Bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 as well as construction method and application of bifunctional fusion protein

Abstract

The invention belongs to the technical field of biological pharmacy, and relates to a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 as well as a construction method and application of the bifunctional fusion protein. The bifunctional fusion protein comprises a PD-1 / PD-L1 antibody and a targeted IL-33 functional fragment, T cells are activated by blocking a PD-1 / PD-L1 signal to kill tumor cells, meanwhile, the level of IL-33 in tumor tissue is reduced, and the activity of IL-33 is inhibited. Researches show that the bifunctional fusion protein can increase infiltration of functional T cells, enhance T cell response and remodel a tumor microenvironment so as to generate an anti-tumor effect superior to that of combined treatment, and has a very good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology and relates to a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33, and particularly to a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33, its construction method and application. Background Technology

[0002] Data shows that malignant tumors are one of the important factors threatening human health. Tumor immunotherapy has become a hot field in tumor treatment in recent years. Among them, immune checkpoint inhibitors (ICIs), especially ICIs targeting programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1), have been approved for the treatment of various cancers (Science, 2013, 342(6165):1432-1433; Immunity, 2013, 39(1):1-10). However, the response rate of PD-1 / PD-L1 monoclonal antibody drugs alone remains low, with an efficacy rate of only 10%-25% in most tumors, especially malignant tumors such as non-small cell lung cancer (Cell, 2017, 168(4):707-723; Transl Oncol, 2020, 13(3):100738; Immunity, 2016, 44(6):1255-1269). Moreover, even tumor patients who initially respond to ICIs may progress in the later stages of the disease. The tumor microenvironment (TME) includes a variety of cellular and non-cellular components. Immunosuppressive TME is one of the important causes of PD-1 / PD-L1 monoclonal antibody resistance (Cancer Cell, 2020, 37(4):443-455; Trends Mol Med, 2016, 22(6):448-451). Among them, immunosuppressive immune cells (such as regulatory T cells (Tregs) and tumor-associated macrophages (TAMs)), cancer-associated fibroblasts (CAFs), and non-cellular components such as TGF-β, various cytokines, and chemokines are all involved in the formation of the immunosuppressive tumor microenvironment (TME) (Cancer Cell, 2023, 41(3):374-403), which is an important factor causing resistance to PD-1 / PD-L1 monoclonal antibodies. Therefore, further "exploring" the "key molecules" that regulate immunosuppressive signals in the tumor microenvironment and breaking the immunosuppressive microenvironment is crucial for enhancing the therapeutic effect of PD-1 / PD-L1 monoclonal antibodies.

[0003] Interleukin-33 (IL-33), a member of the interleukin-1 superfamily, is a multifunctional cytokine released after cell damage, stress, or necrosis (J Allergy Clin Immunol, 2019, 144(1):204-215). IL-33 is constitutively expressed in various cell types, such as epithelial cells, endothelial cells, and matrix fibroblasts. Simultaneously, IL-33 is also expressed in various tumor tissues, participating in the occurrence and development of various tumors (Eur J Immunol, 2021, 51(8):1943-1955). Growth stimulation expressed gene 2 (ST2) proteins are mainly divided into two types: trans-membrane ST2 (ST2L) and soluble ST2 (sST2). The interaction between IL-33 and the ST2 pathway can trigger the activation of NF-κB and MAPK signaling pathways (p38, JNK and ERK1 / 2), mediating a series of subsequent responses in target cells (Cytokine Growth Factor Rev, 2023, 14: S1359-6101).

[0004] IL-33 / ST2 signaling plays a complex role in tumor progression. The specific role of IL-33 in anti-tumor immunotherapy largely depends on factors such as the cellular origin of cytokines, target cell type, expression of active IL-33, and immunogenicity of the tumor microenvironment (TME) (Semin Cancer Biol, 2022, 86(Pt 2):280-295). Within the TME, suppressive immune cells such as Tregs and suppressive molecules such as TGF-β can promote tumor development and play a central role in PD-1 / PD-L1 blockade resistance (Cancers (Basel), 2021, 13(4):663). It has been reported that the IL-33 / ST2 axis can promote the function and proliferation of immunosuppressive cells (such as Tregs or TAMs), remodeling the immunosuppressive TME and promoting tumor progression (Cell Rep, 2019, 29(10):2998-3008; JCI Insight, 2020, 5(9):e136073). Studies have shown that ST2 expression is upregulated in tumor-infiltrating Tregs compared to conventional T cells in tumor tissue or NSCLC patients, as well as in the blood of TC-1 tumor-bearing mice. Furthermore, ST2 plays a crucial role in the proliferation and aggregation of Tregs in tumor tissue mesenchymal stem cells (TME) and can be reduced by ST2-neutralizing antibodies (Cancer Immunol Res, 2020, 8(11):1393-1406). High levels of ST2 expression are also found in Tregs from lung tumors in KrasG12D mice, and treatment with anti-ST2 antibodies can effectively deplete ST2 in lung tumors. + Treg cells enhance anti-tumor immune responses and inhibit lung tumor growth (Oncoimmunology, 2020, 9(1):1682380). Epidermal growth factor receptor (EGFR)-positive CAF cells and ST2 cells specifically expressing amphiregulin (Areg) + Interactions between Treg cells are also regulated by IL-33 signaling, enhancing the immunosuppression of TME and promoting tumor progression (Sci Adv, 2023, 9(34): eadd7399). Furthermore, IL-33 is involved in the regulation of the immunosuppressive molecule TGF-β. Upon release from tumor initiating cells (TICs), IL-33 induces FcεRIα-positive macrophages to differentiate and secrete TGF-β1. In turn, TGF-β1 signaling leads to further release of IL-33 from TICs, thereby promoting the invasiveness and drug resistance of a squamous cell carcinoma mouse subcutaneous tumor model (Science, 2020, 369(6501): eaay1813).

[0005] Based on the current state of the technology, the inventors of this application intend to provide a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33, particularly involving a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33, its construction method, and its applications. This application can downregulate IL-33 levels while blocking PD-L1 activation of T cells to kill tumor cells, which will not only inhibit the effects of Tregs or TAMs and further upregulate T cell killing of tumor cells; it can also inhibit the secretion of TGF-β in the tumor microenvironment, preventing tumor metastasis and drug resistance. Summary of the Invention

[0006] The purpose of this invention is to provide a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33, based on the current state of the technology, particularly relating to a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33, its construction method, and its applications. This invention designs a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 by regulating the tumor microenvironment to overcome the limitations of PD-L1 antibody or PD-1 antibody therapy. The bifunctional fusion protein of this application exhibits better anti-tumor activity compared to single-specific corresponding molecules (combination therapy).

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] One of the technical solutions of the present invention is to provide a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33, wherein the bifunctional fusion protein includes a bifunctional fusion protein targeting PD-1 and IL-33 and a bifunctional fusion protein targeting PD-L1 and IL-33.

[0009] Furthermore, the bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 is characterized in that: the expression form of the bifunctional fusion protein includes PD-L1 antibody-linker peptide-growth-stimulated expression gene 2 protein (ST2), PD-1 antibody-linker peptide-ST2, ST2-linker peptide-PD-L1 antibody, ST2-linker peptide-PD-1 antibody, PD-L1 antibody-linker peptide-IL-33 antibody, PD-1 antibody-linker peptide-IL-33 antibody, IL-33 antibody-linker peptide-PD-L1 antibody, or IL-33 antibody-linker peptide-PD-1 antibody.

[0010] Furthermore, the bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33, wherein the growth-stimulating gene 2 protein ST2 targeting IL-33 comprises the extracellular domains of human ST2 and mouse ST2 proteins, or the full-length soluble ST2 domain. In an embodiment of the present invention, the mouse ST2 is the extracellular domain of the mouse ST2 protein, with the amino acid sequence shown in SEQ ID No. 7. The human ST2 is the full-length soluble human ST2 domain, with the amino acid sequence shown in SEQ ID No. 8.

[0011] Further, the bifunctional fusion protein targeting PD-L1 and IL-33 is a fusion protein of a PD-L1 antibody and a functional molecule targeting IL-33. The PD-L1 antibody includes atezolizumab, avelumab, or durvalumab. In an embodiment of the present invention, the PD-L1 antibody is atezolizumab, and the amino acid sequence of the heavy chain of atezolizumab is shown in SEQ ID No. 1. The amino acid sequence of the light chain of atezolizumab is shown in SEQ ID No. 2.

[0012] Further, the bifunctional fusion protein targeting PD-1 and IL-33 is a fusion protein of a PD-1 antibody and a functional molecule targeting IL-33. The PD-1 antibody includes nivolumab, pembrolizumab, cimiprimab, toripalimab, sintilimab, and camrelizumab. In an embodiment of the present invention, the PD-1 antibody is nivolumab, and the amino acid sequence of the nivolumab heavy chain is shown in SEQ ID No. 3. The amino acid sequence of the atezolizumab light chain is shown in SEQ ID No. 4.

[0013] Furthermore, in the bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33, the amino acid sequence of the linker peptide includes [GlyGlyGlyGlySer]n, [GlySer]n, or Gly[GlySer]n, where n is any integer from 1 to 15. In an embodiment of the present invention, n is 3, and its amino acid sequence is shown in SEQ ID No. 5.

[0014] Furthermore, the bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 further includes a signal peptide linked to the PD-1 / PD-L1 antibody terminus or the ST2 terminus of the growth-stimulating gene 2 protein, the amino acid sequence of which is shown in SEQ ID No. 10.

[0015] Furthermore, the other end of the signal peptide is linked to a sequence, and the Kozak sequence is GCCGCCACC.

[0016] The second technical solution of the present invention is to provide a polynucleotide encoding a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 as described in one of the above technical solutions.

[0017] A third technical solution of the present invention provides an expression vector comprising a polynucleotide containing a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 as described in the second technical solution above, and a host cell containing the expression vector. In an embodiment of the present invention, the vector is pTT5.

[0018] The fourth technical solution of the present invention provides a method for preparing a bifunctional fusion protein containing PD-1 / PD-L1 and IL-33 as described in one of the above technical solutions. The method comprises: (1) expressing the bifunctional fusion protein in host cells and (2) isolating the bifunctional fusion protein from host cells.

[0019] The fifth technical solution of the present invention is to provide a pharmaceutical composition comprising a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 as described in one of the above technical solutions.

[0020] The sixth technical solution of the present invention is to provide a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 as described in one of the above technical solutions, or a pharmaceutical composition as described in the fifth technical solution, for use in the preparation of a kit for detecting or predicting tumors.

[0021] The seventh technical solution of the present invention is to provide a bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 as described in one of the above technical solutions, or the pharmaceutical composition as described in the fifth technical solution, in the preparation of a drug for treating tumors.

[0022] Furthermore, in the above-mentioned technical solutions six and seven, the tumor is a malignant tumor, including lung cancer, breast cancer, ovarian cancer, liver cancer, colorectal cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, prostate cancer, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma.

[0023] Experimental results show that the bifunctional fusion protein of this invention can first block PD-1 / PD-L1 signaling, enhancing the recognition and killing effect of T cells on tumor cells; simultaneously, it delivers the functional fragment targeting IL-33 to the tumor site through PD-1 / PD-L1 antibodies, specifically binding to IL-33 in the tumor site, downregulating the level of IL-33 in the tumor microenvironment, inhibiting the effects of Tregs or TAMs, and further upregulating the killing effect of T cells on tumor cells; it can also inhibit the secretion of TGF-β in the tumor microenvironment, remodel the tumor microenvironment, and prevent tumor metastasis and drug resistance.

[0024] The beneficial effects of this invention are:

[0025] The bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 described in this invention firstly enhances the recognition and killing of tumor cells by T cells through the specific binding of PD-1 or PD-L1 antibodies to PD-1 on the surface of T cells or PD-L1 on the surface of tumor cells via PD-1 or PD-L1 antibodies. Simultaneously, the PD-1 / PD-L1 antibody delivers a functional fragment targeting IL-33 to the tumor site, specifically binding to IL-33 at the tumor site, downregulating the level of IL-33 in the tumor microenvironment (TME), inhibiting the function of Tregs or TAMs, remodeling the tumor microenvironment, and thus inhibiting tumor growth. The bifunctional fusion protein of this invention produces anti-tumor effects superior to combination therapies, significantly prolongs the survival of tumor-bearing mice, and induces immune memory. Attached Figure Description

[0026] Figure 1 To detect the biological activity of the PD-L1 antibody terminus of Anti-PD-L1-sST2 and sST2-anti-PD-L1.

[0027] Figure 2 To detect the biological activity of the sST2 end of Anti-PD-1-sST2 and sST2-anti-PD-1.

[0028] Figure 3 The biological activity of the hsST2 terminus of the bifunctional fusion proteins Anti-PD-L1-hsST2, hsST2-anti-PD-L1, anti-PD-1-hsST2, and hsST2-anti-PD-1 was detected.

[0029] Figure 4 In vivo efficacy of Anti-PD-L1-sST2 and sST2-anti-PD-L1 in a mouse subcutaneous xenograft model of lung cancer. (A) Tumor growth volume and (B) Tumor weight.

[0030] Figure 5 To prolong the survival of LLC metastasis model mice and cultivate immune memory using Anti-PD-L1-sST2. (A) Survival curves of tumor-bearing mice in the LLC lung metastasis model, (B) H&E staining results of lung tissue 3 weeks after re-inoculation with LLC cells in the lung metastasis model.

[0031] Figure 6 To assess the in vivo efficacy of Anti-PD-1-sST2 in a mouse MC38-hPD-L1 subcutaneous xenograft model of colorectal cancer.

[0032] Figure 7UMAP diagrams showing the identification of CD8T cell subsets in tumor tissue and the proportion of CD8T cell subsets in each group.

[0033] Figure 8 UMAP diagrams for identifying CD4T cell subsets in tumor tissue and the proportion of CD4T cell subsets in each group.

[0034] Figure 9 The Anti-PD-L1-sST2 bifunctional fusion protein was used to identify pathways in T cells where genes (A) and (B) were upregulated and downregulated compared to combination therapy.

[0035] Figure 10 This is a schematic diagram illustrating the function of a bifunctional fusion protein that targets PD-L1 and IL-33. Detailed Implementation

[0036] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed in accordance with the techniques or conditions described in the literature in the field, or in accordance with the product manual.

[0037] Example 1: Construction of fusion protein expression vector

[0038] In this invention, the expression vector is pTT5. After all gene coding sequences are successfully constructed, they are digested by EcoRI and BamHI enzymes and then ligated into the pTT5 vector to form the corresponding plasmids.

[0039] Simultaneously, the light chain expression sequence of the bifunctional fusion protein targeting PD-L1 and IL-33 was constructed into the pTT5 vector using the light chain coding sequence of the anti-PD-L1 antibody (SEQ ID No. 2), which is pTT5-anti-PD-1-Light. The heavy chain expression plasmid is as follows: pTT5-anti-PD-L1-sST2-Heavy is constructed by linking the mouse ST2 extracellular domain coding sequence (SEQ ID No. 7) to the C-terminus of the heavy chain coding sequence (SEQ ID No. 1) of the anti-PD-L1 antibody via a linker peptide; pTT5-sST2-anti-PD-L1-Heavy is constructed by linking the mouse ST2 extracellular domain coding sequence (SEQ ID No. 7) to the N-terminus of the heavy chain coding sequence (SEQ ID No. 1) of the anti-PD-L1 antibody; pTT5-hsST2-Heavy is constructed by linking the full-length coding sequence of the human soluble ST2 domain (SEQ ID No. 8) to the C-terminus of the heavy chain coding sequence (SEQ ID No. 1) of the anti-PD-L1 antibody; and pTT5-sST2-heavy is constructed by linking the full-length coding sequence of the human soluble ST2 domain (SEQ ID No. 8) to the C-terminus of the heavy chain coding sequence (SEQ ID No. 1) of the anti-PD-L1 antibody. No.1) The N-terminus is composed of pTT5-hsST2-anti-PD-L1-Heavy.

[0040] Simultaneously, the light chain expression sequence of the bifunctional fusion protein targeting PD-1 and IL-33 was constructed into the pTT5 vector using the light chain coding sequence of the anti-PD-1 antibody (SEQ ID No. 4) as pTT5-anti-PD-1-Light. The heavy chain expression plasmid is constructed by linking the mouse ST2 extracellular domain coding sequence (SEQ ID No. 7) to the C-terminus of the heavy chain coding sequence (SEQ ID No. 3) of the anti-PD-1 antibody via a linker peptide to form pTT5-anti-PD-1-sST2-Heavy; linking the mouse ST2 extracellular domain coding sequence (SEQ ID No. 7) to the N-terminus of the heavy chain coding sequence (SEQ ID No. 3) of the anti-PD-1 antibody to form pTT5-sST2-anti-PD-1-Heavy; linking the full-length coding sequence of the human soluble ST2 domain (SEQ ID No. 8) to the C-terminus of the heavy chain coding sequence (SEQ ID No. 3) of the anti-PD-1 antibody to form pTT5-anti-PD-1-hsST2-Heavy; and linking the full-length coding sequence of the human soluble ST2 domain (SEQ ID No. 8) to the N-terminus of the heavy chain coding sequence (SEQ ID No. 3) of the anti-PD-1 antibody to form pTT5-hsST2-anti-PD-1-Heavy.

[0041] Example 2: Expression and purification of bifunctional fusion protein

[0042] The bifunctional fusion protein was expressed using an expression system transiently transfected into 293F cells. Plasmids were transfected at a concentration of 2 μg / mL. Co-transfection was performed with a heavy chain to light chain plasmid molar ratio of 1:2. The plasmid to PEI-MAX mass ratio was 1:3. The 293F cell density was adjusted to 1.5 × 10⁻⁶ cells one day prior to transfection. 6 The cell density was measured at 3 × 10⁶ cells / mL after overnight culture. 6 Transfection was performed when the cell viability exceeded 95% at a cell density of 1 / mL. Taking a 60mL system as an example, the transfection process is briefly described as follows: The transfection reagent PEI-MAX (1μg / μL) was brought to room temperature. 4mL of serum-free 293F culture medium was transferred to a sterile EP tube, and 120μg of the target plasmid was added. After thorough mixing by pipetting, 360μL of PEI-MAX was added, and immediately mixed by pipetting with a 1mL pipette. The mixture was allowed to stand at room temperature for 10 minutes, and then added to the 293F cells to be transfected. The flask was shaken while adding the plasmid (to avoid excessively high local concentrations). After thorough mixing, the flask was placed on a shaker at 37℃, 5% CO2, and 120rpm.

[0043] After culturing for 7 days, centrifuge at 2000 rpm for 5 min to collect the supernatant, then centrifuge at 8000 rpm for 15 min to collect the supernatant and filter it with a 0.45 μm filter membrane. Store it temporarily at 4℃ for subsequent protein purification: (1) Sample loading purification: Rinse all the tubing used in the protein purification instrument with water, connect the Protein A purification column, and use 1×TBS sample loading buffer to flow through the column to the baseline level. Then slowly load the prepared culture supernatant. This operation is performed on ice. After the sample loading is completed, continue to flow through the column with sample loading buffer until the baseline is maintained. The protein bound on the purification column is eluted with elution buffer (glycine solution at pH 3.2). After collecting the expressed protein, quickly adjust the pH of the protein to about 7 with neutralization buffer for subsequent experiments. After the protein is eluted, flow through the purification column with sample loading buffer to the baseline level and then continue to purify the subsequent protein. After all protein purification is completed, rinse the tubing and column with 0.1M NaOH to remove residual protein, rinse the tubing and column with deionized water, rinse the tubing and column with 20% alcohol, and then disassemble the column for subsequent protein purification. (2) Ultrafiltration buffer replacement: Replace the buffer of the protein solution with PBS in an ultrafiltration tube after pH adjustment, determine the protein concentration by BCA quantification, filter with a sterile 0.22μm filter membrane, aliquot and store at -80℃, avoiding repeated freeze-thaw cycles.

[0044] Example 3: Detection of biological activity at the PD-L1 antibody terminus in a bifunctional fusion protein

[0045] The inhibitory effects of anti-PD-L1-sST2 or sST2-anti-PD-L1 on the PD-1 / PD-L1 pathway in vitro were investigated using a NAFT / luciferase gene reporter system.

[0046] (1) CHOK1-PD-L1 cell plating: Collect CHOK1-PD-L1 cells in the logarithmic growth phase, adjust the cell concentration to 4×10^5 cells / mL, add 100μL of cells to each well of a pure white cell culture plate, and incubate overnight in a cell culture incubator.

[0047] (2) Protein dilution: anti-PD-L1, anti-PD-L1-sST2 and sST2-anti-PD-L1 were diluted to an initial concentration of 50 μg / mL with dilution buffer, and then serially diluted 2.5 times in 96-well plates for a total of 9 dilutions, with 2 replicates for each dilution.

[0048] (3) Jurkat-PD1-NFAT-Luc cell preparation: Jurkat-PD1-NFAT-Luc cells in the logarithmic growth phase were collected and the cell concentration was adjusted to 1.25×10^6 cells / mL for later use.

[0049] (4) Sample addition: Take out the 96-well plate with cells in (1), remove the cell culture medium from the plate, add 40 μL of the protein sample diluted in (2) using a multichannel pipette, and then add 40 μL of Jurkat-PD1-NFAT-Luc cells diluted in (3). After adding the samples, place the plate in a cell culture incubator for further culture.

[0050] (5) Color development: After culturing for 6h±15min, remove the 96-well plate from the cell culture incubator, equilibrate at room temperature for 5-10min, then add 80μL of Bio-Glo Luciferase assay Reagent pre-equilibrated to room temperature to each well, incubate at room temperature for 5-10min, and then read the plate for color development using a fluorescent microplate reader.

[0051] Test results as follows Figure 1As shown, anti-PD-L1-sST2, sST2-anti-PD-L1, and anti-PD-L1 all effectively blocked the binding of PD-1 to PD-L1. The EC50 values ​​of anti-PD-L1-sST2, sST2-anti-PD-L1, and anti-PD-L1 were 0.4 nM, 1.0 nM, and 0.5 nM, respectively. Compared to anti-PD-L1-sST2, sST2-anti-PD-L1 showed weaker biological activity in blocking PD-1 / PD-L1 signaling, while anti-PD-L1-sST2 exhibited comparable biological activity to anti-PD-L1.

[0052] Example 4: Detection of biological activity at the sST2 end of a bifunctional fusion protein

[0053] IL-33 can promote the differentiation and proliferation of Treg cells. To determine the blocking effect of the bifunctional fusion protein on IL-33, we sorted in vitro... CD4-positive T cells were stimulated and intervened. First, SPF-grade C57BL / 6 mice were euthanized by cervical dislocation, and single-cell suspensions of the spleen were extracted after soaking in 75% ethanol solution for 5 minutes. Then, according to the kit, single-cell suspensions of the spleen were used for stimulation and intervention. CD4 + T cell sorting. Sorted cells CD4 + T cells were resuspended in 1640 medium containing 4 μg / mL anti-CD28, 10 ng / mL TGF-β, and 100 U / mL IL-2, and seeded with 2 x 10^5 cells / 50 μL of cell suspension in 96-well plates coated overnight with 5 μg / mL anti-CD3. IL-33 (10 ng / mL), sST2-Fc (10 μg / mL), Anti-PD-1-sST2 (17.6 μg / mL), and sST2-anti-PD-1 (17.6 μg / mL) were added to different wells of the 96-well plates at final concentrations of 50 μL. After 3 days, cells treated with different methods were harvested for flow cytometry analysis of CD4+. + The percentage of Treg cells among T cells. The results showed that ( Figure 2 IL-33 can significantly improve the ability to... CD4 + Foxp3, differentiated from T cells + The percentage of cells, and this effect is completely offset in the presence of sST2-Fc, anti-PD-1-sST2, or sST2-anti-PD-1.

[0054] Example 5: Detection of biological activity at the hsST2 end of a bifunctional fusion protein

[0055] IL-33 can stimulate the proliferation and migration of HUVEC cells (Blood. 2009 Oct1; 114(14):3117-26.). The biological activity of the hsST2 terminus of the bifunctional fusion protein was determined by detecting its effect on IL-33-induced HUVEC cell proliferation. The proliferation assay was performed as follows: HUVEC cells were collected, resuspended in culture medium to 1×10^5 / mL, and 100 μL was added to a 96-well plate. After overnight incubation, IL-33 (20 ng / mL), hsST2-Fc (700 ng / mL), and the bifunctional fusion protein (1.23 μg / mL) were added to the cells. After incubation at 37°C and 5% CO2 for 36 hours, HUVEC cell proliferation was detected using the CCK-8 assay. Figure 3 As shown, the bifunctional fusion proteins can produce effects equivalent to hsST2-Fc. Among them, hsST2-anti-PD-L1 and hsST2-anti-PD-1 have better inhibitory effects on IL-33-induced HUVEC cell proliferation than hsST2-Fc.

[0056] Example 6: In vivo antitumor activity of bifunctional fusion protein

[0057] LLC subcutaneous xenograft model construction and efficacy evaluation: LLC cells, with a total amount of 5 × 10^5, were seeded subcutaneously in the right rib area of ​​C57BL / 6J mice in a resuspended volume of 100 μL. The tumors were transplanted until the tumor volume reached 50-100 mm². 3 Tumor-bearing mice were randomly divided into four groups: Control, anti-PD-L1+sST2-Fc, anti-PD-L1-sST2, and sST2-anti-PD-L1. The dosages were as follows: anti-PD-L1 (7.5 mg / kg), sST2-Fc (6.3 mg / kg), anti-PD-L1-sST2 (11.2 mg / kg), and sST2-anti-PD-L1 (11.2 mg / kg). Administration was via intraperitoneal injection, twice a week for a total of four times. Tumor growth was subsequently recorded using calipers, and the tumors were dissected and weighed at the end of the experiment. Figure 4 As shown, anti-PD-L1-sST2 exhibits superior antitumor activity compared to combination therapy and sST2-anti-PD-L1.

[0058] Construction of an LLC lung metastasis model and evaluation of drug efficacy: LLC cells were injected intravenously into C57BL / 6J mice at a rate of 2×10^5 cells / 100μL, and mice were randomly assigned to different groups for drug administration on the second day after inoculation. The dosages were: anti-PD-L1 (7.5 mg / kg), sST2-Fc (6.3 mg / kg), anti-PD-L1-sST2 (11.2 mg / kg), and sST2-anti-PD-L1 (11.2 mg / kg). Administration was by intraperitoneal injection, twice a week for a total of 4 times. Mice survival was observed and recorded after 4 administrations. Figure 5 As shown in Figure A, mice receiving anti-PD-L1 treatment and combination therapy survived to 72 days. After anti-PD-L1-sST2 treatment, 25% of the mice survived to 100 days. At 100 days, these mice were injected intravenously with a higher number of LLC cells, while a control group was established. Three weeks later, these mice were euthanized, and the formation of lung tumor metastases was observed. The results indicate that ( Figure 5 B) Compared with the control group, the formation of lung tumor foci was significantly reduced in the surviving mice in this group. These results indicate that the use of the bifunctional fusion protein anti-PD-L1-sST2, which simultaneously targets IL-33 and PD-L1, can produce a stronger anti-tumor effect than combination therapy, inhibiting tumor growth and metastasis.

[0059] Construction of MC38-hPD-L1 subcutaneous xenograft tumor model and evaluation of drug efficacy: MC38-hPD-L1 cells, with a total amount of 5 × 10^5, were seeded subcutaneously in the right rib area of ​​C57BL / 6J strain PD-1 humanized mice at a resuspended volume of 100 μL. On day 7 after seeding, the tumor volume reached 50-100 mm. 3 All tumor-bearing mice were randomly divided into 5 groups: control group, anti-PD-1 (5 mg / kg) group, sST2-Fc (4.2 mg / kg) group, anti-PD-1 (5 mg / kg) combined with sST2-Fc (4.2 mg / kg) group, and anti-PD-1-sST2 (7.5 mg / kg) group. Administered intraperitoneally on days 7, 11, and 14 post-inoculation. Tumor growth was subsequently recorded using calipers. Figure 6 As shown, after treatment, 1 / 5 of the mice in the anti-PD-1 treatment group achieved "Tumor free", while the anti-PD-1-sST2 treatment achieved "Tumor free" in 2 / 5 of the mice.

[0060] Example 7: Study on the in vivo antitumor mechanism of bifunctional fusion protein

[0061] Construct an LLC subcutaneous xenograft model until the tumor volume reaches 50-100 mm. 3 At that time, tumor-bearing mice were randomly divided into four groups: Control, anti-PD-L1, anti-PD-L1+sST2-Fc, and anti-PD-L1-sST2. Following the dosage described in Example 6, tumor tissue was harvested 10 days after the first administration for scRNA-seq detection. Cell clustering analysis was performed on the detection results, such as... Figure 7 As shown, for CD8 + Deep clustering of T cells yielded 6 CD8 groups. + T cell subsets. Statistical analysis of the proportions of all T cell subsets showed that, compared with anti-PD-L1 monotherapy and combination therapy, anti-PD-L1-sST2 reduced the proportion of exhausted CD8_S3 T cell subsets. Compared with anti-PD-L1, anti-PD-L1-sST2 increased the proportion of effector CD8 T cell subsets. Figure 8 As shown, compared with anti-PD-L1 monotherapy and combination therapy, anti-PD-L1-sST2 reduced the proportion of Treg cell subsets while increasing the proportion of effector CD4_S1 cell subsets. These results indicate that anti-PD-L1-sST2 can significantly increase the infiltration of effector T cells and reduce the infiltration of suppressor T cells in the TME.

[0062] Furthermore, the changes in T cell function were analyzed compared with the combination therapy of anti-PD-L1-sST2 and ant anti-PD-L1 and sST2-Fc. For example... Figure 9 As shown in Figure A, the upregulated genes in anti-PD-L1-sST2 are mainly enriched in pathways that activate the immune system response (T cell receptor signaling, TNF signaling pathway, Th17 cell differentiation, B cell receptor signaling pathway, and p53 signaling pathway). Meanwhile, the downregulated genes are mainly enriched in pathways that suppress the immune system response, such as the TGF-beta signaling pathway, NF-kappa B signaling pathway, and HIF-1 signaling pathway. Figure 9 B). These results indicate that anti-PD-L1-sST2 treatment promotes the remodeling of tumor-infiltrating immune cells, thereby enhancing anti-tumor immunity, especially CD8. + T cells and CD4 + T cells.

[0063] In summary, the working mode of the bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 of this invention is as follows: Figure 9As shown, the bifunctional fusion protein of the present invention can first block PD-1 / PD-L1 signaling, enhancing the recognition and killing effect of T cells on tumor cells; at the same time, it delivers the functional fragment targeting IL-33 to the tumor site through PD-1 / PD-L1 antibody, specifically binding to IL-33 in the tumor site, downregulating the level of IL-33 in the TME, inhibiting the effect of Tregs or TAM, and further upregulating the killing effect of T cells on tumor cells; it can also inhibit the secretion of TGF-β in the tumor microenvironment, remodel the tumor microenvironment, and prevent tumor metastasis and drug resistance.

[0064] The sequences mentioned above are summarized as follows:

[0065] The amino acid sequence of the heavy chain of atezolizumab (SEQ ID No. 1)

[0066] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0067] The amino acid sequence of the light chain of atezolizumab (SEQ ID No. 2)

[0068] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0069] Amino acid sequence of the heavy chain of nivolumab (SEQ ID No.3)

[0070] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0071] Amino acid sequence of the light chain of nivolumab (SEQ ID No.4)

[0072] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0073] The amino acid sequence of the linker peptide (SEQ ID No. 5)

[0074] GGGGSGGGGSGGGGS

[0075] Amino acid sequence of the extracellular domain of human ST2 (SEQ ID No. 6)

[0076] KFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYST VSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEV EIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPIDHHS

[0077] Amino acid sequence of the extracellular domain of mouse ST2 (SEQ ID No. 7)

[0078] SKSSWGLENEALIVRCPQRGRSTYPVEWYYSDTNESIPTQKRNRIFVSRDRLKFLPARVEDSGIYACVIRSPNLNKTGYLNVTIHKKPPSCNIPDYLMYSTVRGSDKNFKITCPTIDLYNWTAPVQWFKNCKALQEPRFRAHRSYLFIDNVTH DDEGDYTCQFTHAENGTNYIVTATRSFTVEEKGFSMFPVITNPPYNHTMEVEIGKPASIACSACFGKGSHFLADVLWQINKTVVGNFGEARIQEEEGRNESSSNDMDCLTSVLRITGVTEKDLSLEYDCLALNLHGMIRHTIRLRRKQPIDHR

[0079] The full-length amino acid sequence of the human soluble ST2 domain (SEQ ID No. 8)

[0080] KFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMT EDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPSKECF

[0081] The full-length amino acid sequence of the mouse soluble ST2 domain (SEQ ID No. 9)

[0082] SKSSWGLENEALIVRCPQRGRSTYPVEWYYSDTNESIPTQKRNRIFVSRDRLKFLPARVEDSGIYACVIRSPNLNKTGYLNVTIHKKPPSCNIPDYLMYSTVRGSDKNFKITCPTIDLYNWTAPVQWFKNCKALQEPRFRAHRSYLFIDNVTHDDEGDYTCQFTHAENGTNYIVTATRSFTVEEKGFSMFPVITNPPYNHTMEVEIGKPASIACSACFGKGSHFLADVLWQINKTVVGNFGEARIQEEEGRNESSSNDMDCLTSVLRITGVTEKDLSLEYDCLALNLHGMIRHTIRLRRKQPSKECPSHIA

[0083] Amino acid sequence of the signal peptide (SEQ ID No.10)

[0084] MGWSCIILFLVATATGVHS

Claims

1. A bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33, characterized in that, The bifunctional fusion protein is selected from one of the following two structures: Structure one: comprising an antibody fragment binding to PD-1, a functional fragment neutralizing IL-33 activity, and a connecting peptide connecting the two fragments together; in the structure one, PD-1 antibody-connecting peptide-growth stimulating expression gene 2 protein (ST2); ST2-connecting peptide-PD-1 antibody; PD-1 antibody-connecting peptide-IL-33 antibody IL-33 antibody-connecting peptide-PD-1 antibody; Structure two: comprising an antibody fragment binding to PD-L1, a functional fragment neutralizing IL-33 activity, and a connecting peptide connecting the two fragments together; in the structure two, PD-L1 antibody-connecting peptide-ST2; ST2-connecting peptide-PD-L1 antibody; PD-L1 antibody-connecting peptide-IL-33 antibody; IL-33 antibody-connecting peptide-PD-L1 antibody.

2. The bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 according to claim 1, characterized in that, The PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

3. The bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 according to claim 2, characterized in that, The amino acid sequence of the heavy chain of the atezolizumab is shown in SEQ ID No. 1, and the amino acid sequence of the light chain of the atezolizumab is shown in SEQ ID No.

2.

4. The bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 according to claim 1, characterized in that, The PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, tepilumab, sindilimab, and camrelizumab.

5. The bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 according to claim 4, characterized in that, The amino acid sequence of the heavy chain of the nivolumab is shown in SEQ ID No. 3, and the amino acid sequence of the light chain of the atezolizumab is shown in SEQ ID No.

4.

6. The bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 of claim 1, wherein, The amino acid sequence of the connecting peptide comprises [GlyGlyGlyGlySer]n, [GlySer]n, or Gly[GlySer]n, wherein n is any integer from 1 to 15.

7. The bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 of claim 1, wherein, The ST2 comprises an extracellular domain of ST2 protein or a soluble ST2 full-length domain.

8. A polynucleotide sequence, comprising, A nucleotide sequence encoding the bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 according to any one of claims 1-7.

9. An expression vector comprising the polynucleotide sequence according to claim 8.

10. A pharmaceutical composition, characterized by, The pharmaceutical composition is selected from any one of the following (A), (B), (C): (A) comprising the bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 according to any one of claims 1-7, and at least one pharmaceutical adjuvant; (B) comprising the polynucleotide sequence according to claim 8, and at least one pharmaceutical adjuvant; (C) comprising the expression vector according to claim 9, and at least one pharmaceutical adjuvant.

11. Use of the bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 according to any one of claims 1-7, the polynucleotide sequence according to claim 8, the expression vector according to claim 9, or the pharmaceutical composition according to claim 10 in the preparation of a kit for detecting or predicting a tumor.

12. Use of the bifunctional fusion protein targeting PD-1 / PD-L1 and IL-33 according to any one of claims 1-7, the polynucleotide sequence of claim 8, the expression vector of claim 9, or the pharmaceutical composition of claim 10 in the preparation of a medicament for treating a tumor.

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