Bispecific cytokine fusion proteins and uses thereof

Abstract

This invention discloses a bispecific cytokine fusion protein, comprising amino acid sequences of type I and type II cytokines, which are directly linked by a linker peptide or linked in tandem or parallel via a fusion chaperone and a linker peptide. By linking the amino acid sequences of type I and type II cytokines in tandem or parallel, a bispecific cytokine fusion protein is formed, which can synergistically activate the precursor depleted CD8+. + T cells and terminally exhausted CD8 + T cells improve the immunosuppression of the complex tumor microenvironment, unlocking the potential of tumor immunotherapy. This invention also discloses nucleic acids, vectors, cells, drugs, or pharmaceutical compositions related to the above-mentioned bispecific cytokine fusion protein. Furthermore, this invention discloses the application of the above-mentioned bispecific cytokine fusion protein in the preparation of drugs for treating cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to bispecific cytokine fusion proteins and their applications. Background Technology

[0002] Tumor immunotherapy has made significant progress over the past few decades, encompassing cytokines, immune checkpoint inhibitors, oncolytic viruses, tumor vaccines, and adoptive cell therapy. For example, PD-1 / PD-L1 and other immune checkpoint inhibitor (ICB) therapies have shown some success in restoring the immunosuppressive tumor microenvironment. However, the average response rate of ICBs in various tumors is only 20%–30%, with most patients struggling to maintain a sustained response and experiencing adverse reactions such as tissue-specific toxicity. Chimeric antigen receptor T-cell (CAR-T) therapy also shows limited efficacy in solid tumors. Furthermore, more than a dozen cytokines have been used in clinical trials for tumor immunotherapy, yet their efficacy remains unclear. One major reason for this is the depletion of T cells in the tumor microenvironment, manifested by alterations in transcriptional profiles and metabolic pathways, as well as persistently elevated expression of inhibitory receptors and loss of effector function.

[0003] Research aimed at revitalizing exhausted T 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 the transcription factor TCF-1, exhausted CD8+ cells can be revitalized. + TILs are mainly divided into precursor depletion CD8 + T cells (TCF-1) + PD-1 inter TIM-3 - ) and Final Exhaustion CD8 + T cells (TCF-1) - PD-1 high TIM-3 + ).

[0004] In recent years, various drugs have been shown to effectively expand T cells and enhance cytotoxic function and anti-tumor cytokine secretion by targeting certain specific exhausted T cell subsets. For example, PD-1 antibodies have been shown to act on precursor-depleted CD8+ cells in viral infection models and some tumor models. + T cell subsets, however, for terminally exhausted CD8 + T cells have virtually no effect. IL-2, as a pleiotropic type I cytokine, has been shown to amplify activated CD8+ cells. +T cells are induced to differentiate into effector cells and inhibitory receptors such as PD-1 and TIM-3 are upregulated. Reference 1 (Rafi Ahmed et al., PD-1 combination therapy with IL-2 modifies CD8) + The study, *T cell exhaustion program* (Nature, 610, 173-181, 2022, doi:10.1038 / s41586-022-05257-0), disclosed that PD-1 combined with IL-2 altered CD8 expression in chronic lymphocytic choroid plexus meningitis virus (LCMV)-infected mice. + T cell exhaustion program demonstrated in an LCMV mouse model that IL-2 amplification precursors deplete CD8 + T cells, targeting terminally exhausted CD8 cells + T cells are ineffective.

[0005] Final Exhaustion CD8 + T cell impairment hinders the entire field of tumor immunotherapy, and various studies have focused on activating terminally exhausted T cells. (Reference 2: Yugang Guo et al., Metabolic reprogramming of terminally exhausted CD8) + The study "T Cells by IL-10 Enhances Anti-tumor Immunity" (Nat Immunol. 22, 746-756, 2021, doi:10.1038 / s41590-021-00940-2) discloses a typical type II cytokine IL-10 and constructs a recombinant IL-10 and IgG Fc fusion protein with an extended half-life (IL-10–Fc). The IL-10–Fc fusion protein effectively amplifies terminally depleted CD8+ by enhancing mitochondrial pyruvate transporter-dependent oxidative phosphorylation. + T cells are revitalized and their cytotoxic function is restored, enhancing the anti-tumor immune response.

[0006] Reference 3 (Bing Feng et al., The type 2 cytokine Fc-IL-4 revitalizes exhausted CD8) + The study "T cells against cancer" (Nature. 634, 712-720, 2024, doi:10.1038 / s41586-024-07962-4) discloses another typical type II cytokine, interleukin-4, fusion protein (Fc–IL-4), which can directly act on CD8. +T cells, and functionally depleted CD8+ cells are enriched in tumors. + T cells. Fc–IL-4 regulates terminally depleted CD8 cells. + T cell metabolic function improves depleted CD8 cells in a lactate dehydrogenase A-dependent manner. + The killing ability of T cells.

[0007] Research on the expansion of exhausted T cell numbers and functional revitalization is ongoing, but it is limited to exploring single subpopulations and is difficult to effectively eliminate tumors. In current preclinical or clinical studies, there are few strategies that utilize different types of cytokines to synergistically activate different exhausted cell subpopulations. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a bispecific cytokine fusion protein that fuses type I and type II cytokines, thereby achieving the depletion of precursor CD8. + T cells and terminally exhausted CD8 + The synergistic activation of T cells significantly inhibits tumor growth and unlocks the potential of T cell immunotherapy.

[0009] A fusion protein of bispecific cytokines, the fusion protein comprising the amino acid sequences of type I cytokines and type II cytokines.

[0010] Previous studies have suggested that type I and type II cytokines can act on depleted CD8 precursors, respectively. + T cells and terminally exhausted CD8 + T cells. In this invention, a bispecific or multispecific cytokine fusion protein is formed by tandem or parallel connection of the amino acid sequences of type I and type II cytokines. This fusion protein can synergistically activate precursor-depleted CD8+. + T cells and terminally exhausted CD8 + T cells improve the immunosuppression of the complex tumor microenvironment and significantly unleash the potential of tumor immunotherapy dependent on cytotoxic immune cells.

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

[0012] In this invention, the structure of the bispecific cytokine fusion protein can be... Figure 1Any one of the formulas (1) to (12) can also be a parallel connection between the amino acid sequences of type I cytokines and type II cytokines through the carboxyl terminus of the Fc fragment CH2 or CH3 region 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 both 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 combination of formula (12) with the amino or carboxyl terminus of the fusion partner via a linker peptide.

[0013] In this invention, the engineered mutant can be a protein mutant achieved by mutating amino acid sites.

[0014] Type I and type II cytokines are linked by fusion chaperones and linker peptides to form bispecific cytokine fusion proteins. The Fc fragment of human IgG and human serum albumin can increase the molecular volume, making them less susceptible to glomerular filtration, thereby effectively prolonging the half-life of cytokines and overcoming the short half-life of cytokines.

[0015] In this invention, the linker peptide can be a short peptide (amino acid sequence as shown in SEQ ID NO. 1) of the IgG hinge region or a GS linker peptide (amino acid sequence as shown in SEQ ID NO. 2 or SEQ ID NO. 3) composed of extended glycine and serine residues. When a cytokine is linked to the CH2 or CH3 region of the Fc fragment of immunoglobulin IgG, the two are linked through the hinge region of IgG. When a cytokine is linked to the C-terminus of the Fc fragment of immunoglobulin IgG, the two are linked through the GS linker peptide. When a cytokine is linked to human serum albumin (HSA), the two are linked through the GS linker peptide. When cytokines are directly linked, they are linked through the hinge region or the GS linker peptide. When cytokines are linked in parallel and the fusion partner includes the CH3 region, a knock-in-hole (KIH) linker structure is formed between the CH3 regions, as shown in the schematic diagram below. Figure 1 As shown in equation (5) or equation (7) in the text.

[0016] Preferably, the structure of the bispecific cytokine fusion protein is as shown in formula (1), where a and b are type I or type II cytokines, and a and b cannot be both type I and type II cytokines simultaneously; c and d are type I or type II cytokines, and the amino acid sequences of the type I and type II cytokines are linked by a fusion chaperone and a linker peptide, wherein the fusion chaperone 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 linker peptide is shown in SEQ ID NO. 1 to 3.

[0017] In preferred embodiments, the bispecific cytokine fusion protein can achieve complete cure in various solid tumor models (such as colon cancer, melanoma, and head and neck squamous cell carcinoma) and can enhance CD8. + T cell infiltration within the tumor enhances the proliferation and effector capacity of effector T cells, overcoming the incompetence 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, and 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, and IL-33, or an engineered mutant of one of them.

[0019] In this invention, the amino acid sequences of type I 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, and IgG4, or an engineered mutant of one of them.

[0024] In this invention, the amino acid sequence of the human IgG1 Fc fragment is shown in 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 as shown in any one of SEQ ID NO. 9 to 28.

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

[0027] The present invention also provides a carrier carrying the above-mentioned nucleic acid.

[0028] The present invention also provides a cell containing the above-mentioned nucleic acid or vector.

[0029] The present invention also provides a drug or drug composition comprising the above-described bispecific cytokine fusion protein.

[0030] The present invention also provides the use of the above-mentioned bispecific cytokine fusion protein, nucleic acid, carrier, cell, drug or pharmaceutical composition in the preparation of a cancer treatment drug.

[0031] In this invention, a bispecific cytokine fusion protein is constructed by tandem or parallel linking type I and type II cytokines. This fusion protein can synergistically activate the precursor depleted CD8. + T cells and terminally exhausted CD8 + T cells improve the immunosuppression of the complex tumor microenvironment and significantly unleash the potential of tumor immunotherapy dependent on cytotoxic immune cells.

[0032] In this invention, the term "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, stomach 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 this 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 the precursor depleted CD8 + T cells and terminally exhausted CD8 + T cells improve the immunosuppression of the complex tumor microenvironment.

[0036] (2) The bispecific cytokine fusion protein in this 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 tumor-specific CD8 in the tumor microenvironment. + T cell depletion significantly enhances the anti-tumor efficacy of cytokine drugs. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the structure of a bispecific cytokine fusion protein.

[0038] Figure 2 The images show structural diagrams of the IL-10–Fc–IL-2 and IL-10–Fc–H9T fusion proteins, as well as images identifying the expression and biological activity of the IL-10–Fc–IL-2 fusion protein. Specifically, A is a structural diagram of the IL-10–Fc–IL-2 fusion protein; B is a structural diagram of the IL-10–Fc–H9T fusion protein; C is an SDS-PAGE molecular band identification image of the IL-10–Fc–IL-2 fusion protein under reducing and non-reducing conditions; and D is the CD8+ image after treatment with different concentration gradients of the IL-10–Fc–IL-2 fusion protein. + Fold change in T cell count; E represents the STAT5 phosphorylation level of YT-1 cells after 30 minutes of stimulation with different concentration gradients of IL-10–Fc–IL-2 fusion protein; F represents the T cell proliferation test after 3 days of culture with 150 ng / mL IL-10–Fc–IL-2 fusion protein.

[0039] Figure 3 The images show the expression and bioactivity identification of the IL-10–Fc–IL-15SA fusion protein. A is a schematic diagram of the structure of the IL-10–Fc–IL-15SA fusion protein; B is an SDS-PAGE molecular band identification image of the IL-10–Fc–IL-15SA fusion protein and a Western blotting image after incubation with anti-Fc antibody; C is the CD8+ image after treatment with the IL-10–Fc–IL-15SA fusion protein. + A statistical chart of the number of T cells.

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

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

[0042] Figure 6 The graph shows the anti-tumor immunogenicity results of the IL-10–Fc–IL-2 fusion protein in the B16F10 melanoma model. A is a schematic diagram of the tumor invasion assay protocol in C57BL / 6 mice subcutaneously inoculated with B16F10 cells; B is a statistical graph of the number of immune cells after treatment with the IL-10–Fc–IL-2 fusion protein; and C is a graph showing the number of different CD8+ cells after treatment with the IL-10–Fc–IL-2 fusion protein. + Statistical chart of the number of exhausted T cell subsets.

[0043] Figure 7 The figure shows the safety assessment results of the IL-10–Fc–IL-2 fusion protein in C57BL / 6 mice. A is a schematic diagram of the safety assessment experimental protocol; B is the body weight change curve of mice treated with the IL-10–Fc–IL-2 fusion protein; C is the CD8+ concentration in the spleen of mice treated with the IL-10–Fc–IL-2 fusion protein. + Statistical chart of T cell and NK cell counts. Detailed Implementation

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

[0045] All raw materials used in this invention are commercially available.

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

[0047] I. Construction of a Bispecific Cytokine Fusion Protein Expression Vector

[0048] Referring to the schematic diagram of the bispecific cytokine fusion protein molecular structure of formula (1), pcDNA3.1+ was selected as the expression vector, and the gene was synthesized by Nanjing Genscript Biotech Co., Ltd. The 5' end of the synthesized gene was designed with a Hind III restriction site ([AAGCTT]), a Kozak sequence ([ACCACC]), and an H293 signal peptide, and the 3' end of the synthesized gene was designed with a 3'UTR stop codon ([TGA]) and a Not I restriction site ([GCGGCCGC]). Both the pcDNA3.1+ expression vector and the synthesized antibody gene were double-digested with Hind III and Not I. The digestion products of the pcDNA3.1+ expression vector and the antibody gene were subjected to agarose gel electrophoresis and the target fragment was recovered. The recovered fragment was ligated with T4 ligase and transduced into TOP10 competent cells. Cells with correct sequencing were selected for preservation and expansion culture. The expanded cells were used for plasmid extraction.

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

[0050] Using the extracted plasmids, and taking HEK293F human embryonic kidney cells as the expression system, the cell transfection and transient expression techniques are described. Other conventional techniques should also be considered within the scope of this invention. HEK293F cells (Thermo) were thawed in a 37°C water bath, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 1 mL of SMM 293TI (Gibco) medium and added to a 250 mL cell shake flask. 50 mL of SMM 293TI medium was added, along with a final concentration of 1% penicillin-streptomycin (Gibco) and 0.5% serum (Gibco). The 250 mL cell shake flask was then transferred to a 37°C, 5% CO2 cell shaker for culture. After 2-3 days of culture, the cell status was observed. When the cell density reached 1.5 × 10⁻⁶ cells / mL... 6 When the cell density reaches 1.5 × 10⁶ cells / mL or higher, passage 50 mL of the cell suspension from a 250 mL cell shake flask into a 500 mL cell shake flask containing 110 mL of SMM 293TI medium. Then, supplement with 1% penicillin-dip antibiotics and 0.5% serum. Culture for another 2-3 days and observe cell status. When the cell density reaches 1.5 × 10⁶ cells / mL... 6 When the cell density reaches 2 × 10⁶ cells / mL, 160 mL of cell suspension from a 500 mL cell shake flask is evenly passaged into two 1 L cell shake flasks containing 160 mL of SMM 293TI medium. Then, 1% antibiotics and 0.5% serum are added, and the cells are cultured for another 2-3 days. Cell status is observed after 2-3 days, and the cells are cultured until the density reaches 2 × 10⁶ cells / mL. 6 Plasmid transfection can be performed when the number of plasmids per mL is higher than 1. The specific transfection procedure is as follows:

[0051] Sterilize the plasmid by passing it through a 0.22 μm microporous membrane in a biosafety cabinet. 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. Mix thoroughly, then add the liquid from the plasmid tube to the PEI tube, mix thoroughly, and incubate at room temperature for 15 min. Take out a 1 L cell shake flask (containing 240 mL of suspended cells) and add 50 mL of SMM 293TI medium. Add 20 mL of the above transfection solution to the 1 L cell shake flask, and supplement with 1% penicillin antibiotics and 0.5% serum. Return the 1 L cell shake flask to a cell shaker at 37°C and 5% CO2 for 4-5 days, then collect the supernatant.

[0052] The culture supernatant was purified by affinity chromatography. The specific procedure for affinity chromatography is as follows:

[0053] Remove HEK293F cells transfected with plasmids 3-4 days after cell shaking, centrifuge at 4500g for 15 min, collect the supernatant, and filter it sequentially through 0.45μm and 0.22μm filters to remove impurities and prevent column clogging. Prepare Protein A loading buffer (50mM Tris-HCl, 0.15M NaCl, pH 7.4), Protein A elution buffer (50mM sodium citrate dihydrate, pH 3.2), ultrapure water, and 20% ethanol, and filter through a 0.22μm filter to remove impurities.

[0054] The protein was purified using the AKTA protein purification system (GE Healthcare, AKTA purifier). The specific procedure is as follows:

[0055] Start the AKTA protein purification software and set the parameters (column inlet pressure upper limit 0.2 MPa, flow rate 3 mL / min). Rinse pumps A and B with wash bottles containing ultrapure water, then add ultrapure water that has already passed through a membrane to clean the tubing. After cleaning, pack the column (HiTrapr Protein A affinity chromatography column) and clean the column. Rinse pump A with Protein A elution buffer until conductivity equilibrium is reached, and rinse pump B with Protein A loading buffer until conductivity equilibrium is reached. Then rinse pump B with the cell culture supernatant that has already passed through a membrane to begin loading. After loading, rinse pump B with Protein A loading buffer until conductivity equilibrium is reached, then rinse with 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 tubing and column sequentially with purified water and 20% ethanol, collect the column, and turn off the AKTA protein purification instrument. The collected target proteins were concentrated by ultrafiltration through a 50kDa ultrafiltration tube, eluted with PBS, and the protein concentration was determined on a 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 at 4°C.

[0056] Example 1

[0057] The IL-10–Fc–IL-2 fusion protein was obtained according to the above-described method for preparing bispecific cytokine fusion proteins. Its amino acid sequence is SEQ ID NO.9. The expression and bioactivity of the IL-10–Fc–IL-2 fusion protein are shown in the figure below. Figure 2 As shown.

[0058] Figure 2 This image shows structural diagrams of the IL-10–Fc–IL-2 fusion protein and the IL-10–Fc–H9T fusion protein, as well as diagrams illustrating the expression and bioactivity of the IL-10–Fc–IL-2 fusion protein. Figure 2 A in the diagram is a schematic diagram of the structure of the IL-10–Fc–IL-2 fusion protein. The IL-10–Fc–IL-2 fusion protein is obtained by linking 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 linking the amino terminus of human IL-2 to the carboxyl terminus of human IgG1 Fc through the GS linker peptide. Figure 2 C in the image represents the SDS-PAGE molecular band identification of the IL-10–Fc–IL-2 fusion protein under reducing and non-reducing conditions. The protein size is approximately 132 kDa under non-reducing conditions and approximately 66 kDa under reducing conditions. Figure 2 In this context, D represents CD8 after treatment with different concentration gradients of the IL-10–Fc–IL-2 fusion protein.+ Fold change in T cell count. Spleen cells from C57BL / 6 mice were pre-activated with 1 μg / ml anti-CD3 antibody and cultured for 3 days at different IL-10–Fc–IL-2 concentrations. CD8 count was detected by flow cytometry. + The proliferative capacity of T cells was found to be low, with treatment of CD8 cells at relatively low concentrations of IL-10–Fc–IL-2 inducing CD8 cell proliferation. + T cell proliferation. Figure 2 In the figure, E represents the STAT5 phosphorylation level of YT-1 cells 30 minutes after stimulation with different concentration gradients of IL-10–Fc–IL-2 fusion protein. After treating YT-1 cells with different concentration gradients of IL-10–Fc–IL-2 fusion protein, its induction of the downstream signaling pathway STAT5 phosphorylation was examined, where EC... 50 It is 279.5pM. Figure 2 F in the figure represents the T cell proliferation test after culturing with 150 ng / mL IL-10–Fc–IL-2 fusion protein for 3 days. The fusion protein was compared with PBS, and it showed that the fusion protein had the ability to promote proliferation.

[0059] Example 2

[0060] The IL-10–Fc–H9T fusion protein was obtained according to the above-described method for preparing bispecific cytokine fusion proteins. Its amino acid sequence is SEQ ID NO.10. A schematic diagram of the structure of the IL-10–Fc–H9T fusion protein is shown below. 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, in which the carboxyl terminus of human IL-10 is linked to the amino terminus of human IgG1 Fc through the hinge region of the Fc fragment of human IgG, and the amino terminus of human H9T is linked to the carboxyl terminus of human IgG1 Fc through the GS linker peptide to obtain the IL-10–Fc–H9T fusion protein.

[0062] Example 3

[0063] Following the above-described method for preparing bispecific cytokine fusion proteins, the IL-10–Fc–IL-15SA fusion protein was obtained. Its amino acid sequence is SEQ ID NO.12. The expression and bioactivity identification of the IL-10–Fc–IL-15SA fusion protein are shown in the figure below. Figure 3 As shown.

[0064] Figure 3 The diagram shows the expression and bioactivity identification of the IL-10–Fc–IL-15SA fusion protein. Figure 3A is a schematic diagram of the structure of the IL-10–Fc–IL-15SA fusion protein. The IL-15SA fusion protein is constructed by linking human IL-15, human IL-15Rsu (containing the sushi domain), and IgG1Fc, as shown in SEQ ID NO.8. Similarly, another IL-15SA fusion protein can be obtained by combining a mutant human IL-15m3 (sequence shown in SEQ ID NO.5) with human IL-15Rsu. Based on this, we designed the IL-10–Fc–IL-15SA fusion protein. The C-terminus of human IL-10 is linked to the N-terminus of human IgG1 Fc via the hinge region of the Fc fragment of human IgG. The N-terminus of human IL-15SA is linked to the C-terminus of human IgG1 Fc via a GS linker peptide to obtain the fusion protein, named IL-10–Fc–IL-15SA fusion protein. The molecular weight of the IL-10–Fc–IL-15SA fusion protein was verified using SDS-PAGE, and its molecular weight was 170 kDa. The IL-10–Fc–IL-15SA fusion protein was co-incubated with an anti-Fc antibody, and the presence of the fusion protein was confirmed by Western blotting. Figure 3 (B) Further, CD8 was isolated and extracted from the spleen of PMEL mice. + T cells were cultured in vitro for 6 days and then treated with different drugs (PBS, IL-10–Fc 100 ng / ml, IL-15SA 100 ng / ml, IL-10–Fc–IL-15SA 200 ng / ml). After 2 days of culture, flow cytometry analysis was used to verify that the IL-10–Fc–IL-15SA fusion protein significantly amplified CD8+. + T cells, especially PD-1 cells + TIM3 + T cell subsets with an exhausted phenotype exhibit anti-exhaustion potential. Figure 3 (C in the middle).

[0065] Activity test

[0066] I. In vitro activity assessment of bispecific cytokine fusion proteins

[0067] The IL-10–Fc–IL-2 fusion protein obtained in Example 1 was used in CD8+ of OT-1 mice. + In vitro efficacy evaluation of T cell co-incubation with B16F10-OVA tumor cells.

[0068] CD8 in OT-1 mice +T cells were co-incubated with B16F10-OVA tumor cells for 2 days, and different groups of drugs were applied (PBS, IL-2–Fc 10ng / ml, IL-10–Fc 200ng / ml or IL-10–Fc–IL-2 150ng / ml). The number of tumor cells and lymphocytes and the function of cytokine secretion were analyzed.

[0069] CD8 of OT-1 transgenic mice + T cells were co-incubated with B16F10-OVA tumor cells, and CD8 cells from OT-1 transgenic mice were observed. + T cells specifically recognize the OVA antigen and, upon activation in different drug administration groups, kill B16F10 melanoma cells expressing the OVA antigen. Figure 4 (A) Compared with the IL-2–Fc and IL-10–Fc groups, the IL-10–Fc–IL-2 fusion protein can significantly enhance CD8. + T proliferation and tumor-killing ability ( Figure 4 (B) Analysis of cytokines that play an important role in tumor immunity, including CD8 in the IL-10–Fc–IL-2 fusion protein. + T cells secrete more GzmB. + IFN-γ + TNF-α + Cytokines ( Figure 4 In C), the IL-10–Fc–IL-2 fusion protein can enhance CD8. + T cell infiltration within the tumor enhances the proliferation and effector capacity of effector T cells, overcoming the incompetence of exhausted T cells.

[0070] II. In vivo efficacy assessment of bispecific cytokine fusion protein

[0071] Evaluation of the efficacy of the IL-10–Fc–IL-2 fusion protein obtained in Example 1 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.

[0072] C57BL / 6 mice were subcutaneously inoculated with colon cancer cells from MC38 mice (1×10⁻⁶). 6 ), B16F10-OVA melanoma cells (5×10) 5 ) or BALB / c mice subcutaneously inoculated with CT26 colon cancer cells (8×10) 5 Or, 4 MOSC-1 head and neck squamous cell carcinoma cells (4 × 10⁻⁶) were injected into the tip of the tongue of C57BL / 6 mice. 5 Dosing was initiated in different tumor size ranges (MC38 50–60 mm). 2B16F10-OVA 20~30mm 2 CT26 30~40mm 2 ; 4MOSC-1 15~40mm 3 (n=5), and then different drugs (PBS, IL-2–Fc 2μg, IL-10–Fc 20μg, IL-10–Fc–IL-2 15μg (i.e. Example 1)) were injected into the tumor every three days for a total of six times. The growth of the mouse tumor was monitored and the survival time was recorded.

[0073] Figure 5 This image shows the antitumor activity of the IL-10–Fc–IL-2 fusion protein obtained in Example 1 in MC38 mouse colon cancer cell models, B16F10-OVA mouse melanoma models, CT26 mouse colon cancer models, and 4MOSC-1 mouse head and neck squamous cell carcinoma models. In the MC38 mouse colon cancer cell model, treatment was administered every three days within a reasonable dosing window. Figure 5 (A in the text). Experimental results showed that IL-2–Fc or IL-10–Fc monotherapy 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 (B in the original text). We subsequently validated 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. Notably, IL-10–Fc–IL-2 significantly inhibited tumor growth and prolonged the survival of mice in all three models. Figure 5 The D-E, G-H, and J-K groups are used in this study. Three months after tumor recurrence, we conducted a tumor recurrence experiment, inoculating mice in each experimental group with tumor cells but discontinuing other treatments. The results showed that the IL-10–Fc–IL-2 group could resist secondary tumor cell attack (…). Figure 5 The presence of C, F, I, and L in the data demonstrates that it achieves long-term immune memory.

[0074] III. Assessment of the antitumor immunogenicity of bispecific cytokine fusion proteins

[0075] The IL-10–Fc–IL-2 fusion protein obtained in Example 1 was subcutaneously inoculated into C57BL / 6 mice at a rate of 0.5 × 10⁻⁶. 6 Evaluation of the anti-tumor immune capacity of the B16F10 melanoma cell model.

[0076] C57BL / 6 mice were subcutaneously injected with 0.5 × 10⁻⁶ g of water. 6B16F10 melanoma cells, when the tumor grows to an area of ​​25–45 mm 2 The mice were given the first dose within the specified range, and then injected with different drugs (PBS, IL-2–Fc 2μg, IL-10–Fc 20μg, IL-10–Fc–IL-2 15μg (i.e. Example 1)) every 3 days for a total of four injections. On day 21, the spleen and tumor tissue of the mice were removed for immune cell flow cytometry analysis.

[0077] Figure 6 Figure A shows the anti-tumor immunogenicity results of the IL-10–Fc–IL-2 fusion protein in a B16F10 melanoma model, with Figure A illustrating the tumor invasion assay protocol. The results showed that, compared to the IL-2–Fc or IL-10–Fc treatment groups, the IL-10–Fc–IL-2 fusion protein group had significantly higher CD8 counts. + T cells, CD4 + The infiltration of immune cells such as T cells and NK cells increased significantly. Figure 6 B in the middle, which is thought to be CD8 + T cells, as the main subset responding to IL-2 and IL-10, play a major role in killing tumor cells and effectively improve the tumor microenvironment, thereby increasing the number of other immune cells infiltrating the tumor and releasing therapeutic potential. Next, we will further analyze CD8... + The expansion of exhausted T cell subsets in different drug administration groups was consistent with predictions; compared with the single-drug group, the prodrug-depleted CD8 subsets showed increased expansion. + T cells and terminally exhausted CD8 + T cells were significantly expanded, and showed an increasing trend in the proportion of different subsets. Figure 6 (C in the middle).

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

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

[0080] Figure 7 Figure A shows the safety assessment results of the IL-10–Fc–IL-2 fusion protein obtained in Example 1 in C57BL / 6 mice. Figure A is a schematic diagram of the safety assessment experimental protocol. Mouse body weight was monitored during treatment. The IL-10–Fc–IL-2 fusion protein group did not reduce mouse body weight; in fact, it slightly increased it. Figure 7(B) suggests that the mice's health may have improved after tumor control. Treatment with the IL-10–Fc–IL-2 fusion protein reduced CD8 levels in the mouse spleen. + The number of T cells and NK cells did not increase. Figure 7 (C in the text). 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 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 foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a bispecific cytokine fusion protein in the preparation of cancer therapeutic drugs, characterized in that, The bispecific cytokine fusion protein includes the amino acid sequence of a type I cytokine and the amino acid sequence of a type II cytokine. The type I cytokine is IL-2 or IL-15, or an engineered mutant of one of them; the type II cytokine is IL-10, or an engineered mutant of one of them. The amino acid sequence of the bispecific cytokine fusion protein is shown in SEQ ID NO. 9 or SEQ ID NO. 12; The cancers mentioned are colon cancer, melanoma, or squamous cell carcinoma of the head and neck.

2. A nucleic acid, characterized in that, The nucleic acid encodes the bispecific cytokine fusion protein of claim 1.

3. A vector, characterized in that, The vector carries the nucleic acid as described in claim 2.

4. A cell containing the nucleic acid of claim 2 or the vector of claim 3.

5. A drug or pharmaceutical composition comprising the bispecific cytokine fusion protein of claim 1.

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