topic in recent years, especially in the complex tumor microenvironment of solid tumors. + TILs are mainly 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 specific exhausted T cell subsets, effectively expand T cells and enhance cytotoxic function and anti-tumor cytokine secretion. For example, PD-1 antibodies have been shown to act on precursor exhausted CD8 in virus infection models and some tumor models. + T cell subsets, however, are terminally exhausted CD8 + T cells have little effect. IL-2, as a multi-effect type I cytokine, has been shown to amplify activated CD8 +T cells, inducing their differentiation into effector cells and upregulating inhibitory receptors such as PD-1 and TIM-3. Reference 1 (Rafi Ahmed et al., PD-1combination therapy with IL-2modifies CD8 + T cell exhaustion program. Nature. 610, 173-181, 2022, doi: 10.1038 / s41586-022-05257-0) discloses that combined treatment of PD-1 and IL-2 in chronic lymphocytic choriomeningitis virus (LCMV)-infected mice alters CD8 + T cell exhaustion program demonstrated in LCMV mouse model by depletion of CD8 progenitors by IL-2 + T cells, terminally exhausted CD8 + T cells are ineffective.

[0005] Terminal depletion of CD8 + T cell anergy restricts the entire tumor immunotherapy, and various studies focus on activating terminally exhausted T cells. Reference 2 (Yugang Guo et al., 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) disclosed a typical type II cytokine IL-10 and constructed a recombinant IL-10 and IgG Fc fusion protein (IL-10–Fc) with extended half-life. The IL-10–Fc fusion protein effectively amplified terminally exhausted CD8 + T cells and revitalize their cytotoxic function to enhance anti-tumor immune response.

[0006] Reference 3 (Bing Feng et al., The type 2 cytokine Fc-IL-4 revitalizes exhausted CD8 + T cells against cancer. Nature. 634, 712-720, 2024, doi: 10.1038 / s41586-024-07962-4) disclosed another typical type II cytokine interleukin-4 fusion protein (Fc–IL-4), which can directly act on CD8 +T cells and enrichment of functional terminally exhausted CD8 + T cells. Fc–IL-4 regulates terminal exhaustion of CD8 + T cell metabolic function improves exhausted CD8 + The killing ability of T cells.

[0007] Research on the expansion of exhausted T cells and the revitalization of their functions is ongoing, but it is limited to the exploration of a single subpopulation and makes it difficult to effectively eliminate tumors. In current preclinical or clinical studies, there are few strategies that use different types of cytokines to synergistically activate different exhausted cell subpopulations. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a bispecific cytokine fusion protein, which fuses type I cytokines and type II cytokines to achieve precursor depletion of CD8 + T cells and terminal depletion of CD8 + The synergistic activation of T cells significantly inhibits tumor growth and releases the potential of T cell immunotherapy.

[0009] A bispecific cytokine fusion protein comprises the amino acid sequence of type I cytokine and the amino acid sequence of type II cytokine.

[0010] Previous studies have suggested that type I cytokines and type II cytokines can act on precursors to deplete CD8 + T cells and terminal depletion of CD8 + In the present invention, the amino acid sequences of type I and type II cytokines are connected in series or in parallel to form a bispecific or multispecific cytokine fusion protein, which can synergistically activate precursor-exhausted CD8 + T cells and terminal depletion of CD8 + T cells improve the immunosuppression of the complex microenvironment of solid tumors and significantly release the potential of tumor immunotherapy that relies on cytotoxic immune cells.

[0011] Preferably, the amino acid sequence of the type I cytokine and the amino acid sequence of the type II cytokine are directly connected via a connecting peptide or are connected via a fusion partner and a connecting peptide, the fusion partner is the CH2 region, CH3 region of the Fc fragment of human IgG, or any combination thereof, or human serum albumin, or an engineered mutant of one of them, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO.1 to 3.

[0012] In the present invention, the structure of the bispecific cytokine fusion protein can be Figure 1Any one of the formulas (1) to (12) can also be a type I cytokine amino acid sequence and a type II cytokine amino acid sequence connected in parallel through the carboxyl end of the CH2 or CH3 region of the Fc fragment of immunoglobulin IgG; in formulas (1) to (12), a and b are type I cytokines or type II cytokines, and a and b cannot be type I cytokines and type II cytokines at the same time; and in formulas (1) to (3), c and d are type I cytokines or type II cytokines; the bispecific cytokine fusion protein can also be a type I cytokine fusion protein connected to the amino terminus or carboxyl terminus of the fusion partner through a connecting peptide.

[0013] In the present invention, the engineered mutant may be a protein mutant achieved by mutation of amino acid sites.

[0014] The amino acid sequences of type I and type II cytokines are connected by a fusion partner and a connecting peptide to form a bispecific cytokine fusion protein. The Fc fragment of human IgG and human serum albumin can increase the molecular volume, making it less likely to be filtered by the glomerulus, thereby effectively extending the half-life of the cytokine and overcoming the defect of the short half-life of the cytokine.

[0015] In the present invention, the connecting peptide can be a short peptide of the hinge region of IgG (the amino acid sequence is shown in SEQ ID NO.1) or a GS connecting peptide composed of stretched glycine and serine residues (the amino acid sequence is shown in SEQ ID NO.2 or SEQ ID NO.3). When the cytokine is connected to the CH2 region or CH3 region of the amino acid of the Fc fragment of immunoglobulin IgG, the two are connected through the hinge region of IgG. When the cytokine is connected to the carboxyl end of the Fc fragment of immunoglobulin IgG, the two are connected through the GS connecting peptide. When the cytokine is connected to human serum albumin (HSA), the two are connected through the GS connecting peptide. When the cytokines are directly connected to each other, the two are connected through the hinge region or the GS connecting peptide. When the cytokines are connected in parallel and the fusion partner includes a CH3 region, a knob-into-hole (KIH) connection structure is formed between the CH3 regions, and its structural schematic diagram is shown as follows. Figure 1 As shown in formula (5) or formula (7).

[0016] Preferably, the bispecific cytokine fusion protein structure is as shown in formula (1), a and b are type I cytokines or type II cytokines, and a and b cannot be type I cytokines and type II cytokines at the same time; c and d are type I cytokines or type II cytokines, and the amino acid sequence of the type I cytokine and the amino acid sequence of the type II cytokine are connected by a fusion partner and a connecting peptide, the fusion partner is the Fc fragment of human IgG or an engineered mutant of the Fc fragment of human IgG, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO.1~3.

[0017] Preferably, the bispecific cytokine fusion protein can achieve complete cure in tumor models of multiple solid tumors (such as colon cancer, melanoma, and head and neck squamous cell carcinoma) and can enhance CD8 + T cells infiltrate into the tumor, thereby enhancing the proliferation and effector capacity of effector T cells and overcoming the anergy of exhausted T cells.

[0018] Preferably, the type I cytokine is one of IL-2, IL-7, IL-12, IL-15, IL-17, IL-21, H9T, IL-15m3, or an engineered mutant of one of them; the type II cytokine is one of IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-33, or an engineered mutant of one of them.

[0019] 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.

[0020] Table 1: Amino acid sequences of type I and type II cytokines.

[0021]

[0022]

[0023] Preferably, the IgG is at least one of human IgG1, IgG2, IgG3, IgG4, or an engineered mutant of one of them.

[0024] In the present invention, the amino acid sequence of the human IgG1 Fc fragment is shown as SEQ ID NO.7; the GenBank number of human serum albumin in NCBI is AAA98797.1.

[0025] Preferably, the amino acid sequence of the bispecific cytokine fusion protein is shown in any one of SEQ ID NOs. 9 to 28.

[0026] The present invention also provides a nucleic acid encoding the above-mentioned bispecific cytokine fusion protein.

[0027] The present invention also provides a vector, which carries the above nucleic acid.

[0028] The present invention also provides a cell, wherein the cell contains the above nucleic acid or vector.

[0029] The present invention also provides a medicine or a pharmaceutical composition, which comprises the above-mentioned bispecific cytokine fusion protein.

[0030] The present invention also provides the use of the above-mentioned bispecific cytokine fusion protein, nucleic acid, vector, cell, drug or drug composition in the preparation of drugs for treating cancer.

[0031] In the present invention, type I cytokines and type II cytokines are connected in series or in parallel to construct a bispecific cytokine fusion protein, which can synergistically activate precursor-exhausted CD8 + T cells and terminal depletion of CD8 + T cells improve the immunosuppression of the complex microenvironment of solid tumors and significantly release the potential of tumor immunotherapy that relies on cytotoxic immune cells.

[0032] 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, head and neck squamous cell carcinoma and skin cancer.

[0033] Preferably, the cancer is colon cancer, melanoma or head and neck squamous cell carcinoma.

[0034] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0035] (1) In the present invention, type I cytokines and type II cytokines are connected in series or in parallel to construct a bispecific cytokine fusion protein, which can synergistically activate precursor-exhausted CD8 + T cells and terminal depletion of CD8 + T cells, improving the immunosuppression of the complex microenvironment of solid tumors.

[0036] (2) The bispecific cytokine fusion protein of the present invention effectively prolongs the half-life of the fusion protein by introducing a fusion partner. At the same time, the fusion protein can alleviate or overcome the tumor-specific CD8 + Depletion of T cells significantly improves the anti-tumor efficacy of cytokine drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the structure of the bispecific cytokine fusion protein.

[0038] Figure 2 Figure 1 is a schematic diagram of the structure of IL-10–Fc–IL-2 fusion protein and IL-10–Fc–H9T fusion protein, as well as the expression and biological activity identification diagram of IL-10–Fc–IL-2 fusion protein, wherein A is a schematic diagram of the structure of IL-10–Fc–IL-2 fusion protein; B is a schematic diagram of the structure of IL-10–Fc–H9T fusion protein; C is an SDS-PAGE molecular band identification diagram of IL-10–Fc–IL-2 fusion protein under reducing and non-reducing conditions; D is a diagram of CD8 + Fold change of T cell count; E is the STAT5 phosphorylation level of YT-1 cells after stimulation with different concentration gradients of IL-10–Fc–IL-2 fusion protein for 30 minutes; F is a test chart of T cell proliferation ability after culturing with 150 ng / mL IL-10–Fc–IL-2 fusion protein for 3 days.

[0039] Figure 3 Figure 1 is the expression and biological activity identification diagram of IL-10–Fc–IL-15SA fusion protein, where A is the structural schematic diagram of IL-10–Fc–IL-15SA fusion protein; B is the SDS-PAGE molecular band identification diagram of IL-10–Fc–IL-15SA fusion protein and the Western Blot development diagram after incubation with anti-Fc antibody; C is the CD8 + T cell counts.

[0040] Figure 4 CD8 + The results of the efficacy evaluation of IL-10–Fc–IL-2 fusion protein treatment after co-incubation of T cells with B16F10-OVA tumor cells, where A is a schematic diagram of the in vitro OT-1 extraction and co-incubation experimental scheme; B and C are CD8 + T cell count, tumor cell count and CD8 + T cells secrete GzmB + IFN-γ + and TNF-α + Statistical chart of .

[0041] Figure 5Figure 3 is the antitumor activity graph of IL-10–Fc–IL-2 fusion protein in MC38 mouse colon cancer tumor cell model, B16F10-OVA mouse melanoma model, CT26 mouse colon cancer model and 4MOSC-1 mouse head and neck squamous cell carcinoma model. AC, DF, GI and JL are the tumor growth curves, mouse survival curves and mouse survival curves after re-inoculation in the MC38 mouse colon cancer tumor cell model, B16F10-OVA mouse melanoma model, CT26 mouse colon cancer model and 4MOSC-1 mouse head and neck squamous cell carcinoma model after treatment with IL-10–Fc–IL-2 fusion protein, respectively.

[0042] Figure 6 Figure 2 is the anti-tumor immune result of IL-10–Fc–IL-2 fusion protein in the B16F10 melanoma model, where A is a schematic diagram of the tumor infiltration experiment of C57BL / 6 mice subcutaneously inoculated with B16F10 cells; B is a statistical diagram of the number of immune cells after treatment with IL-10–Fc–IL-2 fusion protein; C is a statistical diagram of different CD8 + Statistical graph of the number of T cell exhaustion subsets.

[0043] Figure 7 Figure 1 is a diagram showing the safety evaluation results of IL-10–Fc–IL-2 fusion protein in C57BL / 6 mice, where A is a schematic diagram of the safety evaluation experimental scheme; B is the weight change curve of mice after treatment with IL-10–Fc–IL-2 fusion protein; C is the CD8 + T cell and NK cell count statistics. DETAILED DESCRIPTION

[0044] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited to the following examples.

[0045] The raw materials used in the present invention are all commercially available.

[0046] The preparation method of fusion protein is as follows:

[0047] 1. Construction of bispecific cytokine fusion protein expression vector

[0048] Referring to the schematic diagram of the molecular structure of the bispecific cytokine fusion protein of formula (1), pcDNA3.1+ was selected as the expression vector, and the gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd. The 5' end of the synthetic gene was designed with Hind III restriction site ([AAGCTT]), Kozak sequence ([ACCACC]), and H293 signal peptide, and the 3' end of the synthetic gene was designed with 3'UTR termination codon ([TGA]) and Not I restriction site ([GCGGCCGC]). The pcDNA3.1+ expression vector and the synthetic antibody gene were both subjected to Hind III and Not I double restriction digestion, and the restriction products of the pcDNA3.1+ expression vector and the antibody gene were subjected to agarose gel electrophoresis and target fragment recovery, and the recovered fragments were transduced into TOP10 competent cells after being connected by T4 ligase, and the correct bacterial cells were selected for seed preservation and expansion culture of the bacterial cells, and the expanded bacterial cells were used for plasmid extraction.

[0049] 2. Expression and purification of bispecific cytokine fusion proteins

[0050] The plasmid extracted above is selected, and HEK293F human embryonic kidney cells are used as an expression system as an example to describe the cell transfection and transient expression technical solutions. Other conventional technical processes should also be regarded as the protection scope of the present invention. HEK293F cells (Thermo) are transferred to a 37°C water bath to melt, centrifuged at 1000rpm for 5min, and the supernatant is discarded. The cells are resuspended with 1mL of SMM 293TI (Gibco) culture medium, added to a 250mL cell shake flask, supplemented with 50mL of SMM 293TI culture medium, and a final concentration of 1% penicillin-streptomycin double antibody (Gibco) and 0.5% serum (Gibco) are added. The 250mL cell shake flask is transferred to a 37°C, 5% CO2 cell shaker for culture, and the cell state is observed after 2-3 days of culture. When the cell density reaches 1.5×10 6 When the cell density reaches 1.5×10 / mL, transfer all 50 mL of cell suspension in the 250 mL cell shake flask to a 500 mL cell shake flask that has been supplemented with 110 mL of SMM 293TI medium, and then add 1% double antibody and 0.5% serum. Observe the cell state for another 2-3 days. When the cell density reaches 1.5×10 6 When the cell density reaches 2×10 / mL, 160 mL of cell suspension in the 500 mL cell shake flask is evenly subcultured into two 1 L cell shake flasks that have been added with 160 mL of SMM 293TI medium, and then 1% of double antibody and 0.5% of serum are added to continue the culture. After 2-3 days of culture, observe the cell status and wait until the cell density reaches 2×10 6 Plasmid transfection can be performed when the number of cells / mL is above 100. The specific transfection operation process is as follows:

[0051] In a biosafety cabinet, sterilize the plasmid through a 0.22 μm microporous filter. Prepare two 50 mL centrifuge tubes, one labeled with the corresponding plasmid and the other with the corresponding PEI. Add 10 mL of SMM 293TI medium and 600 μg of plasmid to the plasmid tube, and add 10 mL of SMM 293TI medium and 2 mL of PEI to the PEI tube. After mixing evenly, add the liquid in the plasmid tube to the PEI tube, mix thoroughly and incubate at room temperature for 15 minutes. Take out a 1L cell shake flask (containing 240 mL of suspended cells) and add 50 mL of SMM 293TI medium. Add the above 20 mL of transfection solution to a 1L cell shake flask, supplemented with 1% double antibody and 0.5% serum. Return the 1L cell shake flask to a cell shaker at 37°C and 5% CO2 for 4-5 days and collect the supernatant.

[0052] The culture supernatant was purified by affinity chromatography. The specific operation process of affinity chromatography is as follows:

[0053] HEK293F cells transfected with plasmid for 3-4 days were taken out from the cell shaker, centrifuged at 4500g for 15min, and the supernatant was collected and filtered through 0.45μm and 0.22μm filters to remove impurities to prevent clogging of the chromatography column. Protein A loading buffer (50mM Tris-Hcl, 0.15M Nacl, pH 7.4), Protein A elution buffer (50mM sodium citrate dihydrate, pH3.2), ultrapure water, and 20% ethanol were prepared and filtered through a 0.22μm filter to remove impurities.

[0054] The protein was purified using AKTA protein purifier (GE Healthcare, AKTA purifier). The specific operation process is as follows:

[0055] Start the AKTA protein purification software and set the parameters (upper limit of column pressure 0.2MPa, flow rate 3mL / min), rinse pump A and pump B with a wash bottle filled with ultrapure water, and then put in ultrapure water that has passed through the membrane to clean the pipeline. After cleaning, install the column (HiTraprProtein A affinity chromatography column) and clean the column. Rinse the Protein A elution buffer with pump A to conductivity equilibrium, and rinse the Protein A loading buffer with pump B to conductivity equilibrium. Rinse the cell culture supernatant that has passed through the membrane with pump B to start loading. After loading, flush the Protein A loading buffer with pump B to conductivity equilibrium, and then flush the Protein A elution buffer to elute the target protein, collect the protein peak detected by UV280, and mark the peak position. After the target protein is collected, rinse the pipeline and column with pure water and 20% ethanol in turn, collect the column and turn off the AKTA protein purifier. The collected target proteins were concentrated by ultrafiltration through a 50 kDa ultrafiltration tube, the proteins were eluted with PBS and the protein concentration was measured on Nanodrop. They were named human IL-10–Fc–IL-2 fusion protein, IL-10–Fc–H9T fusion protein and IL-10–Fc–IL-15SA fusion protein, respectively, and stored in a 4 °C refrigerator.

[0056] Example 1

[0057] Referring to the preparation method of the above-mentioned bispecific cytokine fusion protein, an IL-10–Fc–IL-2 fusion protein was obtained, whose amino acid sequence is SEQ ID NO.9. The expression and biological activity identification of the IL-10–Fc–IL-2 fusion protein are shown in FIG. Figure 2 shown.

[0058] Figure 2 The diagram is a schematic diagram of the structure of IL-10–Fc–IL-2 fusion protein and IL-10–Fc–H9T fusion protein, as well as the expression and biological activity identification diagram of IL-10–Fc–IL-2 fusion protein, wherein: Figure 2 A in the figure is a schematic diagram of the structure of the IL-10–Fc–IL-2 fusion protein, which is obtained by connecting the carboxyl terminus of human IL-10 to the amino terminus of human IgG1Fc through the hinge region of the Fc fragment of human IgG, and connecting the amino terminus of human IL-2 to the carboxyl terminus of human IgG1 Fc through a GS connecting peptide. Figure 2 C in the figure is the SDS-PAGE molecular band identification diagram of IL-10–Fc–IL-2 fusion protein under reducing and non-reducing conditions. The protein size under non-reducing conditions is about 132 kDa, and the protein size under reducing conditions is about 66 kDa. Figure 2 D in the figure is the CD8+ Fold changes in T cell counts. Splenocytes from C57BL / 6 mice were preactivated with 1 μg / ml anti-CD3 antibody and cultured for 3 days in the presence of different IL-10–Fc–IL-2 concentrations. CD8 + The expansion capacity of T cells was found to be able to induce CD8 + Proliferation of T cells. Figure 2 E in the figure is the STAT5 phosphorylation level of YT-1 cells after stimulation with IL-10–Fc–IL-2 fusion protein at different concentrations for 30 minutes. After YT-1 cells were treated with IL-10–Fc–IL-2 fusion protein at different concentrations, the STAT5 phosphorylation function of the downstream signaling pathway was detected. 50 It is 279.5pM. Figure 2 F in the figure is a test chart of T cell proliferation ability after culturing with 150 ng / mL IL-10–Fc–IL-2 fusion protein for 3 days. Among them, the IL-10–Fc–IL-2 fusion protein is compared with PBS, and the fusion protein has the ability to promote proliferation.

[0059] Example 2

[0060] Referring to the preparation method of the above-mentioned bispecific cytokine fusion protein, an IL-10–Fc–H9T fusion protein was obtained, whose amino acid sequence is SEQ ID NO.10. The structural schematic diagram of the IL-10–Fc–H9T fusion protein is shown in Figure 2 As shown in B.

[0061] Figure 2 B is a schematic diagram of the structure of the IL-10–Fc–H9T fusion protein, wherein the IL-10–Fc–H9T fusion protein is obtained by connecting the carboxyl terminus derived from human IL-10 to the amino terminus of human IgG1 Fc through the hinge region of the Fc fragment of human IgG, and connecting the amino terminus derived from human H9T to the carboxyl terminus of human IgG1 Fc through a GS connecting peptide.

[0062] Example 3

[0063] Referring to the preparation method of the above-mentioned bispecific cytokine fusion protein, an IL-10–Fc–IL-15SA fusion protein was obtained, whose amino acid sequence is SEQ ID NO.12. The expression and biological activity identification of the IL-10–Fc–IL-15SA fusion protein are shown in FIG. Figure 3 shown.

[0064] Figure 3 The figure shows the expression and biological activity identification of IL-10–Fc–IL-15SA fusion protein, in which: Figure 3A is a schematic diagram of the structure of the IL-10–Fc–IL-15SA fusion protein. Human IL-15, human IL-15Rsu containing the sushi domain, and IgG1Fc are connected to construct the IL-15SA fusion protein, and the sequence is shown in SEQ ID NO.8. Similarly, another IL-15SA fusion protein can be obtained by combining the mutant human IL-15m3 (sequence as SEQ ID NO.5) of human IL-15 with human IL-15Rsu. On this basis, we designed the IL-10–Fc–IL-15SA fusion protein, connecting the carboxyl terminus of human IL-10 to the amino terminus of human IgG1 Fc through the hinge region of the Fc fragment of human IgG, and connecting the amino terminus of human IL-15SA to the carboxyl terminus of human IgG1 Fc through the GS connecting peptide to obtain a fusion protein named IL-10–Fc–IL-15SA fusion protein. The molecular weight of the IL-10–Fc–IL-15SA fusion protein was verified by SDS-PAGE, and the molecular weight was 170 kDa. The IL-10–Fc–IL-15SA fusion protein was co-incubated with anti-Fc antibody, and the presence of the fusion protein was verified by Western Blot ( Figure 3 Furthermore, CD8 + T cells were cultured in vitro for 6 days and treated with different groups of drugs (PBS, IL-10–Fc 100 ng / ml, IL-15SA 100 ng / ml, IL-10–Fc–IL-15SA 200 ng / ml). After culturing for 2 days, flow cytometry analysis was used to verify that the IL-10–Fc–IL-15SA fusion protein could significantly amplify CD8 + T cells, especially PD-1 + TIM3 + Exhausted T cell subsets that exhibit anti-exhaustion potential ( Figure 3 C).

[0065] Activity test

[0066] 1. In vitro activity evaluation of bispecific cytokine fusion proteins

[0067] The IL-10-Fc-IL-2 fusion protein obtained in Example 1 was expressed in the CD8 + In vitro efficacy evaluation of T cells co-cultured with B16F10-OVA tumor cells.

[0068] 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, or IL-10–Fc–IL-2 150 ng / ml), and the number of tumor and lymphocytes and cytokine secretion function were analyzed.

[0069] CD8 + T cells were co-cultured with B16F10-OVA tumor cells, and CD8 + T cells specifically recognized OVA antigen and killed B16F10 melanoma cells expressing OVA antigen under activation in different dosing groups ( Figure 4 A). Compared with IL-2–Fc and IL-10–Fc groups, IL-10–Fc–IL-2 fusion protein could significantly enhance CD8 + T proliferation and tumor killing ability ( Figure 4 B) Analysis of cytokines that play an important role in tumor immunity, CD8 in IL-10–Fc–IL-2 fusion protein + T cells secrete more GzmB + IFN-γ + TNF-α + Cytokines ( Figure 4 C), IL-10–Fc–IL-2 fusion protein can enhance CD8 + T cells infiltrate into the tumor, thereby enhancing the proliferation and effector capacity of effector T cells and overcoming the anergy of exhausted T cells.

[0070] II. In vivo efficacy evaluation of bispecific cytokine fusion proteins

[0071] The efficacy of the IL-10–Fc–IL-2 fusion protein obtained in Example 1 was evaluated in the MC38 mouse colon cancer tumor cell model, the B16F10-OVA mouse melanoma model, the CT26 mouse colon cancer model, and the 4MOSC-1 mouse head and neck squamous cell carcinoma model.

[0072] C57BL / 6 mice were subcutaneously inoculated with MC38 mouse colon cancer cells (1×10 6 ), B16F10-OVA melanoma cells (5×10 5 ) or BALB / C mice were subcutaneously inoculated with CT26 colon cancer cells (8×10 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 (MC38 50~60mm 2; B16F10-OVA 20~30mm 2 ; CT26 30~40mm 2 ; 4MOSC-1 15~40mm 3 ; n=5), and then different drugs (PBS, IL-2–Fc 2 μg, IL-10–Fc 20 μg, IL-10–Fc–IL-2 15 μg (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.

[0073] Figure 5 The anti-tumor activity of the IL-10-Fc-IL-2 fusion protein obtained in Example 1 in the MC38 mouse colon cancer tumor cell model, the B16F10-OVA mouse melanoma model, the CT26 mouse colon cancer model, and the 4MOSC-1 mouse head and neck squamous cell carcinoma model. In the MC38 mouse colon cancer tumor cell model, treatment was performed every three days within a reasonable dosing window ( Figure 5 The experimental results showed that the IL-2–Fc or IL-10–Fc monotherapy group had little inhibitory effect on tumor growth, and most mice died due to increased tumor burden. However, the IL-10–Fc–IL-2 fusion protein achieved complete tumor clearance after 6 treatments and significantly prolonged the survival of mice ( Figure 5 We subsequently verified this in a cold tumor CT26 mouse colon cancer model, a highly invasive B16F10-OVA mouse melanoma model, and an orthotopic tumor 4MOSC-1 mouse head and neck squamous cell carcinoma model. It is noteworthy that IL-10–Fc–IL-2 significantly inhibited tumor growth and prolonged the survival of mice in all three models ( Figure 5 Three months after the tumor was cured, we conducted a tumor recurrence experiment. We inoculated tumor cells into each experimental group of mice but did not give them any more medication. The results showed that the IL-10–Fc–IL-2 group could resist the secondary attack of tumor cells ( Figure 5 C, F, I, L in the figure, proving that it achieves long-lasting immune memory.

[0074] 3. Evaluation of the anti-tumor immune ability of bispecific cytokine fusion proteins

[0075] The IL-10-Fc-IL-2 fusion protein obtained in Example 1 was subcutaneously inoculated into 0.5×10 6 Evaluation of the anti-tumor immunity of the B16F10 melanoma cell model.

[0076] C57BL / 6 mice were subcutaneously inoculated with 0.5×10 6B16F10 melanoma cells, when the tumor grows to an area of ​​25 to 45 mm 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, IL-10–Fc–IL-2 15 μg (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.

[0077] Figure 6 Figure 1 is the anti-tumor immune result of IL-10–Fc–IL-2 fusion protein in the B16F10 melanoma model, where Figure A is a schematic diagram of the tumor infiltration experimental scheme. The results showed that compared with the IL-2–Fc or IL-10–Fc treatment group, the IL-10–Fc–IL-2 fusion protein group had a significantly higher CD8 + T cells, CD4 + The infiltration of immune cells such as T cells and NK cells increased significantly ( Figure 6 B), 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, increasing the number of other immune cells infiltrating the tumor, thereby releasing therapeutic potential. + The expansion of T cell exhaustion subsets under different dosing groups is consistent with the prediction that compared with the single drug group, precursor exhaustion of CD8 + T cells and terminally exhausted CD8 + T cells were significantly expanded, and the proportions of different subsets also showed an increasing trend ( Figure 6 C).

[0078] IV. Safety Assessment of Bispecific Cytokine Fusion Proteins

[0079] C57BL / 6 mice were subcutaneously inoculated with B16F10 melanoma cells (5×10 5 ), different drug combinations (PBS or IL-10-Fc-IL-2 15 μg (ie, Example 1)) were subcutaneously injected every 3 days, and the spleens of the mice were analyzed on the seventh day after two administrations.

[0080] Figure 7 The safety evaluation results of IL-10–Fc–IL-2 fusion protein in C57BL / 6 mice obtained in Example 1 are shown in Figure A, where Figure A is a schematic diagram of the safety evaluation experimental scheme. The weight of mice was monitored during the treatment period. The IL-10–Fc–IL-2 fusion protein group did not reduce the weight of mice, and even slightly increased their weight ( Figure 7B), indicating that the health of mice may have improved after the tumor was controlled. + The number of T cells and NK cells did not increase ( Figure 7 C). These results indicate that the IL-10–Fc–IL-2 fusion protein did not cause significant systemic toxicity in mice.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A bispecific cytokine fusion protein, characterized in that: The bispecific cytokine fusion protein comprises the amino acid sequence of type I cytokine and the amino acid sequence of type II cytokine.

2. The bispecific cytokine fusion protein according to claim 1, characterized in that: The amino acid sequence of the type I cytokine and the amino acid sequence of the type II cytokine are directly connected via a connecting peptide or connected via a fusion partner and a connecting peptide; the fusion partner is the CH2 region, CH3 region of the Fc fragment of human IgG, or any combination thereof, or human serum albumin, or an engineered mutant of one of them; the amino acid sequence of the connecting peptide is shown in SEQ ID NO.1-3.

3. The bispecific cytokine fusion protein according to claim 1, characterized in that: The type I cytokine is one of IL-2, IL-7, IL-12, IL-15, IL-17, IL-21, H9T, IL-15m3, or an engineered mutant thereof; the type II cytokine is one of IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-33, or an engineered mutant thereof.

4. The bispecific cytokine fusion protein according to claim 1, characterized in that: The IgG is at least one of human IgG1, IgG2, IgG3, and IgG4, or an engineered mutant of one of them.

5. The bispecific cytokine fusion protein according to claim 1, characterized in that: The amino acid sequence of the bispecific cytokine fusion protein is shown in any one of SEQ ID NOs. 9 to 28.

6. A nucleic acid, characterized in that The nucleic acid encodes the bispecific cytokine fusion protein according to any one of claims 1 to 5.

7. A carrier, characterized in that The vector carries the nucleic acid according to claim 6.

8. A cell comprising the nucleic acid of claim 6 or the vector of claim 7. 9 . A medicament or a pharmaceutical composition, comprising the bispecific cytokine fusion protein according to any one of claims 1 to 5.

10. Use of the bispecific cytokine fusion protein according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7, the cell according to claim 8, or the drug or pharmaceutical composition according to claim 9 in the preparation of a drug for treating cancer.

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